RU2814653C2 - Novel fusion proteins specific for cd137 and gpc3 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to recombinant production of fusion proteins binding glypican-3 (GPC3) and differentiation cluster 137 (CD137) and can be used in medicine for therapy of GPC3-positive forms of cancer. Disclosed is a fused protein which includes a full-length immunoglobulin which specifically binds to GPC3, connected from the C-terminus by a peptide linker to the N-terminus of a lipocalin mutein which specifically binds to CD137.

EFFECT: invention enables to obtain a fusion protein capable of simultaneously specifically binding both CD137 and GPC3.

15 cl, 11 dwg, 11 tbl, 14 ex

Description

I. Background to the invention

[0001] Glypican-3 (GPC3, also called DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1) is an oncofetal antigen belonging to the glypican family of glycosyl-phosphatidylinositol-anchored heparin sulfate proteoglycans. GPC3 is expressed in various tissues during development, playing important roles in morphogenesis and growth, for example through fibroblast growth factor (FGF), non-canonical Wnt or insulin-like growth factor signaling (Cheng et al., Carcinogenesis, 2008, Song et al. ., J Biol Chem, 2005, Song et al., J Biol Chem, 1997). However, in most healthy adult tissues, GPC3 expression is downregulated or silenced. In addition, GPC3 can either positively or negatively regulate cell growth depending on the cell type. Loss-of-function mutations in GPC3 are responsible for the development of Simpson-Golabi-Bemel syndrome or dysmorphic Simpson syndrome (SGBS), a rare X-linked disorder manifesting as overgrowth, and patients with SGBS have an increased risk of developing embryonal tumors (Pilia et al. al., Nat Genet, 1996), including Wilms tumors.

[0002] GPC3 is expressed in various types of cancer, including liver carcinoma, gastric carcinoma, melanoma, high-grade urothelial carcinoma, testicular cancer, and some forms of uterine and vaginal cancer (Aydin et al., Diagn Pathol, 2015, Ushiku et al. ., Cancer Sci, 2009, Gailey and Bellizzi, Am J Clin Pathol, 2013, Yamanaka et al., Oncology, 2007, Nakatsura et al., Clin Cancer Res, 2004, Zynger et al., Am J Surg Pathol, 2006, Montalbano et al., Int J Oncol, 2016, Midorikawa et al., Int J Cancer, 2003). In particular, GPC3 is highly expressed in hepatocellular carcinoma (HCC) (Capurro et al., Gastroenterology, 2003, Nakatsura et al., Biochem Biophys Res Commun, 2003, Sung et al., Cancer Sci, 2003, Zhu et al. , Gut, 2001), a major form of liver cancer, accounting for 90% of all liver cancer cases and causing at least 500,000 deaths per year (Jelic et al., Ann Oncol, 2010). Extensive research has been conducted to investigate GPC3 as a diagnostic biomarker and therapeutic target in HCC and other types of cancer. Several antibodies targeting GPC3 have been generated, including humanized mouse antibodies YP7 and GC33 and human antibodies HN3 and MDX-1414, but have not been demonstrated to inhibit HCC cell proliferation or induce apoptosis (Feng and Ho, FEBS Lett, 2014) . As an exception, GC33 is currently being evaluated in clinical trials for HCC. The mechanism of GC33 function involves antibody-dependent cell-mediated cytotoxicity (ADCC), but GC33 does not directly inhibit the proliferation of GPC3-positive tumor cells (Takai et al., Cancer Biol Ther, 2009, Nakano et al., Biochem Biophys Res Commun, 2009, Ishiguro et al. , Cancer Res, 2008). However, although GC33 has been shown to have an inhibitory effect on tumor growth in vivo, it has not demonstrated satisfactory clinical efficacy. Meanwhile, the results of knockdown and siRNA experiments indicate that GPC3 is not a lethal gene for HCC cells, making it uncertain whether antibodies to GPC3 can induce effective tumor regression.

[0003] Cluster of differentiation 137 or CD137 (also known as 4-1BB or TNFRS9) is a costimulatory immune receptor and a member of the tumor necrosis factor receptor (TNFR) superfamily. It is predominantly expressed on activated CD4+ and CD8+ T cells, activated B cells and natural killer (NK) cells, but can also occur on resting monocytes and dendritic cells (Li and Liu, Clin Pharmacol, 2013) or endothelial cells (Snell et al., Immunol Rev, 2011). CD137 plays an important role in regulating the immune response and is therefore a target in anticancer immunotherapy. CD137 ligand (CD137L) is the only known natural CD137 ligand and is constitutively expressed on several types of antigen presenting cells, such as activated B cells, monocytes and splenic dendritic cells, and can be induced on T lymphocytes.

[0004] CD137L is a trimeric protein that exists in a membrane-bound form and as a soluble variant. However, the ability of soluble CD137L to activate CD137, for example on CD137-expressing lymphocytes, is limited and large concentrations are required to achieve an effect (Wyzgol et al., J Immunol, 2009). The natural pathway for CD137 activation is through the action of a CD137-positive cell on a CD137L-positive cell. CD137 activation is then thought to be induced by clustering involving CD137L on the opposing cell, leading to signaling through TRAF1, 2 and 3 (Yao et al., Nat Rev Drug Discov, 2013, Snell et al., Immunol Rev, 2011) and additional concomitant downstream effects in CD137-positive T cells. In the case of T cells activated by recognition of their respective cognate targets, the effects caused by CD137 costimulation are further enhanced activation, enhanced survival and proliferation, production of proinflammatory cytokines, and improved killing capacity.

[0005] The benefit of CD137 costimulation in eliminating cancer cells has been demonstrated in a number of in vivo models. Forced expression of CD137L in a tumor, for example, leads to tumor rejection (Melero et al., Eur J Immunol, 1998). Likewise, forced expression of CD137 scFv on tumors results in CD4 ⁺ T cell and NK cell dependent tumor clearance (Yang et al., Cancer Res. 2007, Zhang et al., Mol Cancer Ther. 2006, Ye et al. ., Nat Med, 2002). It has also been demonstrated that systemically administered anti-CD137 antibody results in decreased tumor growth (Martinet et al., Gene Ther, 2002).

[0006] It has been shown that CD137 is an excellent marker of naturally occurring tumor-reactive T cells in human tumors (Ye et al., Clin Cancer Res, 2014), and that antibodies to CD137 can be used to improve expansion and activity of CD8 ⁺ anti-melanoma effector lymphocytes for use in adoptive T-cell therapy (Chacon et al., PLoS One, 2013).

[0007] Preclinical demonstration of the potential therapeutic utility of CD137 costimulation has spurred the development of therapeutic antibodies targeting CD137, including BMS-663513 (described in US Pat. No. 7288638) and PF-05082566 (Fisher et al., Cancer Immunol Immunother, 2012).

[0008] The present invention provides, among other things, novel approaches to simultaneously engage CD137 and GPC3 using one or more fusion proteins having CD137 binding specificity and GPC3 binding specificity.

II. DEFINITIONS

[0009] The following list defines the terms, phrases, and abbreviations used in this specification. All terms listed and defined herein are intended to cover all grammatical forms.

[0010] As used herein, unless otherwise indicated, “CD137” means human CD137 (huCD137). Human CD137 means the full-length protein defined in UniProt Q07011, a fragment or variant thereof. CD137 is also known as 4-1BB, a member of the tumor necrosis factor receptor superfamily 9 (TNFRSF9), and is induced by lymphocyte activation (ILA). In some specific embodiments, CD137 from non-human species is used, for example, cynomolgus CD137 and mouse CD137.

[0011] As used herein, unless otherwise indicated, “glypican-3” or “GPC3” means human GPC3 (huGPC3). Human GPC3 means the full-length protein defined in UniProt P51654, a fragment or variant thereof. Human GPC3 is encoded by the GPC3 gene. GPC3 is also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1. In some specific embodiments, GPC3 from non-human species is used, for example, cynomolgus GPC3 and mouse GPC3.

[0012] As used herein, the term “binding affinity” describes the ability of a biomolecule (e.g., a polypeptide or protein) of the present invention (e.g., a lipocalin mutein, antibody, fusion protein, or any other peptide or protein) to bind and complex with a selected target . Binding affinity is measured using a number of methods known to those skilled in the art, including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA), including direct and competitive ELISA, calorimetric methods such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well known in the art, and some examples of such methods are further described herein. In this case, the binding affinity is recorded as the value of the dissociation constant (K _D ), half-maximal effective concentration (EC ₅₀ ) or half-maximal inhibitory concentration (IC ₅₀ ), measured using these methods. A lower _KD , EC ₅₀ or IC ₅₀ value reflects better (higher) binding capacity (affinity). Accordingly, the binding affinities of two biomolecules for a selected target can be measured and compared. When comparing the binding affinities of two biomolecules to a selected target, the term “about the same,” “substantially the same,” or “substantially similar” means that one biomolecule has a binding affinity, expressed as a _KD , EC ₅₀ , or IC ₅₀ value, that identical or similar to that of another molecule within the range of variation in binding affinity measurement readings from experiment to experiment. The variation in binding affinity measurements from experiment to experiment depends on the specific method used and is known to those skilled in the art.

[0013] As used herein, the term “substantially” can also refer to the qualitative condition of exhibiting a full or nearly full measure or extent of a characteristic or property of interest. A person skilled in the field of biology will understand that biological and chemical phenomena rarely, if ever, reach and/or progress to completion or achieve or fail to achieve an absolute result. Thus, the term “substantially” is used herein to denote the potential lack of completeness inherent in many biological and chemical phenomena.

[0014] As used herein, the term “detect,” “detectable,” “detectable,” or “revealing” is understood at both a quantitative and qualitative level, and combinations thereof. Thus, it includes quantitative, semi-quantitative and qualitative measurements made on the biomolecule of the present invention.

[0015] As used herein, the term “detectable affinity” generally means the binding ability of a biomolecule to its target as measured by a K _D , EC ₅₀ or IC ₅₀ value of a maximum of about 10 ^-5 M or lower. Binding affinities, as measured by K _D , EC ₅₀ or IC ₅₀ values that are greater than 10 ^-5 M, are generally no longer measurable by traditional methods such as ELISA and SPR and are therefore of minor importance.

[0016] It should be noted that the formation of the complex between the biomolecule of the present invention and its target is influenced by many different factors, such as the concentrations of the respective target, the presence of competitors, the pH and ionic strength of the buffer system used, the experimental method used to determine the binding affinity (e.g., fluorescence titration, competitive ELISA (also called competitive ELISA), and surface plasmon resonance), and even a mathematical algorithm used to evaluate experimental data. Therefore, one skilled in the art will appreciate that the binding affinity, as measured by the K _D , EC ₅₀ or IC ₅₀ value, may vary within a certain experimental range, depending on the experimental method and design. This means that there may be slight variation in the measured _KD , EC ₅₀ or IC ₅₀ values or range of acceptable values depending, for example, on whether such values were determined by ELISA (including direct or competitive ELISA), SPR or other method.

[0017] As used herein, “relation specific,” “binding specific,” “specifically binds,” or “binding specificity” refers to the ability of a biomolecule to distinguish a desired target (e.g., CD137 and GPC3) from one or more reference targets (e.g., cellular receptor for lipocalin associated with neutrophil gelatinase). It is clear that such specificity is not an absolute, but a relative property, it can be determined, for example, according to the results of SPR, Western blotting, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric analysis (IRMA), immunohistochemistry (IHC) and peptide scanning.

[0018] When used herein in the context of a fusion protein of the present invention that binds CD137 and GPC3, the term “specific for,” “binding specific,” “specifically binds,” or “binding specificity” means that the fusion protein binds , reacts with or is directed against CD137 and GPC3 as described herein, but does not actually bind another protein. The term “other protein” includes any proteins that are not CD137 or GPC3, or proteins that are closely related to or homologous to CD137 or GPC3. However, the term “other protein” does not exclude CD137 or GPC3 derived from non-human species and fragments and/or variants of CD137 or GPC3. The term “substantially does not bind” means that the fusion proteins of the present invention bind another protein with lower binding affinity than CD137 and/or GPC3, i.e. exhibit cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6 or 5%. Whether a fusion protein is capable of specifically reacting as defined herein above can be readily tested, among other things, by comparing the reaction of the fusion protein of the present invention with CD137 and/or GPC3 and the reaction of said fusion protein with other protein(s). proteins).

[0019] As used herein, the term “lipocalin” refers to a monomeric protein weighing approximately 18-20 kDa having a cylindrical β-sheet sheet supersecondary structure region containing multiple β chains (preferably eight β chains designated A to H) connected in pairs by a plurality of (preferably four) loops at one end so that they ultimately constitute a ligand-binding pocket and define the entrance to the ligand-binding pocket. Preferably, the loops constituting the ligand-binding pocket, in the context of the present invention, are loops connecting the open ends of the β-strands A and B, C and D, E and F and G and H, and are the loops designated AB, CD, E.F. and G.H. It is well known that the diversity of these loops in the otherwise rigid lipocalin framework gives rise to many different binding variants among members of the lipocalin family, each of which is able to adapt to targets of different size, shape and chemical nature (reviewed, for example, in Skerra, Biochim Biophys Acta, 2000, Flower et al., Biochim Biophys Acta, 2000, Flower, Biochem J, 1996). It is clear that the lipocalin family of proteins has naturally evolved to bind a wide range of ligands that exhibit unusually low levels of overall sequence conservation (often with sequence identity values of less than 20%), while maintaining a highly conserved overall folding pattern. The correspondence between positions in different lipocalins is also well known to those skilled in the art (see, for example, US Pat. No. 7,250,297). Proteins falling within the definition of “lipocalin” as used herein include, but are not limited to, human lipocalins, including tear lipocalin (Tlc, Lcn1), lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D ( ApoD), apolipoprotein M, α ₁ -acid glycoprotein 1, α ₁ -acid glycoprotein 2, α ₁ -microglobulin, complement component 8γ, retinol binding protein (RBP), epididymal retinoic acid binding protein, glycodelin, odorant binding protein protein IIa, odorant binding protein IIb, lipocalin 15 (Lcn15) and prostaglandin D synthase.

[0020] As used herein, unless otherwise indicated, “tear lipocalin” refers to human tear lipocalin (hTlc) and further refers to mature human tear lipocalin. The term "mature" when used to characterize a protein means a protein that does not actually contain a signal peptide. "Mature hTlc" as used herein refers to a mature form of human tear lipocalin that does not contain a signal peptide. Mature hTlc is described by residues 19-176 of the sequence deposited in the SWISS-PROT data bank under accession number P31025, and its amino acid sequence is given under SEQ ID NO: 1.

[0021] As used herein, “lipocalin-2” or “neutrophil gelatinase-associated lipocalin” refers to human lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL), and further refers to mature human lipocalin -2 or mature human lipocalin associated with neutrophil gelatinase. The term "mature" when used to characterize a protein means a protein that does not actually contain a signal peptide. “Mature hNGAL” of the present invention refers to the mature form of human lipocalin associated with neutrophil gelatinase, which does not contain a signal peptide. Mature hNGAL is described by residues 21-198 of the sequence deposited in the SWISS-PROT data bank under accession number P80188, and its amino acid sequence is given under SEQ ID NO: 2.

[0022] As used herein, the term “native sequence” refers to a protein or polypeptide having a naturally occurring or wild-type sequence, regardless of how it is produced. A protein or polypeptide with such a native sequence can be isolated from natural sources or can be produced by other methods, such as recombinant or synthetic methods.

[0023] “Native sequence lipocalin” refers to a lipocalin having the same amino acid sequence as the corresponding polypeptide obtained from natural sources. Thus, a native sequence lipocalin may have the amino acid sequence of the corresponding naturally occurring lipocalin (wild type) from any organism, particularly a mammal. The term “native sequence,” when used in the context of lipocalin, specifically covers naturally occurring truncated or secreted forms of lipocalin, naturally occurring variant forms such as alternatively spliced forms, and naturally occurring allelic variants of lipocalin. The terms “native sequence lipocalin” and “wild type lipocalin” are used interchangeably herein.

[0024] As used herein, a "mutein", a "mutant" molecule (whether a protein or a nucleic acid) or a "mutant" refers to a change, deletion or insertion of one or more amino acids or one or more nucleotides from a naturally occurring protein (wild type) or naturally occurring nucleic acid (wild type). The term also includes fragments of the mutein described herein. The present invention explicitly covers the lipocalin muteins described herein having a supersecondary structure region of a cylindrical β-pleated sheet containing eight β-strands connected in pairs by four loops at one end so that they result in a ligand-binding pocket and define entry into the ligand binding pocket, wherein at least one amino acid of each of at least three of the four loops has been mutated from the native sequence lipocalin. The lipocalin muteins of the present invention preferably have CD137 binding function as described herein.

[0025] As used herein, the term “fragment” in connection with the lipocalin muteins of the present invention refers to proteins or polypeptides derived from full-length mature hTlc or hNGAL or lipocalin muteins that are truncated at the N-terminus and/or C-terminus, i.e. .e. which lack at least one of the N-terminal and/or C-terminal amino acids. Such fragments may comprise at least 10 or more, for example 20 or 30 or more, consecutive amino acids of the primary sequence of mature hTlc or hNGAL or the lipocalin mutein from which it is derived, and are typically detectable in a mature hTlc or hNGAL immunoassay. Such a fragment may lack up to 2, up to 3, up to 4, up to 5, up to 10, up to 15, up to 20, up to 25 or up to 30 (including all numbers in between) N-terminal and/or C-terminal amino acids. As an illustrative example, such a fragment may lack one, two, three or four N-terminal (His-His-Leu-Leu) and/or one or two C-terminal amino acids (Ser-Asp) of mature hTlc. It is understood that the fragment is preferably a functional fragment of the mature hTlc or hNGAL or lipocalin mutein from which it is derived, which means that it preferably retains the binding specificity, preferably to CD137, of the mature hTlc/hNGAL or lipocalin mutein from which it is derived. As an illustrative example, such a functional fragment may contain at least amino acids at positions 5-153, 5-150, 9-148, 12-140, 20-135 or 26-133 corresponding to the linear polypeptide sequence of mature hTlc. As another illustrative example, such a functional fragment may contain at least amino acid positions 13-157, 15-150, 18-141, 20-134, 25-134, or 28-134, corresponding to the linear polypeptide sequence of mature hNGAL.

[0026] "Fragment" with respect to the corresponding target CD137 or GPC3 fusion protein of the present invention refers to N-terminally and/or C-terminally truncated CD137 or GPC3 or the protein domains of CD137 or GPC3. The CD137 fragments or GPC3 fragments described herein retain the ability of full-length CD137 or GPC3 to be recognized and/or bound by the fusion protein of the present invention. As an illustrative example, the fragment may be the extracellular domain of CD137 or GPC3. As an illustrative example, such an extracellular domain may contain the amino acids of CD137 extracellular subdomains, such as the amino acid sequences of domain 1 (residues 24-45 from UniProt Q07011), domain 2 (residues 46-86), domain 3 (87-86), individually or in combination. 118) and domain 4 (residues 119-159).

[0027] As used herein, the term “variant” refers to derivatives of a protein or polypeptide that include mutations, for example by substitutions, deletions, insertions and/or chemical modifications, of an amino acid sequence or nucleotide sequence. In some embodiments, such mutations and/or chemical modifications do not reduce the functionality of the protein or peptide. Such substitutions may be conservative, i.e. the amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are substitutions among members of the following groups: 1) alanine, serine, threonine and valine; 2) aspartic acid, glutamic acid, glutamine and asparagine, as well as histidine; 3) arginine, lysine, glutamine, asparagine and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine and proline and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Such variants include proteins or polypeptides in which one or more amino acids have been replaced by their corresponding D-stereoisomers or amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline. Such variants also include, for example, proteins or polypeptides in which one or more amino acid residues have been added or deleted at the N- and/or C-terminus. Typically, the variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, or at least about 98% amino acid sequence identity to a protein or polypeptide with native sequence. The variant preferably retains biological activity, such as binding to the same target, of the protein or polypeptide from which it is derived.

[0028] The term “variant” as used herein in relation to the corresponding CD137 or GPC3 protein ligand of the fusion protein of the present invention refers to CD137 or GPC3 or a fragment thereof, respectively, that has 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 native CD137 or GPC3 sequence (CD137 or wild-type GPC3), such as CD137 deposited under UniProt number Q07011 or GPC3 deposited under UniProt number P51654, as described herein. The CD137 variant or GPC3 variant, respectively, preferably has at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% amino acid identity with wild-type CD137 or GPC3. The CD137 variant or GPC3 variant described herein retains the ability to bind the CD137-GPC3 specific fusion proteins disclosed in the present invention.

