阅读说明：本技术 新型特异性结合多肽及其用途 (Novel specific binding polypeptides and uses thereof ) 是由 M·欣纳 A·维登曼 A·艾乐斯道夫尔 于 2015-05-20 设计创作，主要内容包括：本发明涉及一种针对靶标Swiss Prot Q16552的新型、特异性结合治疗性和/或诊断性多肽,和针对靶标Swiss Prot Q9NPF7的新型、特异性结合治疗性和/或诊断性多肽。此外,本发明涉及针对Swiss Prot Q16552和SwissProt Q9NPF7之一或两者的新型、特异性结合治疗性和/或诊断性多肽。本发明还涉及编码这种多肽的核酸分子及用于产生这种多肽和核酸分子的方法。此外,本发明涉及包含该多肽的组合物及这些多肽的治疗性和/或诊断性用途。(The present invention relates to a novel, specific binding therapeutic and/or diagnostic polypeptide directed to the target Swiss Prot Q16552 and to a novel, specific binding therapeutic and/or diagnostic polypeptide directed to the target Swiss Prot Q9NPF 7. In addition, the present invention relates to novel, specific binding therapeutic and/or diagnostic polypeptides directed against one or both of Swiss Prot Q16552 and SwissProt Q9NPF 7. The invention also relates to nucleic acid molecules encoding such polypeptides and methods for producing such polypeptides and nucleic acid molecules. Furthermore, the invention relates to compositions comprising such polypeptides and to the therapeutic and/or diagnostic use of such polypeptides.)

1. A lipocalin mutein which binds IL-23p19 and/or IL-17A with detectable affinity.

2. The mutein of claim 1, wherein the mutein is a human lipocalin 2(hNGAL) mutein having binding specificity for IL-23p19, wherein the mutein comprises ten or more of the following amino acid residue mutations at sequence positions corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 43): gln 28 → His; leu 36 → Glu; ala 40 → Leu; ile 41 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Ala or Val; lys 75 → Thr; cys76 → Tyr or Arg; asp 77 → Lys; trp79 → Gln or Arg; arg 81 → Gly; cys 87 → Ser; asn 96 → Gly; lys 98 → Glu; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; asn 114 → Asp; met 120 → Ile; lys 125 → Tyr; ser 127 → Tyr; lys 134 → Glu; and Cys175 → Ala.

3. The mutein of claim 2, wherein:

(a) the mutein is capable of binding IL-23p19 with a KD of about 1nM or lower;

(b) the mutant protein with human IL-23 and mouse IL-23 cross reaction; and/or

(c) The mutant protein can inhibit the combination of IL-23 and its receptor IL-23R.

4. The mutein according to claim 2, wherein the mutein comprises one of the following sets of amino acid residue mutations compared to the mature hNGAL linear polypeptide sequence:

(a) gln 28 → His; leu 36 → Glu; ala 40 → Leu; ile 41 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Ala; lys 75 → Thr; cys76 → Tyr; asp 77 → Lys; trp79 → Gln; arg 81 → Gly; asn 96 → Gly; lys 98 → Glu; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; met 120 → Ile; lys 125 → Tyr; ser 127 → Tyr; and Lys 134 → Glu;

(b) gln 28 → His; leu 36 → Glu; ala 40 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Val; lys 75 → Thr; cys76 → Arg; asp 77 → Lys; trp79 → Arg; arg 81 → Gly; asn 96 → Gly; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; lys 125 → Tyr; ser 127 → Tyr; and Lys 134 → Glu; or

(c) Gln 28 → His; leu 36 → Glu; ala 40 → Leu; ile 41 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Val; lys 75 → Thr; cys76 → Tyr; asp 77 → Lys; trp79 → Gln; arg 81 → Gly; asn 96 → Gly; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; asn 114 → Asp; lys 125 → Tyr; ser 127 → Tyr; and Lys 134 → Glu.

5. The mutein of claim 2, wherein the mutein has at least 85% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2 and 45-46.

6. The mutein of claim 2, wherein the mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:2 and 45-46.

7. A fusion protein comprising at least two subunits, wherein one subunit is a mutein of any one of claims 1 to 6, and the other subunit:

(a) has IL-17A binding specificity;

(b) contains an Albumin Binding Domain (ABD) or an albumin binding peptide;

(c) is a human antibody Fc-portion; or

(d) Contains TNF-arrestin.

8. The fusion protein of claim 7, wherein one subunit of the fusion protein comprises a lipocalin mutein that binds IL-17A with a KD of about 1nM or lower.

9. The fusion protein of claim 7, wherein the fusion protein comprises a linker covalently linking one subunit comprising the lipocalin mutein to another subunit, or one subunit of the fusion protein is linked to another subunit of the fusion protein directly or via a chemical linker.

10. The mutein according to any one of claims 1 to 6 or the fusion protein according to any one of claims 7 to 9, wherein:

(a) the mutant protein or fusion protein is conjugated with a compound selected from the group consisting of an organic molecule, an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a cytostatic agent, a toxin, a metal complex, a metal, and colloidal gold;

(b) the mutein or fusion protein is fused at its N-terminus and/or its C-terminus to a moiety, which is a protein, protein domain or peptide; and/or

(c) The mutein or fusion protein is conjugated to a moiety that extends the serum half-life of the mutein or fusion protein.

11. Use of a mutein according to any one of claims 1 to 6 for the preparation of a composition for the diagnosis of diseases or disorders in which binding of IL-23p19 is useful for such diagnosis.

12. A method of detecting the presence of IL-23p19 in a sample, the method comprising: contacting the sample with the mutein of any one of claims 1 to 6 under conditions that allow the formation of a complex of the mutein and IL-23p 19.

13. Use of the mutein of any one of claims 1 to 6 or the fusion protein of any one of claims 7 to 9 for the preparation of a composition for binding IL-23p19 in a subject.

14. A nucleic acid molecule comprising a nucleotide sequence encoding the mutein of any one of claims 1 to 6 or the fusion protein of any one of claims 7 to 9.

15. A host cell comprising the nucleic acid molecule of claim 14.

16. A method of producing the mutein of any one of claims 1 to 6 or the fusion protein of any one of claims 7 to 9, wherein the mutein or the fusion protein is produced starting from a nucleic acid encoding the mutein or the fusion protein.

Background

Muteins of various lipocalins are a class of rapidly expanding therapeutic agents. Indeed, lipocalin muteins can be constructed to exhibit high affinity and specificity for targets that are different from the natural ligand of the wild-type lipocalin (e.g., interleukin-17A or interleukin-23) (e.g., WO 99/16873, WO 00/75308, WO 03/029463, WO 03/029471, and WO 05/19256).

A. Interleukin-17A

Interleukin-17A (IL-17A, synonymous with IL-17) is a cytokine produced from the Th17 line of T cells. IL-17 was originally named "CTL-associated antigen 8" (CTLA-8) (Rouvier et al, J.Immunol, 1505445-. A human equivalent of CTLA-8 was subsequently cloned and designated "IL-17" (Yao et al, J.Immunol,155(12):5483-5486 (1995); Fossiez et al, J.exp.Med.,183(6):2593-2603 (1996)).

Human IL-17A (CTLA-8, also designated IL-17, Swiss Prot Q16552) is a glycoprotein with a Mr of 17,000 daltons (Spriggs et al, J. Clin. Immunol,17: 366-. IL-17A may exist both as homodimer IL-17A/A and as a heterodimer of IL-17A/F that complexes with the homologous IL-17F to form a heterodimer. IL-17F (IL-24, ML-1) has 55% amino acid identity with IL-17A. IL-17A and IL-17F also have the same receptor (IL-17RA) that is expressed on a variety of cells including vascular endothelial cells, peripheral T cells, B cells, fibroblasts, lung cells, bone marrow mononuclear cells and bone stromal cells (Kolls et al, Immunity,21: 467-. Other homologues of IL-17 have been identified (IL-17B, IL-17C, IL-17D and IL-17E). These other family members have less than 30% amino acid identity with IL-17A (Kolls et al, 2004).

IL-17A is expressed primarily by Th17 cells and is present at elevated levels in the synovial fluid of Rheumatoid Arthritis (RA) patients, and has been shown to be involved in early RA development. IL-17A is also overexpressed in the cerebrospinal fluid of patients with Multiple Sclerosis (MS). Furthermore, IL-17 is an inducer of TNF- α and IL-1, the latter being responsible mainly for bone erosion and for the consequences of extreme pain in the affected patients (Lubberts E. (2008) Cytokine, pp 41, 84-91). In addition, inappropriate or excessive production of IL-17A has been associated with the pathology of a variety of other diseases and disorders, such as osteoarthritis, osteoimplant loosening, acute transplant rejection (Antonnysamy et al, (1999) J.Immunol, p.162, 577-.

Although a variety of IL-17A inhibitors have been described, the current approach is not optimal due to the discovery of this key proinflammatory cytokine, such as the necessity of a complex mammalian cell production system, dependence on disulfide stability, tendency of some antibody fragments to aggregate, limited solubility and, last but not least, even when humanized, these IL-17A inhibitors may elicit an undesirable immune response. Thus, there is still a need to develop proteins with IL-17A binding affinity, such as lipocalin muteins.

B. Interleukin-23

Interleukin-23 (also known as IL-23) is a heterodimeric cytokine consisting of two subunits, p19 and p40 (B. Oppmann et al, Immunity 13,715 (2000)). The p19(Swiss Prot Q9NPF7, interchangeably referred to herein as "IL-23 p 19") subunit is structurally related to the p35 subunit of IL-6, granulocyte colony stimulating factor (G-CSF) and IL-12. IL-23 through the combination of IL-23R and IL-12 beta 1 consisting of heterodimeric receptors to mediate signal transduction. IL-12 receptors composed of IL-12 β 1 and IL-12 β 2 share the IL-12 β 1 subunit. Transgenic p19 mice have recently been described to exhibit profound systemic inflammation and neutrophilia (m.t. wiekowski et al, J immunol166,7563 (2001)).

Human IL-23 has been reported to promote proliferation of T cells, particularly memory T cells, and may contribute to differentiation and/or maintenance of Th17 cells (d.m. frucht, Sci STKE 2002Jan 8; 2002(114): PE 1).

Although a variety of selective inhibitors of IL-23 have been described (by binding to the p19 subunit), these prior approaches still have a number of serious drawbacks due to the discovery of this key heterodimeric cytokine, such as the necessity of a complex mammalian cell production system, dependence on disulfide bond stability, tendency of some antibody fragments to aggregate, limited solubility and, last but not least, even when humanized, these selective inhibitors of IL-23 may elicit undesirable immune responses. Thus, there is still a need to develop proteins with IL-23 binding affinity, such as lipocalin muteins.

Definition of

The following list defines terms, phrases and abbreviations used in this specification. All terms listed and defined herein are intended to encompass all grammatical forms.

As used herein, "IL-17A" (including IL-17A/A and IL-17A complexed with IL-17F, also referred to as IL-17A/F) refers to the full-length protein defined by Swiss Prot Q16552, fragments thereof, or variants thereof.

As used herein, "IL-23 p 19" refers to the full-length protein defined by Swiss Prot Q9NPF7, fragments thereof, or variants thereof.

As used herein, "detectable affinity" refers to an affinity generally of at least about 10^-5The ability of the affinity constant of M to bind to a selected target. Lower affinities are generally no longer measurable by conventional methods, such as ELISA, and are therefore of secondary importance. For example, the binding affinity of a lipocalin mutein according to the invention may in some embodiments be a K below 800nM or_DIn some embodiments 30nM or less_DAnd in some embodiments about 50 picomolar (pM) or less.

As used herein, K of a mutein-ligand complex can be measured (and thus can be determined) by a variety of methods known to those skilled in the art_DValue) proteins of the invention (e.g., lipocalins)Muteins of a protein) or a fusion protein thereof with a selected target (in the present invention, IL-17A or IL-23p 19). Such methods include, but are not limited to, fluorescence titration, competition ELISA, calorimetry such as Isothermal Titration Calorimetry (ITC), and surface plasmon resonance (BIAcore). Such methods are well known in the art, and embodiments thereof are also detailed below.

It is also noted that the formation of complexes between the respective binding agents and their ligands is influenced by a number of different factors, such as the concentration of the respective binding partner, the presence of competitors, the pH and the ionic strength of the buffer system employed, and also for determining the dissociation constant K_DSuch as fluorescence titration, competitive ELISA or surface plasmon resonance, to name a few, or even mathematical algorithms for evaluating experimental data.

Thus, K will also be clear to the skilled person_DThe values (dissociation constants of the complexes formed between the respective binders and their targets/ligands) may vary within a certain experimental range, depending on the method and experimental setup used to determine the affinity of a particular lipocalin mutein for a given ligand. This means that the measured K_DThe value may be slightly deviated or within tolerance, for example, depending on the K_DValues were determined by surface plasmon resonance (Biacore), competition ELISA, or "direct ELISA".

As used herein, "mutein", "mutated" entity (protein or nucleic acid) or "mutant" refers to the exchange, deletion or insertion of one or more nucleotides or amino acids compared to the "reference" backbone of a naturally occurring (wild-type) nucleic acid or protein.

The term "fragment" as used herein in relation to the muteins of the invention relates to a protein or peptide derived from full length mature human tear lipocalin shortened at the N-terminus and/or C-terminus (i.e. lacking at least one of the N-terminal and/or C-terminal amino acids). Such fragments may comprise at least 10, more such as 20 or 30 or more consecutive amino acids of the primary sequence of the mature lipocalin and are typically detectable in an immunoassay for the mature lipocalin. The term also includes fragments of the muteins and variants described herein. The lipocalin muteins, fragments or variants thereof of the present invention preferably retain the function of binding IL-17A and/or IL23p19 as described herein.

In general, the term "fragment" as used herein in relation to the corresponding protein ligand IL-17A (including IL-17A/A and IL-17A/F) or IL-23p19 of a lipocalin mutein of the invention or a composition according to the invention or a fusion protein described herein relates to N-terminally and/or C-terminally shortened protein or peptide ligands which retain the ability of the full-length ligand to be recognized and/or bound by a mutein according to the invention.

The term "mutagenesis" as used herein means that the experimental conditions are chosen such that an amino acid naturally occurring at a given position in the mature lipocalin sequence can be replaced by at least one amino acid not occurring at this particular position in the respective native polypeptide sequence. The term "mutagenesis" also includes (additional) changes in the length of a sequence segment by deletion or insertion of one or more amino acids. Therefore, the following is within the scope of the invention: for example, one amino acid at a selected sequence position is replaced by three randomly mutated stretches (stretch), resulting in the insertion of two amino acid residues compared to the length of each segment of the wild-type protein. Such deletions or insertions may be introduced independently of one another into any peptide segment which can be subjected to mutagenesis in the context of the present invention. In an exemplary embodiment of the invention, the insertion of several mutations may be introduced into the AB loop of a selected lipocalin scaffold (see international patent application WO 2005/019256, incorporated herein by reference in its entirety).

The term "random mutagenesis" means that no predetermined single amino acid (mutation) is present at a certain sequence position, but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.

"identity" refers to a sequence property that measures their similarity or relationship. The term "sequence identity" or "identity" as used herein refers to the percentage of paired identical residues relative to the number of residues in the longer of the two sequences after alignment of the polypeptide sequence of the invention with the sequence in question (homology). Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the result by 100.

The term "homology" is used herein in its ordinary sense and includes identical amino acids as well as conservatively substituted amino acids (e.g., aspartic acid residues for glutamic acid residues) considered equivalent positions in the linear amino acid sequence of a polypeptide of the invention (e.g., any lipocalin mutein of the invention).

The percentage of sequence homology or sequence identity can be determined, for example, using the programs BLASTP, BLASTP version 2.2.5 (16.11.2002; see Altschul, S.F. et al (1997) Nucl. acids Res.25, 3389-3402). In this embodiment, the percentage homology is based on a complete polypeptide sequence alignment including the propeptide sequence (matrix: BLOSUM 62; gap weight: 11.1; cut-off set to 10^-3) Preferably, the wild-type protein scaffold is used as a reference in the pairwise comparison. The BLASTP program output is expressed as a percentage calculated as the number of "positives" (homologous amino acids) divided by the total number of amino acids selected by the program for alignment.

In particular, to determine whether an amino acid residue of a lipocalin (mutein) amino acid sequence different from the wild-type lipocalin corresponds to a certain position in the wild-type lipocalin amino acid sequence, the skilled person may use means and methods well known in the art, such as aligning manually or by using a computer program, e.g. using BLAST2.0 (which represents a basic local alignment search tool) or ClustalW or any other suitable program suitable for generating a sequence alignment. Thus, the wild-type lipocalin may serve as "test sequence" or "reference sequence", while the amino acid sequence of a lipocalin different from the wild-type lipocalin described herein serves as "query sequence". The terms "reference sequence" and "wild-type sequence" are used interchangeably herein.

A "gap" is the space in the alignment resulting from the addition or deletion of amino acids. Thus, two identical sequences have 100% identity, but sequences that are less highly conserved and have additions, deletions or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, such as Blast (Altschul et al (1997) Nucleic Acids Res.25,3389-3402), Blast2(Altschul et al (1990) J.mol.biol.215,403-410) and Smith-Waterman (Smith et al (1981) J.mol.biol.147, 195-197).

The term "variant" as used in the present invention relates to a derivative of a protein or peptide comprising an amino acid sequence modification, for example by substitution, deletion, insertion or chemical modification. In some embodiments, such modifications do not reduce the function of the protein or peptide. Such variants include proteins in which one or more amino acids are replaced by their respective D-stereoisomers or amino acids other than the naturally occurring 20 amino acids (such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, pentanine). However, such substitutions may also be conservative, i.e. an amino acid residue is replaced by a chemically similar amino acid residue. Examples of conservative substitutions are substitutions between the following group members: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. The term "variant" as used herein in relation to the lipocalin muteins of the invention or the corresponding protein ligands IL-17A (including IL-17A/a and IL-17A/F) or IL-23p19 of the compositions according to the invention or the fusion proteins described herein, respectively, relates to IL-17 proteins or fragments thereof or IL-23 proteins or fragments thereof having one or more (such as 1,2, 3, 4, 5, 6,7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more) amino acid substitutions, deletions and/or insertions compared to the wild-type IL-17A or IL-23p19 protein (e.g. the SwissProt preserved IL-17A or IL-23p19 reference proteins described herein), respectively. The IL-17A or IL-23p19 variant preferably has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% amino acid identity to the wild-type IL-17A or IL-23p19 protein (e.g., the SwissProt-preserved IL-17A or IL-23p19 reference protein described herein), respectively.

"native sequence" lipocalin refers to a lipocalin having the same amino acid sequence as a corresponding polypeptide derived from nature. Thus, a native sequence lipocalin may have the amino acid sequence of a corresponding naturally occurring lipocalin derived from any organism, in particular a mammal. Such native sequence polypeptides may be isolated from nature or may be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally occurring truncated or secreted forms, naturally occurring variant forms (e.g., spliced forms and naturally occurring allelic variants of a lipocalin) of a lipocalin. A polypeptide "variant" refers to a biologically active polypeptide having at least about 50%, 60%, 70%, or at least about 85% amino acid sequence identity to a native sequence polypeptide. Such variants include, for example, polypeptides having one or more amino acid residues added or deleted from the N-or C-terminus of the polypeptide. Typically, a variant has at least about 70%, including at least about 80%, such as at least about 85%, amino acid sequence identity to a native sequence polypeptide; including at least about 90% amino acid sequence identity or at least about 95% amino sequence identity. As an illustrative example, the first 4N-terminal amino acid residues (HHLA) and the last 2C-terminal amino acid residues (Ser, Asp) may be deleted, for example in the tear lipocalin (Tlc) muteins of the present invention, such as SEQ ID NO:1, without affecting the biological function of the protein.

The term "position" when used in accordance with the present invention refers to a position of an amino acid in an amino acid sequence described herein or a position of a nucleotide in a nucleic acid sequence described herein. In order to understand the term "corresponding" or "corresponding" as used herein in the context of the position of one or more lipocalin mutein amino acid sequences, the corresponding position is not only determined by the number of the aforementioned nucleotides/amino acids. Thus, the position of a given amino acid of the invention that may be substituted may be altered due to amino acid deletions or additions at any position of the (mutant or wild-type) lipocalin. Similarly, the position of a given nucleotide that may be substituted may be altered due to deletion or addition of nucleotides at other positions in the mutein or wild-type lipocalin 5' -untranslated region (UTR), including the promoter and/or any other regulatory sequences or genes, including exons and introns.

Thus, for the corresponding positions of the present invention, it is preferably understood that the positions of nucleotides/amino acids may differ in the indicated numbers, but still have similar adjacent nucleotides/amino acids, but that the corresponding position or positions still comprise the adjacent nucleotides/amino acids, which may be exchanged, deleted or added.

Furthermore, for the corresponding positions of the lipocalin muteins based on the reference scaffold of the present invention, it is preferably understood that the positions of the nucleotides/amino acids correspond in structure to other positions of the (mutant or wild-type) lipocalin, although these positions may differ from the indicated numbers, the skilled person will understand that this is due to the highly conserved overall folded form in the lipocalin.

The term "albumin" includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat serum albumin.

The term "organic molecule" or "small organic molecule" as used herein with respect to a non-natural target means an organic molecule comprising at least two carbon atoms, but preferably, comprising no more than 7 or 12 rotatable carbon bonds; such organic molecule has a molecular weight in the range of 100 to 2000 daltons, preferably 100 to 1000 daltons, and it may optionally contain one or two metal atoms.

The word "detecting/detectable" as used herein is understood to mean at both a quantitative and a qualitative level, as well as combinations thereof. Thus it includes quantitative, semi-quantitative and qualitative determination of the target molecule.

The term "subject" refers to a vertebrate, preferably a mammal, more preferably a human. The term "mammal" as used herein refers to any animal classified as a mammal, including, but not limited to, humans, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgous monkey, to name a few illustrative examples. Preferably, the mammal described herein is a human.

An "effective amount" is an amount sufficient to achieve a beneficial or desired result. An effective amount may be administered in one or more administrations.

A "sample" is defined as a biological sample taken from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue.

A "fusion polypeptide" as described herein comprises at least two subunits, one of which has IL-17A binding specificity or IL-23p19 binding specificity. In the fusion polypeptide, the subunits may be linked by covalent or non-covalent binding (linkage). Preferably, the fusion polypeptide is a translational fusion between two or more subunits. Translational fusions can be generated by genetically engineering the coding sequence of one subunit with the coding sequence of another subunit in frame. Two subunits may be interspersed with a nucleotide sequence encoding a linker. However, the subunits of the fusion polypeptides of the invention may also be linked by chemical linkers.

A "linker" that may be included in a fusion polypeptide of the invention links two or more subunits of a fusion polypeptide described herein. This binding may be covalent or non-covalent. Preferred covalent bonding is through peptide bonds, for example between amino acids. Thus, in preferred embodiments, the linker comprises one or more amino acids, for example 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Preferred linkers are described herein. Other preferred linkers are chemical linkers.

Drawings

FIG. 1: typical measurements of the association-rate and dissociation-rate of the interaction of the lipocalin mutein of SEQ ID NO:1 with human IL-17A by surface plasmon resonance are provided. Immobilization of IL-17A to the sensor Using Standard amine chemistryOn a chip, and the lipocalin mutein of SEQ ID NO:1 was used as the soluble analyte flowing through the chip surface. Resulting dissociation constant (K)_D) It was 0.8 nM.

FIG. 2: typical measurements of the association-rate and dissociation-rate of the interaction of the lipocalin mutein of SEQ ID NO:1 with human IL-17A/F by surface plasmon resonance are provided. Biotinylated SEQ ID NO:1 was captured on a sensor chip using a proprietary assay kit and human IL-17A/F was used as the soluble analyte that flowed across the chip surface. Resulting dissociation constant (K)_D) Is 100 pM.

FIG. 3: the lipocalin mutein of SEQ ID NO:1 was shown to be able to block the interaction between hIL-17A and its receptor hIL-17RA with an IC50 of 75 pM. Biotinylated hIL-17A was pre-incubated with variable concentrations of the lipocalin mutein of SEQ ID NO:1 and the non-neutralized hIL-17A was quantified on ELISA plates with immobilized soluble hIL-17 RA. The negative control SEQ ID NO 41 has NO competitive effect. The data were fitted with a single-site binding model.

FIG. 4: shows the cross-reactivity profile of the lipocalin mutein of SEQ ID NO:1 measured in a competition ELISA format. Compared to hIL-17A, complete cross-reactivity with cynomolgus monkey (cynomolgus monkey) IL-17A and marmoset IL-17A was evident from the almost identical IC 50. The data were fitted with a single-site binding model.

FIG. 5: it was demonstrated that the lipocalin mutein of SEQ ID NO:1 efficiently blocks the binding of hIL-17A to its receptor hIL-17RA in cell-based assays. The assay is based on hIL-17A-induced secretion of G-CSF in U87-MG cells. Cells were incubated with a fixed concentration of hIL-17A and titrated with the lipocalin mutein of SEQ ID NO:1, or as a comparison, with reference antibody 1 (heavy chain SEQ ID NO:53, light chain SEQ ID NO:54), with reference antibody 2 (heavy chain SEQ ID NO:55, light chain SEQ ID NO:56), and SEQ ID NO:2 as a negative control. Will be composed of MSD (Meso Scale)Hereinafter referred to as "MSD") is measured against a G-CSF concentration in arbitrary unitsConcentration of lipocalin muteins or antibody molecules was plotted. The resulting lipocalin mutein of SEQ ID NO:1 had an average IC50 value of 0.13nM (0.17 nM in the first experiment, 0.10nM in the repeated experiments), which was significantly more potent than benchmark 1 showing an average IC50 ═ 2.33(2.65/2.01) nM, and in a similar range compared to benchmark 2 with an average IC50 ═ 0.12(0.14/0.10) nM. The negative control SEQ ID NO. 2 had NO effect on IL-17A-induced cellular G-CSF production. Binding of IL-17A to SEQ ID NO 1 or the reference antibody molecule blocks the binding of IL-17A to cell surface IL-17RA and thus prevents the induction of G-CSF secretion. The data were fitted with a single-site binding model, assuming equal G-CSF concentrations for all molecules were smoothed.

FIG. 6: typical measurements of the association-rate and dissociation-rate of the interaction of the lipocalin mutein of SEQ ID NO:2 with human IL-23 by surface plasmon resonance are provided. The mean dissociation constant determined in three replicates was K_D＝0.35±0.20nM。

FIG. 7: typical measurements of the association-rate and dissociation-rate of the interaction of the lipocalin mutein of SEQ ID NO:2 with human IL-23 by surface plasmon resonance are provided. Biotinylated SEQ ID NO:2 was captured on a sensor chip using a proprietary assay kit and IL-23 was used as the soluble analyte that flowed across the chip surface. Resulting dissociation constant (K)_D) It was 2.9 nM. Note that high non-physiological concentrations of NaCl must be used in order to perform the analysis. Thus, the results do not represent the affinity of SEQ ID NO 2 for IL-23 under physiological conditions. The utility of this assay is that it allows comparison of the affinity between SEQ ID NO:2 and a fusion protein comprising the mutein (see example 11 and Table 1).

FIG. 8: 2 lipocalin muteins were shown to be able to block the interaction between hIL-23 and its receptor hIL-23R with an IC50 of 0.54 nM. Biotinylated hIL-23 was pre-incubated with variable concentrations of the lipocalin mutein of SEQ ID NO. 2, and non-neutralized hIL-23 was quantified on ELISA plates with immobilized soluble hIL-23R. The negative control SEQ ID NO 43 has NO competitive effect. The data were fitted with a single-site binding model.

FIG. 9: shows the cross-reactivity profile and specificity of the lipocalin mutein of SEQ ID NO 2 measured in a competition ELISA format. The lipocalin mutein of SEQ ID NO:2 is fully cross-reactive with human and mouse IL-23 and shows a slightly reduced affinity for cynomolgus monkey and marmoset IL-23. The data were fitted with a single-site binding model.

FIG. 10: it was demonstrated that the lipocalin mutein of SEQ ID NO:2 is able to block the biological activity of hIL-23 in a cell-based proliferation assay. In this assay, SEQ ID NO:2, an IgG isotype negative control, and two reference antibodies (reference 3: heavy chain SEQ ID NO:57 and light chain SEQ ID NO: 58; reference 4: heavy chain SEQ ID NO:59 and light chain SEQ ID NO:60) were pre-incubated with hIL-23, and then Ba/F3 cells transfected with hIL-23R and hIL-12R β 1 were added. Transfected Ba/F3 cells proliferated in response to human IL-23. Experiments have shown that the biological activity is blocked by SEQ ID NO 2 and reference antibodies 3 and 4 with EC50 values of 1.2nM (1.7/0.7), 3.0nM (3.1/2.9), 1.2nM (0.8/1.5), respectively. The negative control had no effect on cell proliferation. The data were fitted with a sigmoidal dose-response model.

FIG. 11: a graphical overview of the constructs characterized in Table 1, SEQ ID NOs 1-13, is provided. SEQ ID NO:1 (abbreviated as "1" in FIG. 11) corresponds to the IL-17A-binding lipocalin mutein. SEQ ID NO:2 (abbreviated as "2") corresponds to the IL-23-binding lipocalin mutein. SEQ ID NO:14 (abbreviated as "14") corresponds to the albumin binding domain of streptococcal protein G. SEQ ID NO:15 (abbreviated "15") is an engineered, deimmunized version of SEQ ID NO: 14. SEQ ID NO:16 (abbreviated as "16") corresponds to the Fc-portion of the human IgG1 antibody.

FIG. 12: it was demonstrated in an exemplary experiment that a multi-specific fusion protein based on the lipocalin muteins disclosed herein was capable of simultaneously binding IL-17A, IL-23 and Human Serum Albumin (HSA) without interference from the respective other target bound. SEQ ID NO 9 is a heterodimeric fusion protein of IL-17A binding lipocalin mutein SEQ ID NO 1, IL-23 binding lipocalin mutein SEQ ID NO 2 and human serum albumin binding peptide derived from the albumin binding domain of streptococcal protein G. In the surface plasmon resonance experiment shown in FIG. 12, biotinylated SEQ ID NO 9 was captured on the sensor chip. To demonstrate simultaneous binding, dilutions of hIL-17AF, hIL-23, and HAS in buffer were applied serially to the prepared chip surface. The application of hIL-17AF, hIL-23 and HAS to immobilized SEQ ID NO. 9 was also performed using a single target to obtain the maximum binding levels obtainable by binding a single target for comparison. Figure 12 shows the measured binding curves and the theoretical binding curves reflecting the expected response to full binding of all three targets. The latter was obtained by pooling the experimental responses of SEQ ID NO 9 to each target. The measured and theoretical curves are almost identical, the differences shown are due to dissociation of the target in the experimental curves. The data indicate that SEQ ID No. 9 is capable of binding all targets simultaneously without loss of signal intensity or kinetic changes compared to binding only a single target.

FIG. 13: typical measurements of the association-rate and dissociation-rate of the lipocalin muteins SEQ ID NO:45 (FIG. 13A) and SEQ ID NO:46 (FIG. 13B) binding to human IL-23 by surface plasmon resonance are provided. Resulting dissociation constant (K)_D) 0.1nM (SEQ ID NO:45) and 0.6nM (SEQ ID NO:46), respectively.

FIG. 14: lipocalin muteins SEQ ID NO:45 and SEQ ID NO:46 were shown to be able to block the interaction between hIL-23 and its receptor hIL-23R with an IC50 of 0.1nM (SEQ ID NO:45) and 1.1nM (SEQ ID NO:46), respectively. Biotinylated hIL-23 was pre-incubated with variable concentrations of the lipocalin mutein and non-neutralized hIL-23 was quantified on ELISA plates with immobilized soluble hIL-23R. The data were fitted with a single-site binding model.