[0029] The term “variant” as used herein in relation to a lipocalin mutein refers to a lipocalin mutein or fragment thereof of the present invention wherein the sequence has mutations, including substitutions, deletions and insertions, and/or chemical modifications. The lipocalin mutein variant described herein retains the biological activity, such as binding to CD137, of the lipocalin mutein from which it is derived. Typically, a lipocalin mutein variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% amino acid sequence identity with the lipocalin mutein from which it is derived. received.

[0030] As used herein, the term "mutagenesis" refers to the introduction of mutations into a polynucleotide or amino acid sequence. Mutations are preferably introduced under experimental conditions such that an amino acid occurring naturally at a given position in a protein or polypeptide sequence can be changed, for example, at least one amino acid can be replaced. The term "mutagenesis" also includes (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. Thus, within the scope of the present invention, for example, one amino acid at a selected position in the sequence is replaced by a stretch of three amino acids, resulting in the addition of two amino acid residues compared to the length of the corresponding segment of the native amino acid sequence of the protein or polypeptide. Such an insertion or deletion can be introduced independently of each other into any of the sequence segments that can be mutagenized in the present invention. In one illustrative embodiment of the present invention, the insertion may be introduced into a segment of the amino acid sequence corresponding to the AB loop of the native sequence lipocalin (see International Patent Publication No. WO 2005/019256, which is incorporated herein by reference in its entirety).

[0031] As used herein, the term "random mutagenesis" means that no predetermined mutation (amino acid change) is present at a specific position in the sequence, but that during mutagenesis, at least two amino acids can be inserted at a predetermined position in the sequence with a certain probability .

[0032] As used herein, the term “sequence identity” or “identity” refers to a property of sequences that measures their similarity or relatedness. The term “sequence identity” or “identity” as used herein means the percentage of pairwise identical residues relative to the number of residues in the longer of the two sequences obtained after (homologous) sequence alignment of a protein or polypeptide of the present invention with the sequence in question. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

[0033] As used herein, the term "sequence homology" or "homology" has its ordinary meaning, and a homologous amino acid includes identical amino acids, as well as amino acids that are considered conservative substitutions at equivalent positions in the linear amino acid sequence of a protein, or a polypeptide herein the invention (eg, any lipocalin fusion proteins or muteins of the present invention).

[0034] One skilled in the art will be aware of available computer programs such as BLAST (Altschul et al., Nucleic Acids Res, 1997), BLAST2 (Altschul et al., J Mol Biol, 1990), and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981) to determine sequence homology or sequence identity using standard parameters. The percentage of sequence homology or sequence identity in this case can be determined, for example, using the BLASTP program, version 2.2.5 dated November 16, 2002 (Altschul et al., Nucleic Acids Res, 1997). In this embodiment, the percentage of homology is based on the results of an alignment of all protein or polypeptide sequences (matrix: BLOSUM 62; gap opening penalties: 11.1; cutoff set to 10 ^-3 ), including propeptide sequences, preferably using a wild-type protein backbone type as a standard for pairwise comparison. It is calculated as the percentage of the number of "positive" (homologous amino acids) indicated by the BLASTP program output divided by the total number of amino acids selected by the program for alignment.

[0035] In particular, to determine whether an amino acid residue from the amino acid sequence of a lipocalin mutein differs from that of wild-type lipocalin corresponding to a particular position in the amino acid sequence of wild-type lipocalin, one skilled in the art can use tools and methods well known in the art. techniques, such as various alignment methods manually or using computer programs such as BLAST 2.0, which means Basic Local Alignment Search Tool, or ClustalW, or any other suitable program that is suitable for obtaining sequence alignment results. Accordingly, a wild-type lipocalin sequence may serve as a “query sequence” or a “reference sequence,” while the amino acid sequence of a lipocalin mutein other than the wild-type lipocalin described herein serves as a “query sequence.” The terms “wild type sequence,” “reference sequence,” and “subject sequence” are used interchangeably herein. The preferred wild-type lipocalin sequence is the hTLc sequence shown under SEQ ID NO: 1, or hNGAL, shown under SEQ ID NO: 2.

[0036] "Gaps" are gaps in the alignment results that result from the addition or deletion of amino acids. Thus, two copies of the same sequence have 100% identity, but sequences that are less conserved and have deletions, additions, or substitutions may have a lower degree of identity.

[0037] As used herein, the term “position” means the position of either an amino acid in an amino acid sequence depicted herein or the position of a nucleotide in a nucleic acid sequence depicted herein. It should be understood that when the term “corresponds” or “corresponding” is used herein in the context of positions in the amino acid sequence of one or more lipocalin muteins, the corresponding position is not determined solely by the number of preceding nucleotides or amino acids. Therefore, the absolute position of a given amino acid in accordance with the present invention may differ from the corresponding position due to the deletion or addition of amino acids elsewhere in the lipocalin (mutant or wild type). Likewise, the absolute position of a given nucleotide in accordance with the present invention may differ from the corresponding position due to deletions or additional nucleotides elsewhere in the 5' untranslated region (UTR) of a lipocalin mutein or wild-type lipocalin, including in the promoter and/or or any other regulatory sequences or in a gene (including exons and introns).

[0038] A “corresponding position” in accordance with the present invention may be a position in the sequence that is compatible with the position in the sequence to which it corresponds in a pairwise or multiple sequence alignment according to the present invention. It is preferably understood that for a "corresponding position" in accordance with the present invention, the absolute positions of nucleotides or amino acids may differ relative to adjacent nucleotides or amino acids, but said adjacent nucleotides or amino acids that may have been replaced, deleted or added may be contained in the same or more "relevant provisions".

[0039] In addition, for a corresponding position in a lipocalin mutein, based on the reference sequence in accordance with the present invention, it is preferably understood that the nucleotide or amino acid positions in the lipocalin mutein may structurally correspond to positions elsewhere in the reference lipocalin (wild type lipocalin) or another lipocalin mutein, even though they may differ in absolute position numbers, as will be understood by those skilled in the art in light of the highly conserved general folding pattern among lipocalins.

[0040] As used interchangeably herein, the terms “conjugate,” “conjugation,” “fusion,” “fusion,” or “connected” refer to the joining together of two or more subunits through all forms of covalent or non-covalent bonding through, without limitation, genetic fusion, chemical conjugation, linking via a linker or cross-linker and non-covalent bonding.

[0041] As used herein, the term “fusion polypeptide” or “fusion protein” refers to a polypeptide or protein containing two or more subunits. In some embodiments, a fusion protein described herein comprises two or more subunits, wherein at least one of the subunits is capable of specifically binding to CD137 and the other subunit is capable of specifically binding to GPC3. In a fusion protein, these subunits can be joined by covalent or non-covalent bonds. Preferably, the fusion protein is the result of a translational fusion of two or more subunits. Translational fusion can be accomplished by genetically engineering the coding sequence for one subunit in frame with the coding sequence of another subunit. Both subunits can be separated by a nucleotide sequence encoding a linker. However, the fusion protein subunits of the present invention can also be joined by chemical conjugation. The subunits that form the fusion protein are usually connected to each other, the C-terminus of one subunit to the N-terminus of another subunit, or the C-terminus of one subunit to the C-terminus of another subunit, or the N-terminus of one subunit to the N-terminus of another subunit, or the N-terminus of one subunit with the C-terminus of another subunit. The fusion protein subunits may be joined in any order and may include more than one of the constituent subunits. If one or more subunits are part of a protein (complex) that consists of more than one polypeptide chain, then the term "fusion protein" may also refer to a protein containing the fusion sequences and all other polypeptide chains of the protein (complex). As an illustrative example, if a full-length immunoglobulin is fused to a lipocalin mutein via an immunoglobulin heavy or light chain, then the term “fusion protein” may refer to a single polypeptide chain comprising the lipocalin mutein and the immunoglobulin heavy or light chain. The term "fusion protein" can also refer to an entire immunoglobulin (both light and heavy chains) and a lipocalin mutein fused to one or both of its heavy and/or light chains.

[0042] As used herein, the term “subunit” of a fusion protein disclosed herein refers to a single protein or single polypeptide chain that can itself form a stable folded structure and determine the unique function of forming a binding motif to a target. In some embodiments, a preferred subunit of the present invention is a lipocalin mutein. In some other embodiments, a preferred subunit of the present invention is a full-length immunoglobulin or an antigen binding domain thereof.

[0043] A “linker,” which may be included in a fusion protein of the present invention, joins together two or more subunits of the fusion protein described herein. The bond may be covalent or non-covalent. A preferred covalent bond is formed through a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. Accordingly, in a preferred embodiment, said linker contains 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 peptide linkers are described herein, including glycine-serine (GS) linkers, glycosylated GS linkers, and proline-alanine-serine polymer (PAS) linkers. In some preferred embodiments, the GS linker is (G ₄ S) ₃ , which is described under SEQ ID NO: 13, and is used to connect the subunits of the fusion protein. Other preferred linkers include chemical linkers.

[0044] As used herein, the term “albumin” includes all mammalian albumins, such as human serum albumin, or bovine serum albumin, or rat serum albumin.

[0045] As used herein, the term "organic molecule" or "small organic molecule" means an organic molecule containing at least two carbon atoms, but preferably no more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range of 100 to 2000 dalton, preferably from 100 to 1000 dalton, and optionally including one or two metal atoms.

[0046] "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, including tumor tissue.

[0047] The "subject" is a vertebrate animal, preferably a mammal, more preferably a human. The term "mammal" is used herein to refer to any animal classified as a mammal, including, without limitation, humans, pets, farm animals, and zoo animals, sporting animals, or domestic animals such as sheep, dogs, horses, cats, cows, rats, pigs, great apes such as the cynomolgus macaque, to name just a few illustrative examples. Preferably, as used herein, "mammal" is a human.

[0048] An "effective amount" is an amount sufficient to obtain useful or desired results. An effective amount may be administered in one or more separate administrations or doses.

[0049] As used herein, an “antibody” refers to a whole antibody or any antigen-binding fragment (ie, “antigen-binding region”) or single chain thereof. By whole antibody is meant a glycoprotein containing at least two heavy chains (HC) and two light chains (LC) connected to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable domain ( _VH or HCVR) and a heavy chain constant region ( _CH ). The heavy chain constant region consists of three domains: _CH1 , _CH2 and _CH3 . Each light chain consists of a light chain variable domain (V _L or LCVR) and a light chain constant region ( _CL ). The light chain constant region consists of a single C _L domain. The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), with inserted regions that are more conserved called framework regions (FRs). Each of V _H and V _L consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with an antigen (eg, GPC3). Antibody constant regions may optionally mediate binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

[0050] As used herein, an “antigen-binding fragment” of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, GPC3). It has been demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen binding fragment” of an antibody include (i) a Fab fragment consisting of the _VH , _VL , _CL , and _CH1 domains; (ii) F(ab') ₂ fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fab' fragment consisting of the V _H , V _L , C _L and C _H1 domains and the region between the _CH1 and _CH2 domains; (iv) Fd fragment consisting of V _H and C _H1 domains; (v) a single-chain Fv fragment consisting of the V _H and V _L domains of one arm of the antibody, (vi) a dAb fragment (Ward et al., Nature, 1989) consisting of a V _H domain; and (vii) a dedicated complementarity determining region (CDR) or a combination of two or more dedicated CDRs, which may optionally be connected by a synthetic linker; (viii) a “diabody” containing V _H and V _L joined into one polypeptide chain by a short linker (see, for example, patent documents EP 404097; WO 93/11161; and Holliger et al., Proc Natl Acad Sci USA, 1993); (ix) an “antibody domain fragment” containing only _VH or _VL , in some cases two or more _VH regions being joined by a covalent bond.

[0051] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic or syngeneic or their modified forms (for example, humanized, chimeric or multispecific). Antibodies can also be entirely human.

[0052] As used herein, “framework” or “FR” refers to variable domain residues other than hypervariable region residues (CDR).

[0053] “Immunoglobulin crystallizing fragment” or “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus, numbered according to the Kabat EU index (Johnson and Wu, Nucleic Acids Res, 2000). The C-terminal lysine (residue 447 according to the Kabat EU index) of the Fc regions can be removed, for example, during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Accordingly, an intact antibody composition may contain antibody populations with K447 residues deleted, antibody populations without K447 residues deleted, and antibody populations containing a mixture of antibodies with and without K447 residue. Suitable native sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

[0054] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.

[0055] As used herein, “isolated antibody” refers to an antibody that is substantially free of its natural environment. For example, an isolated antibody contains substantially no cellular material or other proteins from the cellular or tissue source from which it is derived. “Isolated antibody” further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In this case, the isolated antibody that specifically binds GPC3 contains virtually no antibodies that specifically bind antigens other than GPC3. However, an isolated antibody that specifically binds GPC3 may be cross-reactive with other antigens, such as GPC3 molecules from other species.

[0056] As used herein, “monoclonal antibody” refers to a preparation of antibody molecules with a single molecular composition. A monoclonal antibody composition is characterized by uniform binding specificity and binding affinity for a particular epitope.

[0057] As used herein, a “humanized antibody” refers to an antibody that consists of a CDR of an antibody derived from a non-human mammal, as well as an FR region and a constant region of a human antibody or an antibody derived from a human antibody. In some embodiments, a humanized antibody comprises a variable domain that has a variable region amino acid sequence that, when analyzed as a whole, is closer to human than to other species, as assessed using the DomainGapAlign tool of the Immunogenetics Information System (IMGT) database, as described in Ehrenmann et al. (2010). In some embodiments, a humanized antibody may be useful as an effective component of a therapeutic agent due to its reduced antigenicity. As used herein, the term “therapeutic agent” or “therapeutically active agent” refers to an agent that is therapeutically useful. A therapeutic agent can be any agent for the prevention, amelioration, or treatment of a disease, physiological condition, symptom, or for the evaluation or diagnosis thereof.

[0058] As used herein, a “human antibody” refers to antibodies having variable regions in which both the framework region and the CDR regions are derived from human germline immunoglobulin sequences. Moreover, if the antibody contains a constant region, then the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which CDR sequences derived from the germ line of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

III. Description of graphic materials

[0059] Figure 1 provides an overview of the design of typical fusion proteins described herein that are bispecific for the targets CD137 and GPC3. Exemplary fusion proteins are generated from an antibody specific for GPC3 (e.g., an antibody having a heavy chain of SEQ ID NO: 81, or containing a heavy chain variable domain of SEQ ID NO: 78, or containing CDR sequences of GYTFTDYE ( HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73) and TRFYSYTY (HCDR3; SEQ ID NO: 74), and the light chains are represented by SEQ ID NO: 82, or contain the light chain variable domain of SEQ ID NO: 79, or contain CDR sequences representing QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2, SEQ ID NO: 76) and SQNTHVPPT (LCDR3; SEQ ID NO: 77), and one or more muteins CD137-specific lipocalin muteins (e.g., lipocalin muteins of SEQ ID NO: 40 or a lipocalin mutein having 97% sequence identity with SEQ ID NO: 49 and designated CD137Ac1).One or more lipocalin muteins are genetically fused to The C- and/or N-terminus of either the heavy chain or light chain of a GPC3-specific antibody, as depicted in Figure 1A-1I, resulting in fusion proteins such as SEQ ID NOs: 87 and 82, SEQ ID NO: 88 and 82, SEQ ID NOs: 81 and 89, SEQ ID NOs: 81 and 90, as well as CD137Ac1 fusion protein 1, CD137Ac1 fusion protein 2, CD137Ac1 fusion protein 3, CD137Ac1 fusion protein 4, CD137Ac1 fusion protein 5 , CD137Ac1 fusion protein 6 and CD137Ac1 fusion protein 7 (fusion proteins sharing 97% sequence identity with SEQ ID NOs: 91 and 82, SEQ ID NOs: 92 and 82, SEQ ID NOs: 81 and 93, SEQ ID NO: 81 and 94, SEQ ID NO: 95 and 82 or SEQ ID NO: 96 and 82). In addition, the generated fusion proteins can be divalent with respect to CD137 (for example, as depicted in Figure 1A-1D) or tetravalent with respect to CD137 (for example, as depicted in Figure 1E-1H) or have an even higher valency with respect to CD137 (eg, as depicted in Figure 1I). Additional monospecific fusion proteins were prepared by fusing one or more GPC-3-specific lipocalin muteins or CD137-specific lipocalin muteins to the C-terminus of the Fc region of the provided antibody as described herein through a peptide linker ( for example, as shown in Figure 1J-1K). The resulting monospecific fusion proteins are presented, for example, under SEQ ID NO: 97 and SEQ ID NO: 98.

[0060] Figure 2 shows the results of ELISA experiments in which binding to representative fusion proteins with human CPC3 (Figure 2A), cynomolgus GPC3 (Figure 2B), and human CD137 (Figure 2C) was determined as described in Example 3 GPC3 or C-terminal His-tagged CD137 was layered onto a microtiter plate and the test agents were titrated starting at the highest concentration of 100 nM. Bound agents of interest were detected using horseradish peroxidase (HRP)-conjugated anti-human IgG Fc or HRP-conjugated anti-NGAL, respectively. The data were fit to a 1:1 binding model with EC ₅₀ and maximum signal as free parameters, and the slope was fixed at unity. The obtained EC ₅₀ values are presented in Table 4.

[0061] Figure 3 illustrates the results of exemplary ELISA experiments in which the ability of representative fusion proteins to simultaneously bind both targets, GPC3 and CD137, was determined as described in Example 4. Recombinant huGPC3 (Figure 3A) or huCD137-His (Figure 3B) was layered onto a microtiter plate followed by titration of the fusion proteins. Biotinylated huCD137-His (Figure 3A) or biotinylated huGPC3 was then added at a constant concentration (Figure 3B), respectively, which was detected using the avidin complex and ExtrAvidin-Peroxidase. The data were fit to a 1:1 binding model with EC ₅₀ and maximum signal as free parameters, and the slope was fixed to a single level. The obtained EC ₅₀ values are presented in Table 3.

[0062] Figure 4 shows fusion protein target binding results obtained by flow cytometry using CHO cells expressing human CD137 (Figure 4A) as well as SK-Hep1 cells expressing human GPC3 (Figure 4B) as described in Example 6. No binding was observed when using mock-transfected cells. To calculate the EC ₅₀ values, which are presented in Table 6, geometric mean fluorescence intensity values were used.

[0063] Figure 5 shows the binding of fusion proteins to GPC3-positive tumor cells assessed by flow cytometry. Tumor cell lines with varying levels of GPC3 expression (high to moderate expression: HepG2 (Figure 5A) > Hep3B (Figure 5B) > MKN-45 (Figure 5C)) and the GPC3-negative cell line NCI-N87 (Figure 5D) were incubated with various fusion proteins and controls as described in Example 7, and the corresponding binding affinities (EC ₅₀ values) are summarized in Table 7.

[0064] Figure 6 shows the potential of representative fusion proteins to co-promote T cell activation in a GPC3-targeting dependent manner assessed using the CD137 bioassay. NFκB-luc2/CD137 Jurkat cells were co-cultured with GPC3-expressing tumor cell lines (high to moderate expression: HepG2 > Hep3B > MKN-45, GPC3 negative control: NCI-N87, Figure 6A, 6B, 6C and 6D, respectively) in the presence of varying concentrations of fusion proteins or controls. After 4 h, luciferase assay reagent was added and luminescent signals were measured. Four-parameter logistic curve analysis was performed to calculate EC ₅₀ values (see Table 7). The fusion proteins together stimulate T cell activation only in the presence of excess GPC3 (Figure 6A and 6B) but not in conditions of insufficient or absent GPC3 (Figure 6C and 6D). In contrast, the reference mAb to CD137 (SEQ ID NOs: 26 and 27) exhibited similar activation regardless of the level of GPC3 expression, in the absence of any GPC3-expressing cells and in the absence of any tumor cells.

[0065] Figure 7 demonstrates the ability of a typical fusion protein to co-promote T cell activation in the presence of GPC3. Anti-GPC3 antibody that is part of the fusion protein (SEQ ID NOs: 81 and 82), GPC3-specific lipocalin mutein (Fc fusion product) (SEQ ID NO: 97), already known as a bispecific CD137/GPC3 fusion protein (SEQ ID NO: 83) and isotype control (SEQ ID NO: 24 and 25) were tested in parallel. SK-Hep1 cells, either transfected with human GPC3 (Figure 7A), mock-transfected (human GPC3 negative, Figure 7B), or the GPC3-expressing (Figure 7C) Hep-G2 tumor cell line were seeded onto plates coated with anti-human CD3 antibodies. All T cells, as well as various concentrations of test molecules, were added and incubated for three days. Levels of secreted IL-2 in the supernatant were determined using an electrochemoluminescence assay as described in Example 9. The fusion protein of SEQ ID NOs: 87 and 82 induces a strong dose-dependent increase in IL-2 secretion only in the presence of GPC3, stronger than the already known bispecific for CD137/GPC3 fusion protein with SEQ ID NO: 83.

[0066] Figure 8 shows the ability of typical fusion proteins to co-promote T cell activation in a GPC3-targeting dependent manner. Different tumor cell lines expressing different levels of GPC3 (high to moderate expression: HepG2 > Hep3B > MKN-45, Figure 8A, 8B and 8C, respectively; GPC3 negative control: NCI-N87, Figure 8D) were seeded onto antibody-coated plates to human CD3. All T cells and various concentrations of fusion proteins and individual building blocks were added and incubated for 3 days. Levels of secreted IL-2 were determined using an electrochemoluminescence assay as described in Example 10. The fusion protein is capable of increasing IL-2 secretion in a GPC3-dependent manner. In contrast, IL-2 secretion induced by the reference CD137 antibody tested in parallel (SEQ ID NOs: 26 and 27) was independent of GPC3.

[0067] Figure 9 shows the ability of typical fusion proteins to activate the CD137 co-stimulatory signaling pathway and induce T cell-mediated cytolysis of GPC3-expressing tumor cells as demonstrated in a T cell killing impedance assay. GPC3-expressing HepG2 cells or GPC3-negative NCI-N87 cells were seeded and allowed to adhere to an electronic microtiter plate, followed by the addition of anti-CD3 antibody, test molecule, and nonadherent CD8+ T cells. Impedance was measured as a dimensionless cell coefficient (CI) parameter every 15 minutes for three days as described in Example 11. The fusion protein of SEQ ID NOs: 87 and 82 is capable of activating the CD137 pathway in a GPC3-dependent manner and inducing dose, T cell-mediated lysis of GPC3-expressing tumor cells at a higher level than in the case of the already known bispecific CD137/GPC3 fusion protein of SEQ ID NO: 83.

[0068] Figure 10 shows tumor volume over time following treatment with 0.5, 5, and 20 mg/kg of an exemplary fusion protein, equimolar concentrations (0.39 and 3.9 mg/kg) of the anti-GPC3 antibody that is included in fusion protein, an equimolar concentration (3.9 mg/kg) of the reference CD137 antibody or vehicle control (PBS) in a humanized mouse tumor model as described in Example 12. NOG mice were inoculated subcutaneously with HepG2 tumors and allowed to grow to size 80-100 mm ³ . Mice were then randomized into treatment groups (or control groups) and given intravenous injections of 5 x 10 ⁶ fresh human PBMC and intraperitoneal injections of test molecules at the indicated doses on day 1, day 8 and day 15. Tumor growth was recorded every 3-4 days. Data are presented as median values with interquartile range error bars. The dotted line(s) indicate a decrease in the number of animals in the group compared to previous days. The fusion protein completely inhibited tumor growth in the study at higher dose levels (5 and 20 mg/kg), while at lower concentrations (0.5 or 0.39 mg/kg, respectively) it had a dose-dependent but nonsignificant effect on tumor growth.

[0069] Figure 11 shows the results of pharmacokinetic analyzes in mice of an exemplary fusion protein and an anti-GPC3 antibody that is included in the fusion protein, compared with two already known bispecific CD137/GPC3 fusion proteins (SEQ ID NO: 83 and SEQ ID NO : 84) as described in Example 13. Male CD-1 mice (2 mice at each time point) were intravenously administered fusion proteins at a dose of 2 mg/kg or 10 mg/kg. Drug levels were detected using a sandwich ELISA that detects the full molecule with a GPC3 target and an anti-human Fc antibody or an anti-NGAL antibody. The fusion protein of the present invention (SEQ ID NOs: 87 and 82) exhibits greatly improved pharmacokinetic properties and increased half-lives compared to previously known CD137/GPC3 bispecific fusion proteins (SEQ ID NO: 83 and SEQ ID NO: 84).

IV. Detailed Description of the Invention

[0070] As described herein, the present invention includes the recognition that a divalent CD137-binding construct, such as an antibody, by itself may not be sufficient to cluster CD137 on T cells or NK cells and achieve efficient activation. In addition, recent work studying members of the TNFR family illustrates the mechanisms of action of anti-TNFR antibodies, whereby antibodies interact through their Fc regions with Fc gamma receptors, recruiting activating immune cells expressing the Fc gamma receptor, and promoting their subsequent antitumor activity. activity (Bulliard et al., Immunol Cell Biol, 2014, Bulliard et al., J Exp Med, 2013). Therefore, this suggests that anti-CD137 antibody may trigger CD137 clustering depending on the excess of Fc-gamma receptor-positive cells that are not selectively localized to the tumor but are distributed throughout the body. Accordingly, the efficacy and target specificity of anti-CD137 monotherapy may be of particular interest. It is known that some CD137 antibody therapeutics in clinical trials, such as urelumab and utomilumab, show disappointing results in terms of efficacy at low dose and/or exhibit toxicity at high dose or at the effective dose (Bulliard et al., Immunol Cell Biol, 2014, Bulliard et al., J Exp Med, 2013). Thus, there is an unmet need for CD137-targeted therapeutics that are both effective and safe. An ideal CD137-targeting agent should result in CD137 clustering and should do so in a tumor-site localized manner on antitumor effector lymphocytes. As described herein, to obtain such a CD137-targeting agent, a bispecific agent can be developed to target CD137 at one end and a differentially expressed tumor target at the other end.

[0071] The present invention recognizes that a GPC3-targeting monospecific agent may not be effective enough to radically treat tumors such as HCC, and that the antitumor activity of an anti-GPC3 antibody agent that is capable of recruiting immune cells to the tumor site may be of value. and/or have a direct inhibitory effect on the proliferation and/or survival of cancer cells. Therefore, there is an unmet need for GPC3-targeted therapeutics with complementary antitumor effects and that have the ability to provide enhanced antitumor activity compared to a single GPC3-targeting monospecific agent.

[0072] The present invention provides, among other things, new approaches to simultaneously engage CD137 and GPC3 using fusion proteins having CD137 binding specificity and GPC3 binding specificity. For example, WO2016/184882 described fusion proteins of CD137-specific lipocalin mutein and anti-GPC3 antibody to engage CD137 and GPC3. However, the fusion proteins are still required to induce a GPC3-dependent T cell killing/immune response to suppress the growth of a GPC3-expressing tumor with high efficiency and selectivity. Various aspects of the present invention provide fusion proteins that promote CD137 clustering by bridging CD137-positive T cells to GPC3 expressed in the tumor microenvironment, thereby combining CD137-induced T cell activation and expansion with anti-GPC3 antibody-mediated cytotoxic killing of tumor cells. . The present invention also provides fusion proteins that provide combinatorial therapy capabilities in a single molecule while allowing localized induction of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. In this regard, the fusion proteins of the present invention may provide enhanced IL-2 production, immune responses, T cell mediated cytolysis and/or antitumor effects compared to CD137 or GPC3 monotherapy.

[0073] In addition, since cynomolgus monkeys are widely used in pharmacokinetic or drug safety studies in the development of new therapeutics, including new biological agents, and further such studies may be necessary prerequisites for obtaining regulatory approval, it is desirable to obtain fusion proteins with a GPC3-binding fragment that cross-reacts with both human GPC3 and cynomolgus GPC3. Such fusion proteins with a GPC3-binding fragment having such cross-reactivity characteristics have not been previously described.

[0074] Moreover, since bispecific or multispecific molecules may differ from their structural elements in certain properties, including affinity and target specificity, stability, pharmacokinetics in the blood and mechanism of action (Spiess et al., Mol Immunol, 2015, Sedykh et al., Drug Des Devel Ther., 2018), as provided herein, in some embodiments, careful selection of structural elements and optimization of manufacturing and other practices are provided to obtain quality clinical candidates. For example, bispecific or multispecific molecules may partially or completely lose the binding affinity and specificity of the target structural elements, resulting in unwanted off-target binding. Off-target binding may further affect the pharmacokinetics, tissue distribution, efficacy, and toxicity of such bispecific or multispecific molecules for therapeutic applications. In particular, poor pharmacokinetic profiles can lead to difficulty achieving the dose distribution profile required for therapeutic efficacy and patient non-compliance (Alavijeh and Palmer, IDrugs, 2004), while favorable pharmacokinetics allow dose reduction, allow the development of subcutaneous formulations and reduce COG values, and also extend the dosing interval to, for example, once a week, every 2 weeks, every 3 weeks or every 4 weeks (Ryman and Meibohm, CPT Pharmacometrics Syst Pharmacol, 2017). The pharmacokinetic properties are also influenced by the isoelectric point (pI). An increased pI may result in increased blood clearance and increased tissue retention with a shorter half-life, whereas a lower pI may reduce tissue uptake and result in a longer half-life. Although observations may be inconsistent regarding the correlation between protein clearance and pI, therapeutic antibodies often have pI values slightly above the physiological pH of 7.4 because most cell surfaces are negatively charged. In this regard, various aspects of the present invention provide fusion proteins that are bispecific for CD137 and GPC3, with high affinity and specificity, increased stability, suitable pI and favorable pharmacokinetic properties.

[0075] Such fusion proteins having such features suitable for the uses provided by the present invention have not been previously described. In contrast to the fusion proteins provided herein, previously known fusion proteins targeting both CD137 and GPC3 were characterized by one or more of poor PK, unacceptable degree of off-target binding, unacceptable degree of non-specific (e.g., GPC3-independent) activation, for example, the immune system and/or a reduced or otherwise impaired ability to mediate T cell activation, proliferation and/or infiltration.

A. Exemplary CD137 and GPC3 Specific Fusion Proteins of the Present Invention

[0076] In some embodiments, the provided fusion protein comprises at least two subunits, in any order: (1) a first subunit that provides a full-length immunoglobulin or a GPC3-specific antigen binding domain thereof, and (2) a second subunit that provides a mutein lipocalin specific for CD137.

[0077] In some embodiments, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or antigen binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutein and is specific for CD137, the second the subunit is connected at the N-terminus to the C-terminus of each heavy chain (HC) of the first subunit, optionally via a linker.

[0078] In some embodiments, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or antigen binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutein and is specific for CD137, the second the subunit is connected at the C-terminus to the N-terminus of each HC of the first subunit, optionally via a linker.

[0079] In some embodiments, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or antigen binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutein and is specific for CD137, the second subunit comprising the subunit is connected at the N-terminus to the C-terminus of each light chain (LC) of the first subunit, optionally via a linker.

[0080] In some embodiments, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or antigen binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutein and is specific for CD137, the second subunit comprising the subunit is connected at the C-terminus to the N-terminus of each LC of the first subunit, optionally via a linker.

[0081] In some embodiments, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or antigen binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutein and is specific for CD137, the second subunit comprising the subunit is connected at the N-terminus to the C-terminus of each heavy chain constant region (CH) of the first subunit, optionally via a linker.

[0082] In some embodiments, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or antigen binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutein and is specific for CD137, the second the subunit is connected at the N-terminus to the C-terminus of the constant region of each light chain (CL) of the first subunit, optionally via a linker.

[0083] In some embodiments, the provided fusion protein may also comprise at least one additional subunit, such as a third subunit. For example, the fusion protein may contain a third subunit specific for CD137. In some embodiments, the third subunit may be or include a CD137-specific lipocalin mutein. For example, two lipocalin muteins can be fused to the first immunoglobulin subunit, one at the C-terminus and one at the N-terminus of the immunoglobulin. In some embodiments, lipocalin muteins may be fused to an immunoglobulin heavy chain or light chain.

[0084] In some embodiments, the fusion proteins provided may contain one or more additional subunits (eg, a fourth, fifth, or sixth subunit).

[0085] In some embodiments, at least one subunit may be fused at its N-terminus and/or its C-terminus to another subunit.

[0086] In some embodiments, at least one subunit may be connected to another subunit via a linker. In some further embodiments, the linker is a peptide linker, such as a non-structured glycine serine (GS) linker, a glycosylated GS linker, or a proline alanine serine polymer (PAS) linker. In some embodiments, the GS linker is a (Gly ₄ Ser) ₃ linker ((G ₄ S) ₃ ), which is provided under SEQ ID NO: 13. Other exemplary linkers are provided under SEQ ID NO: 14-23. In some embodiments, the peptide linker may have from 1 to 50 amino acids, such as 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids. For example, if the first subunit provides a full-length immunoglobulin, then the second subunit can be attached via a peptide linker between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of said immunoglobulin. In some further embodiments, the third subunit may be attached via a peptide linker between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the immunoglobulin.

[0087] In some embodiments, one subunit may be fused to another subunit as essentially described in Figure 1. In general, one subunit may be fused at its N-terminus and/or its C-terminus to another subunit. For example, in some embodiments, a lipocalin mutein subunit may be fused at its N-terminus and/or its C-terminus to an immunoglobulin subunit, preferably via a peptide bond. As further examples, a lipocalin mutein subunit may be coupled at its N-terminus to the C-terminus of the heavy chain (HC) domain of an immunoglobulin subunit (Figure 1A), a lipocalin mutein subunit may be coupled at its C -end to the N-terminus of the HC immunoglobulin subunit (Figure 1C), the lipocalin mutein subunit can be linked at its N-terminus to the C-terminus of the light chain (LC) of the immunoglobulin subunit (Figure 1B), and/or the lipocalin mutein subunit may be linked at its C-terminus to the N-terminus of the LC immunoglobulin subunit (Figure 1D).

[0088] In some embodiments, the lipocalin mutein subunit may be fused at its N-terminus and/or its C-terminus to an immunoglobulin moiety. For example, in some embodiments, a lipocalin mutein subunit may be linked, preferably via a peptide linker, at its N-terminus to the C-terminus of a heavy chain constant region (CH) of an immunoglobulin subunit or a lipocalin mutein subunit , may be linked, preferably via a peptide linker, at its N-terminus to the C-terminus of the light chain constant region (CL) of the immunoglobulin subunit.

[0089] In some embodiments, if one subunit provides a full-length immunoglobulin, then a second subunit may be attached between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of said immunoglobulin.

[0090] In some embodiments, the third subunit may be attached between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of said immunoglobulin.

[0091] In some embodiments, with respect to a fusion protein of the present invention, in which at least one subunit can be or comprise a full-length immunoglobulin, the Fc function of the Fc region of the full-length immunoglobulin relative to an Fc receptor positive cell can be while maintaining the fusion protein will simultaneously target CD137 and GPC3.

[0092] In some embodiments, where at least one subunit of the provided fusion protein may be or comprise a full-length immunoglobulin, the Fc function of the Fc region of the full-length immunoglobulin relative to the Fc receptor positive cell can be reduced or completely suppressed through protein engineering , while the fusion protein will simultaneously target CD137 and GPC3. In some embodiments, this can be achieved, for example, by replacing the IgG1 backbone with IgG4, since IgG4 is known to exhibit reduced Fc-gamma receptor interactions compared to IgG1. In some embodiments, mutations such as F234A and L235A can be introduced into the IgG4 backbone to further reduce residual Fc-gamma receptor binding. In some embodiments, the S228P mutation can also be introduced into the IgG4 backbone to minimize replacement of the IgG4 half-antibody (Silva et al., J Biol Chem, 2015). In some embodiments, the F234A and L235A mutations can be administered to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010), and/or the M428L and N434S mutations, or the M252Y, S254T and T256E mutations to prolong serum half-life (Dall'Acqua et al., J Biol Chem, 2006, Zalevsky et al., Nat Biotechnol, 2010). In some embodiments, an additional N297A mutation may be present in the fusion protein to remove a natural glycosylation motif in the immunoglobulin heavy chain.

[0093] In some embodiments, the Fc portion of an immunoglobulin included in a fusion protein of the present invention may help maintain serum levels of the fusion protein. For example, when the Fc moiety binds to Fc receptors on endothelial cells and phagocytes, the fusion protein can be internalized and released back into the bloodstream, increasing its half-life in the body.

[0094] In one aspect, the fusion proteins of the present invention bind CD137 with high affinity. In another aspect, the provided fusion proteins bind GPC3 with high affinity. In some preferred embodiments, the fusion proteins provided simultaneously bind CD137 and GPC3. In some embodiments, simultaneous binding to CD137 and GPC3 allows the provided fusion proteins to exhibit a robust antitumor or anti-infective response.

[0095] In some embodiments, the fusion protein of the present invention may have an isoelectric point (pI) of about 6.5 or higher, such as about 6.8 or higher, about 7.1 or higher, about 7.4 or higher , about 7.5 or higher, about 7.7 or higher, about 8.0 or higher, about 8.5 or higher, or about 9.0 or higher. In some embodiments, the fusion protein of the present invention may have a pI higher than that of a previously known fusion protein that binds both CD137 and GPC3 and contains the amino acid sequence of SEQ ID NO: 83.

[0096] In some embodiments, the fusion protein of the present invention may be capable of binding GPC3 with a K _D value that is at most about 2 nM or lower, such as about 1.5 nM or lower, about 1 nM or lower, about 0.8 nM or lower or about 0.7 nM or lower. In some embodiments, a fusion protein of the present invention may be capable of binding GPC3 with a K _D value comparable to or lower than the K _D value of a GPC3-specific immunoglobulin included in such a fusion protein, such as an antibody with the heavy and light chains represented by under SEQ ID NOs: 81 and 82. The K _D values of the provided fusion proteins can be measured, for example, in a surface plasmon resonance (SPR) assay, such as the SPR assay, which is actually described in Example 2.

[0097] In some embodiments, the fusion protein of the present invention may be capable of binding GPC3 with an EC ₅₀ value that is at most about 1 nM or lower, such as about 0.5 nM or lower, about 0.3 nM or lower, about 0.2 nM or lower, about 0.15 nM or lower, or about 0.1 nM or lower. In some embodiments, a fusion protein of the present invention may be capable of binding GPC3 with an EC ₅₀ value comparable to or lower than the EC ₅₀ value of a GPC3-specific immunoglobulin included in the particular fusion protein, such as an antibody with the heavy and light chains represented by under SEQ ID NOs: 81 and 82. The EC ₅₀ values of the provided fusion proteins can be measured, for example, in an enzyme-linked immunosorbent assay (ELISA), such as the ELISA assay that is actually described in Example 3.

[0098] In some embodiments, the fusion protein of the present invention may be capable of binding CD137 with an EC ₅₀ value that is a maximum of about 5 nM or lower, such as about 3 nM or lower, about 2 nM or lower, about 1 nM, or below, about 0.5 nM or below, about 0.2 nM or below, or about 0.1 nM or below. The EC ₅₀ values of the provided fusion proteins can be measured, for example, in an ELISA assay, such as the ELISA assay that is actually described in Example 3.

[0099] In some embodiments, a fusion protein of the present invention may cross-react with cynomolgus GPC3. In some embodiments, the provided fusion protein may be capable of binding cynomolgus GPC3 with a K _D value that is at most about 2 nM or even lower, such as about 1.5 nM or lower, about 1 nM or lower, about 0.8 nM or below or about 0.7 nM or below. In some embodiments, a fusion protein of the present invention may be capable of binding cynomolgus GPC3 with a _KD value comparable to or lower than the _KD value of a GPC3-specific immunoglobulin included in such a fusion protein, such as a heavy and light chain antibody set forth under SEQ ID NOs: 81 and 82. The K _D values of the provided fusion proteins can be measured, for example, in an SPR assay, such as the SPR assay that is actually described in Example 2. In some embodiments, the provided fusion protein may be capable of bind cynomolgus GPC3 to an EC ₅₀ value that is at most about 1 nM or lower, such as about 0.5 nM or lower, such as about 0.2 nM or lower, about 0.1 nM or lower . The EC ₅₀ values of the provided fusion proteins can be measured, for example measured in an ELISA assay, such as the ELISA assay that is actually described in Example 3.