FIG. 15: it was demonstrated that the lipocalin muteins of SEQ ID NO:45 and SEQ ID NO:46 were able to block the biological activity of hIL-23 in a cell-based proliferation assay. In this assay, SEQ ID NO 45, SEQ ID NO 46 and the negative control SEQ ID NO 43 were preincubated with hIL-23 and then Ba/F3 cells transfected with hIL-23R and hIL-12R β 1 were added. Transfected Ba/F3 cells proliferated in response to human IL-23. Experiments showed that the biological activity was blocked by SEQ ID NO 45, SEQ ID NO 46 with IC50 values of 3.7nM and 5.4nM, respectively. The negative control SEQ ID NO:43 had NO effect on cell proliferation. The data were fitted with a sigmoidal dose-response model.

FIG. 16: representative experiments are provided to determine the specificity of the lipocalin muteins of SEQ ID NO:63 and 62 and SEQ ID NO:1 for the target IL-17A. Biotinylated IL-17A was captured on microtiter plates and the test molecules were titrated. Bound test molecules were detected by HRP-labeled anti-human TLc-specific antibody as described in example 16. Data were fitted with a 1:1 binding model with EC50 values and maximum signal as free parameters (free parameter) and a slope fixed to unity.

FIG. 17: representative experiments are provided to determine the specificity of the lipocalin muteins of SEQ ID NO:64 and 62 and SEQ ID NO:2 for the target IL-23. Biotinylated IL-23 was captured on microtiter plates and the test molecules were titrated. Bound test molecules were detected by HRP-labeled anti-human NGAL-specific antibody as described in example 17. Data were fitted with a 1:1 binding model with EC50 values and maximum signal as free parameters (free parameter) and a slope fixed to unity.

FIG. 18: representative experiments are provided to determine the specificity of polypeptide fusions of SEQ ID NOs 63 and 62 and of polypeptide fusions of SEQ ID NOs 64 and 62 and of antibodies of SEQ ID NOs 61 and 62 for the target TNF- α. TNF- α was coated on a microtiter plate and the test molecules were titrated. Bound test molecules were detected by HRP-labeled anti-human IgG Fc-specific antibody as described in example 18. Data were fitted with a 1:1 binding model with EC50 values and maximum signal as free parameters (free parameter) and a slope fixed to unity.

FIG. 19: representative experiments are provided to determine the ability of the polypeptide fusions of SEQ ID NOs 63 and 62 and the polypeptide fusions of SEQ ID NOs 64 and 62 to bind simultaneously to the targets TNF- α and IL-17A, TNF- α and IL-23, respectively. Recombinant TNF- α was coated onto microtiter plates and the polypeptide fusions were subsequently titrated. Thereafter, a constant concentration of biotinylated IL-17A or IL-23 was added and detected by HRP-labeled extravidin as described in example 19. Data were fitted with a 1:1 binding model with EC50 values and maximum signal as free parameters (free parameter) and a slope fixed to unity.

Detailed Description

The present invention provides to the prior art a polypeptide or protein having binding specificity for IL-17A and/or IL-23p19, wherein said polypeptide comprises a lipocalin mutein that binds IL-17A or IL-23p19 with at least detectable affinity.

In some embodiments, the polypeptide is a lipocalin mutein capable of binding IL-17A with at least detectable affinity. In some embodiments, the polypeptide is a lipocalin mutein capable of binding IL-23p19 with at least detectable affinity. The invention also relates to the use of two polypeptides for binding IL-17A and IL-23p19 in a subject.

In some aspects, the polypeptide is a fusion protein comprising at least two subunits, wherein one subunit has IL-17A binding specificity and the other subunit has IL-23p19 binding specificity. In some further embodiments, the fusion protein may further comprise a subunit, wherein the subunit has IL-23p19 or IL-17A binding specificity. In some still further embodiments, the fusion protein can comprise one IL-17A-specific subunit, one IL-23p 19-specific subunit, and one subunit comprising a bacterial Albumin Binding Domain (ABD).

In some other aspects, the polypeptides of the invention may also be fusion proteins comprising at least two IL-17A-specific subunits, or fusion proteins comprising at least two IL-23p 19-specific subunits.

In some embodiments, the subunit of the fusion protein with binding specificity for IL-17A comprises an IL-17A-specific lipocalin mutein of the invention. In some embodiments, the subunit of the fusion protein having IL-23p19 binding specificity comprises an antibody that binds IL-23p 19. In some other embodiments, the subunit of the fusion protein having IL-23p19 binding specificity comprises a lipocalin mutein of the invention specific for IL-23p 19. In some embodiments, the subunit of the fusion protein having IL-17A binding specificity comprises an antibody that binds IL-17A.

The polypeptide or protein of the invention may be a mutein of a lipocalin, preferably a lipocalin selected from the group consisting of: retinol-binding protein (RBP), bile pigment-binding protein (BBP), apolipoprotein D (APO D), neutrophil gelatinase-associated lipocalin (NGAL), tear lipocalin (TLPC or Tlc), alpha₂-microglobulin-related protein (A2m), 24p3/uterocalin (24p3), von Ebners gland protein 1(VEGP 1), von Ebners gland protein 2(VEGP 2) and major allergen Can f1 precursor (ALL-1).

As used herein, "lipocalin" is defined as a monomeric protein weighing about 18-20kDA, having a cylindrical β -pleated sheet supersecondary structural region comprising a plurality of (preferably 8) β -strands connected pair-by-pair by a plurality of (preferably 4) loops at one end, to thereby define a binding pocket. In members of the lipocalin family, it is this diversity of the loops in the otherwise rigid lipocalin backbone that leads to a variety of different binding patterns, each of which can accommodate targets of different size, shape and chemical characteristics (e.g.Flower, D.R, supra, (1996); Flower, D.R., et al, supra, (2000), or reviewed in Skerra, A. (2000) Biochim. Biophys. acta1482, 337-350). In fact, the lipocalin family of proteins has evolved naturally to bind a wide range of ligands, with very low levels of overall sequence conservation (typically with less than 20% sequence identity), but with the entire folding pattern of high conservation retained. The correspondence between different lipocalin positions is well known to the person skilled in the art. See, for example, U.S. Pat. No.7,25,0297.

As mentioned above, a lipocalin is a polypeptide defined by its supersecondary structure, i.e. comprising 8 β -strands connected pair by 4 loops at one end to thereby define a cylindrical β -pleated sheet supersecondary structure region of the binding pocket. The present invention is not limited to the lipocalin muteins specifically disclosed herein. In this aspect, the invention relates to lipocalin muteins having a cylindrical β -pleated sheet supersecondary structural region comprising 8 β -strands connected pair by 4 loops at one end to thereby define a binding pocket; wherein at least one amino acid of each of at least 3 of the 4 loops has been mutated, and wherein the lipocalin protein is effective to bind IL-17A or IL-23p19 with detectable affinity.

In a particular embodiment, the lipocalin mutein of the invention is a mutein of human tear lipocalin (TLPC or Tlc) (also known as lipocalin-1, pre-lacrimal-albumin or von Ebner gland protein). The term "human tear lipocalin" or "Tlc" or "lipocalin-1" as used herein refers to mature human tear lipocalin with the SWISS-PROT/UniProt database accession number P31025 (isoform 1). The amino acid sequence shown in SWISS-PROT/UniProt database accession number P31025 can be used as a preferred "reference sequence", more preferably the amino acid sequence shown in SEQ ID NO:41 is used as a reference sequence.

The present invention also encompasses a Tlc mutein as defined above, wherein the first 4N-terminal amino acid residues of the mature human tear lipocalin sequence (His-His-Leu-Leu; positions 1-4) and/or the last 2C-terminal amino acid residues (Ser-Asp; position 157-158) of the linear polypeptide sequence of mature human tear lipocalin (SWISS-PROT/UniProt database accession number P31025) are deleted (SEQ ID NO: 2-5). Furthermore, the present invention encompasses a Tlc mutein as defined above, wherein one GH loop amino acid residue (Lys) corresponding to sequence position 108 of the linear polypeptide sequence of mature human tear lipocalin is deleted (SEQ ID NO:1 and SEQ ID NO: 43). Another possible mutation of the wild-type polypeptide sequence of mature human tear lipocalin is the change of the amino acid sequence at sequence positions 5 to 7(Ala Ser Asp) to Gly Asp as described in PCT application WO 2005/019256, which is incorporated herein by reference in its entirety.

The Tlc muteins of the present invention may further comprise the amino acid substitution Arg 111 → Pro. The Tlc muteins of the invention may also comprise the substitution Lys 114 → Trp. It may also comprise the substitution Cys101 → Ser or Cys101 → Thr. In some preferred embodiments, the Tlc muteins of the present invention may further comprise the substitution Cys153 → Ser.

Modification of the amino acid sequence includes direct mutagenesis of individual amino acid positions in order to simplify subcloning of the mutated lipocalin gene or part thereof by introducing cleavage sites for certain restriction enzymes. In addition, can also introduce these mutations to further improve Tlc mutant protein to IL-17A or IL-23p19 affinity. In addition, if desired, mutations can be introduced in order to modulate certain characteristics of the mutein, such as for example to improve folding stability, serum stability, protein resistance or water solubility, or to reduce the tendency to aggregate. For example, a naturally occurring cysteine residue may be mutated to another amino acid to prevent disulfide bridge formation. Exemplary possibilities for such mutations that introduce cysteine residues into the amino acid sequence of the Tlc mutein include the following substitutions: thr 40 → Cys, Glu 73 → Cys, Arg 90 → Cys, Asp 95 → Cys and Glu 131 → Cys. Thiol moieties generated at any of amino acid positions 40, 73, 90, 95 and/or 131 can be used to pegylate or HES the mutein, e.g., to increase the serum half-life of the corresponding Tlc mutein.

In another particular embodiment, the lipocalin mutein disclosed herein is a mutein of human lipocalin 2. The term "human lipocalin 2" or "human Lcn 2" or "human NGAL" as used herein refers to mature human neutrophil gelatinase-associated lipocalin (NGAL) with SWISS-PROT/UniProt database accession number P80188. The invention of the human lipocalin 2 mutant protein can also be named "hNGAL mutant protein". The amino acid sequence shown in SWISS-PROT/UniProt database accession number P80188 is preferably used as a "reference sequence", more preferably the amino acid sequence shown in SEQ ID NO. 43 is used as a reference sequence.

In some embodiments, a lipocalin mutein that binds IL-17A or IL-23p19 with detectable affinity may comprise at least one amino acid substitution of the native cysteine residue with another amino acid (e.g., a serine residue). In some other embodiments, a lipocalin mutein that binds IL-17A or IL-23p19 with detectable affinity may comprise one or more non-native cysteine residues replacing one or more amino acids of the wild-type lipocalin. In a further particular embodiment, the lipocalin mutein of the invention comprises at least two amino acid residue substitutions replacing the natural amino acid with a cysteine residue, thereby forming one or more cysteine bridges. In some embodiments, the cysteine bridge may link at least two loop regions. The definitions of these regions are used herein in accordance with Flower (Flower,1996, supra; Flower et al, 2000, supra) and Breustedt et al (2005, supra).

The proteins of the invention directed against or specific for IL-17A or IL-23p19 comprise any number of specific binding protein muteins based on a defined protein backbone. Preferably, the number of nucleotides or amino acids exchanged, deleted or inserted is 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more (such as 25, 30, 35, 40, 45 or 50), respectively, wherein preferably 1,2, 3, 4, 5, 6,7, 8, 9, 10 or 11 and even more preferably 9, 10 or 11. However, it is preferred that the lipocalin muteins of the invention are still capable of binding IL-17A or IL-23p19, in particular human IL-17A or human IL-23p 19.

In one aspect, the invention comprises various lipocalin muteins that bind IL-17A or IL-23p19 with at least detectable affinity. In this sense, IL-17A or IL-23p19 may be considered to be a non-natural ligand with reference to the wild-type lipocalin, wherein "non-natural ligand" refers to a compound which is unable to bind to the wild-type lipocalin under physiological conditions. By engineering wild-type lipocalins with one or more mutations at certain sequence positions, the inventors of the present invention have demonstrated that high affinity and high specificity for non-natural ligands, such as IL-17A or IL-23p19, is possible. In some embodiments, random mutagenesis may be performed by substitution at certain sequence positions on the coding wild-type lipocalin with a subset of nucleotide triplets (subset) at those positions on 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or even more nucleotide triplets at those positions.

Furthermore, the lipocalin muteins of the invention may have mutated amino acid residues at any one or more (including at least at any 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12) of the sequence positions corresponding to certain sequence positions of the linear polypeptide sequence of the reference lipocalin.

The proteins of the invention may comprise wild type (native) amino acid sequences of the "parent" protein backbone (e.g. lipocalin) outside the mutated amino acid sequence positions. In some embodiments, the lipocalin muteins of the invention may also carry one or more amino acid mutations at sequence positions, as long as such mutations do not hinder or interfere, at least substantially, with the binding activity and folding of the mutein. This mutation can be accomplished very simply at the DNA level using established standard methods (Sambrook, J. et al (2001) Molecular Cloning: A Laboratory Manual,3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Illustrative examples of amino acid sequence alterations are insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e. an amino acid residue is replaced by an amino acid residue that is chemically (particularly with respect to polarity and size) similar. Examples of conservative substitutions are substitutions between members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. Alternatively, non-conservative changes may be introduced into the amino acid sequence. Furthermore, instead of replacing a single amino acid residue, one or more consecutive amino acids may also be inserted or deleted from the primary structure of human tear lipocalin, as long as these deletions or insertions result in a stable folded/functional mutein (e.g., a Tlc mutein with truncated N-and C-termini). In such muteins, for example, one or more amino acid residues are added or deleted at the N-or C-terminus of the polypeptide. Generally, such muteins have an amino acid sequence identity of about at least 70% (including at least about 80%, such as at least about 85%) with the amino acid sequence of mature human tear lipocalin. As an illustrative example, the present invention also encompasses a Tlc mutein as defined above wherein the first 4N-terminal amino acid residues of the mature human tear lipocalin sequence (His-His-Leu-Leu; positions 1-4) and/or the last 2C-terminal amino acid residues of the linear polypeptide sequence of mature human tear lipocalin (Ser-Asp; position 157-158) are deleted (SEQ ID NO:1 and SEQ ID NO: 43).

The amino acid sequences of the lipocalin muteins disclosed herein have high sequence identity with the reference lipocalin when compared to other lipocalins for sequence identity. In this general case, the amino acid sequence of a lipocalin mutein of the invention is at least substantially similar to the amino acid sequence of a reference lipocalin, provided that gaps (as defined below) due to the addition or deletion of amino acids may be present in the alignment. In some embodiments, a corresponding sequence of a lipocalin mutein of the invention that is substantially similar to the sequence of a reference lipocalin has at least 70% identity or sequence homology, at least 75% identity or sequence homology, at least 80% identity or sequence homology, at least 82% identity or sequence homology, at least 85% identity or sequence homology, at least 87% identity or sequence homology, or at least 90% identity or sequence homology (including at least 95% identity or sequence homology) with the sequence of the reference lipocalin mutein, provided that the altered position or sequence is retained and one or more gaps are possible.

As used herein, since binding specificity is not absolute, but rather a relative quality, a lipocalin mutein of the invention is capable of "specifically binding" a target (e.g., IL-17A or IL-23p19) if it is capable of distinguishing such target from one or more reference targets. "specific binding" was determined according to Western blot, ELISA-, RIA-, ECL-, IRMA-assay, FACS, IHC and peptide scan.

In one embodiment, the lipocalin mutein of the invention is fused at its N-terminus and/or its C-terminus to a fusion partner that is a protein domain that extends the serum half-life of the mutein. In further particular embodiments, the protein domain is an Fc portion of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding domain, an albumin binding peptide, and an albumin binding protein.

In another embodiment, the lipocalin muteins of the invention are conjugated to a compound that extends the serum half-life of said mutein. More preferably, the mutein is conjugated to a compound selected from the group consisting of: a polyalkylene glycol molecule, hydroxyethyl starch, an Fc portion of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding domain, an albumin binding peptide, and an albumin binding protein.

In yet another embodiment, the invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding a lipocalin mutein disclosed herein. The invention encompasses host cells comprising the nucleic acid molecules.

The Tlc muteins of the present invention can be obtained by means of mutagenesis of naturally occurring forms of human tear lipocalin. The invention of hNGAL mutant protein can be obtained by mutagenesis of human lipid carrier protein 2 natural forms. In some embodiments of such mutagenesis, the substitution (or substitution) is a conservative substitution. However, any substitution (including non-conservative substitutions or one or more exemplary substitutions derived from below) is envisaged as long as the lipocalin mutein retains its ability to bind IL-17A or IL-23p19 and/or the lipocalin mutein has identity to the sequence which is then substituted, i.e. the lipocalin mutein has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more identity to the amino acid sequence of mature human tear lipocalin or mature human lipocalin 2, respectively.

A. Lipocalin muteins with binding affinity for interleukin-17A (IL-17A, synonymous with IL-17)

In one aspect, the invention provides human lipocalin muteins that bind human IL-17A (identical to "IL-17") and useful uses thereof. The binding proteins described herein can bind to human IL-17A homodimers (identical to "IL-17A/A") and/or heterodimers of human IL-17A and human IL-17F homologs (identical to "IL-17A/F"). The invention also provides methods of making the IL-17A binding proteins described herein, as well as compositions comprising such proteins. The IL-17A binding proteins of the invention and compositions thereof can be used in methods of detecting IL-17A (including IL-17A/A and IL-17A/F) in a sample or in methods of binding IL-17A (including IL-17A/A and IL-17A/F) in a subject. Such human lipocalin muteins having these characteristics of utility provided in connection with the present invention have not been described previously.

One embodiment of the invention relates to a lipocalin mutein which is capable of passing a K of about 1nM or less (e.g., 0.8nM) when measured in an assay essentially as described in example 1_DThe measured affinity binds to interleukin-17A (IL-17A).

In some other embodiments, the lipocalin mutein is capable of inhibiting the binding of IL-17A to its receptor IL-17RA with an IC50 value of about 100pM or less (e.g., 75pM) in a competition ELISA format essentially as described in example 3.

In some particular embodiments, the IL-17A-binding lipocalin mutein cross-reacts with human IL-17A, cynomolgus monkey IL-17A and marmoset IL-17A.

In some yet further embodiments, the lipocalin muteins of the invention are capable of blocking the binding of IL-17A to its receptor IL-17 RA. In some further embodiments, the lipocalin mutein has an average EC50 value that is at least as good as (i.e. differs by less than 0.1nM) or better than the EC50 value of the reference antibody when the lipocalin mutein and the reference antibody are measured in an assay essentially as described in example 5. In some embodiments, the reference antibody is a polypeptide comprising (i) SEQ ID NO:53 or 55 as a first subunit and (ii) SEQ ID NO:54 or 56 as a second subunit. Lipocalin muteins can have an average IC50 value of about 0.13nM or even lower in the assay, while reference antibodies have an EC50 value of about 2.33nM or lower (e.g., about 0.12nM) in the assay.

In some other embodiments, for example, when in an assay essentially as described in example 1, K is passed through a lipocalin mutein of SEQ ID NO:42_DLower K of the first of said lipocalin muteins_DMeasurably, the IL-17A-binding lipocalin muteins of the present invention are capable of binding IL-17A with a higher affinity than the lipocalin mutein of SEQ ID NO: 42. In some further embodiments, for example, the IL-17A-binding lipocalin muteins of the invention are capable of inhibiting the binding of IL-17A to its receptor IL-17RA with a lower EC50 value than the lipocalin mutein of SEQ ID NO:42, when measured in an assay essentially as described in example 5.

1. Exemplary Lipocalin muteins with Interleukin-17A (IL-17A) binding-affinity

In one aspect, the invention relates to novel specific-binding human tear lipocalin muteins that are directed against or specific for interleukin-17A (IL-17A). The human tear lipocalin muteins disclosed herein may be used for therapeutic and/or diagnostic purposes. The human tear lipocalin muteins of the present invention may also be designated herein as "Tlc muteins". As used herein, a Tlc mutein of the invention is capable of "specifically binding" a target (e.g., here IL-17A) if it is capable of distinguishing between such target and one or more reference targets, since binding specificity is not absolute, but rather a relative property. "specific binding" was determined according to Western blot, ELISA-, RIA-, ECL-, IRMA-assay, FACS, IHC and peptide scan.

Thus, the invention provides one or more Tlc muteins capable of passing a K of about 10nM, about 1nM, about 0.1nM or lower_DThe measured affinity binds to interleukin-17A (IL-17A). More preferably, the Tlc mutein may have a K passing through about 1nM, 0.8nM, 0.6nM, 100pM or less_DThe affinity of the measurement.

In some particular embodiments, such a Tlc mutein has mutated amino acid residues at one or more positions corresponding to the linear polypeptide sequence of mature human tear lipocalin (SWISS-PROT database accession number P31025; SEQ ID NO:41) at positions 26-33, 56, 58, 60-61, 64, 92, 101, 104-106, 108, 111, 114 and 153.

In a further particular embodiment, such a Tlc mutein may further have mutated amino acid residues at one or more of the positions corresponding to positions 101, 111, 114 and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 41).

In some further embodiments, the Tlc mutein comprises at least 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or even more mutated amino acid residues at one or more of the sequence positions corresponding to sequence positions 26, 27, 28, 29, 30, 31, 32, 33, 56, 58, 60, 61, 64, 92, 101, 104, 105, 106, 108, 111, 114 and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 41).

In some still further embodiments, the invention relates to a polypeptide, wherein said polypeptide is a Tlc mutein comprising at least 2,3, 4, 5, 6,7, 8, 9, 10, 11, 12 or even more mutated amino acid residues at sequence positions 26-33, 56, 58, 60-61, 64, 92, 101, 104-106, 108, 111, 114 and 153 as compared to the linear polypeptide sequence of mature human tear lipocalin, and wherein said polypeptide binds to IL-17A, in particular to human IL-17A.

In some embodiments, the lipocalin muteins of the invention may comprise at least one amino acid substitution replacing a native cysteine residue with, for example, a serine residue. In some embodiments, the Tlc mutant protein of the present invention comprises an amino acid substitution that replaces a native cysteine residue at position 61 and/or 153 with a serine residue. It is noted in this context that it has been found (at the level of the corresponding native (naive) nucleic acid library) that removal of the structural disulfide bond of wild-type tear lipocalin formed by cysteines 61 and 153 (see breustdet, et al, 2005, supra) can provide tear lipocalin muteins that not only stably fold, but also bind with high affinity to a given non-native ligand. Without wishing to be bound by theory, it is also believed that the elimination of structural disulfide bonds provides the further advantage of allowing (spontaneous) generation of non-native artificial disulfide bonds or their deliberate introduction into the muteins of the invention, thereby increasing the stability of said muteins. For example, in some embodiments, the Tlc muteins of the present invention comprise an amino acid substitution that replaces the native cysteine residue at position 101 with a serine residue. Further, in some embodiments, the muteins of the present invention comprise an amino acid substitution that replaces the native arginine residue at position 111 with a proline residue. In some embodiments, a mutant protein of the invention comprises an amino acid substitution that replaces the native lysine residue at position 114 with a tryptophan residue.

The Tlc muteins of the invention may further comprise one or more (including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13 or at least 14) amino acid substitutions replacing the native amino acid residue with a cysteine residue at any of positions 26-33, 56, 58, 60-61, 64, 92, 101, 104-106, 108, 111, 114 and 153 of the mature human tear lipocalin protein relative to the amino acid sequence of the mature human tear lipocalin protein (WISS-PROT database accession number P31025).

In some embodiments, the muteins of the present invention comprise an amino acid substitution at position 28 or 105 with a cysteine residue instead of the natural amino acid with respect to the amino acid sequence of mature human tear lipocalin. In some embodiments, the muteins of the present invention comprise an amino acid substitution at position 28 or 105 with a cysteine residue instead of the natural amino acid with respect to the amino acid sequence of mature human tear lipocalin. In a further particular embodiment, the mutein of the invention comprises amino acid substitutions at positions 28 and 105 with respect to the amino acid sequence of mature human tear lipocalin, replacing the natural amino acid by two cysteine residues.

In some embodiments, the Tlc muteins of the present invention comprise an amino acid in which at least one or two of the cysteine residues present at each of sequence positions 61 and 153 is replaced by another amino acid, and comprise a mutation of at least three amino acid residues at any of sequence positions 26-33, 56, 58, 60-61, 64, 92, 101, 104 and 106, 108, 111, 114 and 153 of the linear polypeptide sequence of mature human tear lipocalin (SWISS-PROT database accession number P31025). Positions 26-34 are contained in the AB loop, position 55 is at the extreme end of the beta-sheet, and subsequent positions 56-58 and 60-61 and 64 are contained in the CD loop. Position 104-108 is contained in the GH loop in the binding site at the open end of the β -barrel structure of mature human tear lipocalin. The definition of these regions is used herein in accordance with Flower (Flower,1996, supra; Flower et al, 2000, supra) and Breustedt et al (2005, supra). In some embodiments, the Tlc muteins of the invention comprise the amino acid substitutions Cys61 → Ala, Phe, Lys, Arg, Thr, Asn, Gly, gin, Asp, Asn, Leu, Tyr, Met, Ser, Pro, or Trp and Cys153 → Ser or Ala. This substitution has been shown to be useful in preventing the formation of naturally occurring disulfide bridges linking Cys61 and Cys153 and thus facilitating handling of the mutein. However, tear lipocalin muteins that bind IL-17A and have a disulfide bridge formed between Cys61 and Cys153 are also part of the present invention.

In some embodiments, the IL-17A binding Tlc muteins of the present invention comprise one or more of the following mutated amino acid residues at any one or more of sequence positions 26-33, 56, 58, 60-61, 64, 92, 101, 104-106, 108, 111, 114 and 153 of mature human tear lipocalin (SEQ ID NO: 41): arg 26 → Phe; glu 27 → Trpl; phe 28 → Cys; pro 29 → Ser; glu 30 → Gly; met 31 → Ile; asn 32 → His; leu 33 → Glu; leu56 → Asp; ser 58 → Glu; arg 60 → Phe; cys61 → Leu; val 64 → Phe; his92 → Arg; cys101 → Ser; glu 104 → Asp; leu 105 → Cys; his 106 → Pro; lys108 deletion; arg 111 → Pro; lys 114 → Trp; and Cys153 → Ser. In some embodiments, the Tlc muteins of the present invention comprise two or more, such as 3, 4, 5, 6,7, 8, or all mutated amino acid residues at these sequence positions of the mature human tear lipocalin.

In a further particular embodiment, the Tlc muteins of the present invention comprise an amino acid sequence as shown in any one of SEQ ID No. 1 or fragments or variants thereof.

In further particular embodiments, the Tlc muteins of the present invention have at least 75%, at least 80%, at least 85% or more identity to an amino acid sequence selected from SEQ ID No. 1.

The present invention also encompasses structural homologues of a Tlc mutein having an amino acid sequence selected from SEQ ID No. 1, said structural homologues having greater than about 60%, preferably greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92% and most preferably greater than 95% sequence homology or sequence identity to said Tlc mutein.

In some particular embodiments, the invention provides for passing a K of about 1nM or less_DA lipocalin mutein that binds IL-17A with a measured affinity, wherein the lipocalin mutein has at least 90% or more, such as 95% identity with the amino acid sequence of SEQ ID NO: 1.

2. Use of lipocalin muteins with interleukin-17A (IL-17A) binding affinity

IL-17A is a pro-inflammatory cytokine produced by a subset of memory T cells (termed Th17) that is involved in the pathogenesis of a number of disorders, such as Multiple Sclerosis (MS) (Helling, P.W., et al, am.J.Resp.cell mol.biol.28(2003) 42-50; Matusevicius, D.et al, Multiple Sclerosis5(1999)101-104), Rheumatoid Arthritis (RA) (Ziolkovska, M.et al, J.Immunol.164(2000) 32-38; Kotake, S.et al, J.Clin.Invest.103(1999) 1345-52; Helling, P.W., et al, am.J.Resp.cell mol.biol.28(2003) 42-50). IL-17A plays a role in the induction of other inflammatory cytokines, chemokines and adhesion molecules (Komiyama, Y. et al, J.Immunol.177(2006)566-573), psoriasis, Crohn's disease, Chronic Obstructive Pulmonary Disease (COPD), asthma and transplant rejection.

IL-17 is involved in inducing pro-inflammatory responses and in inducing or mediating the expression of a variety of other cytokines, factors and mediators, including tissue necrosis factor-alpha (TNF-alpha), IL-6, IL-8, IL-1 beta, granulocyte colony-stimulating factor (G-CSF), prostaglandin E2(PGE2), IL-10, IL-12, IL-IR antagonists, leukemia inhibitory factor, and stromelysin (Yao et al, J.Immunol,155(12):5483-5486 (1995); Fossiez et al, J.exp.Med.,183(6):2593-2603 (1996); Jovanovic et al, J.Immunol,160:3513-3521 (1998); Tenuissen et al, J.Invig.Imrmatol, 111:645-649 (1998); Chaudba et al, J.Immunol, 414: 409) and 414 (1998)). IL-17A also induces nitric oxide in chondrocytes and human osteoarthritic explants (Shalom-Barak et al, J.biol chem.,273: 27467-. Through the role of IL-17A in T cell-mediated autoimmunity, IL-17A induces the release of cytokines, chemokines and growth factors (as described above), which are important local regulators of neutrophil accumulation and play a role in cartilage and bone destruction. There is increasing evidence that targeting IL-17A signaling may be beneficial in a variety of autoimmune diseases including Rheumatoid Arthritis (RA), psoriasis, crohn's disease, Multiple Sclerosis (MS), psoriatic disease, asthma, and Systemic Lupus Erythematosus (SLE) (see, e.g., agrwal et al, j. leuloc. biol,71(1), (1) -1-8 (2002); luets et al, "Treatment with a lubricating anti-tissue and the set of collagen-induced Arthritis reduction tissue, cartilage breakdown, and bone surgery," Arthritis rheum, 50:650 659 (2004)).

Furthermore, it is known in the art that inflammatory and immunomodulatory processes are involved in the pathogenesis of various forms of cardiovascular disease (Biasucci, L., et al, Circulation 1999,99: 855-. Current studies have established the basis for treating cardiovascular disease by reducing the inflammatory and immunomodulatory response of the disease (Blankenberg, S., et al, Circulation2002,106: 24-30; Mallat, Z., et al, Circulation 2001,104: 1598-. Cardiovascular disease encompasses a variety of disorders that affect the muscles and/or blood vessels of the heart, peripheral blood vessels, muscles, and various organs.

Thus, many possible applications for the Tlc muteins of the present invention exist in medicine. In another aspect, the invention relates to the use of the disclosed Tlc muteins for detecting IL-17A (including IL-17A/A and IL-17A/F) in a sample, as well as corresponding diagnostic methods.

The invention also relates to the use of one or more Tlc muteins as described in for forming a complex with IL-17A.

Thus, in another aspect of the invention, the disclosed muteins are used for the detection of IL-17A. Such use may include the steps of: contacting one or more of said muteins with a sample suspected of containing IL-17A under suitable conditions, thereby allowing a complex to form between said mutein and IL-17A, and detecting said complex by means of a suitable signal.

The detectable signal may be generated by a label as described above, or by a change in physical properties due to binding (i.e., complex formation) itself. One example is plasmon surface resonance, the magnitude of which changes during binding of binding partners, one of which is immobilized on a surface, such as gold foil.

The muteins disclosed herein can also be used to isolate IL-17A. Such use may include the steps of: contacting one or more of said muteins with a sample suspected of containing IL-17A under suitable conditions, thereby allowing a complex to form between said mutein and IL-17A, and isolating said complex from said sample.

In the use of the disclosed muteins for the detection of IL-17A and for the isolation of IL-17A, the muteins and/or IL-17A or domains or fragments thereof can be immobilized on a suitable solid phase.

In yet another aspect, the invention features a diagnostic or analytical kit comprising a Tlc mutein of the invention.

In addition to its use in diagnosis, in a further aspect, the invention encompasses the use of a mutein of the invention or a composition comprising such a mutein for binding IL-17A in a subject and/or inhibiting the binding of IL-17A to its receptor in a subject.

In yet another aspect, the invention features a method of binding IL-17A in a subject, the method comprising administering to the subject an effective amount of one or more lipocalin muteins of the invention or one or more compositions comprising such muteins.

In yet another aspect, the present invention relates to a method of inhibiting the binding of IL-17 to its receptor in a subject, said method comprising administering to said subject an effective amount of one or more lipocalin muteins of the invention or one or more compositions comprising such muteins.

In the context of the present invention, the disclosed lipocalin muteins with IL-17A binding affinity may bind IL-17A present as homodimers, but such muteins may also bind IL-17A present as heterodimers which complex with homologous IL-17F to form heterodimeric IL-17A/F. In a preferred embodiment, a lipocalin mutein of the invention can bind IL-17A complexed with IL-17F with detectable affinity.

B. Lipocalin muteins with binding affinity for interleukin-23 p19(IL-23p19)

Furthermore, the present invention fulfills the need for alternative inhibitors of IL-23p19 by providing human lipocalin muteins that bind to human IL-23p19 and their beneficial uses. Thus, the invention also provides methods of making and using the IL-23p 19-binding proteins described herein, as well as compositions that can be used in methods of detecting IL-23p19 in a sample or in methods of binding IL-23p19 in a subject. Such lipocalin muteins having these characteristics of use provided in connection with the present invention have not been described before.

One embodiment of the present invention relates to a fatA lipocalin mutein capable of passing a K of about 1nM or lower (e.g., about 0.6nM) when measured in an assay essentially as described in example 6 or example 13_DThe affinity measured binds to interleukin-23 p19(IL-23p 19).

In some other embodiments, the lipocalin mutein is capable of inhibiting the binding of IL-23 to its receptor IL-23R with an IC50 value of about 0.55nM or lower in a competition ELISA format essentially as described in example 8 or example 14.

In some particular embodiments, the lipocalin mutein is cross-reactive with both human IL-23 and mouse IL-23.

In some yet further embodiments, the lipocalin muteins of the invention are capable of inhibiting the binding of IL-23 to its receptor IL-23R. In some further embodiments, the lipocalin mutein has an average EC50 value that is at least as good as (i.e. differs by less than 1.0nM) or better than the average EC50 value of the reference antibody when the lipocalin mutein and the reference antibody are measured in an assay essentially as described in example 10 or example 15. In some embodiments, the reference antibody is a polypeptide comprising (i) SEQ ID NO:57 or 59 as a first subunit and (ii) SEQ ID NO:58 or 60 as a second subunit. Lipocalin muteins can have an average EC50 value in the assay of about 1.2nM or even lower, while reference antibodies have an EC50 value in the assay of about 3nM or lower (e.g., about 1.2 nM).

In some other embodiments, the IL-23p 19-binding lipocalin mutein of the invention is more biophysically stable than the lipocalin mutein of SEQ ID NO: 44.

1. Exemplary Lipocalin muteins with binding affinity for Interleukin-23 p19(IL-23p19)

In one aspect, the invention relates to novel specific-binding human lipocalin 2(Lcn2 or NGAL) against or specific for interleukin-23 p19(IL-23p 19). The human lipocalin 2 muteins disclosed herein may be used for therapeutic and/or diagnostic purposes. The human lipocalin 2 muteins of the invention may also be designated herein as "NGAL muteins". As used herein, a Tlc mutein of the invention is capable of "specifically binding" a target (here IL-23p19) if it is capable of distinguishing between the target and one or more reference targets, since the binding specificity is not absolute, but rather a relative property. "specific binding" was determined according to Western blot, ELISA-, RIA-, ECL-, IRMA-assay, FACS, IHC and peptide scan.

In this regard, the invention provides one or more NGAL muteins capable of passing a K of about 10nM or less_DThe affinity measured binds to interleukin-23 p19(IL-23p 19). More preferably, the NGAL mutein may have a K lower by about 1nM_DThe measured affinity.

In some embodiments, the hNGAL mutant protein in accordance with the invention includes substitutions at one or more of the positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134 and 175 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 43).

In a particular embodiment, the lipocalin muteins of the invention comprise at least 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or even more substitutions at sequence positions corresponding to sequence positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134 and 175 of the linear polypeptide sequence of mature hNGAL (SWISS-PROT database accession number P80188; SEQ ID NO: 43). Preferably, the present invention contemplates a lipocalin mutein comprising, in addition to one or more substitutions at positions corresponding to positions 36, 87 and/or 96 of the mature human NGAL linear polypeptide sequence, a substitution at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134 and 175 of the mature hNGAL linear polypeptide sequence.

In some still further embodiments, the invention relates to a polypeptide, wherein the polypeptide is a hNGAL mutein comprising at least 1,2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or even more mutated amino acid residues at sequence positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134, and 175 as compared to the linear polypeptide sequence of mature hNGAL, and wherein the polypeptide binds to IL-23p19, in particular to human IL-23p 19.

In some embodiments, the IL-23p 19-binding hNGAL mutant protein of the invention comprises one or more of the following mutated amino acid residues at any one or more of sequence positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134 and 175 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 43): gln 28 → His; leu 36 → Glu; ala 40 → Leu; ile 41 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Ala or Vla; lys 75 → Thr; asp 77 → Lys; trp79 → Gln or Arg; arg 81 → Gly; asn 96 → Gly; lys 98 → Glu; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; asn 114 → Asp; met 120 → Ile; lys 125 → Tyr; ser 127 → Tyr; and Lys 134 → Glu. In some embodiments, the invention of the hNGAL mutant protein in the mature hNGAL at these sequence positions including two or more, such as 3, 4, 5, 6,7, 8, 9, 10, 11, 12, even more or all of the mutant amino acid residues.

In addition, the invention of the IL-23p 19-binding hNGAL mutant protein can also contain the following replacement: cys 87 → Ser. In addition, the invention of the IL-23p 19-binding hNGAL mutant protein can also contain the following replacement: cys76 → Tyr or Arg. In addition, the invention of the IL-23p 19-binding hNGAL mutant protein can also contain the following replacement: cys175 → Ala.

In some additional embodiments, and mature hNGAL linear polypeptide sequence compared, the invention of the binding of IL-23p19 hNGAL mutant protein includes the following amino acid substitution group of one:

(a) gln 28 → His; leu 36 → Glu; ala 40 → Leu; ile 41 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Ala; lys 75 → Thr; cys76 → Tyr; asp 77 → Lys; trp79 → Gln; arg 81 → Gly; asn 96 → Gly; lys 98 → Glu; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; met 120 → Ile; lys 125 → Tyr; ser 127 → Tyr; and Lys 134 → Glu;

(b) gln 28 → His; leu 36 → Glu; ala 40 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Vla; lys 75 → Thr; cys76 → Arg; asp 77 → Lys; trp79 → Arg; arg 81 → Gly; asn 96 → Gly; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; lys 125 → Tyr; ser 127 → Tyr; and Lys 134 → Glu; or

(c) Gln 28 → His; leu 36 → Glu; ala 40 → Leu; ile 41 → Leu; gln 49 → Arg; tyr 52 → Thr; asn 65 → Asp; ser 68 → Arg; leu 70 → Glu; arg 72 → Gly; lys 73 → Val; lys 75 → Thr; cys76 → Tyr; asp 77 → Lys; trp79 → Gln; arg 81 → Gly; asn 96 → Gly; tyr 100 → Met; leu 103 → Met; tyr 106 → Phe; asn 114 → Asp; lys 125 → Tyr; ser 127 → Tyr; and Lys 134 → Glu.

In the remaining region, i.e. different from the sequence position 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 75-77, 79, 81, 87, 96, 98, 100, 103, 106, 114, 120, 125, 127, 134 and 175, in the mutation of amino acid sequence positions, the hNGAL mutant protein can contain wild-type (natural) amino acid sequence.

In a further particular embodiment, the lipocalin mutein of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2 and 45-46 or a fragment or variant thereof.

The IL-23p 19-binding hNGAL mutant protein amino acid sequence can have a high sequence identity with a sequence selected from SEQ ID NO. 2 and 45-46, such as at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90% identity, including at least 95% identity.

The invention also includes selected from the group consisting of SEQ ID NO:2 and 45-46 amino acid sequence of the hNGAL mutant protein structure homolog, the structural homolog and the hNGAL mutant protein has greater than about 60%, preferably greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92% and most preferably greater than 95% of the amino acid sequence homology or sequence identity.

In some particular embodiments, the invention provides for passing a K of about 1nM or less_DThe measured affinity binds to a lipocalin mutein of IL-23p19, wherein the lipocalin mutein has at least 90% or more, such as 95% identity with the amino acid sequence of SEQ ID NO: 2.

2. Use of lipocalin muteins with interleukin-23 p19(IL-23p19) binding affinity

Interleukin-23 (IL-23) is a heterodimeric cytokine consisting of a unique subunit p19 (interchangeably referred to herein as "IL-23 p 19") and a p40 subunit shared with interleukin-12 (IL-12) (Oppmann, Immunity 13:115 (2000)). It has been found that IL-23 stimulates the production and/or maintenance of IL-17A and IL-17F from activated CD 4T cells, which CD 4T cells are now referred to as a "new" T-helper cell (Th) subset, designated Th 17. Discussion of IL-23 cytokine and receptor biology is reviewed in Holscher, curr, Opin Invest, drugs 6:489(2005) and Langrish et al, Immunol Rev.202:96 (2004). Similar to the Th1 and Th2 lines, Th17 cells most likely evolve to provide adaptive immunity to specific types of pathogens (e.g., extracellular bacteria). However, inappropriate Th17 responses have been strongly implicated in increasing autoimmune disorders, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and psoriasis.

In this regard, IL-23 promotes a diverse CD 4T cell activation state characterized by the production of interleukin-17 (J.biol.chem.278:1910-191 (2003; see also Langrish et al). IL-23 drives a pathogenic T cell population that induces autoimmune inflammation (J.Exp.Med.201: 233-.

Thus, the invention of the IL-23p19 binding affinity of the mutant protein many possible applications exist in medicine. In another aspect, the invention relates to the use of such a mutein as disclosed for the detection of IL-23p19 in a sample as well as a corresponding diagnostic method.

The invention also relates to the use of one or more muteins having binding affinity for IL-23p19 as described for forming a complex with IL-23p 19.

Thus, in a further aspect of the invention, the disclosed muteins are used for the detection of IL-23p 19. Such use may include the steps of: contacting one or more of said muteins with a sample suspected of containing IL-23p19 under suitable conditions, thereby allowing the formation of a complex between said mutein and IL-23p19, and detecting said complex by means of a suitable signal.

The detectable signal may be generated by a label as described above, or by a change in physical properties due to binding (i.e., complex formation) itself. One example is plasmon surface resonance, the magnitude of which changes during binding of binding partners, one of which is immobilized on a surface, such as gold foil.

The muteins disclosed herein can also be used to isolate IL-23p 19. Such use may include the steps of: contacting one or more of said muteins with a sample suspected of containing IL-23p19 under suitable conditions, thereby allowing a complex to form between said mutein and IL-23p19, and isolating said complex from said sample.

In the disclosed use of the muteins for the detection of IL-23p19 and for the isolation of IL-23p19, the muteins and/or IL-23p19 or domains or fragments thereof can be immobilized on a suitable solid phase.

Thus, the presence or absence of a molecule (e.g., IL-23p19) as in a sample, as well as its concentration or level, can be determined.

In yet another aspect, the invention features a diagnostic or analytical kit comprising a mutein of the invention having binding affinity for IL-23p 19.

In addition to its use in diagnosis, in a further aspect, the invention encompasses the use of such a mutein of the invention or a composition comprising such a mutein for binding IL-23p19 in a subject and/or inhibiting the binding of IL-23p19 to its receptor in a subject.

In yet another aspect, the invention features a method of binding IL-23p19 in a subject, the method comprising administering to the subject an effective amount of one or more lipocalin muteins of the invention having IL-23p19 binding affinity or one or more compositions comprising such muteins.

In yet another aspect, the present invention relates to a method for inhibiting the binding of IL-23 to its receptor in a subject, said method comprising administering to said subject an effective amount of one or more lipocalin muteins of the invention having binding affinity for IL-23p19 or one or more compositions comprising such muteins.

C. Compositions comprising IL-17A-binding lipocalin muteins and/or IL-23p 19-binding lipocalin muteins and uses of said lipocalin muteins

IL-17A and IL-23 are cytokines involved in inflammation. Human interleukin 17A (also referred to as "IL-17", including IL-17A/A and IL-17A/F) is a cytokine that stimulates the expression of interleukin-6 (IL-6), intracellular adhesion molecule 1(ICAM-I), interleukin-8 (IL-8), granulocyte macrophage colony stimulating factor (GM-CSF), and the expression of prostaglandin E2, and plays a role in the preferential maturation of CD34+ hematopoietic precursor cells into neutrophils (Yao et al, J.Immunol 755:5483 (1995); Fossiez et al, J.exp.Med.183:2593 (1996)). Human interleukin-23 (also known as IL-23) is a cytokine that has been reported to promote proliferation of T cells, particularly memory T cells.

Both IL-17A (including IL-17A complexed with IL-17F, also known as "IL-17A/F") and IL-23 have been reported to play an important role in a number of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, Crohn's disease and psoriasis. IL-23 and IL-17A are both overexpressed in the central nervous system of humans suffering from multiple sclerosis and in mice undergoing animal models of multiple sclerosis, experimental autoimmune encephalitis myelitis (EAE). This overexpression was observed in mice when EAE was induced by Myelin Oligodendrocyte Glycoprotein (MOG)35-55 peptide-or proteolipid peptide (PLP). In addition, neutralization of IL-23p19 or IL-17A resulted in an improvement in EAE symptoms in mice (Park et al, Immunol 6:1133 (2005); Chen et al, J Clin invest.116:1317 (2006)).

IL-17A and Th17 cells have also been shown to be produced from IL-23-independent sources, and IL-23-independent has been shown to develop in vivo in response to IL-17 effects (Mangan et al, Nature441:231 (2006)). Neutralization of IL-23 would theoretically eliminate existing IL-17A producing cells, but would not completely prevent development of new Th17 cells.

Thus, the present invention relates to the binding of both these pro-inflammatory cytokines IL-17A and IL-23p19, which is beneficial for the effective treatment of inflammatory diseases because the binding of both IL-23 (via p19) and IL-17A is more therapeutically effective than the neutralization of IL-23p19 alone or IL-17A alone.

Although antibodies against IL-17A and/or IL-23p19 have been described, methods based on these antibodies still have a number of serious drawbacks, such as the necessity of complex mammalian cell production systems, dependence on disulfide bond stability, tendency of some antibody fragments to aggregate, limited solubility and, last but not least, even when humanized, these antibodies may elicit undesirable immune responses. Thus, there remains a need to develop small globular proteins (e.g., lipocalins) as scaffolds to generate novel IL-17A or IL-23p19 binding proteins, such as lipocalin muteins with IL-17A or IL-23p19 binding affinity.

It is therefore an object of the present invention to provide human lipocalin muteins which bind IL-17A (including IL17-A/A and IL17-A/F) and/or IL-23p19 and which are useful in pharmaceutical applications. The invention also provides one or more compositions comprising such lipocalin muteins and optionally one or more pharmaceutically or diagnostically acceptable excipients, such as adjuvants, diluents or carriers. The lipocalin muteins and compositions thereof of the present invention may be used in a method for detecting IL-17A (including IL17-A/A and IL17-A/F) and/or IL-23p19 in a sample or in a method for binding IL-17A (including IL17-A/A and IL17-A/F) and/or IL-23p19 in a subject.

As discussed above, simultaneous binding of IL-17A (including IL17-A/A and IL17-A/F) and IL-23p19 with IL-17A (including IL17-A/A and IL17-A/F) or IL-23p19 specific lipocalin muteins, respectively, can overcome some hypoxia-mediated effects that may be induced by binding to IL-17A (including IL17-A/A and IL17-A/F), respectively, or to IL-23p19, respectively. Accordingly, the invention encompasses the use of (i) a first lipocalin mutein specific for IL-17A and (ii) a second lipocalin mutein specific for IL-23p19 for binding IL-17A and IL-23p19 in a subject. Such use comprises the step of administering to the subject an effective amount of (i) a first lipocalin mutein specific for IL-17A and (ii) a second lipocalin mutein specific for IL-23p 19.

In the context of the present invention, IL-17A specific lipocalin muteins can bind to IL-17A (i.e.IL-17A/A) which is present as a homodimer, but such muteins can also bind to IL-17A which is present as a heterodimer which complexes with homologous IL-17F to form heterodimeric IL-17A/F. In a preferred embodiment, the lipocalin mutein binds a complex of IL-17A and IL-17F.

The first lipocalin mutein and the second lipocalin mutein may be administered in combination, which combined administration comprises simultaneous, concomitant or sequential administration. In some embodiments, the first lipocalin mutein and the second lipocalin mutein may be comprised in a composition that may be administered. The composition may comprise an effective amount of the first and second lipocalin muteins as active ingredients, together with at least one pharmaceutically acceptable adjuvant, diluent or carrier. The first lipocalin mutein and the second lipocalin mutein may also be administered independently of each other, including at separate time points at separate intervals.

In some embodiments, the invention also relates to a composition comprising a first lipocalin mutein specific for IL-17A and (ii) a second lipocalin mutein specific for IL-23p19, which composition is useful in a method of binding to IL-17A and IL-23p19, e.g., in a subject. In addition, such compositions may be used in methods for detecting, for example, IL-17A (including IL17-A/A and IL17-A/F) and IL-23p19 in a sample.

In some other embodiments, the invention relates to a combination of a first lipocalin mutein and a second lipocalin mutein. One of these lipocalin muteins can bind IL-17A as a given non-natural target with detectable affinity. Another lipocalin mutein can bind IL-23p19 as a given non-natural target with detectable affinity. Thus, the corresponding lipocalin muteins bind to IL-17A or IL-23p19, respectively, as given non-natural targets. The term "non-natural target" refers to a compound that does not bind to the corresponding lipocalin under physiological conditions. For example, a first lipocalin mutein may bind IL-17A and a second lipocalin mutein may bind IL-23p19, or vice versa. The combination of the first lipocalin mutein and the second lipocalin mutein may be provided in various forms.

In some embodiments, IL-17A specific lipocalin muteins as used in the present invention are capable of binding IL-17A with detectable affinity, i.e., with a dissociation constant of at least 200nM, including about 100nM, about 50nM, about 25nM, or about 15 nM. In some embodiments, IL-23p 19-specific lipocalin muteins as used herein are capable of binding IL-23p19 with detectable affinity, i.e., with a dissociation constant of at least 200nM, including about 100nM, about 50nM, about 25nM or about 15 nM. In some further preferred embodiments, the combined lipocalin muteins of the invention bind to IL-17A or IL-23p19 with a dissociation constant for IL-17A or IL-23p19 of at least about 10nM, about 1nM, about 0.1nM, about 10pM or even lower, respectively. Thus, the present invention provides a combination of (i) a mutein of a lipocalin with particularly high affinity for IL-17A and (ii) a mutein of a lipocalin with particularly high affinity for IL-23p 19.

In some embodiments, the lipocalin mutein having detectable affinity for IL-17A is a mutein of human tear lipocalin. These and further details regarding lipocalin muteins having detectable affinity for IL-17A can be found in section A of the present invention.

In a particularly preferred embodiment, the IL-17A specific lipocalin mutein is shown in SEQ ID NO 1.

In some embodiments, the lipocalin mutein having detectable affinity for IL-23p19 is a mutein of human tear lipocalin or a mutein of human neutrophil gelatinase-associated lipocalin. These and further details regarding lipocalin muteins having detectable affinity for IL-23p19 can be found in section B of the present invention.

In a particularly preferred embodiment, IL-23p19 specific lipocalin muteins are shown in any one of SEQ ID NOs 2, 45 and 46.

In yet another aspect, the invention features a method of binding IL-17A and IL-23 in a subject, the method comprising administering to the subject an effective amount of (i) a first lipocalin mutein specific for IL-17A and (ii) a second lipocalin mutein specific for IL-23p 19.

In yet another aspect, the invention relates to a method for inhibiting the binding of IL-17A and IL-23 to their receptors, respectively, in a subject, said method comprising administering to said subject an effective amount of (i) a first lipocalin mutein specific for IL-17A and (ii) a second lipocalin mutein specific for IL-23p 19.

The invention also relates to the use of (i) a first lipocalin mutein specific for IL-17A and (ii) a second lipocalin mutein specific for IL-23p19 for forming a complex with IL-17A and IL-23p 19.

Thus, in yet another aspect of the invention, the disclosed muteins can be used to detect IL-17A and IL-23p 19. Such use may include the steps of: contacting two or more of said muteins with a sample suspected of containing IL-17A and IL-23p19 under suitable conditions, thereby allowing the formation of a complex between said muteins and IL-17A or between said muteins and IL-23p19, respectively, and detecting said complex by means of a suitable signal.

The detectable signal may be generated by a label as described above, or by a change in physical properties due to binding (i.e., complex formation) itself. One example is plasmon surface resonance, the magnitude of which changes during binding of binding partners, one of which is immobilized on a surface, such as gold foil.

The muteins disclosed herein are also useful for isolating IL-17A and IL-23p 19. Such use may include the steps of: contacting one or more of said muteins with a sample suspected of containing IL-17A and IL-23p19 under suitable conditions, thereby allowing the formation of a complex between said mutein and IL-17A or between said mutein and IL-23, respectively, and isolating said complex from said sample.

In the disclosed use of the muteins for the detection of IL-17A and IL-23p19 and for the isolation of IL-17A and IL-23p19, the muteins and/or IL-17A and IL-23p19 or domains or fragments thereof can be immobilized on a suitable solid phase.

Thus, the presence or absence of IL-17A and/or IL-23p19, e.g., in a sample, as well as the concentration or level thereof, can be determined.

In another aspect, the invention provides kits of parts (kit of parts). The kit includes first and second containers. The first container comprises a first lipocalin mutein and the second container comprises a second lipocalin mutein. In one aspect, the invention relates to a kit comprising, in one or more containers, individually or in a mixture, an IL-17A specific lipocalin mutein. In a further aspect, the invention also relates to a kit comprising in one or more containers, individually or in a mixture, a lipocalin mutein specific for IL-23p 19. In some embodiments, the invention relates to a kit comprising in one or more containers, individually or in a mixture, an IL-17A specific lipocalin mutein and an IL-23p19 specific lipocalin mutein. In some further preferred embodiments, the kit comprises a first container comprising a first lipocalin mutein specific for IL-17A and a second container comprising a second lipocalin mutein specific for IL-23p 19. In some embodiments, the kit further comprises information regarding the content or the use of the kit and the lipocalin mutein, either integral therewith or as one or more separate files. In some embodiments, the kit may include one or more compositions formulated for reconstitution in a diluent. Such diluents, e.g., sterile diluents, may also be included in the kit, e.g., in a container.

D. Fusion proteins with IL-17A and/or IL-23p19 binding affinity and uses thereof

In one aspect, the invention relates to a fusion protein comprising at least two subunits in any order: one subunit has IL-17A binding specificity and the other subunit has IL-23p19 binding specificity.

For example, the invention provides a fusion protein comprising protein portions having binding specificity for IL-17A (including IL-17A/A and IL-17A/F) and IL-23p19, respectively. In this aspect, one subunit of the fusion protein can comprise a lipocalin mutein specific to IL-17A (including IL-17A/A and IL-17A/F) of the invention, while another subunit of the fusion protein can comprise a lipocalin mutein specific to IL-23p19 of the invention.

In another aspect, the invention relates to a fusion protein comprising at least two subunits, whichEach subunit having IL-17A (including IL-17A/A and IL-17A/F) binding specificity. In some embodiments, at least one subunit comprises an IL-17A specific lipocalin mutein. In some embodiments, the fusion protein has a K of about 1nM or less in an assay substantially as described in example 2_DBinding affinity of IL-17A. In other embodiments, the fusion protein is capable of inhibiting the binding of IL-17A to its receptor in a competition ELISA format substantially as described in example 3 or in an assay substantially as described in example 5.

In some further embodiments, each of the two subunits comprises an IL-17A/A specific lipocalin mutein. In some further embodiments, each of the two subunits comprises an IL-17A/F specific lipocalin mutein. The two lipocalin muteins may have different amino acid sequences. Thus, in some embodiments, the two lipocalin muteins bind different epitopes on IL-17A. However, in some other embodiments, the two lipocalin muteins may be identical to each other. For example, such a fusion protein may comprise the amino acid sequences of two SEQ ID NOs: 1. In this aspect, the fusion protein can have an amino acid sequence as shown in SEQ ID NO 10, SEQ ID NO 12, or SEQ ID NO 13.

In some embodiments, fusion proteins of the invention comprising two subunits having binding specificity for IL-17A (including IL-17A/A and IL-17A/F) may exhibit greater potency than a single subunit due to the avidity effect of the two subunits resulting from the dimeric nature of the target (e.g., IL 17A/A). In this aspect, the fusion protein can be a bivalent fusion protein. In yet another aspect, the invention also encompasses fusion proteins comprising at least two subunits having IL-23p19 binding specificity. In some embodiments, at least one subunit comprises a lipocalin mutein specific for IL-23p 19. In some embodiments, the fusion protein has a K of 10nM or less in an assay substantially as described in example 7_DIL-23p19 binding affinity. In some further embodiments, the fusion protein is capable of inhibiting the binding of IL-23 to its receptor in a competition ELISA format substantially as described in example 8 or example 14 or in an assay substantially as described in example 10 or example 15.

In some further embodiments, each of the two subunits comprises an IL-23p 19-specific lipocalin mutein. The two lipocalin muteins may have different amino acid sequences. Thus, in some embodiments, the two lipocalin muteins bind different epitopes on IL-23p 19. However, in some other embodiments, the two lipocalin muteins may be identical to each other.

In yet another aspect, the present application discloses a fusion protein comprising (i) an Fc portion of an immunoglobulin, including a fully human antibody, such as an IgG antibody, and (ii) a lipocalin mutein specific for IL-17A.

In another aspect, the invention discloses a fusion protein comprising (i) an Fc portion of an immunoglobulin, including a fully human antibody, such as an IgG antibody, and (ii) a lipocalin mutein specific for IL-23p 19.

Exemplary IL-17A (including IL-17A/A and IL-17A/F) specific lipocalin muteins include those disclosed in section A of the present invention. In a particularly preferred embodiment, the lipocalin mutein is represented by SEQ ID NO 1.

Exemplary IL-23p19 specific lipocalin muteins include those disclosed in section B of the present invention. In a particularly preferred embodiment, the lipocalin mutein is represented by any one of SEQ ID NO 2, 45 and 46.

In some particular embodiments, the lipocalin mutein may be linked, e.g. by a peptide bond, to the C-terminus and/or N-terminus of the Fc part of a human antibody (see fig. 11). In particular embodiments, the fusion protein of the invention may comprise a lipocalin mutein linked to the Fc part of an IgG antibody. In this aspect, one of the fusion proteins comprises the amino acid sequence shown as SEQ ID NO 16.

In a further preferred embodiment, the fusion protein of the invention comprises the amino acids shown as SEQ ID NO 11, SEQ ID NO 12 or SEQ ID NO 13.

In a related embodiment, one or more fusion proteins of the invention are capable of inhibiting the binding of IL-17A and IL-23 to their receptors, respectively. In some further embodiments, one or more fusion proteins of the invention are capable of simultaneously binding (engage) IL-17A and IL-23p19, and thereby are capable of simultaneously inhibiting the binding of IL-17A and IL-23 to their receptors, respectively.

In this regard, the invention relates to a fusion protein comprising at least two subunits in any order, including one subunit comprising an IL-17A (including IL-17A/A and IL-17A/F) -specific lipocalin mutein and one subunit comprising an IL-23p 19-specific lipocalin mutein. In some further embodiments, the fusion protein may comprise additional subunits comprising lipocalin muteins specific for IL-17A (including IL-17A/A and IL-17A/F) or IL-23p 19. In some embodiments, two IL-17A specific lipocalin muteins comprised in two different subunits of the fusion protein may bind different epitopes on the IL-17A target; alternatively, two IL-17A-specific lipocalin muteins comprised in two different subunits of the fusion protein may have the same amino acid sequence and thus specificity for the same epitope on the IL-17A target. Fusion proteins of the invention having two subunits that bind IL-17A may exhibit stronger binding to IL-17A than fusion proteins having only one subunit that binds IL-17A due to the avidity effect brought about by the dimeric nature of the target. Likewise, two IL-23p 19-specific lipocalin muteins contained in two different subunits of the fusion protein may bind to different epitopes on the IL-23p19 target; alternatively, two IL-23p 19-specific lipocalin muteins comprised in two different subunits of the fusion protein may have the same amino acid sequence and thus specificity for the same epitope on the IL-23p19 target. The fusion protein also includes a linker that links one subunit to another subunit.

In some embodiments, a subunit of a fusion protein of the invention comprises a lipocalin mutein disclosed in part a of the invention. In a particularly preferred embodiment, the subunit comprises a lipocalin mutein as shown in SEQ ID NO 1.

In some embodiments, a subunit of a fusion protein of the invention comprises a lipocalin mutein disclosed in section B of the invention. In a particularly preferred embodiment, the subunit comprises a lipocalin mutein as set forth in any one of SEQ ID NOs 2, 45 and 46.

In some embodiments, the fusion protein of the invention comprises a lipocalin mutein disclosed in part a and a lipocalin mutein disclosed in part B.

In a particular embodiment, the fusion protein of the invention comprises the amino acid sequence shown as SEQ ID NO. 3.

In a particular embodiment, the fusion protein of the invention comprises the amino acid sequence shown as SEQ ID NO. 4.

In yet another preferred embodiment, the fusion protein of the invention comprises the amino acids shown as SEQ ID NO 12 or SEQ ID NO 13.

In another aspect, the invention discloses fusion proteins comprising at least two subunits, wherein one subunit has IL-17A or IL-23p19 binding specificity and the other subunit comprises an Albumin Binding Domain (ABD) or albumin binding peptide. In some embodiments, the subunit with IL-17A or IL-23p19 binding specificity comprises a lipocalin mutein of the invention specific for IL-17A or IL-23p 19. In addition, the fusion protein may comprise, in any order, (i) an IL-17A-specific subunit, (ii) an IL-23p 19-specific subunit, and (iii) a subunit comprising a bacterial albumin binding domain. In some embodiments, the subunit with IL-17A binding specificity comprises an IL-17A specific lipocalin mutein of the invention. In some other embodiments, the subunit with IL-23p19 binding specificity comprises an IL-23p19 specific lipocalin mutein of the invention.

In some embodiments, the Albumin Binding Domain (ABD) may be streptococcal protein G: (i)T.,&Skerra, A. (1998) J.Immunol.Methods218,73-83) or a fragment thereof, for example as shown in SEQ ID NO: 14. In some other embodiments, the albumin binding peptide is a human serum albumin binding peptide derived from the albumin binding domain of streptococcal protein G, such as described in PCT application WO 2012/004384, which is incorporated herein by reference in its entirety. In some further preferred embodiments, the albumin binding peptide comprises the amino acid sequence set forth as SEQ DI NO. 15.