[00100] In some embodiments, a fusion protein of the present invention may be capable of simultaneously binding CD137 and GPC3. In some embodiments, the provided fusion protein may be capable of simultaneously binding CD137 and GPC3 with an EC ₅₀ value that is at most about 10 nM or lower, such as 5 nM or lower, 3 nM or lower, 2 nM or lower, 1 nM or less or 0.5 nM or less. In some other embodiments, the provided fusion protein may be capable of simultaneously binding CD137 and GPC3 with an EC ₅₀ value that is at most about 10 nM or lower, such as 8 nM or lower, 5 nM or lower, 3 nM or lower, or 2 nM or lower. Simultaneous binding can be determined, for example, in an ELISA assay, such as the ELISA assay that is actually described in Example 4.

[00101] In some embodiments, the fusion protein of the present invention may be capable of binding CD137 expressed on a cell with an EC ₅₀ value that is a maximum of about 50 nM or even lower, such as about 30 nM or even lower than about 25 nM or lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, or about 1 nM or lower. The EC ₅₀ value of the provided fusion protein can be measured, for example, in a flow cytometry assay, which is actually described in Example 6. The cell expressing CD137 can be, for example, a CHO cell transfected with human CD137.

[00102] In some embodiments, a fusion protein of the present invention may be capable of binding GPC3 expressed on a cell with an EC ₅₀ value that is at most about 50 nM or even lower, such as about 30 nM or lower than about 25 nM or even lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, or about 1 nM or lower. The EC ₅₀ value of the provided fusion protein can be measured, for example, in a flow cytometry assay, which is actually described in Example 6. The cell expressing GPC3 can be, for example, a SK-Hep1 cell transfected with human GPC3.

[00103] In some embodiments, the fusion proteins of the present invention may be capable of binding GPC3 expressed on tumor cells. In some embodiments, the provided fusion protein may be capable of binding GPC3 expressed on a tumor cell with an EC ₅₀ value that is at most about 50 nM or lower, such as about 30 nM or lower, about 25 nM or lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, or about 1 nM or lower. The EC ₅₀ value of the fusion protein for binding GPC3-expressing tumor cells can be measured, for example, in a flow cytometry assay, which is actually described in Example 7. GPC3-expressing tumor cells can be, for example, HepG2, Hep3B and MKN-45 cells.

[00104] In some embodiments, the fusion proteins of the present invention substantially do not bind targets other than CD137 or GPC3. In some embodiments, the provided fusion protein has reduced off-target binding to targets other than CD137 or GPC3 compared to a previously known fusion protein that binds both CD137 and GPC3 and contains the amino acid sequence of SEQ ID NO: 83. Such evaluation Target binding can be performed using an ELISA assay as described in Example 5.

[00105] In some embodiments, the fusion proteins of the present invention may be capable of co-stimulating T cell responses. In some embodiments, the provided fusion proteins result in comparable or greater activation of T cells compared to the anti-GPC3 antibody included in the fusion protein having the heavy and light chains of SEQ ID NOs: 81 and 82, the reference anti-CD137 antibody, having the heavy and light chains of SEQ ID NOs: 26 and 27, or a pre-existing fusion protein that binds both CD137 and GPC3 comprising the amino acid sequence of SEQ ID NO: 83. In some embodiments, the provided fusion proteins result in activation of T cells with comparable or better potency compared to the anti-GPC3 antibody included in the fusion protein having the heavy and light chains shown under SEQ ID NOs: 81 and 82, the reference anti-CD137 antibody having the heavy and light chains shown under SEQ ID NOs: 26 and 27, or a previously known fusion protein that binds both CD137 and GPC3 containing the amino acid sequence of SEQ ID NO: 83. Stimulated T cell response or T cell activation can be measured, for example, in a bioassay CD137, which is actually described in example 8, or in a functional T cell activation assay, which is actually described in example 9, example 10 and example 11.

[00106] In some embodiments, the fusion proteins of the present invention may be capable of inducing increased secretion of IL-2. In some preferred embodiments, the provided fusion proteins can induce concentration-dependent secretion of IL-2 and/or at higher concentrations tend to induce enhanced secretion of IL-2. In some embodiments, the fusion proteins provided may result in increased secretion of IL-2 with comparable or better potency than the anti-GPC3 antibody of the fusion protein having the heavy and light chains of SEQ ID NOs: 81 and 82, or already known fusion protein that binds both CD137 and GPC3, containing the amino acid sequence of SEQ ID NO: 83. Secretion of IL-2 can be measured, for example, in a functional T-cell activation assay, which is actually described in example 9 and example 10 .

[00107] In some embodiments, the fusion proteins of the present invention may be capable of inducing T cell-mediated cytotoxicity. In some embodiments, the provided fusion proteins may result in dose-dependent T cell-mediated cytolysis. In some embodiments, the provided fusion proteins can induce activation of cytotoxic T cells with comparable or better potency than the anti-GPC3 antibody included in the provided heavy and light chain fusion proteins set forth in SEQ ID NOs: 81 and 82. In some embodiments, In embodiments, the provided fusion proteins can induce activation of cytotoxic T cells with comparable or better potency compared to the anti-CD137 heavy and light chain antibody set forth in SEQ ID NOs: 26 and 27, the reference anti-CD137 heavy and light chain antibody , presented under SEQ ID NO: 26 and 27, or a already known fusion protein that binds both CD137 and GPC3 containing the amino acid sequence under SEQ ID NO: 83. T cell-mediated cytotoxicity can be determined, for example, using impedance analysis T cell killing, which is actually described in example 11.

[00108] In some embodiments, the fusion proteins of the present invention may be capable of co-stimulating T cell responses in a GPC3-dependent manner. In some embodiments, the provided fusion proteins may result in local induction of IL-2 production by T cells in the vicinity of GPC3-positive cells, such as cells transfected with GPC3 or tumor cells expressing GPC3. “In the vicinity of GPC3-positive cells” as used herein refers to a T cell and a GPC3-positive cell that are brought into close proximity to each other by a provided fusion protein that simultaneously binds CD137 and GPC3. In some embodiments, the provided fusion proteins may result in localized T cell-mediated killing of tumor cells expressing GPC3. GPC3-dependent activation of T cells by the provided fusion proteins can be measured, for example, in a CD137 bioassay, which is actually described in example 8, or in a functional T cell activation assay, which is actually described in example 9, example 10 and example 11.

[00109] In some embodiments, the provided fusion proteins are unable to co-stimulate T cell responses in the absence of GPC3. In some embodiments, the provided fusion proteins are unable to co-stimulate T cell responses in the absence of cells expressing GPC3. In some embodiments, the provided fusion protein can recognize the presence of GPC3 and lead to appropriate T cell activation better than the anti-CD137 antibody having the heavy and light chains of SEQ ID NOs: 26 and 27. The GPC3-dependent effect of the fusion proteins can be measured for example, in the CD137 bioassay, which is actually described in example 8, or in a functional T cell activation assay, which is actually described in example 9, example 10 and example 11.

[00110] In some embodiments, the provided fusion proteins may result in a variety of antitumor activities. In some embodiments, the provided fusion proteins can inhibit tumor growth of HepG2 hepatocellular carcinoma cells. In some embodiments, the provided fusion proteins can inhibit tumor growth of HepG2 hepatocellular carcinoma cells with comparable or better potency than an anti-GPC3 antibody included in the heavy and light chain fusion proteins set forth in SEQ ID NOs: 81 and 82. Such varieties antitumor activity can be determined, for example, using the HepG2 xenograft model, as actually described in Example 12.

[00111] In some embodiments, the provided fusion proteins can increase levels of antitumor effector lymphocytes. In some embodiments, the provided fusion proteins may result in increased intratumoral infiltration of CD3, CD4 or CD8 T cells compared to an anti-GPC3 antibody included in the heavy and light chain fusion proteins of SEQ ID NOs: 81 and 82. Antitumor effector lymphocytes can be analyzed, for example, as described in example 13.

[00112] In some embodiments, the provided fusion proteins have favorable stability and pharmacokinetic profiles. In some embodiments, the provided fusion protein has a pharmacokinetic profile comparable to that of an anti-GPC3 antibody included in the fusion protein having the heavy and light chains of SEQ ID NOs: 81 and 82. In some embodiments, the provided fusion protein has pharmacokinetics similar to those of the antibody. In some embodiments, the provided fusion protein has a terminal half-life of about 50 hours or longer, 75 hours or longer, 100 hours or longer, 125 hours or longer, about 150 hours or longer, about 175 hours or longer, about 200 hours or longer, approximately 250 hours or longer or even longer. In some embodiments, the provided fusion protein has a terminal half-life in mice of about 50 hours or longer, 75 hours or longer, 100 hours or longer, 125 hours or longer, about 150 hours or longer, about 175 hours or longer, about 200 hours, or longer, approximately 250 hours or longer or even longer. The pharmacokinetic profiles of the provided fusion proteins can be analyzed as described in Example 13.

[00113] In some embodiments, the provided fusion protein contains the amino acid sequence presented under any of SEQ ID NO: 87-96.

[00114] In some embodiments, the provided fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92% , at least 95%, at least 97%, at least 98% or even higher sequence identity with the amino acid sequences presented under any of SEQ ID NO: 87-96.

[00115] In some embodiments, the provided fusion protein comprises the amino acid sequences set forth in SEQ ID NOs: 87 and 82, SEQ ID NOs: 88 and 82, SEQ ID NOs: 81 and 89, SEQ ID NOs: 81 and 90, SEQ ID NOs: 91 and 82, SEQ ID NOs: 92 and 82, SEQ ID NOs: 81 and 93, SEQ ID NOs: 81 and 94, SEQ ID NOs: 95 and 82, or SEQ ID NOs: 96 and 82.

[00116] In some embodiments, the provided fusion protein contains amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92% at least 95%, at least 97%, at least 98% or greater sequence identity to the amino acid sequences set forth in SEQ ID NOs: 87 and 82, SEQ ID NOs: 88 and 82, SEQ ID NOs: 81 and 89, SEQ ID NO: 81 and 90, SEQ ID NO: 91 and 82, SEQ ID NO: 92 and 82, SEQ ID NO: 81 and 93, SEQ ID NO: 81 and 94, SEQ ID NO: 95 and 82 or SEQ ID NO: 96 and 82.

B. Exemplary Immunoglobulins Included in Fusion Proteins

[00117] In some embodiments, with respect to the fusion protein provided, the first subunit may be or comprise a full-length immunoglobulin or a GPC3-specific antigen binding domain thereof. In some embodiments, the immunoglobulin, for example, may be IgG1, IgG2, or IgG4. In some embodiments, the immunoglobulin is or includes IgG4. In some embodiments, the immunoglobulin is an anti-GPC3 monoclonal antibody.

[00118] Illustrative examples of GPC3-binding antibodies of the present invention may include an antigen-binding region that cross-blocks or binds to the same epitope as a GPC3-binding antibody comprising heavy chain variable domain ( _VH ) and light chain variable domain (VH) regions. V _L ) such known antibodies as codrituzumab (also known as GC33 or RO5137382), YP7 (including humanized YP7), HN3 and HS20. In some embodiments, the GPC3-binding antibody of the present invention may comprise an antigen binding region, such as any of three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of the antibody , selected from the group consisting of codrituzumab, YP7, HN3 and HS20.

[00119] In some embodiments, a provided anti-GPC3 antibody or antigen binding domain thereof may have a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 78, 114, 119, 126 and 129, and/or a variable region light chain (LCVR) selected from the group consisting of SEQ ID NO: 79, 115 and 127.

[00120] In some embodiments, the heavy chain and light chain pair of a contemplated anti-GPC3 antibody or antigen binding domain thereof is or comprises, respectively, the following HCVR and LCVR: SEQ ID NOs: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs : 114 and 115 or SEQ ID NO: 126 and 127.

[00121] In some embodiments, the heavy chain and light chain pair of a contemplated anti-GPC3 antibody or antigen binding domain thereof is or comprises, respectively, HCVR and LCVR, which have a sequence of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or greater sequence identity with the amino acid sequences set forth in SEQ ID NOs: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs: 114 and 115, or SEQ ID NOs: 126 and 127.

[00122] In some embodiments, a provided anti-GPC3 antibody or antigen binding domain thereof may have a heavy chain, which is represented by any of SEQ ID NO: 80 and 81, and/or a light chain, which is represented by SEQ ID NO: 82.

[00123] In some embodiments, the heavy chain and light chain pair of the provided anti-GPC3 antibody is or contains the amino acid sequences set forth in SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82.

[00124] In some embodiments, the heavy chain and light chain pair of a provided anti-GPC3 antibody is or contains a heavy chain and a light chain that have a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or greater sequence identity with the amino acid sequences set forth under SEQ ID NO: 80 and 82 or SEQ ID NO: 81 and 82.

[00125] In some embodiments, a provided anti-GPC3 antibody or antigen binding domain thereof may have an HCVR of at least 70%, of at least 75%, of at least 80%, of at least 85%, of at least 90%, with at least 92%, with at least 95%, with at least 97%, with at least 98%, or even higher sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 78, 114, 119, 126 and 129, and/or LCVR with at least 70%, with at least 75%, with at least 80%, with at least 85%, with at least 90%, with at least 92%, with at least 95%, with at least 97%, with at least 98%, or even greater sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 79, 115, and 127. In other embodiments, the provided anti-GPC3 antibody or antigen binding domain thereof may have a heavy chain of at least 70%, at least 75%, at least 80%, at least 85%, with at least 90%, with at least 92%, with at least 95%, with at least 97%, with at least 98%, or even higher sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 80 and 81, and/or a light chain with at least 70%, with at least 75%, with at least 80%, with at least 85%, with at least 90%, with at least 92% , with at least 95%, with at least 97%, with at least 98%, or even higher sequence identity with the amino acid sequence of SEQ ID NO: 82.

[00126] In some embodiments, the heavy chain variable region of a provided anti-GPC3 antibody or antigen binding domain thereof may have three CDRs having the following sequences: GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), TRFYSYTY (HCDR3; SEQ ID NO: 74). In some embodiments, the heavy chain variable region of a provided anti-GPC3 antibody or antigen binding domain thereof may have three CDRs having the following sequences: GFTFNKNA (HCDR1, SEQ ID NO: 108), IRNKTNNYAT (HCDR2, SEQ ID NO: 109), VAGNSFAY (HCDR3 ; SEQ ID NO: 110). In some embodiments, the heavy chain variable region of a provided anti-GPC3 antibody or antigen binding domain thereof may have three CDRs having the following sequences: YFDFDSYE (HCDR1, SEQ ID NO: 116), IYHSGST (HCDR2, SEQ ID NO: 117), ARVNMDRFDY (HCDR3 ; SEQ ID NO: 118). In some embodiments, the heavy chain variable region of a provided anti-GPC3 antibody or antigen binding domain thereof may have three CDRs having the following sequences: GFTFSSYA (HCDR1, SEQ ID NO: 120), IQKQGLPT (HCDR2, SEQ ID NO: 121), AKNRAKFDY (HCDR3 ; SEQ ID NO: 122).

[00127] In some embodiments, the light chain variable region of a provided anti-GPC3 antibody or antigen binding domain thereof may have three CDRs having the following sequences: QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO: 77). In some embodiments, the light chain variable region of a provided anti-GPC3 antibody or antigen binding domain thereof may have three CDRs having the following sequences: QSLLYSSNQKNY (LCDR1, SEQ ID NO: 111), WAS (LCDR2), QQYYNYPLT (LCDR3; SEQ ID NO: 113 ). In some embodiments, the light chain variable region of a provided anti-GPC3 antibody or antigen binding domain thereof may have three CDRs having the following sequences: QSISSY (LCDR1, SEQ ID NO: 123), NAS (LCDR2), QQNRGFPLT (LCDR3; SEQ ID NO: 125 ).

[00128] In some embodiments, a provided anti-GPC3 antibody or antigen binding domain thereof comprises a heavy chain variable region that has three CDRs having the following sequences: GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73 ), TRFYSYTY (HCDR3; SEQ ID NO: 74), and a light chain variable region which has three CDRs having the following sequences: QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO: 77). In some embodiments, the provided anti-GPC3 antibody or antigen binding domain thereof comprises a heavy chain variable region that has three CDRs having the following sequences: GFTFNKNA (HCDR1, SEQ ID NO: 108), IRNKTNNYAT (HCDR2, SEQ ID NO: 109), VAGNSFAY (HCDR3; SEQ ID NO: 110), and a light chain variable region which has three CDRs having the following sequences: QSLLYSSNQKNY (LCDR1, SEQ ID NO: 111), WAS (LCDR2), QQYYNYPLT (LCDR3; SEQ ID NO: 113 ). In some embodiments, the provided anti-GPC3 antibody or antigen binding domain thereof comprises a heavy chain variable region that has three CDRs having the following sequences: GFTFSSYA (HCDR1, SEQ ID NO: 120), IQKQGLPT (HCDR2, SEQ ID NO: 121), AKNRAKFDY (HCDR3; SEQ ID NO: 122), and a light chain variable region which has three CDRs having the following sequences: QSISSY (LCDR1, SEQ ID NO: 123), NAS (LCDR2), QQNRGFPLT (LCDR3; SEQ ID NO: 125 ).

[00129] Unless otherwise stated, all CDR sequences disclosed herein are determined in accordance with the IMGT method, which is described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999). CDR1 consists of positions 27-38, CDR2 consists of positions 56-65, CDR3 for germline V genes consists of positions 105-116, CDR3 for rearranged V-J genes or V-D-J genes consists of positions 105-117 (the position preceding J -PHE or J-TRP at position 118) with gaps at the top of the loop for a rearranged CDR3-IMGT with less than 13 amino acids or with additional positions 112.1, 111.1, 112.2, 111.2, etc. for a rearranged CDR3-IMGT with more than 13 amino acids. The provisions given in this paragraph correspond to the IMGT numbering described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).

[00130] Antibodies that specifically bind to GPC3 included in the fusion proteins of the present invention may contain an Fc moiety that allows the in vivo half-life of the bispecific binding molecule of the present invention to be increased. In some embodiments, such Fc portion is preferably of human origin, more preferably a human IgG1 or IgG4 antibody Fc portion, even more preferably an engineered human IgG1 or IgG4 Fc portion to activate or silence effector functions. In some embodiments, silencing effector functions may be preferable to activating effector functions. In some embodiments, such an Fc portion is engineered to silence effector functions through mutation(s) at positions 234 and/or 235 numbered according to the Kabat EU index (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations to silence effector functions can be introduced at positions F234 and L235 of a provided anti-GPC3 antibody. In other embodiments, effector function silencing mutations can be introduced at positions D265 and P329 of the provided anti-GPC3 antibody. The numbering of both candidate mutation data sets follows the EU index according to Kabat (Shields et al., J Biol Chem, 2001).

[00131] Various techniques for producing antibodies and fragments thereof are well known in the art and are described, for example, in Altshuler et al. (2010). For example, polyclonal antibodies can be obtained from the blood of an animal after immunization with an antigen in a mixture with additives and adjuvants, and monoclonal antibodies can be obtained using any technique that allows the production of antibodies produced by continuous cultures of cell lines. Examples of such techniques are described, for example, in Harlow and Lane (1999), (1988), and include the hybridoma technique originally described by Köhler and Milstein, 1975, the triome technique, the human B cell hybridoma technique (see, for example, Li et al., Proc Natl Acad Sci U S A, 2006, Kozbor and Roder, Immunol Today, 1983) and the EBV hybridoma technique for producing human monoclonal antibodies (Cole et al., Cancer Res, 1984). Moreover, recombinant antibodies can be produced from monoclonal antibodies or they can be produced de novo using various display methods such as phage, ribosomal, mRNA or cellular display. In some embodiments, a suitable system for expressing recombinant (humanized) antibodies or fragments thereof can be selected, for example, from bacterial, yeast, insect, mammalian, or transgenic animal or plant cell lines (see, for example, US Pat. No. 6,080,560; Holliger and Hudson , Nat Biotechnol, 2005). Additionally, the described methods for producing single chain antibodies (see, in particular, US patent No. 4946778) can be adapted to obtain single chain antibodies specific for the target of the present invention. Surface plasmon resonance, as used in the BIAcore system, can be used to increase the effectiveness of phage antibodies.