In particular, the invention provides a fusion protein capable of simultaneously binding to Human Serum Albumin (HSA) and IL-23p19, said fusion protein comprising an amino acid sequence as set forth, for example, in SEQ DI NO. 7.

The present invention provides a fusion protein capable of simultaneously binding to both HSA and IL-17A, said fusion protein comprising an amino acid sequence as set forth, for example, in SEQ DI NO 8 or SEQ DI NO 10. In some further preferred embodiments, such a fusion protein may comprise two IL-17A-specific lipocalin muteins comprising, for example, the amino acid sequence shown in SEQ DI NO: 10.

In addition, the application features a fusion protein, for example, when in basically the embodiment 12 in the analysis of the measurement of the fusion protein, it can bind to all HAS, IL-17A and IL-23p19 at the same time. In some further embodiments, the fusion protein comprises an amino acid sequence as set forth in SEQ ID NO 5, SEQ ID NO 6, or SEQ ID NO 9. In some yet further preferred embodiments, such a fusion protein may comprise two IL-17A-specific lipocalin muteins comprising, for example, the amino acid sequence shown as SEQ ID NO: 6.

In a further aspect, the invention relates to a fusion protein comprising at least two subunits in any order: one subunit has IL-17A binding specificity or has IL-23p19 binding specificity, and the other subunit has TNF binding specificity, e.g., comprises a TNF-arrestin. Tumor necrosis factor (or TNF family) refers to a group of cytokines (e.g., TNF- α and lymphotoxin- α) that are capable of causing cell death (apoptosis). Exemplary TNF inhibitors include adalimumab (adalimumab), infliximab (infliximab), etanercept (etanercept), certolizumab pegol (certolizumab pegol), and golimumab (golimumab). In some further embodiments, the TNF-specific subunit comprises an anti-TNF- α antibody, e.g., an antibody as set forth in SEQ ID NOS: 61 and 62. In other embodiments, the subunit having IL-17A binding specificity or having IL-23p19 binding specificity comprises a lipocalin mutein of the invention, such as the lipocalin protein of SEQ ID NO:1 or the lipocalin protein of SEQ ID NO: 2. In some still further embodiments, the fusion protein comprises the amino acid sequences of SEQ ID NOs 63 and 62 or the amino acid sequences of SEQ ID NOs 64 and 62.

In some embodiments, the fusion protein is capable of binding 17A, and in some preferred embodiments, the fusion protein may have an average EC50 value that is at least as good as or better than the average EC50 value of the lipocalin mutein comprised in the fusion protein, e.g., when the fusion protein and the lipocalin mutein are measured in an assay essentially as described in example 16. In some embodiments, the fusion protein is capable of binding IL-23, and in some preferred embodiments, the fusion protein may have an average EC50 value that is at least as good as or better than the average EC50 value of the lipocalin mutein comprised in the fusion protein, e.g., when the fusion protein and the lipocalin mutein are measured in an assay essentially as described in example 17. In some embodiments, the fusion protein is capable of binding TNF-a, and in some preferred embodiments, the fusion protein can have an average EC50 value that is at least as good as or better than the average EC50 value of the antibody contained in the fusion protein when the antibody and the fusion protein are measured in an assay substantially as described in example 18. In some further embodiments, for example when the fusion protein is measured in an assay essentially as described in example 19, the fusion protein may be capable of binding all of TNF- α, IL-17A and IL-23p19 simultaneously.

In some embodiments, the fusion proteins of the invention have a K of about 1nM or less_DMeasured IL-17A (including IL-17A/A and IL-17A/F) binding affinity. More preferably, the fusion protein may have a K passing 0.1nM or less_DThe affinity of the measurement. In some further embodiments, one or more IL-17A binding moieties of the fusion proteins of the invention can have as good IL-17A (including IL-17A/A and IL-17A/F) binding affinity or inhibitory ability as when such moieties are stand-alone polypeptides (see Table 1).

In some embodiments, the fusion proteins of the invention have a K passing of about 10nM or less_DMeasured IL-23p19 binding affinity. More preferably, the fusion protein can have a K passing about 1nM or less_DThe affinity of the measurement. In some further embodiments, one or more IL-23p19 binding moieties of the fusion proteins of the invention may have as good IL-23p19 binding affinity or inhibitory ability as when such moieties are stand-alone polypeptides (see Table 1 below).

Table 1: a summary of the activity of the individual lipocalin muteins SEQ ID NO:1 and SEQ ID NO:2 compared to their fusion proteins SEQ ID NO:3-13 in competition ELISA, Surface Plasmon Resonance (SPR) and functional cell-based assays is provided. The value of the interaction with IL-17 and/or IL-23 is determined depending on whether the respective construct contains the IL-17A-binding lipocalin mutein SEQ ID NO 1, the IL-23-binding lipocalin mutein SEQ ID NO 2, or both. To determine the activity towards IL-17 and IL-23, respectively, competition ELISA experiments were performed as described in example 3 and/or example 8, SPR experiments were performed in reverse format (i.e.using protein constructs immobilized on a sensor chip) as described in example 2 and/or example 6, and the cell analysis was based on IL-17A-induced G-CSF secretion (example 5) and/or IL-23-induced Ba/F3 cell proliferation (example 10). It should be noted that the reverse form of the SPR experiment performed to determine IL-23 affinity was performed in the presence of non-physiological high concentrations of NaCl and the resulting values do not reflect the affinity for IL-23 under physiological conditions, but the experiment was used to determine whether the fusion protein of SEQ ID NO:3-13 comprising SEQ ID NO:2 has a different relative affinity for IL-23 compared to the single mutein SEQ ID NO: 2. Table 1 demonstrates that in all assay formats, the IL-17A binding activity of all fusion proteins containing SEQ ID NO:1 is at least as good as the activity of SEQ ID NO:1 itself. Thus, SEQ ID NO 1 can be flexibly used for any fusion protein without loss of activity. In all assay formats, the IL-23 binding activity of all fusion proteins containing SEQ ID NO. 2 is very close to that of SEQ ID NO. 2 itself. Thus, SEQ ID NO 2 can be flexibly used for any fusion protein without significant loss of activity.

In related embodiments, one or more fusion proteins of the invention are capable of inhibiting the binding of IL-17A to its receptor.

In a related embodiment, the fusion protein of the invention is capable of inhibiting the binding of IL-23 to its receptor.

In some embodiments, the fusion protein of the invention may further comprise a linker (e.g., a peptide bond) that covalently links the lipocalin mutein of the invention and the further lipocalin mutein of the invention to each other. This can be achieved, for example, by expressing the linked lipocalin muteins as a single polypeptide linked by a peptide linker. Suitable peptide linkers may be composed of stretches of amino acids of any length containing any amino acid (e.g., as described herein). Preferred linker designs utilize amino acid repeat stretches of glycine and serine according to the formula (GxSy) n, where in a building block repeated n times, x is the number of glycine repeats and y is the number of serine repeats. The values of each of the variables x, y and n may range from 0 to 100, preferably from 0 to 10. Non-limiting examples are provided herein as SEQ ID NOs 18-20.

In some other embodiments, a chemical method of covalent linkage may be applied to link a lipocalin mutein of the invention and another lipocalin mutein of the invention. One example is the use of bifunctional linkers, which allow for a reaction chemistry between the linker and the amino acid side chain, e.g., between maleimide and free cysteine in a lipocalin mutein or between an activated carboxylate and a primary amine in a lipocalin mutein. This includes reactions with non-natural amino acid side chains that may be included during protein expression, and which provide optionally derivatized functionality. In some yet further embodiments, "click" chemistry, such as cycloaddition of azides and alkynes, may be used to link one or more subunits of a fusion protein of the invention.

In some further preferred embodiments, the fusion protein of the invention further comprises an amino acid sequence as set forth in any one of SEQ ID NOs 18-20.

In some further embodiments, in the fusion proteins disclosed herein, one subunit comprising a lipocalin mutein of the invention may be linked to another subunit comprising a lipocalin mutein of the invention, either directly or via a chemical linker.

In some yet further embodiments, a lipocalin mutein of the invention may be fused to the N-or C-terminus or both the N-and C-terminus of another lipocalin mutein.

In some embodiments, each subunit included in a fusion protein of the invention remains thermostable (e.g., can tolerate a T of at least 40 ℃.)_mMelting (temperature) of). In some embodiments, each of the three subunits included in a fusion protein of the invention has a high synergistic unfolding (underfoldi) with respect to one or more other subunitsng) (e.g., eliminating partial unfolding, thereby significantly reducing their degradation rate). The elimination of partial unfolding is called "synergy" because unfolding is an all-or-nothing process. In some further embodiments, one or more lipocalin muteins comprised in the fusion protein may be resistant to a T of at least 50 ℃, at least 55 ℃, at least 60 ℃ or even higher_mMelting (melting) temperature of (a). In some yet further embodiments, one or more HSA components comprised in the fusion protein may tolerate a T of at least 30 ℃, at least 35 ℃, at least 40 ℃ or even higher_mMelting (melting) temperature of (a).

In some embodiments, one or more fusion proteins of the invention comprise a multimer: such as a tetramer, trimer or dimer of a lipocalin mutein of the invention, wherein at least one lipocalin mutein is fused to at least one side (e.g.the N-terminus) of another lipocalin mutein. In some further embodiments, the multimeric fusion protein may preferably correspond to a monomeric fusion protein. For example, the dimeric fusion proteins of the invention that bind IL-17A may exhibit stronger binding to IL-17A due to avidity effects brought about by the dimeric nature of the target.

In some further embodiments, one or more fusion proteins of the invention result in the formation of "Duocalins" as described in Schlehuber, S., and Skerra, A. (2001), Duocalins, engineered ligand-binding proteins with a dual specificity derived from the lipocalin fold. biol. chem.382,1335-1342, the disclosures of which are incorporated herein by reference in their entirety.

In yet another aspect, the invention encompasses the use of one or more fusion proteins of the invention or one or more compositions comprising such proteins for binding IL-17A and/or IL-23p19 in a subject and/or inhibiting the binding of IL-17A and/or IL-23 to their respective receptors in a subject.

In yet another aspect, the invention features a method of binding IL-17A and/or IL-23p19 in a subject, the method including administering to the subject an effective amount of one or more fusion proteins of the invention or one or more compositions including such proteins.

In yet another aspect, the invention relates to a method for inhibiting the binding of IL-17A and/or IL-23 to their respective receptors in a subject, said method comprising administering to said subject an effective amount of one or more fusion proteins of the invention or one or more compositions comprising such proteins.

The fusion protein of the invention may further comprise a signal sequence. The N-terminal signal sequence of the polypeptide directs the polypeptide into a particular cellular compartment, such as the periplasm of E.coli or the endoplasmic reticulum of eukaryotic cells. A large number of signal sequences are known in the art. An exemplary signal sequence for secretion of the polypeptide into the periplasm of E.coli is the OmpA-signal sequence.

The invention also relates to the use of one or more fusion proteins of the invention for forming a complex with IL-17A and/or IL-23p 19.

Thus, in another aspect of the invention, one or more fusion proteins of the invention can be used to detect IL-17A and/or IL-23p 19. Such use may include the steps of: one or more fusion proteins of the invention are contacted with a sample suspected of containing IL-17A and/or IL-23p19 under suitable conditions, thereby allowing a complex to form between the protein and IL-17A and/or between the protein and IL-23p19, respectively, and the complex is detected by a suitable signal.

The detectable signal may be generated by a label as described above, or by a change in physical properties due to binding (i.e., complex formation) itself. One example is plasmon surface resonance, the magnitude of which changes during binding of binding partners, one of which is immobilized on a surface, such as gold foil.

One or more of the fusion proteins disclosed herein may also be used to isolate IL-17A and/or IL-23p19 from a sample containing other substances. Such use may include the steps of: contacting one or more of said fusion proteins with a sample suspected of containing IL-17A and/or IL-23p19 under suitable conditions, thereby allowing the formation of a complex between said protein and IL-17A and/or between said protein and IL-23p19, respectively, and isolating said complex from said sample.

In the disclosed use of the fusion protein for detecting IL-17A and/or IL-23p19 and for isolating IL-17A and/or IL-23p19, the fusion protein, IL-17A, IL-23p19 and/or domains or fragments thereof may be immobilized on a suitable solid phase.

Thus, the presence or absence of molecules (e.g., IL-17A and/or IL-23p19) as in a sample, as well as the concentration or level thereof, can be detected.

In another aspect, the invention provides a kit comprising at least one fusion protein of the invention and one or more instructions for using the kit.

In some embodiments, the kit further comprises information regarding the content or the use of the kit and the fusion protein, either integral therewith or as one or more separate files. In some embodiments, the kit can include one or more fusion proteins of the invention formulated for reconstitution in a diluent. Such diluents, e.g., sterile diluents, may also be included in the kit, e.g., in a container.

In some embodiments, one or more fusion proteins of the invention may be used to treat several conditions where suppression of immune response is desired, such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, crohn's disease, ulcerative colitis, plaque psoriasis, and juvenile idiopathic arthritis.

E. Lipocalin muteins and fusion proteins of the invention

Lipocalins are proteinaceous binding molecules that naturally evolve to bind ligands. Lipocalins are present in many organisms, including vertebrates, insects, plants and bacteria. Members of the lipocalin family (Pervaiz, S., & Brew, K. (1987) FASEB J.1,209-214) are generally small secreted proteins and have a single polypeptide chain. They are characterized by a range of different molecular recognition properties: their ability to bind a variety of molecules, primarily hydrophobic molecules such as retinoids, fatty acids, cholesterol, prostaglandins, biliverdin, pheromones, sweeteners (tastant), and olfactants (odorants); they bind to specific cell surface receptors and form macromolecular complexes. Although they were primarily classified as transporters in the past, it is now clear that lipocalins fulfill a variety of physiological functions. These functions include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins are also involved in the regulation of immune responses and in the mediation of cellular homeostasis (for example, reviewed by Flower, D.R (1996) biochem.j.318,1-14 and Flower, d.r. et al (2000) biochim.biophysis.acta 1482, 9-24).

The overall sequence conservation between lipocalins is very low, typically with less than 20% sequence identity. In strong contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded antiparallel beta sheet that closes upon itself to form a continuous hydrogen-bonded beta barrel. The beta barrel forms a central cavity. One end of the barrel is sterically blocked by an N-terminal peptide segment across its bottom and three peptide loops connecting the beta-sheet strands. The other end of the β -barrel is open to the solvent and contains a target binding site formed by four flexible peptide loops. It is this diversity of the loops in the otherwise rigid lipocalin backbone that results in a variety of different binding patterns, each capable of accommodating targets of different sizes, shapes and chemical characteristics (e.g., Flower, D.R, supra, (1996); Flower, D.R., et al, supra, (2000), or Skerra, A. (2000) reviewed in Biochim. Biophys. acta1482,337-350, supra).

The lipocalin muteins of the invention may be muteins of any alternative lipocalin. Examples of suitable lipocalins (also sometimes designated as "protein 'reference' backbone" or simply "backbone") in which the mutein may be used include, but are not limited to, tear lipocalin (lipocalin-1, von Ebner gland protein), retinol binding protein, neutrophils, lipocalin-type prostaglandin D-synthase, beta-lactoglobulin, posterior bile pigment binding protein (BBP), apolipoprotein D (APO D), neutrophil gelatinase-associated lipocalin (NGAL), tear lipocalin (Tlc), alpha-lactoglobulin₂-microglobulin-related eggsWhite (A2m), 24p3/uterocalin (24p3), von Ebners gland protein 1(VEGP 1), von Ebners gland protein 2(VEGP 2), and the major allergen Can f1 precursor (ALL-1). In related embodiments, the lipocalin mutein is selected from the group consisting of human neutrophil gelatinase-associated lipocalin (NGAL), human tear lipocalin (Tlc), human apolipoprotein d (apo d), and pieria rapa (Pieris brassicca) bile pigment binding protein.

When used herein in the context of a lipocalin mutein of the invention binding IL-17A or IL-23p19, the term "specific for" encompasses that lipocalin mutein pointing to, binding to or reacting with IL-17A or IL-23p19, respectively. Thus, pointing to, binding to or reacting with the lipocalin mutein comprises specific binding to IL-17A or IL-23p19, respectively. As used herein, the term "specifically" in this context means that the lipocalin mutein reacts with the IL-17A protein or the IL-23p19 protein, but at least substantially does not react with the other protein. The term "another protein" includes any non-IL-17A protein or non-IL-23 p19 protein, respectively, including proteins that are closely related or homologous to IL-17A or IL-23p19, to which the lipocalins disclosed herein are directed. However, IL-17A or IL-23p19 proteins, fragments, and/or variants (such as those described in the context of defining a "subject") from species other than human are not excluded by the term "another protein". The term "substantially not bound" means that the lipocalin mutein of the invention does not bind another protein, i.e. shows less than about 30%, preferably 20%, more preferably 10%, particularly preferably less than 9%, 8%, 7%, 6% or 5% cross-reactivity. Whether or not said lipocalin protein as defined above reacts specifically can easily be tested, in particular by comparing the reaction of the lipocalin mutein of the invention with IL-17A or IL-23p19 with the reaction of said lipocalin protein with other (further) proteins. "specific binding" can also be determined, for example, according to Western blot, ELISA-, RIA-, ECL-, IRMA assay, FACS, IHC and peptide scanning.

When compared to sequence identity with another lipocalin, the amino acid sequence of the lipocalin mutein of the invention has a high sequence identity with the corresponding lipocalin (see also above). In this general context, the amino acid sequence of a lipocalin mutein of the combination of the invention is at least substantially similar to the amino acid sequence of the corresponding lipocalin (wild-type or reference lipocalin). The corresponding sequence of a lipocalin mutein of a combination of the invention which is at least substantially similar to the sequence of the corresponding lipocalin has one or more amino acid embodiments, to a certain extent similar to the wild-type (or reference) lipocalin, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, at least 90% identity, including at least 95% identity, with the sequence of the corresponding lipocalin. In this regard, the lipocalin muteins of the invention may of course comprise the comparative substitutions described herein, which enable the lipocalin mutein to bind to IL-17A or IL-23p19, respectively. Typical muteins of lipocalins comprise one or more amino acid mutations in the four loops open to the ligand binding site of the lipocalin relative to the native sequence lipocalin (see above). As described above, these regions are critical in determining the binding specificity of the lipocalin mutein for the desired target. As an illustrative example, a mutein derived from a polypeptide of tear lipocalin, NGAL lipocalin or a homologue thereof may have one, two, three, four or more mutated amino acid residues at any sequence position of the three peptide loops BC, DE, and FG arranged at the end of the N-terminal region and/or the β -barrel structure located opposite the native lipocalin binding pocket. As another illustrative example, a mutein derived from a polypeptide of tear lipocalin or a homologue thereof may have mutated amino acid residues in the peptide loop DE arranged at the end of the β -barrel structure compared to the sequence of wild-type tear lipocalin.

The lipocalin muteins of the invention comprise one or more (e.g. 2,3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even 20) substitutions compared to the corresponding native lipocalin, with the proviso that such lipocalin mutein should be capable of binding to IL-17A or IL-23p19, respectively. For example, a lipocalin mutein may have substitutions at positions corresponding to different positions (i.e. at corresponding positions) of a wild type lipocalin having, for example, the wild type sequence of tear lipocalin, NGAL lipocalin, or any other lipocalin disclosed herein. In some embodiments, the combinatorial lipocalin muteins of the invention comprise at least two amino acid substitutions, including at least two amino acid substitutions of 2,3, 4 or 5, sometimes even more, of the natural amino acid with an arginine residue. Thus, with the aim of generating nucleic acids capable of binding to IL-17A or IL-23p19, respectively, the proteins described herein 'reference' backbones were subjected to mutagenesis.

Furthermore, the lipocalin muteins of the invention may comprise a heterologous amino acid sequence (e.g.strep-tag, such as Strep II-tag) at its N-or C-terminus, preferably at its C-terminus, without affecting the biological activity of the lipocalin mutein (binding to its target, such as IL-17A or IL-23p 19). A preferred example of a tag is shown in SEQ ID NO 17.

Likewise, a lipocalin mutein of the invention may have 1,2, 3, 4 or more amino acids deleted from its N-terminus and/or 1,2 or more amino acids deleted from its C-terminus compared to the corresponding wild-type lipocalin; such as SEQ ID NOs 2-7 and 12-14.

In particular, to determine whether an amino acid residue of a lipocalin mutein amino acid sequence that is different from the wild-type lipocalin corresponds to a certain position in the wild-type lipocalin amino acid sequence, the skilled person may use means and methods well known in the art, such as aligning manually or by using a computer program, such as BLAST2.0 (which represents a basic local alignment search tool) or ClustalW, or any other suitable program suitable for generating a sequence alignment. Thus, the wild-type lipocalin may serve as "test sequence" or "reference sequence", while the amino acid sequence of a lipocalin different from the wild-type lipocalin described herein serves as "query sequence". The terms "reference sequence" and "wild-type sequence" are used interchangeably herein.

In some embodiments, the substitution (or substitution) is a conservative substitution. However, any substitution (including non-conservative substitutions or one or more resulting from the exemplary substitutions listed below) can be envisaged as long as the lipocalin mutein retains its ability to bind to IL-17A or IL-23p19, respectively, and/or the lipocalin mutein has identity to the sequence that is then substituted, i.e. the lipocalin mutein has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more identity to the "original" sequence.

Conservative substitutions are generally the following substitutions, each of which is followed by one or more substitutions that may be conservative, according to the amino acid list to be mutated: ala → Gly, Ser, Val; arg → Lys; asn → Gln, His; asp → Glu; cys → Ser; gln → Asn; glu → Asp; gly → Ala; his → Arg, Asn, Gln; ile → Leu, Val; leu → Ile, Val; lys → Arg, Gln, Glu; met → Leu, Tyr, Ile; phe → Met, Leu, Tyr; ser → Thr; thr → Ser; trp → Tyr; tyr → Trp, Phe; val → Ile, Leu. Other substitutions are also possible and can be determined empirically based on other known conservative or non-conservative substitutions. As another orientation, each of the following eight groups contains amino acids, which can generally be used to define conservative substitutions for another amino acid:

a. alanine (Ala), glycine (Gly);

b. aspartic acid (Asp), glutamic acid (Glu);

c. asparagine (Asn), glutamine (Gln);

d. arginine (Arg), lysine (Lys);

e. isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val);

f. phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp);

g. serine (Ser), threonine (Thr); and

h. cysteine (Cys), methionine (Met).

If such a substitution results in an alteration in biological activity, more substantial changes can be introduced (as described below or further below with respect to amino acid classes) and the product screened for the desired characteristic. Examples of such more substantial changes are: ala → Leu, Ile; arg → Gln; asn → Asp, Lys, Arg, His; asp → Asn; cys → Ala; gln → Glu; glu → Gln; his → Lys; ile → Met, Ala, Phe; leu → Ala, Met, norleucine; lys → Asn; met → Phe; phe → Val, Ile, Ala; trp → Phe; tyr → Thr, Ser; val → Met, Phe, Ala.

Substantial modifications in the biological properties of lipocalins are accomplished by selecting substitutions that differ significantly in their maintenance of the following effects: (a) the structure of the polypeptide backbone in the replacement region, e.g., sheet (sheet) or helical conformation; (b) the charge or hydrophobicity of the molecule at the target; or (c) the volume of the side chain (bulk). Based on general side chain properties, naturally occurring residues are classified into the following groups: (1) hydrophobicity: norleucine, methionine, alanine, valine, leucine, isoleucine; (2) neutral hydrophilicity: cysteine, serine, threonine; (3) acidity: aspartic acid, glutamic acid (4) basic: asparagine, glutamine, histidine, lysine, arginine; (5) residues that influence chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine.

Non-conservative substitutions entail replacing members of one of these classes with another. Any cysteine residues not involved in maintaining the correct conformation of the corresponding lipocalin can also be replaced with serine in general to improve the oxidative stability of the molecule and to prevent abnormal cross-linking. Instead, cysteine bonds may be added to the lipocalin to increase its stability.

Any mutation (including the insertions discussed above) can be accomplished very simply on the nucleic acid (e.g., DNA level) using standard methods that are already established. Illustrative examples of amino acid sequence alterations are insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e. an amino acid residue is replaced by an amino acid residue that is chemically (particularly with respect to polarity and size) similar. Examples of conservative substitutions are substitutions between members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. Alternatively, non-conservative changes may be introduced into the amino acid sequence. Furthermore, instead of replacing a single amino acid residue, it is also possible to insert or delete one or more consecutive amino acids from the primary structure of tear lipocalin, as long as these deletions or insertions result in a stable folded/functional mutein.

Modification of the amino acid sequence includes direct mutagenesis of individual amino acid positions in order to simplify subcloning of the mutated lipocalin gene or part thereof by introducing cleavage sites for certain restriction enzymes. In addition, these mutations can also be introduced to further improve the affinity of the lipocalin muteins or fusion proteins for a given target. In addition, if desired, mutations can be introduced in order to modulate certain characteristics of the mutein or fusion protein, for example to improve folding stability, serum stability, protein resistance or water solubility or to reduce the tendency toward aggregation. For example, a naturally occurring cysteine residue may be mutated to another amino acid to prevent disulfide bridge formation. Other amino acid sequence positions may also be intentionally mutated to cysteine in order to introduce new reactive groups, for example for conjugation with other compounds such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for formation of non-naturally occurring disulfide bonds (links). The thiol moiety produced can be used to pegylate or HES the mutein or fusion protein, e.g., in order to increase the serum half-life of the corresponding lipocalin mutein or fusion protein.

In some embodiments, conjugation to an amino acid side chain may be beneficial if one of the above moieties is conjugated to a lipocalin mutein or fusion protein of the invention. Suitable amino acid side chains may be naturally present in the amino acid sequence of human lipocalin or may be introduced by mutagenesis. If a suitable binding site is introduced by mutagenesis, one possibility is to replace the amino acid at the appropriate position with a cysteine residue.

For example, such mutations include at least one of the substitutions Thr 40 → Cys, Glu 73 → Cys, Arg 90 → Cys, Asp 95 → Cys or Glu 131 → Cys in the wild type sequence of human tear lipocalin. Newly generated cysteine residues at any of these positions can then be used to conjugate the mutein or fusion protein with a moiety (e.g., PEG or an activated derivative thereof) that extends the serum half-life of the mutein or fusion protein thereof.

For muteins of human lipocalin 2, an exemplary possibility of such a mutation that introduces a cysteine residue into the amino acid sequence of a lipocalin, including the human lipocalin 2 mutein, comprises the introduction of a cysteine (Cys) residue at least one of the sequence positions corresponding to sequence position 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 or 158 of the wild-type sequence of human NGAL. In some embodiments, wherein the human lipocalin 2 mutein of the invention has a sequence in which a cysteine has been substituted with another amino acid residue in comparison with the sequence of SWISS-PROT/UniProt database accession number P80188, the corresponding cysteine may be reintroduced into the sequence. As an illustrative example, in this case, a cysteine residue can be introduced at amino acid position 87 by reduction to the cysteine originally present in the sequence of SWISS-PROT accession number P80188. Thiol moieties generated at the flanks of any of amino acid positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146 and/or 158 can be used to pegylate or HES the mutein, e.g., in order to increase the serum half-life of the corresponding human lipocalin 2 mutein or fusion protein thereof.

In another embodiment, artificial amino acids may be introduced by mutagenesis in order to provide suitable amino acid side chains for conjugating one of the above moieties to the lipocalin mutein or fusion protein of the invention. In general, these artificial amino acids are designed to be more reactive, thereby facilitating conjugation with the desired compound. An example of such an artificial amino acid that can be introduced via an artificial tRNA is para-acetyl-phenylalanine.

For several applications of the muteins or fusion proteins disclosed herein, it may be advantageous to use them in the form of conjugates (e.g., fused to moieties that are proteins or protein domains or peptides). In some embodiments, a lipocalin mutein or a fusion protein thereof is fused to a protein, protein domain or peptide, e.g. a single sequence and/or affinity tag, at the N-terminus or C-terminus of the lipocalin mutein (including comprised in the fusion protein of the invention).

Further examples of suitable fusion partners are, for exampleOrII (Schmidt, T.G.M. et al (1996) J.mol.biol.255,753-766), myc-tag, FLAG-tag, His₆-a tag or an HA-tagged affinity tag or a protein such as glutathione-S-transferase which also allows easy detection and/or purification of the recombinant protein. Finally, proteins with chromogenic or fluorescent properties, such as Green Fluorescent Protein (GFP) or Yellow Fluorescent Protein (YFP), are also suitable fusion partners for the lipocalin muteins of the invention.

In general, the lipocalin muteins or fusion proteins of the invention may be labeled with a compound comprising any suitable chemical substance or enzyme that directly or indirectly generates a detectable compound or a chemical, physical, optical or enzymatic reaction signal. Examples of physical reactions and simultaneous optical reactions/labels are fluorescence emission upon irradiation or X-ray emission when using radioactive labels. Alkaline phosphatase, horseradish peroxidase and beta-galactosidase are examples of enzyme labels (and at the same time optical labels) that catalyze the formation of a chromogenic reaction product. In general, all labels commonly used for antibodies (except those specifically used with the carbohydrate moiety in the Fc portion of an immunoglobulin) can also be used for conjugation to the lipocalin muteins or fusion proteins of the invention. The lipocalin muteins or fusion proteins of the invention may also be conjugated with any suitable therapeutically active agent, e.g. for targeted delivery of such an agent to a given cell, tissue or organ or for selective targeting of cells like tumor cells without affecting the surrounding normal cells. Examples of such therapeutically active agents include radionuclides, toxins, small organic molecules, and therapeutic peptides (e.g., peptides that act as agonists/antagonists of cell surface receptors or peptides that compete for protein binding sites on a given cellular target). However, the lipocalin muteins or fusion proteins of the invention may also be conjugated to therapeutically active nucleic acids (e.g. antisense nucleic acid molecules, small interfering RNAs, micrornas or ribozymes). Such conjugates can be produced using methods well known in the art.

As noted above, in some embodiments, a lipocalin mutein or fusion protein of the invention may be conjugated with a moiety that extends the serum half-life of the mutein or fusion protein (see also PCT publication WO 2006/56464 in this regard, where this conjugation strategy is described with reference to a mutein of human neutrophil gelatinase-associated lipocalin having CTLA-4 binding affinity). The moiety that increases serum half-life may be a polyalkylene glycol molecule, hydroxyethyl starch, such as palmitic acid (Vajo)&Duckworth 2000, pharmacol. rev.52,1-9), the Fc portion of an immunoglobulin, the CH3 domain of an immunoglobulin, the CH4 domain of an immunoglobulin, the albumin binding domain, albumin binding peptides or albumin binding proteins, transferrin, to name a few. The albumin binding protein may be a bacterial albumin binding protein, an antibody fragment comprising a domain antibody (see, e.g., U.S. Pat. No. 6,696,245), or a lipocalin mutein having albumin binding activity. Thus, suitable conjugation partners for extending the half-life of the lipocalin muteins or fusion proteins of the invention include bacterial albumin binding domains, such as streptococcal protein G (C: (R))T.,&Skerra, A. (1998) J.Immunol.methods218,73-83) or one of the groups shown in SEQ ID NO: 39. Furthermore, examples of albumin binding peptides which can be used as conjugation partners are, for example, those having Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-those of Cys consensus sequences, like, among others, us patent application 2003/0069395 or Dennis et al (Dennis, m.s., Zhang, m., Meng, y.g., Kadkhodayan, m., Kirchhofer, D.&Damico, L.A, (2002) J Biol Chem277,35035-35043) described in, Xaa₁Asp, Asn, Ser, Thr or Trp; xaa₂Is Asn, Gln, His, Ile, Leu or Lys; xaa₃Is Ala, Asp, Phe, Trp or Tyr; and Xaa₄Asp, Gly, Leu, Phe, Ser or Thr.