C. Exemplary Lipocalin Muteins of the Present Invention

[00132] Lipocalins are protein binding molecules that have naturally evolved the ability to bind ligands. Lipocalins are found in many organisms, including vertebrates, insects, plants and bacteria. Members of the lipocalin family of proteins (Pervaiz and Brew, FASEB J, 1987) are typically small secreted proteins and have a single polypeptide chain. They are characterized by a spectrum of different molecular recognition properties: their binding to various, predominantly hydrophobic, small molecules (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants and odorants) and their binding to specific cell surface receptors, as well as formation of macromolecular complexes with them. Although in the past they were classified primarily as transport proteins, it is now clear that lipocalins have a number of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins are also involved in regulating the immune response and mediating cellular homeostasis (reviewed, for example, in Flower et al., Biochim Biophys Acta, 2000, Flower, Biochem J, 1996).

[00133] Lipocalins have unusually low levels of overall sequence conservation, often with sequence identity values of less than 20%. In contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded antiparallel β-sheet closed on itself to form a continuously hydrogen-bonded β-barrel. This β-barrel forms the central cavity. One end of the barrel is sterically blocked by an N-terminal peptide segment that extends through its bottom, as well as three peptide loops connecting the β-strands. The other end of the β-barrel is solvent-exposed and encompasses the target-binding site, which is formed by four flexible peptide loops (AB, CD, EF, and GH). It is the diversity of loops in the remainder of the rigid lipocalin framework that gives rise to many different binding variants, each capable of adapting to targets of different size, shape and chemical nature (reviewed, for example, in Skerra, Biochim Biophys Acta, 2000, Flower et al., Biochim Biophys Acta, 2000, Flower, Biochem J, 1996).

[00134] The lipocalin mutein of the present invention can be any lipocalin mutein. Examples of suitable lipocalins (also sometimes referred to as "reference lipocalin", "wild type lipocalin", "reference protein scaffolds" or simply "scaffolds") from which the mutein can be used include, but are not limited to, tear lipocalin (lipocalin-1, Tlc or protein von Ebner glands), retinol binding protein, neutrophil prostaglandin D synthase, β-lactoglobulin, bilin binding protein (BBP), apolipoprotein D (APOD), neutrophil gelatinase-associated lipocalin (NGAL), protein-related α2-microglobulin (A2m), 24p3/uterocalin (24p3), von Ebner's gland protein 1 (VEGP 1), von Ebner's gland protein 2 (VEGP 2) and major allergen Can f 1 (ALL-1). In related embodiments, the lipocalin mutein is derived from the group of lipocalins consisting of human tear lipocalin (hTlc), human neutrophil gelatinase-associated lipocalin (hNGAL), human apolipoprotein D (hAPOD), and bilin-binding protein from Pieris brassicae.

[00135] The amino acid sequence of a lipocalin mutein according to the present invention may have high sequence identity compared to the reference lipocalin (or wild type lipocalin) from which it is derived, such as hTlc or hNGAL, compared to sequence identity values with another lipocalin (see also higher). In this general context, the amino acid sequence of a lipocalin mutein according to the present invention is at least substantially similar to the amino acid sequence of the corresponding reference lipocalin (wild type lipocalin), provided that gaps (as defined herein) may occur in the alignment that result from adding or removing amino acids. The corresponding lipocalin mutein sequence of the present invention, while being substantially similar to the sequences of the corresponding reference lipocalin (wild type lipocalin), in some embodiments is at least 60%, 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 mutein of the present invention may, of course, contain substitutions described herein that render the lipocalin mutein capable of binding to CD137.

[00136] Typically, a lipocalin mutein contains one or more mutated amino acid residues relative to the amino acid sequence of wild-type lipocalin or reference lipocalin, such as hTlc and hNGAL, in four open-end loops that constitute the ligand-binding pocket and define entry into the ligand-binding pocket. binding pocket (see above). As explained above, these regions are required to determine the binding specificity of the lipocalin mutein to the desired target. In some embodiments, the lipocalin mutein of the present invention may also contain regions of mutated amino acid residues outside the four loops. In some embodiments, the lipocalin mutein of the present invention may contain one or more mutant amino acid residues in one or more of the three peptide loops (denoted BC, DE and FG) connecting the β-strands at the closed end of the lipocalin. In some embodiments, a mutein derived from tear lipocalin, NGAL lipocalin, or a homolog thereof may have 1, 2, 3, 4 or more mutated amino acid residues at any sequence position in the N-terminal region and/or in the three peptide loops BC, DE and FG, located at the end of the β-barrel structure, which is located opposite the natural binding pocket of lipocalin. In some embodiments, a mutein derived from tear lipocalin, NGAL lipocalin, or a homolog thereof may lack mutated amino acid residues in the DE peptide loop located at the end of the β-barrel structure compared to the wild-type tear lipocalin sequence.

[00137] In some embodiments, the lipocalin mutein of the present invention may include one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more mutated amino acid residues compared to the amino acid sequence of the corresponding reference lipocalin (wild-type lipocalin), provided that such lipocalin mutein is capable of binding to CD137. In some embodiments, the lipocalin mutein of the present invention includes at least two, including 2, 3, 4, 5 or even more, mutant amino acid residues wherein the native amino acid residue of the corresponding reference lipocalin (wild type lipocalin) is replaced by an arginine residue.

[00138] Any types and numbers of mutations are contemplated, including substitutions, deletions, and insertions, as long as the provided lipocalin mutein retains its ability to bind CD137 and/or it has at least 60% sequence identity, such as , at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or greater identity to the amino acid sequence of reference lipocalin (wild type lipocalin), e.g. mature hTlc or mature hNGAL.

[00139] In some embodiments, the replacement is a conservative replacement. In some embodiments, the substitution is a non-conservative substitution or one or more of the following illustrative substitutions.

[00140] In particular, to determine whether an amino acid residue from the amino acid sequence of a lipocalin mutein differs from that of a reference lipocalin (wild-type lipocalin) corresponding to a specific position in the amino acid sequence of a reference lipocalin (wild-type lipocalin), one skilled in the art may use tools and methods well known in the art, for example various alignment methods manually or using computer programs such as BLAST 2.0, which means Basic Local Alignment Search Tool, or ClustalW, or any other suitable program that is suitable for obtaining sequence alignment results . Accordingly, the amino acid sequence of a reference lipocalin (wild-type lipocalin) may serve as a “query sequence” or “reference sequence,” while the amino acid sequence of a lipocalin mutein serves as a “query sequence” (see also above).

[00141] Conservative substitutions are typically the following substitutions, listed according to the amino acid to be mutated, each followed by one or more substitutions that may be considered conservative: Ala → Ser, Thr or Val; Arg → Lys, Gln, Asn or His; Asn → Gln, Glu, Asp or His; Asp → Glu, Gln, Asn or His; Gln → Asn, Asp, Glu or His; Glu → Asp, Asn, Gln or His; His → Arg, Lys, Asn, Gln, Asp or Glu; Ile → Thr, Leu, Met, Phe, Val, Trp, Tyr, Ala or Pro; Leu → Thr, Ile, Val, Met, Ala, Phe, Pro, Tyr or Trp; Lys → Arg, His, Gln or Asn; Met → Thr, Leu, Tyr, Ile, Phe, Val, Ala, Pro or Trp; Phe → Thr, Met, Leu, Tyr, Ile, Pro, Trp, Val or Ala; Ser → Thr, Ala or Val; Thr → Ser, Ala, Val, Ile, Met, Val, Phe, Pro or Leu; Trp → Tyr, Phe, Met, Ile or Leu; Tyr → Trp, Phe, Ile, Leu or Met; Val → Thr, Ile, Leu, Met, Phe, Ala, Ser or Pro. Other substitutions are also possible and can be determined empirically or according to other known conservative or non-conservative substitutions. As an additional orientation, each of the following groups contains amino acids that can usually be taken to determine conservative substitutions for each other:

(a) alanine (Ala), serine (Ser), threonine (Thr), valine (Val);

(b) aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), asparagine (Asn), histidine (His);

(c) arginine (Arg), lysine (Lys), glutamine (Gln), asparagine (Asn), histidine (His);

(d) isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val), alanine (Ala), phenylalanine (Phe), threonine (Thr), proline (Pro);

(e) isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp).

[00142] If such conservative substitutions result in a change in biological activity, then more significant changes, such as those presented below or further described below with respect to amino acid classes, can be made, and the products can be screened for the desired characteristic. Examples of such more significant changes are: Ala → Leu or Phe; Arg → Glu; Asn → Ile, Val or Trp; Asp → Met; Cys → Pro; Gln → Phe; Glu → Arg; His → Gly; Ile → Lys, Glu or Gln; Leu → Lys or Ser; Lys → Tyr; Met → Glu; Phe → Glu, Gln or Asp; Trp → Cys; Tyr → Glu or Asp; Val → Lys, Arg, His.

[00143] In some embodiments, significant modifications to the physical and biological properties of the lipocalin (mutein) are accomplished by selecting substitutions that vary significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, e.g., a sheet or helical conformation, ( b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk characteristics of the side chain.

[00144] Naturally occurring residues are classified into groups based on general side chain properties: (1) hydrophobic: methionine, alanine, valine, leucine, isoleucine; (2) neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues that affect chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine. In some embodiments, substitutions may involve replacing a representative of one of the given classes with a representative of another class.

[00145] Any cysteine residue not involved in maintaining the proper conformation of the corresponding lipocalin can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent miscrosslinking. Conversely, a cysteine bond(s) can be added to lipocalin to improve its stability.

D. Exemplary CD137-specific lipocalin muteins of the present invention

[00146] As noted above, lipocalin is a polypeptide defined by its supersecondary structure, namely a region with a supersecondary structure of a cylindrical β-pleated sheet containing eight β-strands linked in pairs by four loops at one end, thereby defining a binding pocket. The present invention is not limited specifically to the lipocalin muteins disclosed herein. In this regard, the present invention relates to a lipocalin mutein having a supersecondary structure region of a cylindrical β-pleated sheet containing eight β-strands connected in pairs by four loops at one end so that they thereby define a binding pocket, wherein at least at least one amino acid of each of at least three of the four loops has been mutated, and wherein said lipocalin is effective in binding to CD137 with detectable affinity.

[00147] In some embodiments, the lipocalin muteins disclosed herein may be or include a mature human tear lipocalin (hTlc) mutein. Mature hTlc mutein may be referred to herein as “hTlc mutein.” In some other embodiments, the lipocalin mutein disclosed herein is a human neutrophil gelatinase-associated lipocalin (hNGAL) mutein. Mature hNGAL mutein may be referred to herein as “hNGAL mutein.”

[00148] In one aspect, the present invention provides any number of lipocalin muteins derived from a reference lipocalin (wild-type lipocalin), preferably derived from mature hTlc or mature hNGAL, that bind CD137 with detectable affinity. In a related aspect, the present invention provides various lipocalin muteins that are capable of activating downstream CD137 signaling pathways by binding to CD137. In this sense, CD137 can be considered as a non-natural target of a reference lipocalin (wild-type lipocalin), preferably hTlc or hNGAL, where "non-natural target" refers to a substance that does not bind to reference lipocalins (wild-type lipocalin) under physiological conditions. By engineering modification of reference lipocalins (wild-type lipocalins) to introduce one or more mutations at specific sequence positions, we have demonstrated that high affinity and high specificity for a non-natural target, CD137, is possible. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more nucleotide triplets encoding specific positions in wild-type lipocalin sequences can be subjected to random mutagenesis by substitution at these positions into a subset of nucleotide triplets to produce a lipocalin mutein capable of binding CD137.

[00149] In some embodiments, the lipocalin muteins of the present invention may have mutant(s), including replaced, deleted, and inserted amino acid residue(s) in one or more sequence positions corresponding to the linear polypeptide sequence of the reference lipocalin, preferably hTlc or hNGAL. In some embodiments, the number of amino acid residues of the lipocalin mutein of the present invention that are mutated from the amino acid sequence of the reference lipocalin, preferably hTlc or hNGAL, 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, with 1, 2, 3, 4 being preferred 5, 6, 7, 8, 9, 10 or 11, and even more preferred are 9, 10 or 11. However, it is preferable that the lipocalin mutein of the present invention is still capable of binding CD137.

[00150] In some embodiments, the lipocalin mutein of the present invention may lack 1, 2, 3, 4 or more amino acids at its N-terminus and/or 1, 2 or more amino acids at its C-terminus compared to the corresponding reference lipocalin (wild type lipocalin); for example, SEQ ID NO: 32-38. In some embodiments, the present invention includes hTlc muteins as defined above in which the first four, one, two, three N-terminal amino acid residues of the mature hTlc sequence (His-His-Leu-Leu; positions 1-4) and/or have been deleted. or the last one or two C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of mature hTlc (eg, SEQ ID NO: 32-38). In some embodiments, the present invention includes hNGAL muteins, as defined above, from which amino acid residues (Lys-Asp-Pro, positions 46-48) of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 43) are removed. Additionally, the lipocalin mutein of the present invention may include the wild-type amino acid sequence (natural amino acid sequence) of the reference lipocalin (wild-type lipocalin), preferably hTlc or hNGAL, outside of the mutated amino acid sequence positions.

[00151] In some embodiments, one or more mutant amino acid residues included in the lipocalin mutein of the present invention substantially do not impede or interfere with the target binding activity and folding of the mutein. Such mutations, including substitution, deletion and insertion, can be carried out at the DNA level using generally accepted standard methods (Sambrook and Russell, 2001, Molecular cloning: a laboratory manual). In some embodiments, mutant amino acid residue(s) at one or more sequence positions corresponding to the linear polypeptide sequence of a reference lipocalin (wild-type lipocalin), preferably hTlc or hNGAL, are introduced by random mutagenesis by nucleotide substitution ( -th) triplet(s) encoding the corresponding positions of the reference lipocalin sequence, into a subset of nucleotide triplets.

[00152] In some embodiments, a provided lipocalin mutein that binds CD137 with detectable affinity may include at least one amino acid substitution of a native cysteine residue with another amino acid, such as a serine residue. In some embodiments, a lipocalin mutein that binds CD137 with detectable affinity may include one or more non-native cysteine residues replacing one or more amino acids of a reference lipocalin (wild-type lipocalin), preferably hTlc or hNGAL. In some embodiments, the lipocalin mutein of the present invention includes at least two amino acid substitutions of a native amino acid for a cysteine residue, resulting in one or more cysteine bridges. In some embodiments, said cysteine bridge may connect at least two loop regions. This document uses the definition of these regions according to (Biochim Biophys Acta, 2000), Flower (1996) and Breustedt et al. (2005).

[00153] Typically, a lipocalin mutein of the present invention may have at least 70%, including at least about 80%, such as at least about 85%, amino acid sequence identity with the amino acid sequence of mature hTlc (SEQ ID NO: 1) or mature hNGAL (SEQ ID NO: 2).

[00154] In some aspects, the present invention provides CD137 binding hTlc muteins. In this regard, the present invention provides one or more hTlc muteins that are capable of binding CD137 with an affinity, measured by K _D , of approximately 300 nM, 200 nM, 150 nM, 100 nM, or lower. In some embodiments, the provided hTlc muteins are capable of binding CD137 with an EC ₅₀ value of approximately 250 nM, 150 nM, 100 nM, 50 nM, 20 nM, or even lower. In some other embodiments, hTlc CD137 binding muteins may be cross-reactive with cynomolgus CD137 (cyCD137).

[00155] In some embodiments, the hTlc mutein of the present invention may interfere with the binding of CD137L to CD137.

[00156] In some embodiments, the provided hTlc muteins may contain a mutant amino acid residue at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71 , 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[00157] In some embodiments, the provided hTlc muteins may contain a mutant amino acid residue at one or more positions corresponding to positions 26-34, 55-58, 60-61, 65, 104-106 and 108 of the linear mature hTlc polypeptide sequence. (SEQ ID NO: 1).

[00158] In some embodiments, the provided hTlc muteins may further comprise a mutant amino acid residue at one or more positions corresponding to positions 101, 111, 114 and 153 of the linear mature hTlc polypeptide sequence (SEQ ID NO: 1).

[00159] In some embodiments, the provided hTlc muteins may contain a mutant amino acid residue at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or even more provisions corresponding to provisions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65 , 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In some preferred embodiments, the provided hTlc muteins are capable of binding CD137, in particular human CD137.

[00160] In some embodiments, the provided hTlc muteins may contain a mutant amino acid residue at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more positions corresponding to positions 26-34 , 55-58, 60-61, 65, 104-106 and 108 linear polypeptide sequence of mature hTlc. (SEQ ID NO: 1). In some preferred embodiments, the provided hTlc muteins are capable of binding CD137, in particular human CD137.

[00161] In some embodiments, the lipocalin mutein of the present invention may include at least one amino acid substitution of a native cysteine residue, such as a serine residue. In some embodiments, the hTlc mutein of the present invention comprises an amino acid replacement of a native cysteine residue at positions corresponding to positions 61 and/or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) with another amino acid, such as a serine residue. In this context, it should be noted that removal of the structural disulfide bond (at the level of the corresponding intact nucleic acid library) of wild-type hTlc, which is formed by cysteine residues 61 and 153 (see Breustedt et al., J Biol Chem, 2005), has been found to can lead to the production of hTlc muteins, which are not only characterized by stable folding, but are also capable of binding with high affinity to a given non-natural target. In some embodiments, elimination of the structural disulfide bond may provide the additional benefit of allowing non-natural disulfide bonds to be created or intentionally introduced into the muteins of the present invention, thereby increasing the stability of the muteins. However, hTlc muteins that bind CD137 and that have a disulfide bridge formed between Cys 61 and Cys 153 are also part of the present invention.

[00162] In some specific embodiments, the hTlc mutein of the present invention may include one or more amino acid substitutions Cys 61 → Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser , Pro or Trp and/or Cys 153 → Ser or Ala at positions corresponding to positions 61 and/or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1).

[00163] In some embodiments, two or all three cysteine codons at positions corresponding to positions 61, 101 and 153 of the linear mature hTlc polypeptide sequence (SEQ ID NO:1) are replaced by a codon of another amino acid. Additionally, in some embodiments, the hTlc mutein of the present invention comprises an amino acid replacement of a native cysteine residue at position corresponding to position 101 of the mature hTlc linear polypeptide sequence (SEQ ID NO:1) with a serine residue or a histidine residue.

[00164] In some embodiments, the mutein of the present invention comprises an amino acid substitution of a native amino acid for a cysteine residue at positions corresponding to positions 28 or 105 of the linear mature hTlc polypeptide sequence (SEQ ID NO: 1). Additionally, in some embodiments, the mutein of the present invention comprises an amino acid replacement of the native arginine residue at position corresponding to position 111 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) with a proline residue. Additionally, in some embodiments, the mutein of the present invention comprises an amino acid substitution of a native lysine residue at position corresponding to position 114 of the linear polypeptide sequence of mature hTlc (SEQ ID NO:1) with a tryptophan residue or glutamic acid.

[00165] In some embodiments, the provided CD137 binding hTlc muteins may contain at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the mature hTlc linear polypeptide sequence (SEQ ID NO: 1), one or more of the following mutant amino acid residues: Ala 5 → Val or Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Thr 42 → Ser; Gly 46 → Asp; Lys 52 → Glu; Leu 56 →Ala; Ser 58 → Asp; Arg 60 →Pro; Cys 61 → Ala; Lys 65 → Arg or Asn; Thr 71 → Ala; Val 85 → Asp; Lys 94 → Arg or Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile and Cys 153 → Ser. In some embodiments, the hTlc mutein of the present invention contains two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more or even all of the mutant amino acid residues at these positions in the mature hTlc sequence (SEQ ID NO: 1).

[00166] In some embodiments, the provided hTlc CD137 binding muteins may contain one of the following sets of mutant amino acid residues compared to the linear mature hTlc polypeptide sequence (SEQ ID NO: 1):

(a) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp and Cys 153 → Ser;

(b) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg; Val 85 → Asp; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 →Glu; Ala 133 → Thr and Cys 153 → Ser;

(c) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Asn; Lys 94 → Arg; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr and Cys 153 → Ser;

(d) Ala 5 → Val; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg; Lys 94 → Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr and Cys 153 → Ser;

(e) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 →Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Thr 42 → Ser; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile and Cys 153 → Ser;

(f) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Lys 52 → Glu; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile and Cys 153 → Ser and

(g) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Gly 46 → Asp; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile and Cys 153 → Ser.