In other embodiments, albumin itself (Osborn, B.L. et al, 2002, J.Pharmacol. Exp. Ther.303,540-548) or a biologically active fragment of albumin may be used as a conjugation partner for a lipocalin mutein or fusion protein of the invention. The term "albumin" includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat albumin.

If the albumin binding protein is an antibody fragment, it may be a domain antibody. Domain antibodies (dabs) are engineered to allow precise control of biophysical properties and in vivo half-lives, resulting in optimal safety and efficacy product profiles. Domain antibodies are commercially available, for example, from Domantis Ltd. (Cambridge, UK and MA, USA).

When transferrin is used as a moiety to extend the serum half-life of a lipocalin mutein or fusion protein of the invention, the mutein or fusion protein may be genetically fused to the N-or C-terminus or both of the non-glycosylated transferrin. Non-glycosylated transferrin has a half-life of 14-17 days, and transferrin-conjugated muteins or fusion proteins will similarly have an extended half-life. The transferrin carrier also provides high bioavailability, biodistribution and circulatory stability. This technique is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA). For use as protein stabilisers/half-life extending partnersRecombinant human transferrin (DeltaFerrin)^TM) Commercially available from Novozymes Delta Ltd. (Nottingham, UK).

If the Fc part of an immunoglobulin is used for the purpose of extending the serum half-life of a lipocalin mutein or fusion protein of the invention, Synfusion, commercially available from Syntonix Pharmaceuticals, Inc (MA, USA), may be used^TMProvided is a technique. The use of this Fc-fusion technology allows for the production of longer lasting biopharmaceuticals and may for example consist of two copies of a mutein linked to the Fc region of an antibody to improve pharmacokinetics, solubility and production efficiency.

Another option for extending the half-life of the lipocalin muteins or fusion proteins of the invention is to fuse a long, unstructured, flexible glycine-rich sequence (e.g.polyglycine having about 20 to 80 consecutive glycine residues) to the N-or C-terminus of the mutein (comprised in the fusion protein of the invention). Such a method, for example also referred to as "rPEG" (recombinant PEG), is disclosed in WO 2007/038619.

If a polyalkylene glycol is used as a conjugation partner, the polyalkylene glycol may be substituted, unsubstituted, linear or branched. It may also be an activated polyalkylene derivative. Examples of suitable compounds are polyethylene Glycol (PEG) molecules as described in WO 99/64016, us patent 6,177,074 or us patent 6,403,564 for interferons, or polyethylene Glycol (PEG) molecules as described for other Proteins, such as PEG-Modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (see, e.g., Fuertges et al (1990) The Clinical Efficacy of Poly (Ethylene Glycol) -Modified Proteins j.control. release11,139-148). Such polymers (e.g., polyethylene glycol) can have a molecular weight of from about 300 to about 70.000 daltons, including, for example, polyethylene glycol having a molecular weight of about 10.000, about 20.000, about 30.000, or about 40.000 daltons. Furthermore, carbohydrate oligo-and polymers such as starch or hydroxyethyl starch (HES) may be conjugated to the muteins or fusion proteins of the present invention for the purpose of extending the serum half-life, as described for example in us patent 6,500,930 or 6,620,413.

Furthermore, the lipocalin muteins or fusion proteins disclosed herein may be conjugated to a moiety that may confer a novel characteristic (e.g., enzymatic activity or binding affinity to other molecules) to the lipocalin muteins or fusion proteins of the invention. Examples of suitable moieties include alkaline phosphatase, horseradish peroxidase, glutathione-S-transferase, the albumin binding domain of protein G, protein A, antibody fragments, oligomerization domains, or toxins.

Furthermore, the lipocalin muteins or fusion proteins disclosed herein may be fused to separate enzymatic active sites, such that the two "components" of the resulting fusion protein act together on a given therapeutic target. For example, the binding domain of a lipocalin mutein (including that comprised in the fusion protein of the invention) may be attached to a pathogenic target, thereby allowing the enzyme domain to eliminate the biological function of the target.

In some embodiments, a lipocalin mutein or fusion protein of the invention may be conjugated to the moiety via a linker (e.g. a peptide bond) that covalently links the lipocalin mutein of the invention and the disclosed further moiety to each other. This can be achieved, for example, by expressing the linked lipocalin muteins as a single polypeptide linked by a peptide linker. Suitable peptide linkers may consist of stretches of amino acids of any length containing any amino acid. Preferred linker designs utilize amino acid repeat stretches of glycine and serine according to the formula (GxSy) n, where in a building block repeated n times, x is the number of glycine repeats and y is the number of serine repeats. The values of each of the variables x, y and n may range from 0 to 100, preferably from 0 to 10. Non-limiting examples are provided herein as SEQ ID NO 18 and SEQ ID NO 36-38.

In some other embodiments, covalent linkage chemistry can be applied to link the lipocalin muteins of the invention and another moiety disclosed. One example is the use of bifunctional linkers, which allow for a reaction chemistry between the linker and the amino acid side chain, e.g. between maleimide and free cysteine in a lipocalin mutein or between an activated carboxylate and a primary amine in a lipocalin mutein. This includes reactions with non-natural amino acid side chains that may be included during protein expression, and which provide optionally derivatized functionality. In some yet further embodiments, "click" chemistry, such as cycloaddition of azides and alkynes, may be used to link one or more subunits of a fusion protein of the invention.

The invention also relates to nucleic acid molecules (DNA and RNA) comprising a nucleotide sequence encoding a lipocalin mutein or fusion protein of the invention. Since the degeneracy of the genetic code allows the substitution of certain codons by other codons specifying the same amino acid, the invention is not limited to a specific nucleic acid molecule encoding a lipocalin mutein or fusion protein as described herein, but encompasses all nucleic acid molecules comprising a nucleotide sequence encoding a functional mutein or functional fusion protein. In this aspect, the invention provides nucleotide sequences encoding some exemplary lipocalin muteins, some exemplary fusion proteins, as generally shown in SEQ ID NOS: 23-35, 45-49 and 54.

In one embodiment of the invention, the method includes the nucleic acid molecules at nucleotide triplets encoding at least one, and sometimes even more, of the sequence positions corresponding to 28, 36, 40-41, 49, 52, 68, 70, 72-73, 75, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132 and 134 of the sequence position of the linear polypeptide sequence of human NGAL (SEQ ID NO:8) are subjected to mutagenesis.

In another embodiment of the method of the invention, the nucleic acid molecule encoding human tear lipocalin is first subjected to mutagenesis at one or more of the amino acid sequence positions 26-34, 55-58, 60-61, 64, 104-108 of the linear polypeptide sequence of human tear lipocalin (SEQ ID NO: 1). Second, the nucleic acid molecule encoding human tear lipocalin is also subjected to mutagenesis at one or more of amino acid sequence positions 101, 111, 114 and 153 of the linear polypeptide sequence of mature human tear lipocalin.

The invention also encompasses nucleic acid molecules encoding the lipocalin muteins and fusion proteins of the invention, which comprise further mutations outside the sequence positions specified by the experimental mutagenesis. Such mutations are often tolerated or may even prove beneficial, for example if they contribute to an improved folding efficiency, serum stability, thermostability or ligand binding affinity of the muteins and fusion proteins.

The nucleic acid molecules disclosed herein can be "operably linked" to one or more regulatory sequences to allow expression of the nucleic acid molecule.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing the nucleic acid molecule" or capable of "allowing expression of the nucleotide sequence" if it includes sequence elements that contain information about transcriptional and/or translational regulation, and such sequence is "operably linked" to the nucleotide sequence encoding the polypeptide. The operable connection is such that: in this connection, the regulatory sequence elements and the sequence to be expressed are linked in such a way that the gene is expressible. The exact nature of the regulatory regions required for gene expression may vary from species to species, but generally these regions include promoters which, in prokaryotes, include both the promoter itself (i.e., the DNA element which directs the initiation of transcription) and DNA elements which signal the initiation of translation when transcribed into RNA. Such promoter regions typically include 5 'non-coding sequences involved in initiation of transcription and translation, such as the-35/-10 box, as well as the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences and 5' -capping elements in eukaryotes. These regions may also include enhancer or repressor elements as well as translational signals and leader sequences for targeting the native polypeptide to a particular compartment of the host cell.

In addition, the 3' non-coding sequence may comprise regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences do not function satisfactorily in a particular host cell, they may be replaced by signals functional in that cell.

Thus, the nucleic acid molecules of the invention may comprise regulatory sequences, such as promoter sequences. In some embodiments, the nucleic acid molecules of the invention include a promoter sequence and a transcription termination sequence. Suitable prokaryotic promoters are, for example, the tet promoter, the lacUV5 promoter or the T7 promoter. Examples of promoters which can be used for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

The nucleic acid molecule of the invention may also be part of a vector or any other type of cloning vector, such as a plasmid, phagemid, phage, baculovirus, cosmid or artificial chromosome.

In one embodiment, the nucleic acid molecule is comprised in a plasmid. Plasmid vector refers to a vector encoding a temperate phage (e.g., M13 or f1) intergenic region or functional portion thereof fused to a cDNA of interest. Upon superinfection of bacterial host cells with such a phagemid vector and a suitable helper phage (e.g. M13K07, VCS-M13 or R408), intact phage particles are produced, thereby allowing the physical coupling of the encoded heterologous cDNA to its corresponding polypeptide displayed on the phage surface (see e.g. Lowman, H.B, (1997) Annu. Rev. Biophys. Biomol. struct.26, 401-424, or Rodi, D.J. and Makowski, L. (1999) curr. Opin. Biotechnol.10, 87-93).

In addition to the regulatory sequences described above and the nucleic acid sequences encoding the lipocalin muteins or fusion proteins described herein, such cloning vectors may comprise replication and control sequences derived from a species compatible with the host cell used for expression, as well as selection markers conferring a selectable phenotype on the transformed or transfected cells. A large number of suitable cloning vectors are known in the art and are commercially available.

DNA molecules encoding the lipocalin muteins or fusion proteins described herein, in particular cloning vectors containing the coding sequence of such muteins, can be transformed into host cells capable of expressing the gene. Transformation can be carried out using standard techniques. Thus, the invention also relates to host cells containing the nucleic acid molecules disclosed herein.

The transformed host cell is cultured under conditions suitable for expression of the nucleotide sequence encoding the lipocalin mutein or fusion protein of the invention. Suitable host cells may be prokaryotic cells, such as e.coli (e.coli) or Bacillus subtilis, or eukaryotic cells, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), SF9 or High5 insect cells, immortalized mammalian cell lines (e.g. HeLa cells or CHO cells) or primary mammalian cells.

The invention also relates to a method for producing a polypeptide as described herein, wherein the lipocalin mutein or fusion protein is produced starting from the nucleic acid encoding the lipocalin mutein or fusion protein by means of genetic engineering methods. The method can be performed in vivo, and the lipocalin muteins or fusion proteins can be produced, for example, in a bacterial or eukaryotic host organism and then isolated from the host organism or its culture. Proteins can also be produced in vitro, for example using an in vitro translation system.

In the in vivo production of lipocalin muteins, fusion proteins or fragments, the nucleic acid encoding the mutein is introduced into a suitable bacterial or eukaryotic host organism by recombinant DNA techniques (already outlined above). For this purpose, the host cell is first transformed with a cloning vector comprising a nucleic acid molecule encoding a lipocalin mutein or fusion protein according to the invention using established standard methods. The host cell is then cultured under conditions which allow the expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Thereafter, the polypeptide is recovered from the cells or the culture medium.

In some embodiments, a nucleic acid molecule disclosed herein, such as DNA, can be "operably linked" to another nucleic acid molecule of the invention to allow for expression of a fusion protein of the invention. In this respect, an operable connection is one in which: in such a linkage, the sequence elements of the first nucleic acid molecule and the sequence elements of the second nucleic acid molecule are linked in such a way that the fusion protein can be expressed as a single polypeptide.

Furthermore, in some embodiments, the naturally occurring disulfide bond between Cys76 and Cys175 may be removed in the NGAL muteins of the invention (including those comprised in the fusion proteins of the invention). In some embodiments of the Tlc muteins of the invention (including those comprised in the fusion proteins of the invention), the disulfide bond naturally occurring between Cys61 and Cys153 may be removed. Thus, such a mutein or fusion protein may be produced in a cell compartment having a reducing redox environment, e.g. in the cytoplasm of gram-negative bacteria.

In case the lipocalin muteins or fusion proteins of the invention comprise intramolecular disulfide bonds, it may be preferred to direct the nascent polypeptide to a cell compartment with an oxidative redox environment using a suitable signal sequence. Such an oxidative environment may be provided by the periplasm of gram-negative bacteria (e.g., E.coli), in the extracellular environment of gram-positive bacteria, or in the endoplasmic reticulum lumen of eukaryotic cells, and is generally favorable for the formation of structural disulfide bonds.

However, it is also possible to produce the lipocalin muteins or fusion proteins of the invention in the cytosol of a host cell, preferably E.coli. In this case, the polypeptide may be obtained directly in the soluble and folded state, or may be recovered in the form of inclusion bodies and then renatured in vitro. Other options are to use specific host strains with an oxidizing intracellular environment which can thus allow the formation of disulfide bonds in the cytosol (Venturi et al (2002) J.mol.biol.315, 1-8.).

However, the lipocalin muteins or fusion proteins described herein may not necessarily be generated or produced solely by using genetic engineering. Rather, such lipocalin muteins or fusion proteins can also be obtained by chemical synthesis, such as Merrifield solid phase polypeptide synthesis, or by in vitro transcription and translation. For example, it is possible to identify promising mutations using molecular modeling, then synthesize the desired (designed) polypeptide in vitro, and then study the binding activity for IL-17A. Methods for solid and/or liquid phase synthesis of proteins are well known in the art (see, e.g., Bruckdorfer et al (2004) curr. pharm. Biotechnol.5, 29-43).

In another embodiment, the lipocalin muteins or fusion proteins of the invention can be produced by in vitro transcription/translation using established methods known to the person skilled in the art.

One skilled in the art will appreciate methods that can be used to prepare lipocalin muteins contemplated by the present invention, but whose protein or nucleic acid sequences are not specifically disclosed herein. As an overview, such modifications of the amino acid sequence include, for example, site-directed mutagenesis of individual amino acid positions by introducing cleavage sites for certain restriction enzymes in order to simplify subcloning of the mutated lipocalin gene or a part thereof. In addition, these mutations can also be introduced to further improve the lipocalin mutein or fusion protein affinity for a given target (such as IL-17A or IL-23p19, respectively). Furthermore, mutations may be introduced, if necessary, to modulate certain characteristics of the mutein or fusion protein, such as improving folding stability, serum stability, protein resistance or water solubility, or reducing the tendency towards aggregation. For example, naturally occurring cysteine residues may be mutated to other amino acids to prevent disulfide bridges from forming.

The lipocalin muteins or fusion proteins disclosed herein as well as derivatives thereof can be used in many fields like antibodies or fragments thereof. For example, the lipocalin muteins or fusion proteins as well as their corresponding derivatives can be used for labeling with enzymes, antibodies, radioactive substances or any other group with biochemical activity or defined binding characteristics. In this way, their respective targets can be detected or contacted with them. For example, the lipocalin muteins and/or fusion proteins of the invention can be used for the detection of chemical structures by established analytical methods (e.g., ELISA or western blotting) or by microscopy or immunosensors. In this regard, the detection signal may be generated directly by using a suitable mutein or a suitable fusion protein, or indirectly by immunochemical detection of the bound mutein via an antibody.

Other protein backbones that can be engineered to provide protein muteins that bind IL-17 and/or IL-23 with detectable affinity according to the present invention include: EGF-like domain, Kringle-domain, fibronectin type I domain, fibronectin type II domain, fibronectin type III domain, PAN domain, G1a domain, SRCR domain, Kunitz/Bovine pancreatic trypsin inhibitor domain, Amylase-inhibitor (tenamistat), Kazal-type serine protease inhibitor domain, Trefoil (p-type) domain, von Willebrand factor type C domain, anaphylatoxin-like domain, CUB domain, thyroglobulin type I repeat, LDL receptor type A domain, Sushi domain, Link domain, thrombospondin type I domain, immunoglobulin domain or immunoglobulin-like domain (e.g., domain antibody or camel heavy chain antibody), C-type lectin domain, MAM domain, von Willebrand factor type A domain, somatomedin B domain, WAP-type four disulfide core domains, F5/8C-type domain, hemoglobin-binding Protein domain, SH2 domain, SH3 domain, laminin-type EGF-like domain, C2 domain, "kappa-antibodies" (III. et al, "Design and construction of a carbohydrate domain with properties of bone and light chain variable regions," Protein Eng 10:949-57(1997), "Minibodies" (Martin et al "The affinity-selection of a carbohydrate inhibitor of carbohydrate Protein kinase-6," EMBO J13: 5303-BO 9 (3659), "Home et al" collagen et al, "molecular proteins polypeptide binding Protein and Protein kinase-6" (6455) and "collagen Protein binding Protein J6455" (6455) coding Protein of Protein kinase-6, EMBO J13: 5303-BO 19, and "collagen et al" (6455: viral gene coding Protein J3648) (Jassing 5: 2) and "collagen binding Protein J35 and Protein binding Protein J.)," collagen Protein binding Protein J "(6455, 2. mu. 52(1992), Nanobodies, adnectins, tetranectins (tetranectins), microbodies, affifiin, affibody or ankyrin, crystallin, knottin, ubiquitin, zinc finger proteins, autofluorescent proteins, ankyrin or ankyrin repeat proteins or leucine rich repeat proteins, avimer (SIIverman, Lu Q, Bakker A, To W, Duguy A, Alba BM, Smith R, Rivas A, Li P, Le H, Whitehorn E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Steer WP 2005, Nat biomtech, Dec; 23(12):1556-61, E-Publication in Nat Biotech.2005Nov 20 edition); and multivalent avimer proteins evolved by exon shuffling of the human receptor domain family, also described in Silverman J, Lu Q, Bakker a, To W, Duguay a, Alba BM, Smith R, Rivas a, Li P, Le H, Whitehorn E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Stemmer WP, Nat Biotech, Dec; 23(12) 1556-61, E-Publication in nat. Biotechnology.2005Nov 20 edition).

Further objects, advantages and features of the present invention will become apparent to those skilled in the art upon examination of the following examples and figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosure herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

Examples

Example 1: affinity of lipocalin muteins for IL-17A as measured by SPR

To measure the binding affinity of the lipocalin mutein of SEQ ID NO:1 for IL-17A, a Surface Plasmon Resonance (SPR) based analysis was performed using a Biacore T200 instrument (GE Healthcare). In this SPR affinity assay (FIG. 1), standard amine chemistry is used to immobilize IL-17A on the sensor chip: the surface of the chip was activated using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Subsequently, a 5. mu.g/mL solution of IL-17A in 10mM pH5 acetate was applied at a flow rate of 10. mu.L/min until a fixed level of 279 Resonance Units (RU) was reached. The residual activating groups were quenched with ethanolamine. The reference channel was blank fixed by EDC/NHS treatment after ethanolamine.

For determination of affinity, four dilutions of SEQ ID NO:1 were prepared in HBS-EP + buffer and applied to the prepared chip surface. The binding assay was performed with a contact time of 300 seconds, a dissociation time of 1200 seconds and a flow rate of 30 μ L/min. All measurements were performed at 25 ℃. Regeneration of IL-17A immobilized surfacesObtained by the following method: continuous injection of 10mM H₃PO₄Aqueous solution (30 seconds) and 10mM glycine hydrochloride pH 1.5(15 seconds), followed by additional washes with running buffer and a 30 second stationary phase. Before protein measurements, three start-up cycles were performed for regulatory purposes. Data were evaluated using Biacore T200 evaluation software (V1.0). A double reference is used. The 1:1 binding model was used to fit the raw data.

The resulting fitted curve is shown in fig. 1. The data show that SEQ ID NO 1 has high affinity (k)_a＝7.0×10⁴M^-1sec^-1And k is_d＝5.3×10^-5sec^-1Dissociation rate constant of (1) to give K_DDissociation constant of 0.8nM) binds IL-17A.

Example 2: affinity of lipocalin muteins for IL-17A/F as measured by SPR in another format

To measure the binding affinity of the lipocalin mutein of SEQ ID NO:1 for IL-17AF in a Surface Plasmon Resonance (SPR) based assay in another assay format (capturing SEQ ID NO:1 as ligand and applying human IL-17A/F as analyte), a Biacore T200 instrument (GE Healthcare) was used. In this format, human IL-17A/F (as opposed to homodimeric IL-17A used in example 1) was used to avoid potential avidity effects in the assay. The SEQ ID NO:1 was biotinylated at room temperature for 2 hours by applying a suitable excess of EZ-Link NHS-PEG 4-biotin (Thermo, Cat #21329), followed by separation of unreacted biotin using a Zeba Spin desating Plate (Thermo, Cat #21329) according to the manufacturer's instructions.

In this SPR affinity assay, biotinylated SEQ ID NO:1 was captured on a sensor chip CAP using the biotin CAPture kit (GE Healthcare): the sensor chip CAP is pre-immobilized with ssDNA oligonucleotides. Undiluted biotin CAPture reagent (streptavidin conjugated to complementary ss-DNA oligonucleotides) was applied at a flow rate of 2. mu.L/min for 300 seconds. Then, 0.4. mu.L/mL to 10. mu.L/mL biotinylated SEQ ID NO was applied at a flow rate of 5. mu.L/min for 1300 seconds. The reference channel was loaded with biotin CAPture reagent only.

To determine binding affinity, five (0.2-20nM) dilutions of human IL-17A/F were prepared in HBS-EP + buffer and applied to the prepared chip surface. A flow rate of 30. mu.L/min was applied, using the single cycle kinetics method with a sample contact time of 300 seconds and a dissociation time of 2400 seconds. After ligand immobilization, all 5 concentrations were applied consecutively in ascending order before monitoring dissociation. All measurements were performed at 25 ℃. The regeneration of the sensor chip CAP surface is obtained by: 6M guanidine-HCl and 0.25M NaOH were injected, followed by additional washes with running buffer and a stabilization period of 120 seconds. Data were evaluated using Biacore T200 evaluation software (V1.0). A double reference is used. A binding model of single cycle kinetics 1:1 was used to fit the raw data.

The resulting fitted curve is shown in fig. 2. The data show that SEQ ID NO 1 has high affinity (k)_a＝1.2×10⁴M^-1sec^-1And k is_d＝1.2×10^-4sec^-1Dissociation rate constant of (1) to give K_DCalculated equilibrium dissociation constant of 100 pM) binds IL-17A/F. Comparison with example 1 shows that the results of the SPR analysis depend to some extent on the assay format. However, in both forms, SEQ ID NO 1 has high affinity for human IL-17A and human IL-17A/F, respectively, and is in the sub-nanomolar range. This analysis is very important to allow comparison between SEQ ID NO:1 and fusion proteins comprising such muteins (see example 11 below).

Example 3: competitive mode of action of lipocalin muteins binding to IL-17A

In vitro, the competition ELISA format was used to test whether SEQ ID NO:1 binds IL-17A in a competition mode (FIG. 3). In this experiment, a constant concentration of biotinylated human IL-17A was incubated with a variable concentration of SEQ ID NO:1 for 1 hour. After this pre-incubation in solution, aliquots of the lipocalin mutein/IL-17A mixture were transferred to ELISA plates coated with human IL-17RA to measure the concentration of hIL-17A that was not blocked but bound to hIL-17RA (FIG. 3).

All incubation steps were performed with shaking at 300rpm, and after each incubation step, the plates were washed 5 times with 800 μ Ι PBS-T buffer (PBS, 0.05% tween 20), with the CW washer selected using Biotek EL 405. In the first step, using 1 u g/mL concentration of 20L soluble IL-17RA in PBS directly at 4 degrees C coated 384 hole MSD plate overnight. After washing, the receptor-coated wells were blocked with 60 μ l PBS-T/BSA (2% BSA in PBS containing 0.1% tween 20) for 1 hour at room temperature.

A fixed concentration of 0.01nM human IL-17A was incubated in solution with variable concentrations of SEQ ID NO:1 or SEQ ID NO:41 as a negative control, using appropriate starting concentrations of SEQ ID NO:1 and SEQ ID NO:41 serially diluted to the picomolar range in PBS-T/BSA buffer at a ratio of 1: 4. After 1 hour incubation at room temperature, 20 μ l of the reaction mixture was transferred to the hIL-17RA coated MSD plate to capture unbound (free) or non-competitively bound hIL-17A for 20 minutes at room temperature. To convert the ELISA read-out to absolute free hIL-17A concentration (see below), standard curves containing different concentrations of hIL-17A were prepared in PBS-T/BSA and also incubated on the same MSD plate for 20 min.

To allow detection and quantification of bound biotinylated hIL-17A, the residual supernatant was removed and 20. mu.l of streptavidin sulfo-tag was added at a concentration of 1. mu.g/mL in PBS-T/BSA and incubated for 1 hour at room temperature. After washing, 60 μ l MSD reading buffer T (2 ×) was added to each well and the plates were read over 15 minutes.

The resulting Electrochemiluminescence (ECL) signal was measured using a SECTOR Imager 2400(Meso Scale Discovery). The evaluation was carried out as follows: (based on a parallel standard curve) calculating the free IL-17A concentration c (IL-17A)_{Free form}And plotted against the concentration c of SEQ ID NO:1 (SEQ ID NO: 1). To obtain the concentration of SEQ ID NO:1 at which IL-17A/IL-17 RA-complex formation is blocked by 50% (IC50), according to c (IL-17A)_{Free form}＝c(IL-17A)_{General assembly}/(1+ c (SEQ ID NO:1)/IC50)), curve fitting was performed by non-linear regression using a single-site binding model, with total tracer concentration c (IL-17A)_{General assembly}And IC50 values as free parameters. Curve fitting was performed using GraphPad Prism 4 software.

FIG. 3 shows that negative control SEQ ID NO:41 does not bind hIL-17A; in contrast, SEQ ID NO 1 showed strong competitive binding to hIL-17A with a fitted IC50 value of 75 pM.

Example 4: specificity and species Cross-reactivity of the lipocalin mutein with IL-17A the specificity and species cross-reactivity of the lipocalin mutein of SEQ ID NO:1 (FIG. 4) was determined by a "solution competition ELISA" on the following principle: constant concentrations of SEQ ID NO:1 were incubated with variable concentrations of ligands (human IL-17A, cynomolgus monkey IL-17A (cIL-17A) and marmoset IL-17A ligand) for 1 hour. After this pre-incubation in solution, an aliquot of this mutein/ligand mixture was transferred to an ELISA plate with biotinylated hIL-17A immobilized by neutravidin to measure the residual concentration of SEQ ID NO:1 (FIG. 3). The concentration of free (non-ligand bound) SEQ ID NO:1 was determined by a quantitative ELISA device (FIG. 4). Note that this analysis relies on all ligands targeting the same binding site on SEQ ID NO. 1, i.e., ligands that bind to SEQ ID NO. 1 compete with each other.

In the following detailed experimental protocol, incubation and washing steps were performed as described above in the competition ELISA protocol. A384 well plate suitable for fluorescence measurements (Greiner FLUOTRAC) was coated with 20. mu.l of neutravidin at a concentration of 5. mu.g/ml in PBS at 4 ℃^TM600, black flat bottom, high binding) overnight. After washing, the neutravidin-coated wells were blocked with 100. mu.l of blocking buffer (PBS-T/BSA) for 1 hour at room temperature. After washing again, 20. mu.l of biotinylated hIL-17A at a concentration of 1. mu.g/ml in PBS was added for 1 hour at room temperature and excess reagent was removed.

A fixed concentration of 0.25nM SEQ ID NO:1 was incubated in solution with variable concentrations of ligands (hIL-17A, cIL-17A and marmoset IL-17A) using appropriate starting concentrations serially diluted to the picomolar range in PBS-T/BSA at a ratio of 1: 3. After 1 hour incubation at room temperature, 20. mu.l of the reaction mixture was transferred to a 384 well plate with biotinylated hIL-17A immobilized thereon to capture unbound (free) SEQ ID NO for 120 minutes at room temperature. To convert the ELISA read-out to absolute free SEQ ID NO:1 concentration (see below), standard curves containing different concentrations of SEQ ID NO:1 were prepared in PBS-T/BSA and also incubated on the same ELISA plates for 20 min.

The residual supernatant was removed and 20. mu.l of HRP-labeled anti-lipocalin antibody was added at the predetermined optimal concentration in PBS-T/BSA and incubated for 1 hour at room temperature. Anti-lipocalin antibodies were obtained by immunizing rabbits with a mixture of lipocalin muteins and subsequently coupled to HRP using a kit (EZ-link Plus Activated Peroxidase, Thermo Scientific) according to the manufacturer's instructions to obtain antibody-HRP conjugates. After washing, 20 μ l of fluorescent HRP substrate (Quantablue, Pierce) was added to each well and the reaction was allowed to proceed for 60 minutes. Fluorescence intensity was read for each well on the plate using a Genios Plus microplate reader (Tecan). To evaluate the data, the free SEQ ID NO:1 concentration c (SEQ ID NO:1) was calculated based on the standard curve results_{Free form}And plotted against ligand concentration c (ligand). To obtain the ligand concentration at which IL-17A/SEQ ID NO:1 complex formation was blocked by 50% (IC50), according to c (SEQ ID NO:1)_{Free form}＝c(SEQ ID NO:1)_{General assembly}/(1+ c (ligand)/IC 50)), curve fitting was performed by non-linear regression using a single-site binding model, with total tracer concentration c (SEQ ID NO:1)_{General assembly}And IC50 values as free parameters. Curve fitting was performed using GraphPad Prism 4 software.

In summary, the curve fitting yields the following results: IC50_hIL-17A＝0.1nM，IC50_cIL-17A0.1nM and IC50_{Marmoset IL-17A}0.2 nM. FIG. 4 shows that the data demonstrate that SEQ ID NO:1 shows high affinity for IL-17A from human, cynomolgus and marmoset monkeys, thus indicating that SEQ ID NO:1 is fully cross-reactive with cynomolgus and marmoset IL-17A.

Example 5: lipocalin mutein-mediated blockade of IL-17A-induced G-CSF secretion in cell-based assays

Cell-based assays were used to demonstrate the ability of SEQ ID NO 1 to block IL-17A bioactivity. The assay is based on IL-17A-induced G-CSF secretion in U87-MG cells (ATCC catalog # HTB-14). This highly sensitive functional assay was used to compare the potency of SEQ ID NO 1 to two clinically developed benchmark antibodies. In this assay, recombinant hIL-17A was preincubated with SEQ ID NO 1, a standard antibody molecule or control and added to the cells. In addition to SEQ ID NO:1, the following references and controls were also included in the assay: standard antibody 1 (heavy chain SEQ ID NO: 53; light chain SEQ ID NO:54), standard antibody 2 (heavy chain SEQ ID NO: 55; light chain SEQ ID NO:56) and SEQ ID NO:2 and the human IgG isotype antibody (Dianova, CAT #009-000-002) were used as negative controls. The concentration of G-CSF in the supernatant was then measured by ELISA.

Dulbecco's modified Eagle Medium DMEM (PAN Biotech GmbH) at 37 ℃, 5% CO under standard conditions (containing 10% fetal calf serum FCS (PAA laboratories)), 5% CO₂Atmosphere) U87-MG cells were cultured in cell culture flasks.

On day 1 of the experiment, adherent cells were detached from the substrate using accutase (paa laboratories) according to the manufacturer's instructions. The cells were then centrifuged at 1000rpm for 5 minutes, resuspended in culture medium and filtered through a 100 μm cell filter (Falcon) to remove cell aggregates. Cells were then seeded at a density of 5000 cells per well in 96-well flat-bottomed tissue culture plates (Greiner) using a final volume of 100 μ Ι. These cells were incubated overnight under standard conditions.