[00167] In some embodiments, the remaining area, i.e. area different from the provisions corresponding to provisions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the mature hTlc linear polypeptide sequence (SEQ ID NO: 1) of the hTlc mutein of the present invention may contain the (natural amino acid sequence) wild-type amino acid sequence of the mature hTlc linear polypeptide sequence outside the positions of the mutant amino acid sequence .

[00168] In some embodiments, the hTlc mutein of the present invention has at least 70% sequence identity or at least 70% sequence homology to the mature hTlc sequence (SEQ ID NO: 1). As an illustrative example, the mutein of SEQ ID NO: 32 has approximately 84% amino acid sequence identity or sequence homology with the amino acid sequence of mature hTlc.

[00169] In some embodiments, the hTlc mutein of the present invention contains the amino acid sequence set forth in any of SEQ ID NOs: 32-38, or a fragment or variant thereof.

[00170] In some embodiments, the hTlc mutein of the present invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more high sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 32-38.

[00171] The present invention also provides structural homologs of an hTlc mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-38, wherein the structural homologues have amino acid sequence homology or sequence identity of 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% relative to said hTlc mutein.

[00172] In some aspects, the present invention provides CD137-binding hNGALs. In this regard, the present invention provides one or more hNGAL muteins that are capable of binding CD137 with an affinity, measured by K _D , of about 800 nM, 700 nM, 200 nM, 140 nM, 100 nM or lower, preferably about 70 nM, 50 nM, 30 nM, 10 nM, 5 nM, 2 nM or even lower. In some embodiments, the provided hNGAL muteins are capable of binding CD137 with an EC ₅₀ value of approximately 1000 nM, 500 nM, 100 nM, 80 nM, 50 nM, 25 nM, 18 nM, 15 nM, 10 nM, 5 nM, or lower.

[00173] In some embodiments, the provided hNGAL CD137 binding muteins may be cross-reactive with cynomolgus CD137. In some embodiments, the provided hNGAL muteins are capable of binding cynomolgus CD137 with an affinity, measured by K _D , of about 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, or even lower. In some embodiments, the provided hNGAL muteins are capable of binding cynomolgus CD137 with an EC ₅₀ value of approximately 100 nM, 80 nM, 50 nM, 30 nM, or even lower.

[00174] In some embodiments, the hNGAL mutein of the present invention may interfere with or compete for binding of CD137L to CD137. In some other embodiments, the hNGAL mutein of the present invention may be capable of binding CD137 in the presence of CD137L and/or binding the CD137/CD137L complex.

[00175] In some embodiments, the provided hNGAL muteins may contain a mutant amino acid residue at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00176] In some embodiments, the provided hNGAL muteins may contain a mutant amino acid residue at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or even more provisions corresponding to provisions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96 , 100, 103, 106, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding CD137, in particular human CD137.

[00177] In some embodiments, the provided hNGAL muteins may contain a mutant amino acid residue at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81 87, 96, 100, 103, 106, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding CD137, in particular human CD137.

[00178] In some embodiments, the provided hNGAL muteins may contain a mutant amino acid residue at one or more positions corresponding to positions 36, 87 and 96 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), and at one or more positions corresponding to positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 94, 100, 103, 106, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00179] In some other embodiments, the provided hNGAL muteins may contain a mutant amino acid residue at one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70- 73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00180] In other embodiments, the provided hNGAL muteins may contain a mutant amino acid residue at one or more positions corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100 , 103, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), and at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101 and 122 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00181] In some embodiments, the lipocalin mutein of the present invention may contain at least one amino acid substitution of a native cysteine residue, for example, a serine residue. In some embodiments, the hNGAL mutein of the present invention may comprise an amino acid substitution of a native cysteine residue at positions corresponding to positions 76 and/or 175 of the linear hNGAL polypeptide sequence (SEQ ID NO: 2) with another amino acid, such as a serine residue. In this context, it should be noted that removal of the structural disulfide bond (at the level of the corresponding intact nucleic acid library) of wild-type hNGAL, which is formed by cysteine residues 76 and 175 (see Breustedt et al., J Biol Chem, 2005), has been found to can lead to the production of hNGAL muteins, which are not only characterized by stable folding, but are also capable of binding a given non-natural target with high affinity. In some embodiments, elimination of the structural disulfide bond may provide the additional benefit of allowing the creation or intentional introduction of unnatural disulfide bonds into the muteins of the present invention, thereby increasing the stability of the muteins. However, also part of the present invention are hNGAL muteins that bind CD137 and that have a disulfide bridge formed between Cys 76 and Cys 175.

[00182] In some embodiments, the provided CD137 binding muteins hNGAL may contain at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutant amino acid residues: Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg or Lys; Gln 49 → Val, Ile, His, Ser or Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met, Ala or Gly; Leu 70 → Ala, Lys, Ser or Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met, Arg, Thr or Asn; Trp 79 → Ala or Asp; Arg 81 → Met, Trp or Ser; Phe 83 → Leu; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr. In some embodiments, the hNGAL mutein of the present invention contains two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, even more, such as 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24 or all mutant amino acid residues at these positions in the mature hNGAL sequence (SEQ ID NO: 2).

[00183] In some embodiments, the provided CD137 binding muteins hNGAL may contain at one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutant amino acid residues: Gln 20 → Arg; Asn 25 → Tyr or Asp; Gln 28 → His; Val 33 → Ile; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Val or Asp; Gln 49 → His; Tyr 52 → Ser or Gly; Lys 59 → Asn; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln or His; Tyr 78 → His; Trp 79 → Ile; Ile 80 → Asn; Arg 81 → Trp or Gln; Thr 82 → Pro; Cys 87 → Ser; Phe 92 → Leu or Ser; Asn 96 → Phe; Lys 98 → Arg; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → His or Pro; Phe 122 → Tyr; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly. In some embodiments, the hNGAL mutein of the present invention contains two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34, mutant amino acid residues at these positions in the mature hNGAL sequence (SEQ ID NO: 2).

[00184] In some embodiments, the provided CD137 binding hNGAL muteins may comprise one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, provisions corresponding to provisions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutant amino acid residues: Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser or Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln or His; Trp 79 → Ile; Arg 81 → Trp or Gln; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His or Pro; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly. In some embodiments, the provided CD137 binding hNGAL muteins may further comprise at one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, positions corresponding positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101 and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutant amino acid residues: Gln 20 → Arg; Asn 25 → Tyr or Asp; Val 33 → Ile; Glu 44 → Val or Asp; Lys 59 → Asn; Phe 71 → Leu; Tyr 78 → His; Ile 80 → Asn; Thr 82 → Pro; Phe 92 → Leu or Ser; Lys 98 → Arg; Pro 101 → Leu and Phe 122 → Tyr.

[00185] In some embodiments, the provided hNGAL CD137 binding muteins may contain one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(b) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ile; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met; Leu 70 → Lys; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(c) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Ala; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(d) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Ala; Arg 72 →Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 →Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(e) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Ser; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Ser; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Met; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 →Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(f) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 →Val; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Arg; Trp 79 → Asp; Arg 81 → Ser; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(g) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → His; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(h) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Phe 83 → Leu; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr or

(i) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ser; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Asn; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr.

[00186] In some additional embodiments, in the remaining area, i.e. area different from positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127 , 132 and 134 of the linear hNGAL polypeptide sequence (SEQ ID NO: 2) of the hNGAL mutein of the present invention may include the (natural amino acid sequence) wild-type amino acid sequence of mature hNGAL outside the positions of the mutant amino acid sequence.

[00187] In some other embodiments, the provided hNGAL CD137 binding muteins may contain one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(b) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Lys 98 → Arg; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(c) Asn 25 → Tyr; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Gln; Phe 92 → Ser; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(d) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 →Asp; Asp 77 → Gln; Tyr 78 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(e) Asn 25 → Asp; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(f) Val 33 → Ile; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 →Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(g) Gln 20 → Arg; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Val; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Phe 122 → Tyr; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(h) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Ile 80 → Asn; Arg 81 → Trp; Thr 82 → Pro; Asn 96 → Phe; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → Pro; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(i) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Lys 59 → Asn; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly and

(j) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Asp; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly.

[00188] In some embodiments, the provided CD137 binding mutein of hNGAL may contain the following set of mutant amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ile; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met; Leu 70 → Lys; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr, and/or the provided mutein may have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or greater sequence identity to the amino acid sequence of SEQ ID NO: 40.

[00189] In some embodiments, the remaining area, i.e. area different from positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103 122, 125, 127, 132 and 134 of the linear hNGAL polypeptide sequence (SEQ ID NO: 2) of the hNGAL mutein of the present invention may include the (natural amino acid sequence) wild-type amino acid sequence of mature hNGAL outside the positions of the mutant amino acid sequence.

[00190] In some embodiments, the hNGAL mutein of the present invention has at least 70% sequence identity or at least 70% sequence homology with the mature hNGAL sequence (SEQ ID NO: 2). As an illustrative example, the mutein of SEQ ID NO: 40 has an amino acid sequence identity or sequence homology of approximately 87% with the amino acid sequence of mature hNGAL.

[00191] In some embodiments, the hNGAL mutein of the present invention contains the amino acid sequence set forth in any of SEQ ID NOs: 39-57, or a fragment or variant thereof.

[00192] In some embodiments, the hNGAL mutein of the present invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more high sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 39-57.

[00193] The present invention also provides structural homologues of an hNGAL mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-57, wherein the structural homologues have amino acid sequence homology or sequence identity of 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% relative to said hNGAL mutein.

[00194] In some embodiments, the present invention provides a lipocalin mutein that binds CD137 with an affinity, measured by K _D , of about 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or greater sequence identity to the amino acid sequence of SEQ ID NO: 40.

[00195] In some embodiments, the present invention provides a lipocalin mutein that binds CD137 with an affinity, measured by K _D , of approximately 5 nM or lower, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or greater sequence identity to the amino acid sequence of SEQ ID NO: 49.

[00196] In some embodiments, the lipocalin mutein of the present invention may contain a heterologous amino acid sequence at its N- or C-terminus, preferably at the C-terminus, such as a Strep II streptavidin tag (SEQ ID NO: 12) or a cleavage site sequence for certain restriction enzymes, which does not affect the biological activity (binding to its target, for example CD137) of lipocalin mutein.

[00197] In some embodiments, additional modifications of the lipocalin mutein can be introduced to modulate certain characteristics of the mutein, such as increasing folding stability, serum stability, resistance to other proteins, or water solubility, or to reduce the tendency to aggregate, or to introduce into the mutein new characteristics. In some embodiments, the modification(s) may result in modulation of two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10) characteristics of the provided mutein.

[00198] For example, one or more positions in the amino acid sequence of the lipocalin mutein can be changed 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 to form non-naturally occurring disulfide bonds. Conjugated compounds such as PEG and HES may, in some cases, increase the serum half-life of the corresponding lipocalin mutein.

[00199] In some embodiments, a reactive group on a lipocalin mutein may occur naturally in its amino acid sequence, such as in naturally occurring cysteine residues in the amino acid sequence. In other embodiments, such a reactive group can be introduced through mutagenesis. In the case of introducing a reactive group by mutagenesis, one possibility is to mutate the amino acid at the appropriate position to a cysteine residue. Illustrative variants of such a mutation to introduce a cysteine residue into the amino acid sequence of the hTlc mutein include the substitutions Thr 40 → Cys, Glu 73 → Cys, Arg 90 → Cys, Asp 95 → Cys and Glu 131 → Cys of the wild-type hTlc sequence (SEQ ID NO: 1 ). Exemplary variants of such a mutation to introduce a cysteine residue into the amino acid sequence of the hNGAL mutein include the introduction of a cysteine residue at one or more sequence positions that correspond to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146, or 158 wild type hNGAL sequences (SEQ ID NO: 2). The resulting thiol moiety can be used to PEGylate or gekylate a mutein, for example to increase the serum half-life of the corresponding lipocalin mutein.

[00200] In some embodiments, artificial amino acids may be introduced into the amino acid sequence of the lipocalin mutein to provide suitable amino acid side chains as new reactive groups for conjugating one of the above compounds to a lipocalin mutein. Typically, such artificial amino acids are designed to be more reactive and therefore facilitate conjugation with the desired compound. Such artificial amino acids can be introduced by mutagenesis, for example using artificial tRNA, which is para-acetyl-phenylalanine.

[00201] In some embodiments, the lipocalin mutein of the present invention is fused at its N-terminus or its C-terminus to a protein, protein domain, or peptide, such as an antibody, signal sequence, and/or affinity tag. In some other embodiments, the lipocalin mutein of the present invention is conjugated at its N-terminus or at its C-terminus to a partner that is a protein, protein domain, or peptide; for example an antibody, a signal sequence and/or an affinity tag.

[00202] Examples of suitable fusion partners are affinity tags such as the streptavidin tags Strep tag or Strep tag II (Schmidt et al., J Mol Biol, 1996), c-myc tag, FLAG tag, His tag or HA tag or proteins such as glutathione S-transferase, which allow easy detection and/or purification of recombinant proteins. Suitable fusion partners for the lipocalin muteins of the present invention also include proteins with chromogenic or fluorescent properties, such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP). In general, the lipocalin muteins of the present invention can be labeled with any suitable chemical or enzyme that directly or indirectly produces a detectable compound or signal in a chemical, physical, optical or enzymatic reaction. For example, a fluorescent or radioactive label can be conjugated to the lipocalin mutein to produce fluorescence or x-rays as a detectable signal. Examples of enzyme tags (and at the same time optical tags) that catalyze the formation of chromogenic reaction products are alkaline phosphatase, horseradish peroxidase and β-galactosidase. In general, all labels typically used in the case of antibodies (with the exception of labels exclusively used with the sugar moiety in the Fc portion of immunoglobulins) can also be used for conjugation with the lipocalin muteins of the present invention.

[00203] In some embodiments, the lipocalin mutein of the present invention may be fused or conjugated to a moiety that extends the serum half-life of the mutein (on this subject, see also International Patent Publication No. WO 2006/056464, where such strategies are described in relation to human neutrophil gelatinase-associated lipocalin (hNGAL) muteins with binding affinity for CTLA-4). The moiety that prolongs the serum half-life may be a PEG molecule, a HES molecule, a fatty acid molecule such as palmitic acid (Vajo and Duckworth, Pharmacol Rev, 2000), an immunoglobulin Fc portion, an immunoglobulin C _H 3 domain, a C Immunoglobulin _H 4 domain, albumin binding peptide, albumin binding protein or transferrin, to name a few.

[00204] In some embodiments, when using PEG as a conjugation partner, the PEG molecule may be substituted, unsubstituted, linear, or branched. It may also be an activated polyethylene derivative. Examples of suitable compounds are PEG molecules which are described in International Patent Publication No. WO 1999/64016, US Patent No. 6177074 or US Patent No. 6403564 for interferon or which are described for other proteins such as PEG modified asparaginase, PEG adenosine deaminase (PEG-ADA) or PEG superoxide dismutase (Fuertges and Abuchowski, Journal of Controlled Release, 1990). The molecular weight of such a polymer, such as polyethylene glycol, may range from about 300 to about 70,000 daltons, including, for example, polyethylene glycol with a molecular weight of about 10,000, about 20,000, about 30,000, or about 40,000 daltons. Moreover, as described, for example, in US Pat. No. 6,500,930 or US Pat. No. 6,620,413, carbohydrate oligomers and polymers, such as HES, can be conjugated to the mutein of the present invention to extend serum half-life.

[00205] In some embodiments, SynFusion™ technology, which is commercially available from Syntonix Pharmaceuticals, Inc., can be used when using the Fc portion of an immunoglobulin to extend the half-life of the lipocalin muteins of the present invention in serum. (Massachusetts, USA). The use of this Fc fusion technology allows the creation of long-acting biopharmaceuticals, and the product obtained using this technology can, for example, consist of two copies of a mutein associated with the Fc region of the antibody to improve pharmacokinetics, solubility and production efficiency.

[00206] Examples of albumin-binding peptides that can be used to increase the serum half-life of lipocalin mutein include, for example, peptides having the consensus sequence Cys-Xaa ₁ -Xaa ₂ -Xaa ₃ -Xaa ₄ -Cys, where Xaa ₁ represents is Asp, Asn, Ser, Thr or Trp; Xaa ₂ is Asn, Gln, His, Ile, Leu or Lys; Xaa ₃ is Ala, Asp, Phe, Trp or Tyr; and Xaa ₄ is Asp, Gly, Leu, Phe, Ser or Thr, as described in US Patent Publication No. 20030069395 or Dennis et al. (2002). The albumin binding protein fused or conjugated to a lipocalin mutein to increase serum half-life may be a bacterial albumin binding protein, an antibody, an antibody fragment including domain antibodies (see, for example, US Pat. No. 6,696,245), or a lipocalin mutein with binding activity to albumin. Examples of bacterial albumin-binding proteins include streptococcal protein G (Konig and Skerra, J Immunol Methods, 1998).

[00207] In some embodiments, if the albumin binding protein is an antibody fragment, it may be a domain antibody. Domain-specific antibodies (dAbs) are engineered to provide precise control of biophysical properties and in vivo half-life to create an optimal product safety and efficacy profile. Domain antibodies, for example, are commercially available from Domantis Ltd. (Cambridge, UK and Massachusetts, USA).

[00208] In some embodiments, albumin itself (Osborn et al., J Pharmacol Exp Ther, 2002) or a biologically active fragment of albumin can be used as a partner of the lipocalin mutein of the present invention to increase serum half-life. The term "albumin" includes all mammalian albumins, such as human serum albumin, or bovine serum albumin, or rat albumin. Albumin or a fragment thereof can be produced by recombinant techniques as described in US Pat. No. 5,728,553 or European Patent Publications No. EP0330451 and No. EP0361991. Accordingly, recombinant human albumin (eg, Recombumin® from Novozymes Delta Ltd., Nottingham, UK) can be conjugated or fused to the lipocalin mutein of the present invention.

[00209] In some embodiments, when using transferrin as a partner to increase the serum half-life of the lipocalin muteins of the present invention, the muteins can be genetically fused to the N- or C-terminus, or both the first or second, of non-glycosylated transferrin. Non-glycosylated transferrin has a half-life of 14-17 days, and a transferrin fusion protein will similarly have an extended half-life. The transferrin carrier also ensures high bioavailability, biodistribution and circulation stability in the bloodstream. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, Pennsylvania, USA). Recombinant human transferrin (DeltaFerrin™) for use as a protein stabilizer/half-life extension partner is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

[00210] Another alternative for increasing the half-life of the lipocalin muteins of the present invention is to fuse long, unstructured, flexible glycine-rich sequences (eg, polyglycine with approximately 20-80 consecutive glycine residues) to the N- or C-terminus of the mutein. This approach, disclosed for example in International Patent Publication No. WO2007/038619, is also called “rPEG” (recombinant PEG).

E. Exemplary Uses and Applications of Novel CD137-GPC3 Specific Fusion Proteins

[00211] In some embodiments, the fusion proteins of the present invention may produce a synergistic effect by dual targeting CD137 and GPC3. In some embodiments, the fusion proteins of the present invention can produce a localized antitumor effect by dual targeting CD137 and GPC3. Consequently, numerous possible applications for the fusion proteins of the present invention exist in medicine.

[00212] In some embodiments, the present invention covers the use of one or more fusion proteins disclosed herein, or one or more compositions containing such fusion proteins, to simultaneously bind CD137 and GPC3.

[00213] The present invention also provides for the use of one or more of the disclosed fusion proteins to form a complex with CD137 and/or GPC3.

[00214] Therefore, in one aspect of the present invention, the provided fusion proteins can be used to detect CD137 and GPC3. Such use may involve the steps of contacting one or more of said fusion proteins under suitable conditions with a sample suspected of containing CD137 and/or GPC3, thereby allowing the formation of a complex between the fusion proteins and CD137 and/or GPC3, and detecting the complex using a suitable signal. The detectable signal may be due to a label, as explained above, or a change in physical properties due to binding, i.e. formation of the complex itself. One example is surface plasmon resonance, the value of which changes during the binding of binding partners, one of which is immobilized on a surface such as gold foil.