SEQ ID NO 1, SEQ ID NO 2, human IgG isotype antibody (Dianova, CAT # 009-. The following day, the medium of cells grown in 96-well plates was replaced with 80 μ l of fresh medium, and then a dilution series of fixed concentrations of 0.1nM recombinant hIL-17A and MUS solution was added to the cells. This procedure was performed in triplicate for each MUS or control. The cells were incubated under standard conditions for an additional 20-24 hours. The wells were visually inspected with a microscope before collecting the supernatant for measuring G-CSF levels. Wells exhibiting significant cell death or the presence of cell aggregates were excluded from the evaluation. G-CSF levels were determined using a G-CSF hypersensitive kit from MSD. To evaluate the data, G-CSF concentrations in arbitrary units were plotted against MUS concentrations c (MUS). To obtain a MUS concentration at which the induction of G-CSF production by U-87MG cells was reduced to 50% (IC50)According to c (G-CSF) ═ c (G-CSF)_{Minimum size}+[c(G-CSF)_{Maximum of}-c(G-CSF)_{Minimum size}]/[1+c(MUS)/IC50]Curve fitting was performed by non-linear regression using a single-site binding model, where the free parameters are I C50, induced G-CSF concentration c (G-CSF)_{Maximum of}And non-induced G-CSF concentration c (G-CSF)_{Minimum size}. Here, assume c (G-CSF)_{Maximum of}And c (G-CSF)_{Minimum size}Independent of the antagonist or control molecules under investigation, they fit to common values for all molecules.

Figure 5 shows a representative example of this experiment, which was performed in duplicate. The resulting average EC50 value for SEQ ID NO:1 was 0.13nM (0.17 nM in the first experiment, 0.10nM in the repeat experiments), which was significantly more effective than baseline 1 showing EC50 ═ 2.33(2.65/2.01) nM, and in a similar range compared to baseline 2 with EC50 ═ 0.12(0.14/0.10) nM. The negative control consisting of SEQ ID NO:2 and the human IgG isotype antibody (Dianova, CAT # 009-.

Example 6: affinity of lipocalin muteins for IL-23 as measured by SPR

To measure the binding affinity of the lipocalin mutein of SEQ ID NO:2 to human IL-23, a Surface Plasmon Resonance (SPR) based analysis was performed using a Biacore T200 instrument (GE Healthcare). In this SPR affinity assay (FIG. 6), hIL-23 was immobilized on a sensor chip using standard amine chemistry: the surface of the chip was activated using EDC and NHS. Subsequently, a 5. mu.g/mL solution of hIL-23 in 10mM pH 4.5 acetate was applied at a flow rate of 10. mu.L/min until a sufficient fixation level was reached. The residual activating groups were quenched with ethanolamine. The reference channel was treated with EDC/NHS (blank immobilization) after ethanolamine.

To determine the binding affinity of SEQ ID NO. 2, five dilutions of SEQ ID NO. 2 were prepared in HBS-EP + buffer and applied to the prepared chip surface. The binding assay was performed with a contact time of 300 seconds, a dissociation time of 1200 seconds and a flow rate of 30 μ L/min. All measurements were performed at 25 ℃. Watch with fixed hIL-23Regeneration of the facets is obtained by: injection of 10mM H₃PO₄Aqueous solution (30 seconds), followed by additional washing with running buffer and a stabilization period of 30 seconds. Before protein measurements, three start-up cycles were performed for regulatory purposes. Data were evaluated using Biacore T200 evaluation software (V1.0). A double reference is used. The 1:1 binding model was used to fit the raw data.

Figure 6 shows a representative example of this experiment, which was performed in triplicate. The resulting fitted curve demonstrates that SEQ ID NO 2 has high affinity (k)_a＝5.0×10⁴M^-1sec^-1And k is_d＝1.9×10^-5sec^-1Dissociation rate constant) binds to hIL-23. The mean dissociation constant determined in three replicates was equal to K_D0.35 ± 0.20nM, indicating a high affinity interaction between SEQ ID No. 2 and human IL-23.

Example 7: affinity of lipocalin muteins for IL-23, measured in another format by SPR

To measure the binding affinity of SEQ ID NO:2 to human IL-23 using Surface Plasmon Resonance (SPR) in another assay format (capture of SEQ ID NO:2 as ligand and application of hIL-23 as analyte), a Biacore T200 instrument (GE Healthcare) was used. The SEQ ID NO 2 and control were biotinylated as described in example 2. In this SPR affinity assay, biotinylated SEQ ID NO:2 was captured on a sensor chip CAP using the biotin CAPture kit (GE Healthcare). To this end, undiluted biotin CAPture reagent was applied at a flow rate of 2 μ L/min for 300 seconds. Then, 0.4. mu.L/mL to 10. mu.L/mL biotinylated SEQ ID NO:2 or control was applied at a flow rate of 5. mu.L/min for 300 seconds. The reference channel was loaded with biotin CAPture reagent only.

To determine binding affinity, five dilutions of hIL-23(7-600nM) were prepared in HBS-EP + buffer supplemented with 350mM NaCl and applied to the prepared chip surface. 350mM NaCl needs to be added to minimize non-specific interaction of hIL-23 with the chip surface. A flow rate of 30 μ L/min was applied using a single cycle kinetic method with a sample contact time of 300 seconds, a dissociation time of 4000 seconds or 7200 seconds. After ligand immobilization, all 5 concentrations were applied consecutively in ascending order before monitoring dissociation. All measurements were performed at 25 ℃. The regeneration and data evaluation of the sensor chip CAP surface is done as described in example 2.

The resulting fitted curve is shown in fig. 7. The data show that SEQ ID NO 2 has a rather high affinity (k)_a＝1.23×10⁴M^-1sec^-1And k is_d＝3.55×10^-5sec^-1Dissociation rate constant of (1) to give K_DCalculated equilibrium dissociation constant of 2.9 nM) binds to hIL-23. Comparison with example 6 shows a strong decrease in affinity when using this SPR assay format due to the high and non-physiological concentration of NaCl (350mM) that must be included in the buffer to minimize non-specific interactions of hIL-23 with the chip surface and thus facilitate the performance of this format of assay. This analysis is very important to allow comparison between SEQ ID NO:2 and fusion proteins comprising such muteins (see example 11 below).

Example 8: competitive mode of action of lipocalin muteins binding to IL-23

Similar to example 3, but using hIL-23 as a target, the lipocalin mutein SEQ ID NO:2 was tested in vitro for binding to hIL-23 in a competition mode using a competition ELISA format (FIG. 8).

All incubation steps were performed under shaking at 300rpm, and after each incubation step, the plates were washed 5 times with 80 μ Ι pbs/0.05% tween 20, with the CW washer selected using Biotek EL 405. A384-well MSD plate was coated directly overnight at 4 ℃ with 20. mu.l of soluble human IL-23 receptor at a concentration of 1. mu.g/mL in PBS. After washing, the receptor-coated wells were blocked with 60 μ l PBS-T/BSA at room temperature for 1 hour.

A fixed concentration of 0.01nM biotinylated human IL-23 was incubated in solution with variable concentrations of SEQ ID NO:2 or SEQ ID NO:43 as a negative control, using appropriate starting concentrations serially diluted to the picomolar range in PBS-T/BSA at a ratio of 1: 4. After 1 hour incubation at room temperature, 20 μ l of the reaction mixture was transferred to an IL-23 receptor coated MSD plate to capture unbound (free) or non-competitively bound hIL-2320 minutes at room temperature. To convert the ELISA read-out to absolute free hIL-23 concentration (see below), standard curves containing different concentrations of hIL-23 starting at 100nM (1: 4 serial dilutions in 11 steps) were prepared in PBS-T/BSA and also incubated on the same MSD plates for 20 min. To allow detection and quantification of bound biotinylated hIL-23, the residual supernatant was removed and 20. mu.l of streptavidin sulfo-tag was added at a concentration of 1. mu.g/mL in PBS-T/BSA and incubated for 1 hour at room temperature. After washing, 60 μ l MSD reading buffer T (2 ×) was added to each well and the plates were read over 15 minutes.

The resulting ECL signal was measured using a SECTOR Imager 2400(Meso Scale Discovery). Evaluation was carried out analogously to example 3. As shown in FIG. 8, the negative control SEQ ID NO 43 did not bind hIL-23; in contrast, it was demonstrated that SEQ ID NO 2 competes for binding with hIL-23 with a fitted IC50 value of 0.54 nM.

Example 9: specificity and species Cross-reactivity of Lipocalin muteins with IL-23

Similar to example 4, but different ligands, i.e.IL-23 from human, cynomolgus monkey (cIL-23), marmoset and mouse were investigated, and the specificity and species cross-reactivity of the lipocalin mutein of SEQ ID NO:2 was determined by "solution competition ELISA" (FIG. 9).

In the following detailed experimental protocol, incubation and washing steps were performed as described above in the competition ELISA protocol. A384 well plate suitable for fluorescence measurements (Greiner FLUOTRAC) was coated with 20. mu.l of neutravidin at a concentration of 5. mu.g/ml in PBS at 4 ℃^TM600, black flat bottom, high binding) overnight. After washing, the neutravidin coated wells were blocked with 100. mu.l PBS-T/BSA for 1 hour at room temperature. After washing again, 20. mu.l of biotinylated hIL-23-Bio at a concentration of 0.25. mu.g/ml in PBS was added for 1 hour at room temperature and excess reagents were removed.

A fixed concentration of 0.5nM SEQ ID NO:2 was incubated in solution with variable concentrations of ligands (hIL-23, cIL-23 and marmoset IL-23 and mouse IL-23) using appropriate starting concentrations serially diluted to the picomolar range in PBS-T/BSA at a ratio of 1: 3. After 20 min incubation at room temperature, 20. mu.l of the reaction mixture was transferred to a 384 well plate coated with hIL-23 to capture unbound (free) SEQ ID NO at room temperature for 220 min. To convert the ELISA read-out to absolute free SEQ ID NO:2 concentration (see below), standard curves containing different concentrations of SEQ ID NO:2 were prepared in PBS-T/BSA and also incubated on the same MSD plate for 20 minutes.

To quantify plate-captured SEQ ID NO 2, residual supernatant was removed and 20. mu.l of HRP-labeled anti-lipocalin antibody was added at a predetermined optimal concentration in PBS-T/BSA and incubated at room temperature for 1 hour. Anti-lipocalin antibodies were obtained by immunizing rabbits with a mixture of lipocalin muteins and subsequently coupled to HRP using a kit (EZ-link Plus Activated Peroxidase, Thermo Scientific) according to the manufacturer's instructions to obtain antibody-HRP conjugates. Further processing of the plates, data acquisition and evaluation were performed as described in example 4.

As shown in FIG. 9, the results demonstrate that SEQ ID NO 2 is fully cross-reactive with human and mouse IL-23 and shows a certain reduced affinity for cynomolgus monkey and marmoset IL-23, where IC50_hIL-23＝0.9nM、IC50_cIL-23＝4.8nM、IC50_marmIL-2312nM and IC50_mIL-23＝0.5nM。

Example 10: lipocalin mutein-mediated blockade of IL-23 in cell-based proliferation assays

The ability of the lipocalin mutein of SEQ ID NO:2 to neutralize the biological activity of hIL-23 was evaluated by applying a short-term proliferation bioassay using recombinant cells expressing the human IL-23 receptor. Ba/F3 transfected cell lines expressed the two subunits of the receptor hIL-23R and hIL-12R β 1 and responded to both human IL-23 and cynomolgus IL-23. Ba/F3 cells proliferated in a dose-dependent manner in response to hIL-23, and this proliferation could be inhibited by hIL-23 neutralizing agents. In this assay, different concentrations of SEQ ID NO:2 were preincubated with constant amounts of hIL-23, and the mixture was then added to Ba/F3 cells in culture. After three days of culture, the degree of proliferation was evaluated by quantifying the number of viable cells. This was done using CellTiter-Glo Luminescent Cell Viability Assay (Promega CAT # G7571) to measure ATP levels related to the number of metabolically active cells. The ability of SEQ ID NO:2 to neutralize hIL-23 was assessed by its IC50 value (i.e., the concentration of the lipocalin mutein that results in half-maximal inhibition of hIL-23-induced proliferation).

The detailed procedure for establishing this analysis is described below. Ba/F3 transfectants were maintained in RPMI-1640 medium (10% fetal bovine serum, 0.05mM 2-mercaptoethanol, 500. mu.g/mL geneticin (G418), 1ng/mL mIL-3, 2. mu.g/mL puromycin, and 200. mu.g/mL bleomycin (zeocin)). Ba/F3 proliferation assay was performed in RPMI-1640 medium (10% fetal bovine serum and 0.05mM 2-mercaptoethanol). The analysis was performed in 384 well white transparent flat-bottom plates (Greiner) at 25 μ L/well.

On the first day, cells from Ba/F3 suspension cell cultures were counted, pelleted, washed twice in assay medium and seeded at a density of 2500 cells/well. 50pM of hIL-23(CAT # HZ-1254, HumanZyme) (corresponding to the predetermined EC50 required for inducing proliferation of Ba/F3 cells) and dilution series of SEQ ID NO:2, negative control (human IgG isotype antibody, CAT # 009-. All titration series were performed in the assay medium at the series 1:3 dilution and appropriate starting concentrations. Subsequently, the cells were propagated at 37 ℃ for 72 hours. To ensure that the potency of hIL-23 was not affected by day-to-day variability, the dose-dependent proliferative response of Ba/F3 cells to hIL-23 was examined by adding dilution series of hIL-23 alone to Ba/F3 cells, using a 1:3 dilution step initiated at 50nM in assay media. To quantify cell proliferation after 72 hours, 25 μ L of CellTiter-Glo reagent was added to the cells in each well and incubated on an orbital shaker for 2 minutes to induce cell lysis and luminescence was measured using a PheraStar FS reader.

EC50 values were determined using GraphPad Prism Software (GraphPad Software inc., San Diego, California, USA) by plotting luminescence signal against sample concentration and using non-linear regression of the data using a sigmoidal dose-response model.

The results of this experiment are shown in fig. 10. The proliferation assay disclosed above represents two independent experiments. SEQ ID NO:2 showed an average EC50 value of 1.2nM (1.7 nM in the first experiment and 0.7nM in the repeat experiments), an EC50 of 3.0nM (3.1/2.9) for reference 3 and an EC50 of 1.2nM (0.8/1.5) for reference 4. Negative controls had no effect on proliferation. Thus, the data demonstrate that SEQ ID NO 2 and the reference molecule exhibit comparable potency in this functional assay.

Example 11: design and characterization of fusion proteins

Various fusion proteins containing one or both of IL-17A and IL-23 binding lipocalin muteins were produced, expressed and characterized as follows, and are shown in FIG. 11.

SEQ ID NO 8 is a fusion protein of SEQ ID NO 1 with a deimmunized albumin binding peptide (dABD, SEQ ID NO 15) derived from the albumin binding domain of streptococcal protein G.

SEQ ID NO.7 is a fusion protein of SEQ ID NO. 2 and SEQ ID NO. 15.

SEQ ID NO 10 is a homodimeric fusion protein of two SEQ ID NO 1 sequences in a row linked by a linker SEQ ID NO 18 and fused to SEQ ID NO 15.

SEQ ID NO 3 is a heterodimer fusion protein of SEQ ID NO 1 and SEQ ID NO 2 linked by a linker SEQ ID NO 18; whereas SEQ ID NO 4 is a heterodimeric fusion protein with the reverse lipocalin mutein sequences SEQ ID NO 2 and SEQ ID NO 1, linked with a slightly different linker SEQ ID NO 19.

SEQ ID NO 5 is a fusion protein corresponding to SEQ ID NO 3 but having an additional C-terminal fusion to the albumin binding domain of streptococcal protein G (SEQ ID NO 14).

SEQ ID NO 9 is a fusion protein corresponding to SEQ ID NO 3 but having an additional C-terminal fusion to dABD (SEQ ID NO 15).

SEQ ID NO 6 is a trimeric fusion protein consisting of two SEQ ID NO 1 sequences fused C-terminally to SEQ ID NO 2 and dABD (SEQ ID NO 15) in a row. The linker between the lipocalins consists of SEQ ID NO 18.

We also generated one or more fusion proteins of part SEQ ID No. 1 with the Fc portion of IgG1, corresponding to part SEQ ID No. 16:

SEQ ID NO 11 is a fusion protein corresponding to SEQ ID NO 1 linked by a peptide linker SEQ ID NO 19 fused at the C-terminus to SEQ ID NO 16.

SEQ ID NO. 12 is a fusion protein corresponding to the double fusion of the N-and C-termini of SEQ ID NO. 1 such that the Fc molecule of SEQ ID NO. 16 is conferred on both the N-and C-termini of SEQ ID NO. 1.

SEQ ID NO. 13 is a fusion protein corresponding to the C-terminus of the homodimeric fusion protein of SEQ ID NO. 10 fused to the Fc molecule of SEQ ID NO. 16.

Where appropriate, all fusion proteins were fully characterized by the analysis exactly as described above for SEQ ID NO:1 and SEQ ID NO: 2: competitive ELISA assays for hIL-17A (example 3) and hIL-23 (example 8); SPR assay in which fusion proteins are immobilized on SPR chips for hIL-17AF (example 2) and hIL-23 (example 6); and functional cell-based assays for hIL-17A (example 5) and hIL-23 (example 10) as described.

The results of the experiments are summarized in table 1. The table provides an overview of the activity of the individual lipocalin muteins SEQ ID NO:1 and SEQ ID NO:2 compared to their fusion proteins SEQ ID NO:3-13 in competition ELISA, Surface Plasmon Resonance (SPR) and functional cell-based assays. The value of the interaction with IL-17 and/or IL-23 is determined depending on whether the respective construct contains the IL-17A-binding lipocalin mutein SEQ ID NO 1, the IL-23-binding lipocalin mutein SEQ ID NO 2 or both. To determine the activity on IL-17 and IL-23, respectively, a competition ELISA experiment was performed as described in example 3 and/or example 8; SPR analysis was performed in reverse format (i.e. protein construct immobilized on sensor chip) as described in example 2 and/or example 6; and the cell analysis was based on IL-17A-induced G-CSF secretion (example 5) and/or IL-23-induced Ba/F3 cell proliferation (example 10). It should be noted that the reverse form of the SPR experiment performed to determine IL-23 affinity was performed in the presence of non-physiologically high concentrations of NaCl and the resulting values do not reflect the affinity for IL-23 under physiological conditions, but the experiment was used to determine whether the relative affinities for IL-23 of the fusion proteins SEQ ID NO:3-13 were different compared to the respective lipocalin muteins SEQ ID NO:1 and SEQ ID NO: 2. Table 1 demonstrates that the IL-17A binding activity of all fusion proteins containing SEQ ID NO:1 is at least as good as the activity of SEQ ID NO:1 itself in all assay formats. Thus, SEQ ID NO 1 can be flexibly used for any fusion protein without loss of activity. In all assay formats, the IL-23 binding activity of all fusion proteins containing SEQ ID NO. 2 is very close to that of SEQ ID NO. 2 itself. Thus, SEQ ID NO 2 can be flexibly used for any fusion protein without significant loss of activity.

Example 12: SPR experiment designed to bind all targets simultaneously via fusion proteins

To demonstrate simultaneous binding of the fusion protein of SEQ ID NO:9 to hIL-17A, hIL-23 and Human Serum Albumin (HSA), a Surface Plasmon Resonance (SPR) based assay was performed using a Biacore T200 instrument (GE Healthcare). The SEQ ID NO 9 was biotinylated as described in example 2. In this SPR affinity assay, biotinylated SEQ ID NO:9 was captured on a sensor chip CAP using the biotin CAPture kit (GE Healthcare). For this, undiluted biotin CAPture reagent was applied at a flow rate of 2 μ L/min for 300 seconds. Then, 0.4. mu.L/mL to 10. mu.L/mL biotinylated SEQ ID NO: 9300 seconds was applied at a flow rate of 5. mu.L/min. The reference channel was loaded with biotin CAPture reagent only.

To demonstrate simultaneous binding, dilutions of hIL-17A/F, hIL-23 and HAS (200nM, 1000nM and 2000nM) were prepared in HBS-EP + buffer supplemented with 350mM NaCl and applied to the prepared chip surface continuously. hIL-17A/F, hIL-23 and HAS were injected sequentially at a sample contact time of 300 seconds, with a flow rate of 30. mu.L/min applied. Single ligands hIL-17A/F, hIL-23 and HSA were also used for targeting to immobilized SEQ ID NO:9 to obtain the maximum binding level obtainable by binding a single target for comparison.

Figure 12 compares the measured binding curve with the theoretical binding curve reflecting complete binding of all ligands. The latter was obtained by pooling the experimental responses of SEQ ID NO 9 to each ligand. The measured and theoretical curves are almost identical, the differences shown are due to dissociation of the target in the experimental curves. The data indicate that SEQ ID NO 9 is capable of binding both hIL-17A, hIL-23 and HSA as the full targets without loss of signal intensity or kinetic changes, compared to binding only a single target.

Example 13: affinity of additional lipocalin muteins for IL-23

To measure the binding affinity of the lipocalin muteins SEQ ID NO:45 and SEQ ID NO:46 to human IL-23, a Surface Plasmon Resonance (SPR) based analysis was performed using a Biacore T200 instrument (GE Healthcare). In this SPR affinity assay (FIG. 13), hIL-23 was immobilized on a sensor chip using standard amine chemistry: chip activation, hIL-23 immobilization, SPR measurement and data evaluation were performed as described in example 6.

As shown in FIG. 13, the resulting fitted curve demonstrates that SEQ ID NO:45 has high affinity (k)_a＝3.0×10⁵M^-1sec^-1And k is_d＝3×10^-5sec^-1Dissociation rate constant of (1) to give K_DDissociation constant of 100 pM) binds to hIL-23. Similarly, as shown in FIG. 13, SEQ ID NO:46 is shown to have high affinity (k)_a＝7.0×10⁴M^-1sec^-1And k is_d＝4.0×10^-5sec^-1Dissociation rate constant of (1) to give K_DDissociation constant of 0.6nM) binds to hIL-23.

Example 14: competitive mode for the Effect of lipocalin muteins on IL-23

Lipocalin muteins SEQ ID NO:45 and SEQ ID NO:46 were tested in vitro for binding to human hIL-23 in a competition mode using a competition ELISA format (FIG. 14). The experiment and evaluation were performed in the same manner as in example 8.

The results of this experiment are shown in fig. 14. SEQ ID No. 45 showed competitive binding to hIL23 with a fitted IC50 value of 0.1nM, and SEQ ID No. 46 also exhibited competitive binding to hIL23 with a fitted IC50 value of 1.1 nM.

Example 15: lipocalin mutein-mediated blockade of IL-23 in cell-based proliferation assays

The ability of the lipocalin muteins SEQ ID NO:45 and SEQ ID NO:46 to neutralize the biological activity of hIL-23 was evaluated by applying a short-term proliferation bioassay using recombinant human IL-23 receptor expressing cells. The experiment and evaluation were carried out similarly to example 10. SEQ ID NO 43 was used as a negative control.

The results of this experiment are shown in fig. 15. SEQ ID NO 45 showed an average EC50 value of 3.7nM and SEQ ID NO 46 showed an average EC50 value of 5.4 nM. Negative controls had no effect on proliferation. Thus, the data demonstrate that the lipocalin muteins of SEQ ID NO:2 and SEQ ID NO:45 and SEQ ID NO:46 show comparable potency in this functional assay.

Example 16: specificity of the fusion protein for IL-17A

We used ELISA analysis to determine the specificity of the fusion proteins of SEQ ID NO:63 and SEQ ID NO:62 for IL-17A. Neutravidin was dissolved in PBS (5. mu.g/mL) and coated on a microtiter plate overnight at 4 ℃. After each incubation step, plates were washed 5 times with 100 μ l PBS (PBS-T) supplemented with 0.1% (v/v) Tween 20. The plates were blocked with 2% BSA (w/v) in PBS-T (PBS-TB) for 1 hour at room temperature, then washed. Biotinylated IL-17A (Peprotech) was captured on neutravidin at a concentration of 1. mu.g/ml for 20 minutes. Unbound protein is washed away. Subsequently, different concentrations of the lipocalin mutein or fusion protein of SEQ ID NO:1 were added to the wells and incubated for 1 hour at room temperature, followed by a washing step. Bound fusion proteins or lipocalin muteins were detected after incubation with HRP-conjugated anti-human TLc antibody diluted 1:2000 in PBS-TB. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

The results of the experiment are shown in fig. 16 along with a fitted curve from 1:1 in combination with sigmoid fitting, where EC50 values and maximum signal are free parameters and the slope is fixed to one. The resulting EC50 values are provided in table 2, including the error of the sigmoid fit of the data. Within experimental error, the EC50 values observed for the antibody-lipocalin mutein fusion protein and the lipocalin mutein are very similar. Experiments have shown that the lipocalin mutein of SEQ ID NO:1 comprised in the fusion protein can be fused with antibodies of SEQ ID NO:61 and 62 without loss of activity on IL-17A.

TABLE 2 ELISA data for IL-17A binding

Example 17: specificity of the fusion protein for IL-23

We used ELISA analysis to determine the specificity of the fusion proteins of SEQ ID NO:64 and SEQ ID NO:62 for IL-23. Neutravidin was dissolved in PBS (5. mu.g/mL) and coated on a microtiter plate overnight at 4 ℃. After each incubation step, plates were washed 5 times with 100 μ l PBS (PBS-T) supplemented with 0.1% (v/v) Tween 20. The plates were blocked with 2% BSA (w/v) in PBS-T (PBS-TB) for 1 hour at room temperature, then washed. Biotinylated IL-23 was captured on neutravidin at a concentration of 1. mu.g/ml for 20 minutes. Unbound protein is washed away. Subsequently, different concentrations of the lipocalin mutein or fusion protein of SEQ ID NO:2 were added to the wells and incubated for 1 hour at room temperature, followed by a washing step. Bound fusion or lipocalin was determined after incubation with HRP-conjugated anti-human NGAL antibody diluted 1:1000 in PBS-TB supplemented with 2% (w/v) BSA (PBS-TB). After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

The results of the experiment are shown in fig. 17 along with a fitted curve from 1:1 in combination with sigmoid fitting, where EC50 values and maximum signal are free parameters and the slope is fixed to one. The resulting EC50 values are provided in table 3. Within experimental error, the EC50 values observed for the antibody-lipocalin mutein fusion protein and the lipocalin mutein are very similar. This demonstrates that the lipocalin mutein of SEQ ID NO:2 comprised in the fusion protein can be fused with antibodies of SEQ ID NO:64 and 62 without loss of activity on IL-23.

TABLE 3 ELISA data for IL-23 binding

Example 18: specificity of fusion proteins for TNF-alpha

We used ELISA analysis to determine the specificity of the fusion proteins of SEQ ID NO:63 and SEQ ID NO:62 and of the fusion proteins of SEQ ID NO:64 and SEQ ID NO:65 for TNF-. alpha.. Antibodies of SEQ ID NO 61 and SEQ ID NO 62 served as positive controls. Recombinant TNF-. alpha. (R & DSystem, 210-TA-100/CF) was dissolved in PBS (1. mu.g/mL) and applied overnight at 4 ℃ onto microtiter plates. After each incubation step, plates were washed 5 times with 100 μ L PBS-T. The plates were blocked with 2% BSA in PBS-T (w/v) for 1 hour at room temperature and then washed. Different concentrations of TNF-alpha specific parent antibody or fusion protein were added to the wells and incubated for 1 hour at room temperature, followed by a washing step. Bound fusion protein or antibody was detected after 1 hour incubation with goat anti-human IgG Fab antibody conjugated to hrp (jackson laboratories) diluted 1:5000 in PBS-TB at room temperature. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

The results of the experiment are shown in fig. 18 along with a fitted curve from 1:1 in combination with sigmoid fitting, where EC50 values and maximum signal are free parameters and the slope is fixed to one. The resulting EC50 values are provided in table 4. EC50 values observed for all tested proteins were very similar. This demonstrates that antibodies contained in the fusion protein can be fused to different lipocalin muteins without impairing their activity on TNF- α.

TABLE 4 ELISA data for TNF-alpha binding

Example 19: demonstration of simultaneous target binding of fusion proteins in an ELISA-based setting

To demonstrate that the fusion proteins of SEQ ID NO:63 and 62 and the fusion proteins of SEQ ID NO:64 and 62 bind TNF-. alpha.and hIL-17A or IL-23, respectively, simultaneously, a dual binding ELISA format was used. Recombinant TNF-. alpha. (R & D System, 210-TA-100/CF) in PBS (1. mu.g/mL) was coated overnight at 4 ℃ on a microtiter plate. After each incubation step, plates were washed 5 times with 100 μ L PBS-T. The plates were blocked with 2% BSA in PBS-T (w/v) for 1 hour at room temperature, then washed again. Different concentrations of the fusion protein were added to the wells and incubated at room temperature for 1 hour, followed by a washing step. Subsequently, biotinylated IL-17A (in the case of SEQ ID NO:63 and 62) or biotinylated IL-23 (in the case of SEQ ID NO:64 and 62) was added at a constant concentration of 1. mu.g/mL in PBS-TB for 1 hour. After washing, Extravidin-HRP (Sigma-Adrich, 1:5000 in PBS-TB) was added to the wells for 1 hour. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

The results of the experiment are shown in fig. 19 along with a fitted curve from 1:1 in combination with sigmoid fitting, where EC50 values and maximum signal are free parameters and the slope is fixed to one. The resulting EC50 values are provided in table 5. All fusion proteins showed clear binding signals with EC50 values in the single digit nanomolar range, indicating that the fusion proteins were capable of binding TNF-a and IL-17A or IL-23 simultaneously.

TABLE 5 ELISA data for simultaneous target binding

Name (R)	EC50 double binding [ nM]
		63 and 62 of SEQ ID NO	2.70±0.22
64 and 62 SEQ ID NOS	1.54±0.16

The embodiments exemplarily described herein may be implemented in the presence or absence of any one or more elements, one or more limitations not specifically disclosed herein, as appropriate. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and without limitation. Additionally, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although these embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variation thereof may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. All patents, patent applications, textbooks, and peer review publications described herein are incorporated by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Further, where features are described in terms of markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the markush group. Further embodiments will become apparent from the claims below.