[00215] The fusion proteins of the present invention can also be used to separate CD137 and/or GPC3. Such use may involve the steps of contacting one or more of said fusion proteins under suitable conditions with a sample that is believed to contain CD137 and/or GPC3, thereby allowing the formation of a complex between the fusion proteins and CD137 and/or GPC3, and separating the complex from the sample. .

[00216] In some embodiments, the present invention provides diagnostic and/or assay kits containing one or more fusion proteins of the present invention.

[00217] In addition to their use in diagnostics, in another aspect, the present invention provides pharmaceutical compositions comprising one or more fusion proteins of the present invention and a pharmaceutically acceptable excipient.

[00218] Moreover, in some embodiments, the present invention provides fusion proteins that simultaneously bind CD137 and/or GPC3 for the use of such proteins as antitumor and/or anti-infective agents, as well as immunomodulators. In some embodiments, the fusion proteins of the present invention are provided for use in a method of preventing, ameliorating, or treating human diseases, such as various forms of cancer, including GPC3-positive cancers such as hepatocellular carcinoma (HCC), melanoma, Merkel cells, Wilm's tumor and hepatoblastoma. Accordingly, methods are also provided for preventing, reducing, or treating human diseases, such as various forms of cancer, including GPC3-positive cancer, such as hepatocellular carcinoma (HCC), melanoma, Merkel cell carcinoma, in a subject in need thereof, comprising: administering to said subject a therapeutically effective amount of one or more fusion proteins of the present invention.

[00219] Other examples of forms of cancer that can be treated using the fusion proteins of the present invention include liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma , kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma , non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, induced by external factors forms of cancer, including asbestos-induced, hematological malignancies, including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma/primary B-cell lymphoma of the mediastinum, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large-cell b-cell lymphoma, berkite lymphoma, immunoblastic large-cell lymphoma, v-lymphoblastic lymphoma of predecessor cells, mantle cell lymphoma, acute lymphoblastic leycosis, fungoid mycosis, anaplastic cerebellar lymphone mu, T-cell lymphoma and T-lymphoblastic lymphoma from progenitor cells and any combination of these cancers. In some embodiments, the present invention is also useful for treating metastatic cancers.

[00220] In some embodiments, the fusion proteins of the present invention can simultaneously target tumor cells that express GPC3, such as HCC cells, melanomas, Merkel cell carcinomas, Wilm tumors, and hepatoblastomas, and activate host immune system lymphocytes located near such tumor cells. In some embodiments, the fusion proteins of the present invention can enhance targeted antitumor activity of T cells, enhance antitumor immunity, induce T cell-mediated cytolysis, and/or have a direct inhibitory effect on tumor growth, thereby producing antitumor results. In some embodiments, the fusion proteins of the present invention can activate immune responses in the tumor microenvironment. In some embodiments, the fusion proteins of the present invention can reduce the side effects of effector lymphocytes on healthy cells, i.e. off-target toxicity, for example, through local inhibition of oncogene activity and induction of cell-mediated cytotoxicity carried out by NK cells and/or T cells.

[00221] In some embodiments, the present invention covers the use of a fusion protein of the present invention, or a composition comprising the provided fusion protein, to induce a localized lymphocytic response in close proximity to GPC3-positive tumor cells, such as HCC cells, melanomas, Merkel cell carcinomas , Wilm's tumor and hepatoblastoma. Accordingly, in some embodiments, the present invention provides methods for inducing a localized lymphocytic response in close proximity to GPC3-positive tumor cells, such as HCC cells, melanomas, Merkel cell carcinomas, Wilms tumors, and hepatoblastomas, using one or more fusion proteins of the present invention. the invention or one or more compositions containing such fusion proteins. “Localized” means that by simultaneously binding to T cells via CD137 and targeting GPC3-positive tumor cells, the T cells produce cytokines, particularly IL-2 and/or IFN-gamma, in close proximity to the GPC3-positive cells. Such cytokines reflect activation of T cells, which may then be able to kill GPC3-positive cells either directly or indirectly by activating other killer cells such as T cells or NK cells.

[00222] In some embodiments, the present invention covers the use of a fusion protein of the present invention or a composition comprising such a fusion protein to co-stimulate T cells and/or activate downstream CD137 signaling pathways. Preferably, the provided fusion protein provides co-stimulation of T cells and/or activates downstream CD137 signaling pathways when exposed to tumor cells that express GPC3. Accordingly, the present invention provides methods for inducing T cell proliferation and/or activation of downstream CD137 signaling pathways, preferably by targeting tumor cells that express GPC3, such as HCC cells, melanomas, Merkel cell carcinomas, Wilms tumors and hepatoblastomas, providing the use of one or more fusion proteins of the present invention and/or one or more compositions containing such fusion proteins.

[00223] In some embodiments, the present invention covers the use of a fusion protein of the present invention, or a composition comprising such a fusion protein, to induce clustering and activation of CD137 on T cells and direct such T cells to tumor cells that express GPC3, such such as HCC cells, melanoma, Merkel cell carcinoma, Wilm's tumor and hepatoblastoma.

[00224] Additional objects, advantages and features of the present invention will become apparent to those skilled in the art upon examination of the following examples and the accompanying drawings, which are not intended to be limiting. Thus, it is to be understood that while the present invention has been specifically disclosed by way of illustrative embodiments and additional features, modifications and variations to the illustrated embodiments of the present invention may be contemplated by those skilled in the art, and that such modifications and variations are considered to fall within the scope of the present invention.

F. Preparation of Exemplary Novel CD137 and GPC3 Specific Fusion Proteins Provided

[00225] In some embodiments, the present invention provides nucleic acid molecules (DNA and RNA) that include nucleotide sequences encoding the provided fusion proteins. In some embodiments, the present invention includes a host cell containing the provided nucleic acid molecule. Because the degeneracy of the genetic code allows for the substitution of certain codons for other codons specifying the same amino acid, the present invention is not limited to the specific nucleic acid molecule encoding the fusion protein described herein, but rather embraces all nucleic acid molecules that include nucleotide sequences encoding a functional fusion protein. In this regard, the present invention also relates to nucleotide sequences encoding the provided fusion proteins.

[00226] A nucleic acid molecule, such as DNA, is said to be “capable of expressing a nucleic acid molecule” or “capable of allowing the expression of a nucleotide sequence” if it includes sequence elements that contain information relating to the regulation of transcription and/or translation, and such sequences "operably linked" to a nucleotide sequence encoding a protein. A functional link is a link in which the regulatory sequence elements and the sequence to be expressed are linked in such a way as to allow gene expression. The exact nature of the regulatory regions required for gene expression may vary between species, but in general these regions include the promoter, which in prokaryotes contains both the promoter itself, i.e. DNA elements that control the start of transcription, and DNA elements that, when transcribed into RNA, will signal the start of translation. Such promoter regions typically include 5' non-coding sequences involved in triggering transcription and translation, such as -35/-10 boxes and the Shine-Dalgarno element in prokaryotes or TATA box, CAAT sequences and 5' capping elements in eukaryotes. These regions may also include enhancer or repressor elements, as well as translated signal and leader sequences for targeting the native protein to a specific compartment of the host cell.

[00227] In addition, 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these transcription termination sequences do not function satisfactorily in a particular host cell, then they can be replaced by signals that are functional in such a cell.

[00228] Therefore, a nucleic acid molecule of the present invention may be "operably linked" to one or more regulatory sequences, such as a promoter sequence, to enable expression of the nucleic acid molecule. In some embodiments, a nucleic acid molecule of the present invention includes 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 useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

[00229] In some embodiments, a nucleic acid molecule encoding a lipocalin mutein disclosed herein may be "operably linked" to another nucleic acid molecule encoding an immunoglobulin of the present invention to allow expression of a fusion protein disclosed herein. .

[00230] In some embodiments, the methods provided may include subjecting at least one nucleic acid molecule encoding mature hTlc to mutagenesis at one or more triplets encoding one or more positions corresponding to positions 5, 26-31, 33-34, 42 , 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 hTlc linear polypeptide sequence (SEQ ID NO : 1), to obtain lipocalin muteins, which are included in the provided fusion proteins. In some embodiments, the methods provided may include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at one or more triplets encoding one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the hNGAL linear polypeptide sequence (SEQ ID NO: 2), producing muteins lipocalin, which are included in the provided fusion proteins. In some embodiments, the method provided may include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at one or more triplets encoding one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 linear polypeptide sequence of hNGAL (SEQ ID NO: 2), producing lipocalin muteins that are included in the provided fusion proteins.

[00231] Additionally, with respect to the hTlc muteins or hNGAL muteins of the present invention that are included in fusion proteins, in some embodiments the naturally occurring disulfide bond between Cys 61 and Cys 153 or Cys 76 and Cys 175, respectively, may be removed. Accordingly, such muteins can be produced in a cell compartment that has a reducing redox environment, for example, in the cytoplasm of Gram-negative bacteria.

[00232] With respect to further contemplated hTlc muteins or hNGAL muteins of the present invention that are included in fusion proteins, the present invention also includes nucleic acid molecules encoding such muteins, which in some embodiments may include one or more additional mutations outside of the specified sequence positions experimental mutagenesis. Such mutations are often tolerated or may even be advantageous, for example, if they contribute to improved folding efficiency, serum stability, heat stability, or ligand binding affinity of lipocalin muteins and/or fusion proteins.

[00233] In some embodiments, the provided nucleic acid molecules may also be part of a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome.

[00234] In some embodiments, the provided nucleic acid molecule may be included in a phagemid. As used herein, a phagemid vector refers to a vector encoding the intergenic region of a temperate phage, such as M13 or f1, or a functional portion thereof fused to a cDNA of interest. For example, in some embodiments, following infection through superinfection of bacterial host cells with such a provided phagemid vector and a suitable helper phage (e.g., M13K07, VCS-M13, or R408), intact phage particles are obtained, thereby allowing physical binding of the encoded heterologous cDNA to its corresponding polypeptide , exposed on the surface of the phage (Lowman, Annu Rev Biophys Biomol Struct, 1997, Rodi and Makowski, Curr Opin Biotechnol, 1999).

[00235] In accordance with various embodiments, cloning media may include, in addition to the regulatory sequences described above and nucleic acid sequences encoding the fusion protein described herein, replication and control sequences obtained from species compatible with the host cell being used for expression, as well as selection markers that give transformed or transfected cells a selectable phenotype. Numerous suitable cloning vectors are known and commercially available in the prior art.

[00236] The present invention, in some embodiments, also provides methods for producing the fusion proteins of the present invention from a nucleic acid encoding the fusion protein or any subunits thereof using genetic engineering methods. In some embodiments, the present method can be performed in vivo, wherein the provided fusion protein can, for example, be produced in a bacterial or eukaryotic host organism and then isolated from that host organism or culture thereof. The fusion protein of the present invention can also be produced in vitro, for example, using an in vitro translation system.

[00237] When producing a fusion protein in vivo, the nucleic acid encoding the fusion protein can be introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA technology, which is well known in the art. In some embodiments, a DNA molecule encoding a fusion protein described herein, and, in particular, a cloning vector containing a sequence encoding such a fusion protein, can be introduced by transformation into a host cell capable of expressing the gene. The transformation can be accomplished using standard techniques. Thus, the present invention also relates to host cells containing the nucleic acid molecule disclosed herein.

[00238] In some embodiments, the transformed host cells can be cultured under conditions suitable for expression of a nucleotide sequence encoding a fusion protein of the present invention. In some embodiments, the host cells may be prokaryotic, such as Escherichia coli (E. coli) or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9, or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.

[00239] In some embodiments, if the lipocalin mutein of the present invention, including that contained in a fusion protein described herein, includes intramolecular disulfide bonds, it may be advantageous to direct the nascent protein, via an appropriate signal sequence, to a cell compartment having redox environment. This oxidative environment may be provided by the periplasm of Gram-negative bacteria such as E. coli, the extracellular environment of Gram-positive bacteria, or the lumen of the endoplasmic reticulum of eukaryotic cells and typically promotes the formation of structural disulfide bonds.

[00240] In some embodiments, it is also possible to produce the fusion protein of the present invention in the cytosol of a host cell, preferably E. coli. In this case, the provided fusion protein can either be directly produced in a soluble and folded state, or isolated in the form of inclusion bodies followed by renaturation in vitro. An additional option is the use of specific host strains that have an oxidizing intracellular environment, which can thus allow the formation of disulfide bonds in the cytosol (Venturi et al., J Mol Biol, 2002).

[00241] In some embodiments, the fusion protein of the present invention described herein may not be obtained or produced, in whole or in part, by genetic engineering. Rather, such a protein can also be produced using any of many traditional and well-known techniques, such as standard organic synthesis strategies, solid phase synthesis techniques, commercially available automated synthesizers, or by in vitro transcription and translation. For example, it is possible for promising lipocalin fusion proteins or muteins included in such fusion proteins to be identified by molecular modeling, synthesized in vitro, and screened for binding activity to the target(s) of interest. Methods for solid-phase and/or liquid-phase protein synthesis are well known in the art (see, for example, Bruckdorfer et al., Curr Pharm Biotechnol, 2004).

[00242] In some embodiments, the fusion protein of the present invention can be produced by in vitro transcription/translation using conventional methods known to those skilled in the art.

[00243] In some additional embodiments, fusion proteins described herein can also be produced using conventional recombinant techniques alone or in combination with conventional synthesis techniques.

[00244] Moreover, in some embodiments, the fusion protein of the present invention can be prepared by conjugating together individual subunits, such as immunoglobulins and muteins, included in the fusion protein. Such conjugation can be accomplished, for example, through all forms of covalent or non-covalent binding using conventional methods.

[00245] One skilled in the art will be aware of methods useful for producing the fusion proteins provided by the present invention, but the protein or nucleic acid sequences of which are not explicitly disclosed herein. By way of overview, such amino acid sequence modifications include, for example, site-directed mutagenesis of individual amino acid positions to facilitate subcloning of a protein gene or parts thereof by incorporating cleavage sites for specific restriction enzymes. These mutations can also be included to further improve the affinity of the fusion protein for its targets (eg, CD137 and GPC3). Moreover, if necessary, mutations can be introduced to modulate one or more characteristics of the protein, such as increasing folding stability, serum stability, resistance to other proteins or water solubility, or to reduce the tendency to aggregate.

[00246] The present invention can be further characterized by the following points.

[00247] Claim 1. A fusion protein capable of binding both CD137 and GPC3, where the fusion protein contains at least two subunits in any order, where the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific for GPC3, and where the second subunit contains a lipocalin mutein and is specific for CD137.

[00248] Claim 2. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen binding domain thereof and is specific for GPC3, and where the second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is connected at the N-terminus to the C-terminus of each heavy chain of the first subunit, optionally via a linker.

[00249] Claim 3. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen binding domain thereof and is specific for GPC3, and where the second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is connected at the C-terminus to the N-terminus of each heavy chain of the first subunit, optionally via a linker.

[00250] Claim 4. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen binding domain thereof and is specific for GPC3, and where the second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is connected at the N-terminus to the C-terminus of each light chain of the first subunit, optionally via a linker.

[00251] Claim 5. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen binding domain thereof and is specific for GPC3, and where the second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is connected at the C-terminus to the N-terminus of each light chain of the first subunit, optionally via a linker.

[00252] Claim 6. The fusion protein of any one of claims 1 to 5, wherein the fusion protein is capable of binding GPC3 with a K _D value of at most about 1 nM or comparable to or lower than the K _D value of an immunoglobulin or an antigen binding domain thereof that is included exclusively in the first subunit.

[00253] Claim 7. The fusion protein of claim 6, wherein the K _D value is determined by surface plasmon resonance (SPR) analysis.

[00254] Claim 8. The fusion protein of any one of claims 1 to 7, wherein the fusion protein is capable of binding GPC3 with an EC ₅₀ value of at most about 0.5 nM or comparable to or lower than the EC ₅₀ value of the immunoglobulin or an antigen binding domain thereof that is included exclusively in the first subunit.

[00255] Claim 9. The fusion protein of any one of claims 1 to 8, wherein the fusion protein is capable of binding CD137 with an EC ₅₀ value of at most about 3 nM or comparable to or lower than the EC ₅₀ value of a CD137-specific lipocalin mutein that is exclusively included into the second subunit.

[00256] Claim 10. The fusion protein of any one of clauses 8-9, wherein the EC ₅₀ value is determined using an enzyme-linked immunosorbent assay (ELISA) assay.

[00257] Claim 11. The fusion protein of any one of claims 1-10, wherein the fusion protein cross-reacts with cynomolgus GPC3.

[00258] Claim 12. The fusion protein of any one of claims 1-11, wherein the fusion protein is capable of simultaneously binding CD137 and GPC3 with EC ₅₀ values of a maximum of approximately 10 nM when measured by the fusion protein in an ELISA assay.

[00259] Claim 13. The fusion protein of any one of claims 1-12, wherein the fusion protein is capable of binding CD137 expressed on a cell with EC ₅₀ values of a maximum of about 30 nM when measured by a flow cytometric assay.

[00260] Claim 14. The fusion protein of any one of claims 1-13, wherein the fusion protein is capable of binding GPC3 expressed on a cell with EC ₅₀ values of a maximum of approximately 30 nM when the fusion protein is measured in a flow cytometry assay.

[00261] Claim 15. The fusion protein of any one of claims 1-14, wherein the fusion protein is capable of binding GPC3-expressing tumor cells.

[00262] Claim 16. The fusion protein of any one of claims 1-15, wherein the fusion protein is capable of stimulating T cell responses.

[00263] Claim 17. The fusion protein of any one of claims 1-16, wherein the fusion protein is capable of inducing increased secretion of IL-2.

[00264] Claim 18. The fusion protein of any one of claims 1 to 17, wherein the fusion protein is capable of inducing increased secretion of IL-2 to a level greater than SEQ ID NO: 83 and/or with better potency than SEQ ID NO: : 83.

[00265] Claim 19. The fusion protein of any one of claims 1-18, wherein the fusion protein is capable of inducing lymphocyte-mediated cytotoxicity.

[00266] Claim 20. The fusion protein of any one of claims 1-19, wherein the fusion protein is capable of inducing enhanced T cell-mediated killing of GPC3-expressing tumor cells compared to SEQ ID NO: 83 and/or inducing activation of cytotoxic T cells with better efficiency compared to SEQ ID NO: 83.

[00267] Claim 21. The fusion protein of any one of claims 1-20, wherein the fusion protein is capable of co-stimulating T cell responses in a GPC3-dependent manner.

[00268] Claim 22. The fusion protein of any one of claims 1-21, wherein the fusion protein is capable of co-stimulating T cell responses in a tumor microenvironment.

[00269] Claim 23. The fusion protein of any one of claims 1-22, wherein the fusion protein does not co-stimulate T cell responses in the absence of GPC3.

[00270] Claim 24. The fusion protein of any one of claims 1-23, wherein the fusion protein has a pharmacokinetic profile similar to that of the antibody.

[00271] Claim 25. The fusion protein of any one of claims 1-24, wherein the fusion protein has a half-life in mice of at least 50 hours, at least 75 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 175 hours, at least 200 hours, at least 250 hours or even longer, and/or wherein the fusion protein has a half-life in mice that is longer than SEQ ID NO: 83.

[00272] Claim 26. The fusion protein of any one of claims 1-25, wherein the fusion protein has an isoelectric point of at least 6.5, at least 6.8, at least 7.1, at least 7, 4, at least 7.5, at least 7.7 or even higher, and/or where the fusion protein has an isoelectric point that is higher than SEQ ID NO: 83.

[00273] Claim 27. The fusion protein of any one of claims 1-26, wherein the lipocalin mutein contains one or more mutant amino acid residues at positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58 , 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1).

[00274] Claim 28. The fusion protein of any one of claims 1-27, wherein the amino acid sequence of a lipocalin mutein contains at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), one or more of the following mutant amino acid residues: Ala 5 → Val or Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Thr 42 → Ser; Gly 46 → Asp; Lys 52 → Glu; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg or Asn; Thr 71 → Ala; Val 85 → Asp; Lys 94 → Arg or Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile and Cys 153 → Ser.