Equivalents: those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is intended that the following claims cover such equivalents. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

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<211> 406

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 5

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

1 5 10 15

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

20 25 30

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

35 40 45

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

50 55 60

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

65 70 75 80

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

85 90 95

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

100 105 110

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

115 120 125

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

130 135 140

Ser Glu Thr Ser Ser Pro Gly Ser Asp Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu

165 170 175

Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly

180 185 190

Lys Trp Tyr Val Val Gly Glu Ala Gly Asn Leu Leu Leu Arg Glu Asp

195 200 205

Lys Asp Pro Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu Lys Glu Asp

210 215 220

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

225 230 235 240

Tyr Gln Ile Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr

245 250 255

Leu Gly Gly Ile Glu Ser Met Pro Gly Met Thr Ser Phe Leu Val Arg

260 265 270

Val Val Ser Thr Asn Tyr Asn Gln His Ala Ile Val Phe Phe Lys Tyr

275 280 285

Val Tyr Gln Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr Gly Arg Thr

290 295 300

Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys

305 310 315 320

Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp

325 330 335

Gln Ala Ile Asp Gly Ser Ala Gly Ala Val Asp Ala Asn Ser Leu Ala

340 345 350

Glu Ala Lys Val Leu Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser

355 360 365

Asp Tyr Tyr Lys Asn Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val

370 375 380

Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro Ser Ala Trp Ser

385 390 395 400

His Pro Gln Phe Glu Lys

405

<210> 6

<211> 556

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 6

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

1 5 10 15

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

20 25 30

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

35 40 45

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

50 55 60

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

65 70 75 80

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

85 90 95

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

100 105 110

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

115 120 125

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

130 135 140

Ser Glu Thr Ser Ser Pro Gly Ser Asp Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp

165 170 175

Tyr Leu Lys Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu

180 185 190

Glu Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Ile Phe Tyr Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp

260 265 270

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

275 280 285

Glu Lys Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile

290 295 300

Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Asp Gly Gly Gly Gly

305 310 315 320

Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala

325 330 335

Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln

340 345 350

Phe His Gly Lys Trp Tyr Val Val Gly Glu Ala Gly Asn Leu Leu Leu

355 360 365

Arg Glu Asp Lys Asp Pro Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu

370 375 380

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

385 390 395 400

Thr Tyr Lys Tyr Gln Ile Gly Thr Phe Val Pro Gly Ser Gln Pro Gly

405 410 415

Glu Phe Thr Leu Gly Gly Ile Glu Ser Met Pro Gly Met Thr Ser Phe

420 425 430

Leu Val Arg Val Val Ser Thr Asn Tyr Asn Gln His Ala Ile Val Phe

435 440 445

Phe Lys Tyr Val Tyr Gln Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr

450 455 460

Gly Arg Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg

465 470 475 480

Phe Ser Lys Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val

485 490 495

Pro Ile Asp Gln Ala Ile Asp Gly Ser Gly Gly Gly Gly Ser Leu Ala

500 505 510

Glu Ala Lys Glu Ala Ala Asn Ala Glu Leu Asp Ser Tyr Gly Val Ser

515 520 525

Asp Phe Tyr Lys Arg Leu Ile Asp Lys Ala Lys Thr Val Glu Gly Val

530 535 540

Glu Ala Leu Lys Asp Ala Ile Leu Ala Ala Leu Pro

545 550 555

<210> 7

<211> 240

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 7

Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val

1 5 10 15

Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr

20 25 30

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

35 40 45

Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asp Val Thr Arg Val Glu Phe Gly Ala Lys Thr Tyr Lys Tyr Gln Ile

65 70 75 80

Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Gly

85 90 95

Ile Glu Ser Met Pro Gly Met Thr Ser Phe Leu Val Arg Val Val Ser

100 105 110

Thr Asn Tyr Asn Gln His Ala Ile Val Phe Phe Lys Tyr Val Tyr Gln

115 120 125

Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu

130 135 140

Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly

145 150 155 160

Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Ala Ile

165 170 175

Asp Gly Ser Gly Gly Gly Gly Ser Leu Ala Glu Ala Lys Glu Ala Ala

180 185 190

Asn Ala Glu Leu Asp Ser Tyr Gly Val Ser Asp Phe Tyr Lys Arg Leu

195 200 205

Ile Asp Lys Ala Lys Thr Val Glu Gly Val Glu Ala Leu Lys Asp Ala

210 215 220

Ile Leu Ala Ala Leu Pro Ser Ala Trp Ser His Pro Gln Phe Glu Lys

225 230 235 240

<210> 8

<211> 215

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 8

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

1 5 10 15

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

20 25 30

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

35 40 45

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

50 55 60

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

65 70 75 80

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

85 90 95

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

100 105 110

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

115 120 125

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

130 135 140

Ser Glu Thr Ser Ser Pro Gly Ser Asp Ser Gly Gly Gly Gly Ser Leu

145 150 155 160

Ala Glu Ala Lys Glu Ala Ala Asn Ala Glu Leu Asp Ser Tyr Gly Val

165 170 175

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

180 185 190

Val Glu Ala Leu Lys Asp Ala Ile Leu Ala Ala Leu Pro Ser Ala Trp

195 200 205

Ser His Pro Gln Phe Glu Lys

210 215

<210> 9

<211> 403

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 9

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

1 5 10 15

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

20 25 30

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

35 40 45

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

50 55 60

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

65 70 75 80

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

85 90 95

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

100 105 110

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

115 120 125

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

130 135 140

Ser Glu Thr Ser Ser Pro Gly Ser Asp Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu

165 170 175

Ser Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly

180 185 190

Lys Trp Tyr Val Val Gly Glu Ala Gly Asn Leu Leu Leu Arg Glu Asp

195 200 205

Lys Asp Pro Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu Lys Glu Asp

210 215 220

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

225 230 235 240

Tyr Gln Ile Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr

245 250 255

Leu Gly Gly Ile Glu Ser Met Pro Gly Met Thr Ser Phe Leu Val Arg

260 265 270

Val Val Ser Thr Asn Tyr Asn Gln His Ala Ile Val Phe Phe Lys Tyr

275 280 285

Val Tyr Gln Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr Gly Arg Thr

290 295 300

Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys

305 310 315 320

Ser Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp

325 330 335

Gln Ala Ile Asp Gly Ser Gly Gly Gly Gly Ser Leu Ala Glu Ala Lys

340 345 350

Glu Ala Ala Asn Ala Glu Leu Asp Ser Tyr Gly Val Ser Asp Phe Tyr

355 360 365

Lys Arg Leu Ile Asp Lys Ala Lys Thr Val Glu Gly Val Glu Ala Leu

370 375 380

Lys Asp Ala Ile Leu Ala Ala Leu Pro Ser Ala Trp Ser His Pro Gln

385 390 395 400

Phe Glu Lys

<210> 10

<211> 378

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 10

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

1 5 10 15

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

20 25 30

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

35 40 45

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

50 55 60

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

65 70 75 80

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

85 90 95

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

100 105 110

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

115 120 125

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

130 135 140

Ser Glu Thr Ser Ser Pro Gly Ser Asp Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp

165 170 175

Tyr Leu Lys Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu

180 185 190

Glu Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Ile Phe Tyr Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp

260 265 270

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

275 280 285

Glu Lys Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile

290 295 300

Pro Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Asp Ser Gly Gly Gly

305 310 315 320

Gly Ser Leu Ala Glu Ala Lys Glu Ala Ala Asn Ala Glu Leu Asp Ser

325 330 335

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

340 345 350

Val Glu Gly Val Glu Ala Leu Lys Asp Ala Ile Leu Ala Ala Leu Pro

355 360 365

Ser Ala Trp Ser His Pro Gln Phe Glu Lys

370 375

<210> 11

<211> 390

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 11

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

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

115 120 125

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

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Asp

225 230 235 240

Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys Ala Met Thr

245 250 255

Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val Thr Pro Met

260 265 270

Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys Val Thr Met

275 280 285

Asp Ile Glu Gly Phe Leu Gln Glu Phe Lys Ala Val Leu Glu Lys Thr

290 295 300

Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His Val Ala Tyr

305 310 315 320

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

325 330 335

Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly Arg Asp Pro

340 345 350

Lys Asn Asn Leu Glu Ala Leu Glu Asp Phe Glu Lys Ala Ala Gly Ala

355 360 365

Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln Ser Glu Thr

370 375 380

Ser Ser Pro Gly Ser Asp

385 390

<210> 12

<211> 553

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 12

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

1 5 10 15

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

20 25 30

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

35 40 45

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

50 55 60

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

65 70 75 80

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

85 90 95

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

100 105 110

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

115 120 125

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

130 135 140

Ser Glu Thr Ser Ser Pro Gly Ser Asp Gly Gly Gly Gly Ser Gly Gly

145 150 155 160

Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

165 170 175

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

180 185 190

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

195 200 205

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

210 215 220

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

225 230 235 240

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

245 250 255

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

260 265 270

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

275 280 285

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

290 295 300

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

305 310 315 320

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

325 330 335

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

340 345 350

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

355 360 365

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

370 375 380

Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

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

405 410 415

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

420 425 430

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

435 440 445

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

450 455 460

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

465 470 475 480

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

485 490 495

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

500 505 510

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

515 520 525

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

530 535 540

Ser Glu Thr Ser Ser Pro Gly Ser Asp

545 550

<210> 13

<211> 553

<212> PRT

<213> Artificial

<220>

<223> fusion protein

<400> 13

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

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

115 120 125

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

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Asp

225 230 235 240

Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys Ala Met Thr

245 250 255

Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val Thr Pro Met

260 265 270

Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys Val Thr Met

275 280 285

Asp Ile Glu Gly Phe Leu Gln Glu Phe Lys Ala Val Leu Glu Lys Thr

290 295 300

Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His Val Ala Tyr

305 310 315 320

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

325 330 335

Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly Arg Asp Pro

340 345 350

Lys Asn Asn Leu Glu Ala Leu Glu Asp Phe Glu Lys Ala Ala Gly Ala

355 360 365

Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln Ser Glu Thr

370 375 380

Ser Ser Pro Gly Ser Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

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

405 410 415

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

420 425 430

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

435 440 445

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

450 455 460

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

465 470 475 480

Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile Phe Tyr

485 490 495

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

500 505 510

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

515 520 525

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

530 535 540

Ser Glu Thr Ser Ser Pro Gly Ser Asp

545 550

<210> 14

<211> 55

<212> PRT

<213> Artificial

<220>

<223> Albumin binding Domain

<400> 14

Ser Ala Gly Ala Val Asp Ala Asn Ser Leu Ala Glu Ala Lys Val Leu

1 5 10 15

Ala Asn Arg Glu Leu Asp Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn

20 25 30

Leu Ile Asn Asn Ala Lys Thr Val Glu Gly Val Lys Ala Leu Ile Asp

35 40 45

Glu Ile Leu Ala Ala Leu Pro

50 55

<210> 15

<211> 52

<212> PRT

<213> Artificial

<220>

<223> Albumin binding Domain

<400> 15

Ser Gly Gly Gly Gly Ser Leu Ala Glu Ala Lys Glu Ala Ala Asn Ala

1 5 10 15

Glu Leu Asp Ser Tyr Gly Val Ser Asp Phe Tyr Lys Arg Leu Ile Asp

20 25 30

Lys Ala Lys Thr Val Glu Gly Val Glu Ala Leu Lys Asp Ala Ile Leu

35 40 45

Ala Ala Leu Pro

50

<210> 16

<211> 227

<212> PRT

<213> Artificial

<220>

<223> Fc portion

<400> 16

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

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

115 120 125

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

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 17

<211> 10

<212> PRT

<213> Artificial

<220>

<223> protein tag

<400> 17

Ser Ala Trp Ser His Pro Gln Phe Glu Lys

1 5 10

<210> 18

<211> 12

<212> PRT

<213> Artificial

<220>

<223> synthetic linker

<400> 18

Ser Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial

<220>

<223> synthetic linker

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 20

<211> 2

<212> PRT

<213> Artificial

<220>

<223> synthetic linker

<400> 20

Ser Asp

1

<210> 21

<211> 453

<212> DNA

<213> Artificial

<220>

<223> lipocalin muteins

<400> 21

gcctcagacg aggagattca ggatgtgtca gggacgtggt atctgaaggc catgacggtg 60

gatttttggt gttctgggat tcatgaggag tctgttacgc caatgactct gactaccctt 120

gaaggcggca atctggaggc taaggtcacc atggatattg agggatttct tcaagagttt 180

aaggcagtgt tagagaagac agatgaaccg ggtaaatata cggccgatgg cggtaaacat 240

gttgcctata tcattcgcag ccgtgtgaaa gatcattaca tcttttatag cgagggagat 300

tgtcctggtc cggttccagg ggtgtggctc gtgggcagag accccaagaa caacctggaa 360

gccttggagg actttgagaa agccgcagga gcccgcggac tcagcacgga gagcatcctc 420

atccccaggc agagcgaaac cagctctcca ggg 453

<210> 22

<211> 534

<212> DNA

<213> Artificial

<220>

<223> lipocalin muteins

<400> 22

caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60

aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcgaggc cggaaatctt 120

ttgctgcgtg aggataagga tccgaggaaa atgacggcga ccatttacga gttgaaagaa 180

gataaatcat atgacgtcac ccgggtggag tttggggcta agacatacaa gtaccagatt 240

gggacctttg tgccggggag ccagccgggc gagtttactt taggcggtat tgaaagtatg 300

ccgggcatga catcattttt ggtccgtgtc gtgagcacca actacaacca gcatgccata 360

gtgttcttca agtatgtgta tcagaaccgc gagtattttg agatcacact gtacgggcgc 420

acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480

ctccctgaaa accacatcgt cttccctgtc ccaatcgacc aggctatcga cggc 534

<210> 23

<211> 1053

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 23

gcctcagacg aggagattca ggatgtgtca gggacgtggt atctgaaggc catgacggtg 60

gatttttggt gttctgggat tcatgaggag tctgttacgc caatgactct gactaccctt 120

gaaggcggca atctggaggc taaggtcacc atggatattg agggatttct tcaagagttt 180

aaggcagtgt tagagaagac agatgaaccg ggtaaatata cggccgatgg cggtaaacat 240

gttgcctata tcattcgcag ccgtgtgaaa gatcattaca tcttttatag cgagggagat 300

tgtcctggtc cggttccagg ggtgtggctc gtgggcagag accccaagaa caacctggaa 360

gccttggagg actttgagaa agccgcagga gcccgcggac tcagcacgga gagcatcctc 420

atccccaggc agagcgaaac cagctctcca gggagcgacg gtggtggtgg ttctggtggt 480

ggtggatcgc aggactccac ctcagacctg atcccagccc cacctctgag caaggtccct 540

ctgcagcaga acttccagga caaccaattc catgggaaat ggtatgtcgt gggcgaggcc 600

ggaaatcttt tgctgcgtga ggataaggat ccgaggaaaa tgacggcgac catttacgag 660

ttgaaagaag ataaatcata tgacgtcacc cgggtggagt ttggggctaa gacatacaag 720

taccagattg ggacctttgt gccggggagc cagccgggcg agtttacttt aggcggtatt 780

gaaagtatgc cgggcatgac atcatttttg gtccgtgtcg tgagcaccaa ctacaaccag 840

catgccatag tgttcttcaa gtatgtgtat cagaaccgcg agtattttga gatcacactg 900

tacgggcgca cgaaagaact gacaagcgag ctgaaggaaa attttatccg cttttccaaa 960

tctctgggcc tccctgaaaa ccacatcgtc ttccctgtcc caatcgacca ggctatcgac 1020

ggcagcgctt ggtctcaccc gcagttcgaa aaa 1053

<210> 24

<211> 1047

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 24

caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60

aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcgaggc cggaaatctt 120

ttgctgcgtg aggataagga tccgaggaaa atgacggcga ccatttacga gttgaaagaa 180

gataaatcat atgacgtcac ccgggtggag tttggggcta agacatacaa gtaccagatt 240

gggacctttg tgccggggag ccagccgggc gagtttactt taggcggtat tgaaagtatg 300

ccgggcatga catcattttt ggtccgtgtc gtgagcacca actacaacca gcatgccata 360

gtgttcttca agtatgtgta tcagaaccgc gagtattttg agatcacact gtacgggcgc 420

acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480

ctccctgaaa accacatcgt cttccctgtc ccaatcgacc aggctatcga cggcggcggt 540

ggtggttctg gtggtggtgg atcggcctca gacgaggaga ttcaggatgt gtcagggacg 600

tggtatctga aggccatgac ggtggatttt tggtgttctg ggattcatga ggagtctgtt 660

acgccaatga ctctgactac ccttgaaggc ggcaatctgg aggctaaggt caccatggat 720

attgagggat ttcttcaaga gtttaaggca gtgttagaga agacagatga accgggtaaa 780

tatacggccg atggcggtaa acatgttgcc tatatcattc gcagccgtgt gaaagatcat 840

tacatctttt atagcgaggg agattgtcct ggtccggttc caggggtgtg gctcgtgggc 900

agagacccca agaacaacct ggaagccttg gaggactttg agaaagccgc aggagcccgc 960

ggactcagca cggagagcat cctcatcccc aggcagagcg aaaccagctc tccagggagc 1020

gcttggtctc acccgcagtt cgaaaaa 1047

<210> 25

<211> 1218

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 25

gcctcagacg aggagattca ggatgtgtca gggacgtggt atctgaaggc catgacggtg 60

gatttttggt gttctgggat tcatgaggag tctgttacgc caatgactct gactaccctt 120

gaaggcggca atctggaggc taaggtcacc atggatattg agggatttct tcaagagttt 180

aaggcagtgt tagagaagac agatgaaccg ggtaaatata cggccgatgg cggtaaacat 240

gttgcctata tcattcgcag ccgtgtgaaa gatcattaca tcttttatag cgagggagat 300

tgtcctggtc cggttccagg ggtgtggctc gtgggcagag accccaagaa caacctggaa 360

gccttggagg actttgagaa agccgcagga gcccgcggac tcagcacgga gagcatcctc 420

atccccaggc agagcgaaac cagctctcca gggagcgacg gtggtggtgg ttctggtggt 480

ggtggatcgc aggactccac ctcagacctg atcccagccc cacctctgag caaggtccct 540

ctgcagcaga acttccagga caaccaattc catgggaaat ggtatgtcgt gggcgaggcc 600

ggaaatcttt tgctgcgtga ggataaggat ccgaggaaaa tgacggcgac catttacgag 660

ttgaaagaag ataaatcata tgacgtcacc cgggtggagt ttggggctaa gacatacaag 720

taccagattg ggacctttgt gccggggagc cagccgggcg agtttacttt aggcggtatt 780

gaaagtatgc cgggcatgac atcatttttg gtccgtgtcg tgagcaccaa ctacaaccag 840

catgccatag tgttcttcaa gtatgtgtat cagaaccgcg agtattttga gatcacactg 900

tacgggcgca cgaaagaact gacaagcgag ctgaaggaaa attttatccg cttttccaaa 960

tctctgggcc tccctgaaaa ccacatcgtc ttccctgtcc caatcgacca ggctatcgac 1020

ggcagcgctg gtgccgtcga cgctaactct ctggctgaag ctaaagttct ggctaaccgt 1080

gaactggaca aatacggtgt ttccgactac tacaaaaacc tcatcaacaa cgctaaaacc 1140

gttgaaggtg ttaaagctct gatcgacgaa attctcgcag cactgccgag cgcttggtct 1200

cacccgcagt tcgaaaaa 1218

<210> 26

<211> 1671

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 26

gcaagtgatg aagaaattca ggatgttagc ggcacctggt atctgaaagc aatgaccgtt 60

gatttttggt gcagcggtat tcatgaagaa agcgttaccc cgatgaccct gaccaccctg 120

gaaggtggta atctggaagc aaaagttacc atggatattg agggttttct gcaagaattt 180

aaagccgtgc tggaaaaaac cgatgaaccg ggtaaatata ccgcagatgg tggtaaacat 240

gtggcctata ttatccgtag ccgtgtgaaa gatcactata tcttttatag cgaaggtgat 300

tgtccgggtc cggttccggg tgtttggctg gttggtcgtg atccgaaaaa taacctggaa 360

gcactggaag attttgaaaa agcagccggt gcacgtggtc tgagcaccga aagcattctg 420

attccgcgtc agagcgaaac cagcagtcct ggatccgacg gtggtggtgg ttctggtggt 480

ggtggatcgg cctcagacga ggagattcag gatgtgtcag ggacgtggta tctgaaggcc 540

atgacggtgg atttttggtg ttctgggatt catgaggagt ctgttacgcc aatgactctg 600

actacccttg aaggcggcaa tctggaggct aaggtcacca tggatattga gggatttctt 660

caagagttta aggcagtgtt agagaagaca gatgaaccgg gtaaatatac ggccgatggc 720

ggtaaacatg ttgcctatat cattcgcagc cgtgtgaaag atcattacat cttttatagc 780

gagggagatt gtcctggtcc ggttccaggg gtgtggctcg tgggcagaga ccccaagaac 840

aacctggaag ccttggagga ctttgagaaa gccgcaggag cccgcggact cagcacggag 900

agcatcctca tccccaggca gagcgaaacc agctctccag ggagcgacgg cggaggtggc 960

tcaggaggtg gcggatccca ggactccacc tcagacctga tcccagcccc acctctgagc 1020

aaggtccctc tgcagcagaa cttccaggac aaccaattcc atgggaaatg gtatgtcgtg 1080

ggcgaggccg gaaatctttt gctgcgtgag gataaggatc cgaggaaaat gacggcgacc 1140

atttacgagt tgaaagaaga taaatcatat gacgtcaccc gggtggagtt tggggctaag 1200

acatacaagt accagattgg gacctttgtg ccggggagcc agccgggcga gtttacttta 1260

ggcggtattg aaagtatgcc gggcatgaca tcatttttgg tccgtgtcgt gagcaccaac 1320

tacaaccagc atgccatagt gttcttcaag tatgtgtatc agaaccgcga gtattttgag 1380

atcacactgt acgggcgcac gaaagaactg acaagcgagc tgaaggaaaa ttttatccgc 1440

ttttccaaat ctctgggcct ccctgaaaac cacatcgtct tccctgtccc aatcgaccag 1500

gctatcgacg gcagcggcgg cggcggctct ctggctgaag ctaaagaagc ggctaacgcg 1560

gaactggact cttacggtgt ttccgacttt tacaaacgtc tcatcgataa agctaaaacc 1620

gttgaaggtg ttgaagctct gaaagacgcg attctcgcag cactgccgaa a 1671

<210> 27

<211> 720

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 27

caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60

aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcgaggc cggaaatctt 120

ttgctgcgtg aggataagga tccgaggaaa atgacggcga ccatttacga gttgaaagaa 180

gataaatcat atgacgtcac ccgggtggag tttggggcta agacatacaa gtaccagatt 240

gggacctttg tgccggggag ccagccgggc gagtttactt taggcggtat tgaaagtatg 300

ccgggcatga catcattttt ggtccgtgtc gtgagcacca actacaacca gcatgccata 360

gtgttcttca agtatgtgta tcagaaccgc gagtattttg agatcacact gtacgggcgc 420

acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480

ctccctgaaa accacatcgt cttccctgtc ccaatcgacc aggctatcga cggcagcggc 540

ggcggcggct ctctggctga agctaaagaa gcggctaacg cggaactgga ctcttacggt 600

gtttccgact tttacaaacg tctcatcgat aaagctaaaa ccgttgaagg tgttgaagct 660

ctgaaagacg cgattctcgc agcactgccg agcgcttggt ctcacccgca gttcgaaaaa 720

<210> 28

<211> 645

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 28

gcctcagacg aggagattca ggatgtgtca gggacgtggt atctgaaggc catgacggtg 60

gatttttggt gttctgggat tcatgaggag tctgttacgc caatgactct gactaccctt 120

gaaggcggca atctggaggc taaggtcacc atggatattg agggatttct tcaagagttt 180

aaggcagtgt tagagaagac agatgaaccg ggtaaatata cggccgatgg cggtaaacat 240

gttgcctata tcattcgcag ccgtgtgaaa gatcattaca tcttttatag cgagggagat 300

tgtcctggtc cggttccagg ggtgtggctc gtgggcagag accccaagaa caacctggaa 360

gccttggagg actttgagaa agccgcagga gcccgcggac tcagcacgga gagcatcctc 420

atccccaggc agagcgaaac cagctctcca gggagcgata gcggcggcgg cggctctctg 480

gctgaagcta aagaagcggc taacgcggaa ctggactctt acggtgtttc cgacttttac 540

aaacgtctca tcgataaagc taaaaccgtt gaaggtgttg aagctctgaa agacgcgatt 600

ctcgcagcac tgccgagcgc ttggtctcac ccgcagttcg aaaaa 645

<210> 29

<211> 1209

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 29

gcctcagacg aggagattca ggatgtgtca gggacgtggt atctgaaggc catgacggtg 60

gatttttggt gttctgggat tcatgaggag tctgttacgc caatgactct gactaccctt 120

gaaggcggca atctggaggc taaggtcacc atggatattg agggatttct tcaagagttt 180

aaggcagtgt tagagaagac agatgaaccg ggtaaatata cggccgatgg cggtaaacat 240

gttgcctata tcattcgcag ccgtgtgaaa gatcattaca tcttttatag cgagggagat 300

tgtcctggtc cggttccagg ggtgtggctc gtgggcagag accccaagaa caacctggaa 360

gccttggagg actttgagaa agccgcagga gcccgcggac tcagcacgga gagcatcctc 420

atccccaggc agagcgaaac cagctctcca gggagcgacg gcggaggtgg ctcaggaggt 480

ggcggatccc aggactccac ctcagacctg atcccagccc cacctctgag caaggtccct 540

ctgcagcaga acttccagga caaccaattc catgggaaat ggtatgtcgt gggcgaggcc 600

ggaaatcttt tgctgcgtga ggataaggat ccgaggaaaa tgacggcgac catttacgag 660

ttgaaagaag ataaatcata tgacgtcacc cgggtggagt ttggggctaa gacatacaag 720

taccagattg ggacctttgt gccggggagc cagccgggcg agtttacttt aggcggtatt 780

gaaagtatgc cgggcatgac atcatttttg gtccgtgtcg tgagcaccaa ctacaaccag 840

catgccatag tgttcttcaa gtatgtgtat cagaaccgcg agtattttga gatcacactg 900

tacgggcgca cgaaagaact gacaagcgag ctgaaggaaa attttatccg cttttccaaa 960

tctctgggcc tccctgaaaa ccacatcgtc ttccctgtcc caatcgacca ggctatcgac 1020

ggcagcggcg gcggcggctc tctggctgaa gctaaagaag cggctaacgc ggaactggac 1080

tcttacggtg tttccgactt ttacaaacgt ctcatcgata aagctaaaac cgttgaaggt 1140

gttgaagctc tgaaagacgc gattctcgca gcactgccga gcgcttggtc tcacccgcag 1200

ttcgaaaaa 1209

<210> 30

<211> 1134

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 30

gcaagtgatg aagaaattca ggatgttagc ggcacctggt atctgaaagc aatgaccgtt 60

gatttttggt gcagcggtat tcatgaagaa agcgttaccc cgatgaccct gaccaccctg 120

gaaggtggta atctggaagc aaaagttacc atggatattg agggttttct gcaagaattt 180

aaagccgtgc tggaaaaaac cgatgaaccg ggtaaatata ccgcagatgg tggtaaacat 240

gtggcctata ttatccgtag ccgtgtgaaa gatcactata tcttttatag cgaaggtgat 300

tgtccgggtc cggttccggg tgtttggctg gttggtcgtg atccgaaaaa taacctggaa 360

gcactggaag attttgaaaa agcagccggt gcacgtggtc tgagcaccga aagcattctg 420

attccgcgtc agagcgaaac cagcagtcct ggatccgacg gtggtggtgg ttctggtggt 480

ggtggatcgg cctcagacga ggagattcag gatgtgtcag ggacgtggta tctgaaggcc 540

atgacggtgg atttttggtg ttctgggatt catgaggagt ctgttacgcc aatgactctg 600

actacccttg aaggcggcaa tctggaggct aaggtcacca tggatattga gggatttctt 660

caagagttta aggcagtgtt agagaagaca gatgaaccgg gtaaatatac ggccgatggc 720

ggtaaacatg ttgcctatat cattcgcagc cgtgtgaaag atcattacat cttttatagc 780

gagggagatt gtcctggtcc ggttccaggg gtgtggctcg tgggcagaga ccccaagaac 840

aacctggaag ccttggagga ctttgagaaa gccgcaggag cccgcggact cagcacggag 900

agcatcctca tccccaggca gagcgaaacc agctctccag ggagcgatag cggcggcggc 960

ggctctctgg ctgaagctaa agaagcggct aacgcggaac tggactctta cggtgtttcc 1020

gacttttaca aacgtctcat cgataaagct aaaaccgttg aaggtgttga agctctgaaa 1080

gacgcgattc tcgcagcact gccgagcgct tggtctcacc cgcagttcga aaaa 1134

<210> 31

<211> 1173

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 31

gacaaaaccc acacctgccc accttgtcct gcccctgaac tgctgggagg accttctgtg 60

tttctgttcc caccaaaacc aaaagatacc ctgatgatct ctagaacccc tgaggtgaca 120

tgtgtggtgg tggatgtgtc tcatgaggac cctgaggtca aattcaactg gtacgtggat 180

ggagtggaag tccacaatgc caaaaccaag cctagagagg aacagtacaa ttcaacctac 240

agagtggtca gtgtgctgac tgtgctgcat caggattggc tgaatggcaa ggaatacaag 300

tgtaaagtct caaacaaggc cctgcctgct ccaattgaga aaacaatctc aaaggccaag 360

ggacagccta gggaacccca ggtctacacc ctgccacctt caagagagga aatgaccaaa 420

aaccaggtgt ccctgacatg cctggtcaaa ggcttctacc cttctgacat tgctgtggag 480

tgggagtcaa atggacagcc tgagaacaac tacaaaacaa ccccccctgt gctggattct 540

gatggctctt tctttctgta ctccaaactg actgtggaca agtctagatg gcagcagggg 600

aatgtctttt cttgctctgt catgcatgag gctctgcata accactacac tcagaaatcc 660

ctgtctctgt ctcctggcaa aggcggcgga ggatccggcg gaggaggtag cgcatcagac 720

gaggaaatcc aggacgtgtc agggacctgg tacctgaaag ccatgaccgt ggatttttgg 780

tgctccggca tccatgagga gtcagtcact cccatgaccc tgacaaccct agaaggtggg 840

aatctggagg ccaaagtgac aatggatatt gagggctttc tccaggagtt caaagccgtc 900

ctcgagaaga cagacgagcc tggaaagtat actgctgatg ggggaaaaca cgtagcctat 960

atcattcgat ctcgggtgaa ggatcattat atcttctatt ccgagggcga ctgccccggc 1020

cctgtgccag gtgtctggct agttgggagg gaccccaaga acaatctcga ggctctggag 1080

gacttcgaga aggcagctgg tgccagggga ttgagcactg agtctatcct tatcccacgc 1140

cagagcgaga cctcaagccc agggtccgac tga 1173

<210> 32

<211> 1659

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 32

gcatcagacg aggaaatcca ggacgtgtca gggacctggt acctgaaagc catgaccgtg 60

gatttttggt gctccggcat ccatgaggag tcagtcactc ccatgaccct gacaacccta 120

gaaggtggga atctggaggc caaagtgaca atggatattg agggctttct ccaggagttc 180

aaagccgtcc tcgagaagac agacgagcct ggaaagtata ctgctgatgg gggaaaacac 240

gtagcctata tcattcgatc tcgggtgaag gatcattata tcttctattc cgagggcgac 300

tgccccggcc ctgtgccagg tgtctggcta gttgggaggg accccaagaa caatctcgag 360

gctctggagg acttcgagaa ggcagctggt gccaggggat tgagcactga gtctatcctt 420

atcccacgcc agagcgagac ctcaagccca gggtccgacg gcggtggagg atccggtgga 480

ggcggttctg acaaaaccca cacctgccca ccttgtcctg cccctgaact gctgggagga 540

ccttctgtgt ttctgttccc accaaaacca aaagataccc tgatgatctc tagaacccct 600

gaggtgacat gtgtggtggt ggatgtgtct catgaggacc ctgaggtcaa attcaactgg 660

tacgtggatg gagtggaagt ccacaatgcc aaaaccaagc ctagagagga acagtacaat 720

tcaacctaca gagtggtcag tgtgctgact gtgctgcatc aggattggct gaatggcaag 780

gaatacaagt gtaaagtctc aaacaaggcc ctgcctgctc caattgagaa aacaatctca 840

aaggccaagg gacagcctag ggaaccccag gtctacaccc tgccaccttc aagagaggaa 900

atgaccaaaa accaggtgtc cctgacatgc ctggtcaaag gcttctaccc ttctgacatt 960

gctgtggagt gggagtcaaa tggacagcct gagaacaact acaaaacaac cccccctgtg 1020

ctggattctg atggctcttt ctttctgtac tccaaactga ctgtggacaa gtctagatgg 1080

cagcagggga atgtcttttc ttgctctgtc atgcatgagg ctctgcataa ccactacact 1140

cagaaatccc tgtctctgtc tcccgggaaa gggggtgggg gatccggcgg aggaggtagc 1200

gcatcagacg aggaaatcca ggacgtgtca gggacctggt acctgaaagc catgaccgtg 1260

gatttttggt gctccggcat ccatgaggag tcagtcactc ccatgaccct gacaacccta 1320

gaaggtggga atctggaggc caaagtgaca atggatattg agggctttct ccaggagttc 1380

aaagccgtcc tcgagaagac agacgagcct ggaaagtata ctgctgatgg gggaaaacac 1440

gtagcctata tcattcgatc tcgggtgaag gatcattata tcttctattc cgagggcgac 1500

tgccccggcc ctgtgccagg tgtctggcta gttgggaggg accccaagaa caatctcgag 1560

gctctggagg acttcgagaa ggcagctggt gccaggggat tgagcactga gtctatcctt 1620

atcccacgcc agagcgagac ctcaagccca gggtccgac 1659

<210> 33

<211> 1659

<212> DNA

<213> Artificial

<220>

<223> fusion protein

<400> 33

gacaaaaccc acacctgccc accttgtcct gcccctgaac tgctgggagg accttctgtg 60

tttctgttcc caccaaaacc aaaagatacc ctgatgatct ctagaacccc tgaggtgaca 120

tgtgtggtgg tggatgtgtc tcatgaggac cctgaggtca aattcaactg gtacgtggat 180

ggagtggaag tccacaatgc caaaaccaag cctagagagg aacagtacaa ttcaacctac 240

agagtggtca gtgtgctgac tgtgctgcat caggattggc tgaatggcaa ggaatacaag 300

tgtaaagtct caaacaaggc cctgcctgct ccaattgaga aaacaatctc aaaggccaag 360

ggacagccta gggaacccca ggtctacacc ctgccacctt caagagagga aatgaccaaa 420

aaccaggtgt ccctgacatg cctggtcaaa ggcttctacc cttctgacat tgctgtggag 480

tgggagtcaa atggacagcc tgagaacaac tacaaaacaa ccccccctgt gctggattct 540

gatggctctt tctttctgta ctccaaactg actgtggaca agtctagatg gcagcagggg 600

aatgtctttt cttgctctgt catgcatgag gctctgcata accactacac tcagaaatcc 660

ctgtctctgt ctcctggcaa aggcggcgga ggatccggcg gaggaggtag cgcatcagac 720

gaggaaatcc aggacgtgtc agggacctgg tacctgaaag ccatgaccgt ggatttttgg 780

tgctccggca tccatgagga gtcagtcact cccatgaccc tgacaaccct agaaggtggg 840

aatctggagg ccaaagtgac aatggatatt gagggctttc tccaggagtt caaagccgtc 900

ctcgagaaga cagacgagcc tggaaagtat actgctgatg ggggaaaaca cgtagcctat 960

atcattcgat ctcgggtgaa ggatcattat atcttctatt ccgagggcga ctgccccggc 1020

cctgtgccag gtgtctggct agttgggagg gaccccaaga acaatctcga ggctctggag 1080

gacttcgaga aggcagctgg tgccagggga ttgagcactg agtctatcct tatcccacgc 1140

cagagcgaga cctcaagccc agggtccgac ggcggcggtg ggtccggcgg cggtggctcc 1200

gcgtccgatg aagaaatcca ggatgtgagc ggcacctggt atctgaaagc aatgaccgtt 1260

gacttctggt gctccggcat ccatgaagaa agcgtgaccc caatgacctt gaccacgctg 1320

gaaggcggta atttagaagc caaagtaact atggatatcg aaggcttcct gcaggaattt 1380

aaagcggtgc tggaaaaaac tgatgagcca ggtaaataca ccgccgacgg tggcaaacac 1440

gtggcctata ttatccgttc tcgtgtcaaa gaccattata tcttctactc tgaaggcgat 1500

tgccccggtc cggttccggg cgtctggctt gtcggtcgtg acccgaaaaa caacctggaa 1560

gcactcgaag acttcgaaaa agcggcaggc gcgcgtggtc tgtctaccga gagcatcctt 1620

atcccacgtc agagcgaaac ctccagccct ggttccgat 1659

<210> 34

<211> 165

<212> DNA

<213> Artificial

<220>

<223> Albumin binding Domain

<400> 34

agcgctggtg ccgtcgacgc taactctctg gctgaagcta aagttctggc taaccgtgaa 60

ctggacaaat acggtgtttc cgactactac aaaaacctca tcaacaacgc taaaaccgtt 120

gaaggtgtta aagctctgat cgacgaaatt ctcgcagcac tgccg 165

<210> 35

<211> 156

<212> DNA

<213> Artificial

<220>

<223> Albumin binding Domain

<400> 35

agcggcggcg gcggctctct ggctgaagct aaagaagcgg ctaacgcgga actggactct 60

tacggtgttt ccgactttta caaacgtctc atcgataaag ctaaaaccgt tgaaggtgtt 120

gaagctctga aagacgcgat tctcgcagca ctgccg 156

<210> 36

<211> 681

<212> DNA

<213> Artificial

<220>

<223> Fc portion

<400> 36

gacaaaaccc acacctgccc accttgtcct gcccctgaac tgctgggagg accttctgtg 60

tttctgttcc caccaaaacc aaaagatacc ctgatgatct ctagaacccc tgaggtgaca 120

tgtgtggtgg tggatgtgtc tcatgaggac cctgaggtca aattcaactg gtacgtggat 180

ggagtggaag tccacaatgc caaaaccaag cctagagagg aacagtacaa ttcaacctac 240

agagtggtca gtgtgctgac tgtgctgcat caggattggc tgaatggcaa ggaatacaag 300

tgtaaagtct caaacaaggc cctgcctgct ccaattgaga aaacaatctc aaaggccaag 360

ggacagccta gggaacccca ggtctacacc ctgccacctt caagagagga aatgaccaaa 420

aaccaggtgt ccctgacatg cctggtcaaa ggcttctacc cttctgacat tgctgtggag 480

tgggagtcaa atggacagcc tgagaacaac tacaaaacaa ccccccctgt gctggattct 540

gatggctctt tctttctgta ctccaaactg actgtggaca agtctagatg gcagcagggg 600

aatgtctttt cttgctctgt catgcatgag gctctgcata accactacac tcagaaatcc 660

ctgtctctgt ctcctggcaa g 681

<210> 37

<211> 30

<212> DNA

<213> Artificial

<220>

<223> protein tag

<400> 37

agcgcttggt ctcacccgca gttcgaaaaa 30

<210> 38

<211> 36

<212> DNA

<213> Artificial

<220>

<223> synthetic linker

<400> 38

agcgacggtg gtggtggttc tggtggtggt ggatcg 36

<210> 39

<211> 30

<212> DNA

<213> Artificial

<220>

<223> synthetic linker

<400> 39

ggtggtggtg gttctggtgg tggtggatcg 30

<210> 40

<211> 6

<212> DNA

<213> Artificial

<220>

<223> synthetic linker

<400> 40

agcgac 6

<210> 41

<211> 158

<212> PRT

<213> human

<400> 41

His His Leu Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr

1 5 10 15

Trp Tyr Leu Lys Ala Met Thr Val Asp Arg Glu Phe Pro Glu Met Asn

20 25 30

Leu Glu Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn

35 40 45

Leu Glu Ala Lys Val Thr Met Leu Ile Ser Gly Arg Cys Gln Glu Val

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Ile Phe Tyr Cys Glu Gly Glu Leu His Gly Lys Pro Val Arg Gly

100 105 110

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

115 120 125

Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile

130 135 140

Leu Ile Pro Arg Gln Ser Glu Thr Cys Ser Pro Gly Ser Asp

145 150 155

<210> 42

<211> 151

<212> PRT

<213> Artificial

<220>

<223> lipocalin muteins

<400> 42

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

1 5 10 15

Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu Ser Val

20 25 30

Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys

35 40 45

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

50 55 60

Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His

65 70 75 80

Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His Tyr Ile Phe Tyr

85 90 95

Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu Val Gly

100 105 110

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

115 120 125

Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg Gln

130 135 140

Ser Glu Thr Ser Ser Pro Gly

145 150

<210> 43

<211> 178

<212> PRT

<213> human

<400> 43

Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val

1 5 10 15

Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr

20 25 30

Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro

35 40 45

Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile

65 70 75 80

Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Asn

85 90 95

Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser

100 105 110

Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln

115 120 125

Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu

130 135 140

Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly

145 150 155 160

Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile

165 170 175

Asp Gly

<210> 44

<211> 178

<212> PRT

<213> Artificial

<220>

<223> lipocalin muteins

<400> 44

Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val

1 5 10 15

Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr

20 25 30

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

35 40 45

Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Arg Val Glu Phe Gly Val Lys Thr Tyr Lys Tyr Gln Ile

65 70 75 80

Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Gly

85 90 95

Ile Lys Ser Met Pro Gly Met Thr Ser Phe Leu Val Arg Val Val Ser

100 105 110

Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Tyr Val Tyr Gln

115 120 125

Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu

130 135 140

Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly

145 150 155 160

Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Ala Ile

165 170 175

Asp Gly

<210> 45

<211> 178

<212> PRT

<213> Artificial

<220>

<223> lipocalin muteins

<400> 45

Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val

1 5 10 15

Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr

20 25 30

Val Val Gly Glu Ala Gly Asn Leu Ile Leu Arg Glu Asp Lys Asp Pro

35 40 45

Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asp Val Thr Arg Val Glu Phe Gly Val Lys Thr Arg Lys Tyr Arg Ile

65 70 75 80

Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Gly

85 90 95

Ile Lys Ser Met Pro Gly Met Thr Ser Phe Leu Val Arg Val Val Ser

100 105 110

Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Tyr Val Tyr Gln

115 120 125

Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu

130 135 140

Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly

145 150 155 160

Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Ala Ile

165 170 175

Asp Gly

<210> 46

<211> 178

<212> PRT

<213> Artificial

<220>

<223> lipocalin muteins

<400> 46

Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val

1 5 10 15

Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr

20 25 30

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

35 40 45

Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu Arg Glu Asp Lys Ser Tyr

50 55 60

Asp Val Thr Arg Val Glu Phe Gly Val Lys Thr Tyr Lys Tyr Gln Ile

65 70 75 80

Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Gly

85 90 95

Ile Lys Ser Met Pro Gly Met Thr Ser Phe Leu Val Arg Val Val Ser

100 105 110

Thr Asp Tyr Asn Gln His Ala Met Val Phe Phe Lys Tyr Val Tyr Gln

115 120 125

Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu

130 135 140

Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly

145 150 155 160

Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Ala Ile

165 170 175

Asp Gly

<210> 47

<211> 456

<212> DNA

<213> human

<400> 47

gcctcagacg aggagattca ggatgtgtca gggacgtggt atctgaaggc catgacggtg 60

gacagggagt tccctgagat gaatctggaa tcggtgacac ccatgaccct cacgaccctg 120

gaagggggca acctggaagc caaggtcacc atgctgataa gtggccggtg ccaggaggtg 180

aaggccgtcc tggagaaaac tgacgagccg ggaaaataca cggccgacgg gggcaagcac 240

gtggcataca tcatcaggtc gcacgtgaag gaccactaca tcttttactg tgagggcgag 300

ctgcacggga agccggtccg aggggtgaag ctcgtgggca gagaccccaa gaacaacctg 360

gaagccttgg aggactttga gaaagccgca ggagcccgcg gactcagcac ggagagcatc 420

ctcatcccca ggcagagcga aacctgctct ccaggg 456

<210> 48

<211> 453

<212> DNA

<213> Artificial

<220>

<223> lipocalin muteins

<400> 48

gcctcagacg aggagattca ggatgtgtca gggacgtggt atctgaaggc catgacggtg 60

gatttttggt gttctgggat tcatgaggag tctgttacgc caatgactct gactaccctt 120

gaaggcggca atctggaggc taaggtcacc atggatattg agggatttct tcaagagttt 180

aaggcagtgt tagagaagac agatgaaccg ggtaaatata cggccgatgg cggtaaacat 240

gttgcctata tcattcgcag ccatgtgaaa gatcattaca tcttttatag cgagggagat 300

tgtcctggtc cggttccagg ggtgtggctc gtgggcagag accccaagaa caacctggaa 360

gccttggagg actttgagaa agccgcagga gcccgcggac tcagcacgga gagcatcctc 420

atccccaggc agagcgaaac cagctctcca ggg 453

<210> 49

<211> 534

<212> DNA

<213> human

<400> 49

caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60

aacttccagg acaaccaatt ccaggggaag tggtatgtgg taggcctggc agggaatgca 120

attctcagag aagacaaaga cccgcaaaag atgtatgcca ccatctatga gctgaaagaa 180

gacaagagct acaatgtcac ctccgtcctg tttaggaaaa agaagtgtga ctactggatc 240

aggacttttg ttccaggttg ccagcccggc gagttcacgc tgggcaacat taagagttac 300

cctggattaa cgagttacct cgtccgagtg gtgagcacca actacaacca gcatgctatg 360

gtgttcttta agaaagtttc tcaaaacagg gagtacttca agatcaccct ctacgggaga 420

accaaggagc tgacttcgga actaaaggag aacttcatcc gcttctccaa atctctgggc 480

ctccctgaaa accacatcgt cttccctgtc ccaatcgacc agtgtatcga cggc 534

<210> 50

<211> 534

<212> DNA

<213> Artificial

<220>

<223> lipocalin muteins

<400> 50

caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60

aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcgaggc cggaaatctt 120

ttgctgcgtg aggataagga tccgaggaaa atgacggcga ccatttacga gttgaaagaa 180

gataaatcat ataacgtcac ccgggtggag tttggggtta agacatacaa gtaccagatt 240

gggacctttg tgccggggag ccagccgggc gagtttactt taggcggtat taaaagtatg 300

ccgggcatga catcattttt ggtccgcgtc gtgagcacca actacaacca gcatgccatg 360

gtgttcttca agtatgtgta tcagaaccgc gagtattttg agatcacact gtacgggcgc 420

acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480

ctccctgaaa accacatcgt cttccctgtc ccaatcgacc aggctatcga cggc 534

<210> 51

<211> 534

<212> DNA

<213> Artificial

<220>

<223> lipocalin muteins

<400> 51

caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60

aacttccagg acaaccaatt ccatgggaaa tggtacgtag taggtgaggc cggaaatctg 120

attctgcgtg aggataagga tccgagaaaa atgactgcga ccatttacga gttgaaagaa 180

gataaatcat atgacgtcac cagggtggag tttggggtga agacgcgtaa gtaccggatt 240

gggacctttg tgccggggag ccagccgggc gagtttactt taggcggtat taaaagtatg 300

ccgggcatga catcattttt ggtccgcgtc gtgagcacca actacaacca gcatgccatg 360

gtgttcttca agtacgtgta tcagaaccgc gagtattttg agatcacact gtacgggcgc 420

acgaaagaac tgacaagcga gctgaaggaa aattttatcc gcttttccaa atctctgggc 480

ctccctgaaa accacatcgt cttccctgtc ccaatcgacc aggctatcga cggc 534

<210> 52

<211> 534

<212> DNA

<213> Artificial

<220>

<223> lipocalin muteins

<400> 52

caggactcca cctcagacct gatcccagcc ccacctctga gcaaggtccc tctgcagcag 60

aacttccagg acaaccaatt ccatgggaaa tggtatgtcg tgggcgaggc cggaaatctt 120

ttgctgcgtg aggataagga tccgaggaaa atgacggcga ccatttacga gttgagagaa 180

gataaatcat atgacgtcac ccgggtggag tttggggtta agacatacaa gtaccagatt 240

gggacctttg tgccggggag ccagccgggc gagtttactt taggcggtat taaaagtatg 300

ccgggcatga catcattttt ggtccgcgtc gtgagcaccg actacaacca gcatgccatg 360

gtgttcttca agtatgtgta tcagaaccgc gagtattttg agatcacact gtacgggcgc 420

acgaaagaac tgacaagcga gttgaaggaa aattttatcc gcttttccaa atctctgggc 480

ctccctgaaa accacatcgt cttccctgtc ccaatcgacc aggctatcga cggc 534

<210> 53

<211> 457

<212> PRT

<213> Artificial

<220>

<223> heavy chain of reference antibody

<400> 53

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

1 5 10 15

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

20 25 30

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

35 40 45

Ala Ala Ile Asn Gln Asp Gly Ser Glu Lys Tyr Tyr Val Gly Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Val Arg Asp Tyr Tyr Asp Ile Leu Thr Asp Tyr Tyr Ile His Tyr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

130 135 140

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

145 150 155 160

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

165 170 175

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

180 185 190

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

195 200 205

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

210 215 220

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

225 230 235 240

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

245 250 255

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

260 265 270

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

275 280 285

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

290 295 300

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

305 310 315 320

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

325 330 335

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

340 345 350

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

355 360 365

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

370 375 380

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

385 390 395 400

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

405 410 415

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

420 425 430

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 54

<211> 215

<212> PRT

<213> Artificial

<220>

<223> reference antibody light chain

<400> 54

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

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

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Cys Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

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

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 55

<211> 446

<212> PRT

<213> Artificial

<220>

<223> heavy chain of reference antibody

<400> 55

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr

20 25 30

His Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Val Ile Asn Pro Glu Tyr Gly Thr Thr Asp Tyr Asn Gln Arg Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

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

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 56

<211> 219

<212> PRT

<213> Artificial

<220>

<223> reference antibody light chain

<400> 56

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Lys His Ser

20 25 30

Arg Gly Asn Thr Phe Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ile Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 57

<211> 449

<212> PRT

<213> Artificial

<220>

<223> heavy chain of reference antibody

<400> 57

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Lys Thr Phe Trp Ser Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Ile Gly Thr Gly Tyr Thr Glu Pro Asn Pro Lys Tyr

50 55 60

Lys Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Ile Gly Gly Tyr Tyr Gly Asn Phe Asp Gln Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

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

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

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

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 58

<211> 219

<212> PRT

<213> Artificial

<220>

<223> reference antibody light chain

<400> 58

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Ile Ser

20 25 30

Gly Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Gln Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

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

85 90 95

Thr Tyr Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 59

<211> 447

<212> PRT

<213> Artificial

<220>

<223> heavy chain of reference antibody

<400> 59

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

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Asn Tyr

20 25 30

Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asp Pro Ser Asn Ser Tyr Thr Arg Tyr Ser Pro Ser Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Trp Tyr Tyr Lys Pro Phe Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

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

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

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

210 215 220

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

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

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

340 345 350

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

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 60

<211> 217

<212> PRT

<213> Artificial

<220>

<223> reference antibody light chain

<400> 60

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu

35 40 45

Leu Ile Tyr Gly Asn Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Thr Asp Gly

85 90 95

Leu Ser Leu Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

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

115 120 125

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

130 135 140

Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val

145 150 155 160

Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys

165 170 175

Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser

180 185 190

His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu

195 200 205

Lys Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 61

<211> 451

<212> PRT

<213> Artificial

<220>

<223> reference antibody heavy chain

<400> 61

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

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

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

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

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 62

<211> 214

<212> PRT

<213> Artificial

<220>

<223> reference antibody light chain

<400> 62

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 63

<211> 619

<212> PRT

<213> Artificial

<220>

<223> fusion polypeptide

<400> 63

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

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

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

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

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

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

465 470 475 480

Leu Lys Ala Met Thr Val Asp Phe Trp Cys Ser Gly Ile His Glu Glu

485 490 495

Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu

500 505 510

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

515 520 525

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

530 535 540

Lys His Val Ala Tyr Ile Ile Arg Ser Arg Val Lys Asp His Tyr Ile

545 550 555 560

Phe Tyr Ser Glu Gly Asp Cys Pro Gly Pro Val Pro Gly Val Trp Leu

565 570 575

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

580 585 590

Lys Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro

595 600 605

Arg Gln Ser Glu Thr Ser Ser Pro Gly Ser Asp

610 615

<210> 64

<211> 644

<212> PRT

<213> Artificial

<220>

<223> fusion polypeptide

<400> 64

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

1 5 10 15

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

20 25 30

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

35 40 45

Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

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

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

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

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

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

340 345 350

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

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

450 455 460

Gly Ser Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser

465 470 475 480

Lys Val Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys

485 490 495

Trp Tyr Val Val Gly Glu Ala Gly Asn Leu Leu Leu Arg Glu Asp Lys

500 505 510

Asp Pro Arg Lys Met Thr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys

515 520 525

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

530 535 540

Gln Ile Gly Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu

545 550 555 560

Gly Gly Ile Glu Ser Met Pro Gly Met Thr Ser Phe Leu Val Arg Val

565 570 575

Val Ser Thr Asn Tyr Asn Gln His Ala Ile Val Phe Phe Lys Tyr Val

580 585 590

Tyr Gln Asn Arg Glu Tyr Phe Glu Ile Thr Leu Tyr Gly Arg Thr Lys

595 600 605

Glu Leu Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser

610 615 620

Leu Gly Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln

625 630 635 640

Ala Ile Asp Gly

<210> 65

<211> 1353

<212> DNA

<213> Artificial

<220>

<223> reference antibody heavy chain

<400> 65

gaagtgcagc tggtcgaaag tggtggtggt ctggtgcagc ccggtagaag tctgcgtctg 60

tcttgtgccg catctggttt tacattcgac gattacgcaa tgcattgggt gagacaggcc 120

cccggcaagg gactggagtg ggtctccgct atcacctgga acagcgggca tattgactac 180

gcagattccg tggaaggcag gttcacaatc tctcgggaca acgccaagaa tagtctgtat 240

ctgcagatga attcactgag ggccgaggat accgccgtgt actattgcgc taaagtctct 300

tatctgtcta ccgcatcatc tctggattac tggggtcagg gaacactggt cactgtctcc 360

tctgctagca caaagggccc tagtgtgttt cctctggctc cctcttccaa atccacttct 420

ggtggcactg ctgctctggg atgcctggtg aaggattact ttcctgaacc tgtgactgtc 480

tcatggaact ctggtgctct gacttctggt gtccacactt tccctgctgt gctgcagtct 540

agtggactgt actctctgtc atctgtggtc actgtgccct cttcatctct gggaacccag 600

acctacattt gtaatgtgaa ccacaaacca tccaacacta aagtggacaa aaaagtggaa 660

cccaaatcct gtgacaaaac ccacacctgc ccaccttgtc ctgcccctga actgctggga 720

ggaccttctg tgtttctgtt cccaccaaaa ccaaaagata ccctgatgat ctctagaacc 780

cctgaggtga catgtgtggt ggtggatgtg tctcatgagg accctgaggt caaattcaac 840

tggtacgtgg atggagtgga agtccacaat gccaaaacca agcctagaga ggaacagtac 900

aattcaacct acagagtggt cagtgtgctg actgtgctgc atcaggattg gctgaatggc 960

aaggaataca agtgtaaagt ctcaaacaag gccctgcctg ctccaattga gaaaacaatc 1020

tcaaaggcca agggacagcc tagggaaccc caggtctaca ccctgccacc ttcaagagag 1080

gaaatgacca aaaaccaggt gtccctgaca tgcctggtca aaggcttcta cccttctgac 1140

attgctgtgg agtgggagtc aaatggacag cctgagaaca actacaaaac aaccccccct 1200

gtgctggatt ctgatggctc tttctttctg tactccaaac tgactgtgga caagtctaga 1260

tggcagcagg ggaatgtctt ttcttgctct gtcatgcatg aggctctgca taaccactac 1320

actcagaaat ccctgtctct gtctcctggc aaa 1353

<210> 66

<211> 642

<212> DNA

<213> Artificial

<220>

<223> reference antibody light chain

<400> 66

gacatccaga tgacccagag cccaagttcc ctgagcgcaa gcgtcggaga tcgtgtgact 60

attacctgta gagcaagcca gggcatcaga aactacctgg catggtatca gcagaagccc 120

ggtaaagccc ctaagctgct gatctacgcc gcttccactc tgcagtctgg cgtgccaagc 180

aggttctctg gcagtggatc agggaccgac tttaccctga caatttccag cctgcagccc 240

gaggatgtcg ctacatacta ttgccagcgg tacaatcggg caccttatac attcggtcag 300

gggactaaag tggaaatcaa gagaactgtc gcggcgcctt ctgtgttcat tttcccccca 360

tctgatgaac agctgaaatc tggcactgct tctgtggtct gtctgctgaa caacttctac 420

cctagagagg ccaaagtcca gtggaaagtg gacaatgctc tgcagagtgg gaattcccag 480

gaatctgtca ctgagcagga ctctaaggat agcacatact ccctgtcctc tactctgaca 540

ctgagcaagg ctgattacga gaaacacaaa gtgtacgcct gtgaagtcac acatcagggg 600

ctgtctagtc ctgtgaccaa atccttcaat aggggagagt gc 642

<210> 67

<211> 1857

<212> DNA

<213> Artificial

<220>

<223> fusion polypeptide

<400> 67

gaagtgcagc tggtcgaaag tggtggtggt ctggtgcagc ccggtagaag tctgcgtctg 60

tcttgtgccg catctggttt tacattcgac gattacgcaa tgcattgggt gagacaggcc 120

cccggcaagg gactggagtg ggtctccgct atcacctgga acagcgggca tattgactac 180

gcagattccg tggaaggcag gttcacaatc tctcgggaca acgccaagaa tagtctgtat 240

ctgcagatga attcactgag ggccgaggat accgccgtgt actattgcgc taaagtctct 300

tatctgtcta ccgcatcatc tctggattac tggggtcagg gaacactggt cactgtctcc 360

tctgctagca caaagggccc tagtgtgttt cctctggctc cctcttccaa atccacttct 420

ggtggcactg ctgctctggg atgcctggtg aaggattact ttcctgaacc tgtgactgtc 480

tcatggaact ctggtgctct gacttctggt gtccacactt tccctgctgt gctgcagtct 540

agtggactgt actctctgtc atctgtggtc actgtgccct cttcatctct gggaacccag 600

acctacattt gtaatgtgaa ccacaaacca tccaacacta aagtggacaa aaaagtggaa 660

cccaaatcct gtgacaaaac ccacacctgc ccaccttgtc ctgcccctga actgctggga 720

ggaccttctg tgtttctgtt cccaccaaaa ccaaaagata ccctgatgat ctctagaacc 780

cctgaggtga catgtgtggt ggtggatgtg tctcatgagg accctgaggt caaattcaac 840

tggtacgtgg atggagtgga agtccacaat gccaaaacca agcctagaga ggaacagtac 900

aattcaacct acagagtggt cagtgtgctg actgtgctgc atcaggattg gctgaatggc 960

aaggaataca agtgtaaagt ctcaaacaag gccctgcctg ctccaattga gaaaacaatc 1020

tcaaaggcca agggacagcc tagggaaccc caggtctaca ccctgccacc ttcaagagag 1080

gaaatgacca aaaaccaggt gtccctgaca tgcctggtca aaggcttcta cccttctgac 1140

attgctgtgg agtgggagtc aaatggacag cctgagaaca actacaaaac aaccccccct 1200

gtgctggatt ctgatggctc tttctttctg tactccaaac tgactgtgga caagtctaga 1260

tggcagcagg ggaatgtctt ttcttgctct gtcatgcatg aggctctgca taaccactac 1320

actcagaaat ccctgtctct gtctcctggc aaaggcggcg gaggatccgg cggaggaggt 1380

agcggcggag gaggtagcgc atcagacgag gaaatccagg acgtgtcagg gacctggtac 1440

ctgaaagcca tgaccgtgga tttttggtgc tccggcatcc atgaggagtc agtcactccc 1500

atgaccctga caaccctaga aggtgggaat ctggaggcca aagtgacaat ggatattgag 1560

ggctttctcc aggagttcaa agccgtcctc gagaagacag acgagcctgg aaagtatact 1620

gctgatgggg gaaaacacgt agcctatatc attcgatctc gggtgaagga tcattatatc 1680

ttctattccg agggcgactg ccccggccct gtgccaggtg tctggctagt tgggagggac 1740

cccaagaaca atctcgaggc tctggaggac ttcgagaagg cagctggtgc caggggattg 1800

agcactgagt ctatccttat cccacgccag agcgagacct caagcccagg gtccgac 1857

<210> 68

<211> 1932

<212> DNA

<213> Artificial

<220>

<223> fusion polypeptide

<400> 68

gaagtgcagc tggtcgaaag tggtggtggt ctggtgcagc ccggtagaag tctgcgtctg 60

tcttgtgccg catctggttt tacattcgac gattacgcaa tgcattgggt gagacaggcc 120

cccggcaagg gactggagtg ggtctccgct atcacctgga acagcgggca tattgactac 180

gcagattccg tggaaggcag gttcacaatc tctcgggaca acgccaagaa tagtctgtat 240

ctgcagatga attcactgag ggccgaggat accgccgtgt actattgcgc taaagtctct 300

tatctgtcta ccgcatcatc tctggattac tggggtcagg gaacactggt cactgtctcc 360

tctgctagca caaagggccc tagtgtgttt cctctggctc cctcttccaa atccacttct 420

ggtggcactg ctgctctggg atgcctggtg aaggattact ttcctgaacc tgtgactgtc 480

tcatggaact ctggtgctct gacttctggt gtccacactt tccctgctgt gctgcagtct 540

agtggactgt actctctgtc atctgtggtc actgtgccct cttcatctct gggaacccag 600

acctacattt gtaatgtgaa ccacaaacca tccaacacta aagtggacaa aaaagtggaa 660

cccaaatcct gtgacaaaac ccacacctgc ccaccttgtc ctgcccctga actgctggga 720

ggaccttctg tgtttctgtt cccaccaaaa ccaaaagata ccctgatgat ctctagaacc 780

cctgaggtga catgtgtggt ggtggatgtg tctcatgagg accctgaggt caaattcaac 840

tggtacgtgg atggagtgga agtccacaat gccaaaacca agcctagaga ggaacagtac 900

aattcaacct acagagtggt cagtgtgctg actgtgctgc atcaggattg gctgaatggc 960

aaggaataca agtgtaaagt ctcaaacaag gccctgcctg ctccaattga gaaaacaatc 1020

tcaaaggcca agggacagcc tagggaaccc caggtctaca ccctgccacc ttcaagagag 1080

gaaatgacca aaaaccaggt gtccctgaca tgcctggtca aaggcttcta cccttctgac 1140

attgctgtgg agtgggagtc aaatggacag cctgagaaca actacaaaac aaccccccct 1200

gtgctggatt ctgatggctc tttctttctg tactccaaac tgactgtgga caagtctaga 1260

tggcagcagg ggaatgtctt ttcttgctct gtcatgcatg aggctctgca taaccactac 1320

actcagaaat ccctgtctct gtctcctggc aaaggcggcg gaggatccgg gggtggggga 1380

agcggcggag gaggtagcca ggattcaacc agcgatctga ttccagcacc gccactgtcg 1440

aaagtgccac tgcaacaaaa ctttcaagat aaccagtttc acggcaagtg gtatgtggtc 1500

ggggaggccg gtaacctgct gctgagggaa gacaaagatc cacggaaaat gaccgccacc 1560

atctacgagc tgaaagagga taagtcctac gacgtgactc gggtggagtt cggcgcaaaa 1620

acctacaagt accagatcgg caccttcgtg cccggctctc agcctggcga gtttaccctg 1680

ggcggcatcg aatctatgcc cggcatgacc agctttctcg tgcgggtggt gtccaccaac 1740

tacaaccagc acgccatcgt gttcttcaaa tacgtgtacc agaaccgcga gtacttcgag 1800

atcaccctgt acggccggac caaagagctg acctccgaac tgaaagagaa cttcatccgg 1860

ttctccaagt ccctgggcct gcccgagaac cacatcgtgt tccccgtgcc tatcgaccag 1920

gccatcgacg gc 1932