[00275] Claim 29. The fusion protein of any one of clauses 1-28, wherein the amino acid sequence of the lipocalin mutein contains one of the following sets of mutant amino acid residues compared to the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1):

(a) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp and Cys 153 → Ser;

(b) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg; Val 85 → Asp; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr and Cys 153 → Ser;

(c) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Asn; Lys 94 → Arg; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr and Cys 153 → Ser;

(d) Ala 5 → Val; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Lys 65 → Arg; Lys 94 → Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr and Cys 153 → Ser;

(e) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Thr 42 → Ser; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile and Cys 153 → Ser;

(f) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Lys 52 → Glu; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile and Cys 153 → Ser and

(g) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Gly 46 → Asp; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile and Cys 153 → Ser.

[00276] Claim 30. The fusion protein of any one of claims 1-29, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-38, or a fragment or variant thereof.

[00277] Claim 31. The fusion protein of any one of claims 1-30, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-38.

[00278] Claim 32. The fusion protein of any one of claims 1-26, wherein the lipocalin mutein contains one or more mutant amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72 -73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 human neutrophil gelatinase-associated mature lipocalin (hNGAL) linear polypeptide sequence (SEQ ID NO: 2 ).

[00279] Claim 33. The fusion protein of any one of claims 1-26 and 32, wherein the amino acid sequence of a lipocalin mutein is contained at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73 , 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 human neutrophil gelatinase-associated mature lipocalin (hNGAL) linear polypeptide sequence (SEQ ID NO: 2), one or more of the following mutant amino acid residues: Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg or Lys; Gln 49 → Val, Ile, His, Ser or Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met, Ala or Gly; Leu 70 → Ala, Lys, Ser or Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met, Arg, Thr or Asn; Trp 79 → Ala or Asp; Arg 81 → Met, Trp or Ser; Phe 83 → Leu; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr.

[00280] Claim 34. The fusion protein of any one of claims 1-26 and 33, wherein the lipocalin mutein contains one or more mutant amino acid residues at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49 , 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 of the linear polypeptide sequence of mature lipocalin associated with human neutrophil gelatinase ( hNGAL) (SEQ ID NO: 2).

[00281] Claim 35. The fusion protein of any one of claims 1-26 and 34, wherein the amino acid sequence of a lipocalin mutein is contained at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59 , 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more from the following mutant amino acid residues: Gln 20 → Arg; Asn 25 → Tyr or Asp; Gln 28 → His; Val 33 → Ile; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Val or Asp; Gln 49 → His; Tyr 52 → Ser or Gly; Lys 59 → Asn; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln or His; Tyr 78 → His; Trp 79 → Ile; Ile 80 → Asn; Arg 81 → Trp or Gln; Thr 82 → Pro; Cys 87 → Ser; Phe 92 → Leu or Ser; Asn 96 → Phe; Lys 98 → Arg; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → His or Pro; Phe 122 → Tyr; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly.

[00282] Claim 36. The fusion protein of any one of claims 1-26 and 32-35, wherein the amino acid sequence of the lipocalin mutein contains one of the following sets of mutant amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(b) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ile; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met; Leu 70 → Lys; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(c) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Ala; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(d) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Ala; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(e) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Ser; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Ser; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Met; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(f) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Val; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Arg; Trp 79 → Asp; Arg 81 → Ser; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(g) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → His; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(h) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Lys; Gln 49 → Asn; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Ala; Arg 81 → Ser; Phe 83 → Leu; Cys 87 → Ser; Leu 94 → Phe; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(i) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ser; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Asn; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr;

(j) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(k) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Lys 98 → Arg; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(l) Asn 25 → Tyr; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Gln; Phe 92 → Ser; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(m) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Tyr 78 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(n) Asn 25 → Asp; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(o) Val 33 → Ile; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 →Trp and Lys 134 →Gly;

(p) Gln 20 → Arg; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Val; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Phe 122 → Tyr; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(q) Leu 36 →Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Ile 80 → Asn; Arg 81 → Trp; Thr 82 → Pro; Asn 96 → Phe; Tyr 100 → Asp; Pro 101 → Leu; Leu 103 → Pro; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly;

(r) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Lys 59 → Asn; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp and Lys 134 → Gly and

(s) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Asp; Gln 49 → His; Tyr 52 → Ser; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 →Ile; Tyr 132 → Trp and Lys 134 → Gly.

[00283] Claim 37. The fusion protein of any one of claims 1-26 and 32-36, wherein the amino acid sequence of the lipocalin mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-57, or a fragment or variant thereof.

[00284] Claim 38. The fusion protein of any one of claims 1-26 and 32-36, wherein the amino acid sequence of the lipocalin mutein has at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 39-57 .

[00285] Claim 39. The fusion protein of any one of clauses 1-26 and 32-38 wherein the amino acid sequence of the lipocalin mutein contains the following set of mutant amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ile; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met; Leu 70 → Lys; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu and Lys 134 → Tyr, and/or wherein the lipocalin mutein has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 40.

[00286] Clause 40. The fusion protein of any one of claims 1-39, wherein one subunit is attached to another subunit via a linker.

[00287] Clause 41. The fusion protein of any one of clauses 1-40, wherein the second subunit is connected at the N-terminus by a linker to the N- or C-terminus of the constant region of each heavy chain (CH) of the first subunit or to the N- or C- the end of the constant region of each light chain (CL) of the first subunit.

[00288] Claim 42. The fusion protein of any one of clauses 1-41, wherein the third subunit is connected at the N-terminus by a linker to the N- or C-terminus of the constant region of each heavy chain (CH) of the first subunit, the N- or C-terminus the constant region of each light chain (CL) of the first subunit or the C-terminus of each second subunit.

[00289] Claim 43. The fusion protein of any one of clauses 40-42, wherein the linker is an unstructured (Gly-Gly-Gly-Gly-Ser) ₃ linker (SEQ ID NO: 13).

[00290] Claim 44. The fusion protein of any one of clauses 40-43, wherein the linker is an unstructured glycine-serine linker, a polyproline linker, a proline-alanine-serine polymer, or a linker selected from the group consisting of SEQ ID NO: 13- 23.

[00291] Claim 45. The fusion protein of any one of claims 1-44, wherein the first subunit is an antibody.

[00292] Claim 46. The fusion protein of any one of claims 1-45, wherein the antibody heavy chain variable region is selected from the group consisting of SEQ ID NOs: 78, 114, 119, 126 and 129 or a sequence having at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or more high sequence identity to the amino acid sequences set forth under SEQ ID NOs: 78, 114, 119, 126 and 129, and wherein the antibody light chain variable region is selected from the group consisting of SEQ ID NOs: 79, 115 and 127 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or greater sequence identity to the amino acid sequences set forth in SEQ ID NOs: 79, 115 and 127.

[00293] Claim 47. The fusion protein of any one of claims 1 to 46, wherein the antibody comprises a heavy chain that is any of SEQ ID NOs: 80 and 81, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or higher sequence identity to amino acid sequences, represented by SEQ ID NO: 80 or 81, and a light chain of SEQ ID NO: 82 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, at least 92%, at least 95%, at least 97%, at least 98% or greater sequence identity with the amino acid sequences presented under SEQ ID NO: 82.

[00294] Claim 48. The fusion protein of any one of claims 1-47, wherein the antibody comprises, respectively, the following heavy chain variable region and the following light chain variable region: SEQ ID NOs: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs: 114 and 115 or SEQ ID NOs: 126 and 127, or a heavy chain variable region and a light chain variable region, which have a sequence having at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or greater sequence identity to the amino acid sequences set forth in SEQ ID NO: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs: 114 and 115, or SEQ ID NOs: 126 and 127.

[00295] Claim 49. The fusion protein of any one of claims 1-48, wherein the antibody comprises, respectively, the following heavy chain and the following light chain: SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82, or a heavy chain and a light chain that have a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least at least 97%, at least 98%, or greater sequence identity to the amino acid sequences set forth in SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82.

[00296] Clause 50. The fusion protein of claim 36, wherein the antibody heavy chain contains one of the following sets of CDR sequences:

(a) GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), TRFYSYTY (HCDR3; SEQ ID NO: 74);

(b) GFTFNKNA (HCDR1, SEQ ID NO: 108), IRNKTNNYAT (HCDR2, SEQ ID NO: 109), VAGNSFAY (HCDR3, SEQ ID NO: 110);

(c) YFDFDSYE (HCDR1, SEQ ID NO: 116), IYHSGST (HCDR2, SEQ ID NO: 117), ARVNMDRFDY (HCDR3, SEQ ID NO: 108) or

(d) GFTFSSYA (HCDR1, SEQ ID NO: 120), IQKQGLPT (HCDR2, SEQ ID NO: 121), AKNRAKFDY (HCDR3, SEQ ID NO: 122).

[00297] Clause 51. The fusion protein of any one of claims 1-50, wherein the antibody light chain contains one of the following sets of CDR sequences:

(a) QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO: 77);

(b) QSLLYSSNQKNY (LCDR1, SEQ ID NO: 111), WAS (LCDR2), QQYYNYPLT (LCDR3, SEQ ID NO: 113) or

(c) QSISSY (LCDR1, SEQ ID NO: 123), NAS (LCDR2), QQNRGFPLT (LCDR3, SEQ ID NO: 125).

[00298] Clause 52. The fusion protein of any one of clauses 1-51, wherein the antibody heavy chain comprises the following set of CDR sequences: GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), TRFYSYTY ( HCDR3; SEQ ID NO: 74), and the light chain of the antibody contains the following set of CDR sequences: QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO: 77).

[00299] Clause 53. The fusion protein of any one of claims 1-51, wherein the antibody comprises the following set of CDR sequences:

(a) GFTFNKNA (HCDR1, SEQ ID NO: 108), IRNKTNNYAT (HCDR2, SEQ ID NO: 109), VAGNSFAY (HCDR3, SEQ ID NO: 110), QSLLYSSNQKNY (LCDR1, SEQ ID NO: 111), WAS (LCDR2 ), QQYYNYPLT (LCDR3, SEQ ID NO: 113) or

(b) GFTFSSYA (HCDR1, SEQ ID NO: 120), IQKQGLPT (HCDR2, SEQ ID NO: 121), AKNRAKFDY (HCDR3, SEQ ID NO: 122), QSISSY (LCDR1, SEQ ID NO: 123), NAS (LCDR2 ), QQNRGFPLT (LCDR3, SEQ ID NO: 125).

[00300] Claim 54. The fusion protein of any one of claims 1-53, wherein the antibody has an IgG4 backbone.

[00301] Claim 55. The fusion protein of claim 54, wherein the IgG4 backbone has one or more of the following mutations: S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.

[00302] Claim 56. The fusion protein of any one of claims 1-55, wherein the fusion protein contains an amino acid sequence represented by any of SEQ ID NO: 87-96, or where the fusion protein contains an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or higher sequence identity with the amino acid sequences presented under any of SEQ ID NO: 87-96.

[00303] Clause 57. The fusion protein of any one of claims 1 to 56, wherein the fusion protein comprises the amino acids set forth in SEQ ID NOs: 87 and 82, the amino acids set forth in SEQ ID NOs: 88 and 82, and the amino acids set forth in SEQ ID NOs: NOs: 81 and 89, amino acids presented under SEQ ID NOs: 81 and 90, amino acids presented under SEQ ID NOs: 91 and 82, amino acids presented under SEQ ID NOs: 92 and 82, amino acids presented under SEQ ID NO: 81 and 93, the amino acids presented under SEQ ID NOs: 81 and 94, the amino acids presented under SEQ ID NOs: 95 and 82, or the amino acids presented under SEQ ID NOs: 96 and 82.

[00304] Clause 58. The fusion protein of any one of clauses 1-57, wherein the fusion protein contains amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or greater sequence identity to the amino acid sequences set forth in SEQ ID NO: 87 and 82, SEQ ID NO: 88 and 82, SEQ ID NO: 81 and 89, SEQ ID NO: 81 and 90, SEQ ID NO: 91 and 82, SEQ ID NO: 92 and 82, SEQ ID NO: 81 and 93, SEQ ID NO: 81 and 94, SEQ ID NO: 95 and 82 or SEQ ID NO: 96 and 82.

[00305] Clause 59. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein according to any one of clauses 1-58.

[00306] Clause 60. The nucleic acid molecule of claim 59, wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of said nucleic acid molecule.

[00307] Clause 61. The nucleic acid molecule of claim 59 or claim 60, wherein the nucleic acid molecule is contained in a vector or phagemid vector.

[00308] Clause 62. A host cell containing the nucleic acid molecule of any one of claims 59-61.

[00309] Clause 63. The method of producing a fusion protein according to any one of claims 1 to 58, wherein the fusion protein is derived from a nucleic acid encoding the fusion protein.

[00310] Clause 64. The method of claim 63, wherein the fusion protein is produced in a bacterial or eukaryotic host organism and isolated from the host organism or culture thereof.

[00311] Clause 65. Use of a fusion protein according to any one of claims 1 to 58, or a composition containing such a fusion protein, to simultaneously activate downstream CD137 signaling pathways and target GPC3-positive tumor cells.

[00312] Clause 66. A method of simultaneously activating downstream CD137 signaling pathways and targeting GPC3-positive tumor cells, comprising the use of one or more fusion proteins according to any of claims 1-58 or one or more compositions containing such a fusion protein with respect to tissue containing the tumor.

[00313] Clause 67. A method of simultaneous co-stimulation of T cells and targeting GPC3-positive tumor cells, comprising the use of one or more fusion proteins according to any of claims 1-58 or one or more compositions containing such a fusion protein with respect to tissue containing the tumor.

[00314] Clause 68. A method of simultaneously inducing lymphocyte activity and targeting GPC3-positive tumor cells, comprising using one or more fusion proteins according to any one of clauses 1-58 or one or more compositions containing such a fusion protein to tissue, containing a tumor.

[00315] Clause 69. A method of inducing clustering and activation of CD137 on T cells and directing said T cells to GPC3-positive tumor cells, comprising the use of one or more fusion proteins according to any of claims 1-58 or one or more compositions containing such a fusion protein in relation to the tissue containing the tumor.

[00316] Clause 70. A method of inducing a localized lymphocytic response in the vicinity of GPC3-positive tumor cells, comprising the use of one or more fusion proteins according to any of claims 1-58 or one or more compositions containing such a fusion protein to the tissue containing a tumor.

[00317] Clause 71. A method of inducing increased secretion of IL-2 by T cells in close proximity to GPC3-positive tumor cells, comprising the use of one or more fusion proteins according to any one of claims 1-58 or one or more compositions containing such a fusion protein , in relation to the tissue containing the tumor.

[00318] Clause 72. A method of inducing increased lymphocyte-mediated cytolysis of GPC3-positive tumor cells, comprising the use of one or more fusion proteins according to any of claims 1-58 or one or more compositions containing such a fusion protein to tissue containing a tumor .

[00319] Clause 73. A pharmaceutical composition comprising one or more fusion proteins according to any of claims 1-58.

[00320] Clause 74. A method of preventing, ameliorating, or treating GPC3-positive cancers, comprising administering the fusion protein of any one of claims 1 to 58, or one or more compositions containing such a fusion protein, to tissue containing a tumor.

[00321] Clause 75. A method of preventing, ameliorating, or treating hepatocellular carcinoma, comprising administering the fusion protein of any one of claims 1 to 58, or one or more compositions containing such a fusion protein, to tissue containing a tumor.

[00322] Clause 76. The fusion protein of any one of claims 1-58 for use in therapy.

[00323] Clause 77. A fusion protein for use in claim 76, wherein the use is in the treatment of cancer.

[00324] Clause 78. Use of the fusion protein of any of claims 1-58 for the production of a medicinal product.

[00325] Clause 79. Use as defined in Clause 78, wherein the medicinal product is intended to treat cancer.

V. EXAMPLES

[00326] Example 1: Expression and Analysis of Representative Fusion Proteins

[00327] In this example, representative antibody-lipocalin mutein fusion proteins were created by fusing together a GPC3-specific antibody having the heavy chain of SEQ ID NO: 81 or containing the heavy chain variable domain of SEQ ID NO: 78. or containing the CDRs GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73) and TRFYSYTY (HCDR3; SEQ ID NO: 74), and the light chains presented under SEQ ID NO: 82, or containing light chain variable domain under SEQ ID NO: 79, or containing the CDRs QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2, SEQ ID NO: 76) and SQNTHVPPT (LCDR3; SEQ ID NO: 77), and CD137- a specific lipocalin mutein of SEQ ID NO: 40 or a CD137-specific lipocalin having 97% sequence identity to SEQ ID NO: 49 and designated CD137Ac1, through a linker such as the unstructured (G ₄ S) ₃ linker of SEQ ID NO: 13, to simultaneously target GPC3 and CD137. Various formats that have been created are depicted in Figure 1. For example, such fusion proteins, for example, SEQ ID NO: 87 and 82, SEQ ID NO: 88 and 82, SEQ ID NO: 81 and 89, SEQ ID NO: 81 and 90, as well as CD137Ac1 fusion protein 1, CD137Ac1 fusion protein 2, CD137Ac1 fusion protein 3, CD137Ac1 fusion protein 4, CD137Ac1 fusion protein 5, CD137Ac1 fusion protein 6 and CD137Ac1 fusion protein 7 (fusion proteins having 97% sequence identity to SEQ ID NOs: 91 and 82, SEQ ID NOs: 92 and 82, SEQ ID NOs: 81 and 93, SEQ ID NOs: 81 and 94, SEQ ID NOs: 95 and 82 or SEQ ID NO : 96 and 82) were created by fusing one or more lipocalin muteins with SEQ ID NO: 40 or having 97% sequence identity with SEQ ID NO: 49, with one or more of the four ends of an antibody containing a heavy chain, represented by a heavy chain, presented under SEQ ID NO: 81, or containing the heavy chain variable domain under SEQ ID NO: 78, or containing the CDRs GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73) and TRFYSYTY (HCDR3; SEQ ID NO: 74), and the light chains represented by SEQ ID NO: 82, or containing the light chain variable domain of SEQ ID NO: 79, or containing the CDR QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2, SEQ ID NO: 76) and SQNTHVPPT (LCDR3; SEQ ID NO: 77). The generated fusion proteins can be divalent with respect to CD137 (for example, as depicted in Figure 1A-1D) or tetravalent with respect to CD137 (for example, as depicted in Figure 1E-1H) or have an even higher valency with respect to CD137 (for example , as depicted in Figure 1I).

[00328] The GPC3-specific antibodies, as well as all antibody-lipocaine mutein fusion proteins described in this example, had an engineered IgG4 backbone that contained the S228P mutation to minimize IgG4 half-antibody turnover in in-vitro and in-vivo conditions (Silva et al. al., J Biol Chem, 2015). In all antibodies and fusion proteins described herein, additional mutations in the IgG4 backbone may also exist, including any one or more of the F234A, L235A, M428L, N434S, M252Y, S254T, and T256E mutations. The F234A and L235A mutations can be introduced to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010). The M428L and N434S mutations or the M252Y, S254T and T256E mutations can be introduced to increase serum half-life (Dall'Acqua et al., J Biol Chem, 2006, Zalevsky et al., Nat Biotechnol, 2010). To avoid heterogeneity, all antibodies were expressed without the lysine at the carboxy terminus.

[00329] In addition, monospecific lipocalin mutein Fc fusion proteins were created by fusing one or more CD137-specific lipocalin mutein SEQ ID NO: 40 or GPC3-specific lipocalin mutein SEQ ID NO: 64 via a linker, such as unstructured (G4S) ₃ linkers with SEQ ID NO: 13, with the C-terminus of the Fc region of the antibody presented under SEQ ID NO: 28, as depicted in figure 1J-1K. The resulting constructs are provided under SEQ ID NO: 98.

[00330] The present invention also provides asymmetric antibody-lipocalin mutein fusion formats, where, for example, one light chain of an antibody may be fused with a lipocalin mutein and the other cannot.

[00331] Fusion protein constructs were generated by gene synthesis and cloned into a mammalian expression vector. They were then transiently expressed in Expi293FTM cells (Life Technologies). The concentration of fusion proteins in the cell culture medium was measured by HPLC (Agilent Technologies) using a POROS® Protein A affinity column (Applied Biosystems). The titers of the fusion proteins are summarized in Table 1.

[00332] The fusion proteins were purified using Protein A chromatography followed by size exclusion chromatography (SEC) in phosphate buffered saline (PBS). After purification by SEC, fractions containing monomeric protein were pooled and analyzed again using analytical SEC and percentage of monomers.

[00333] The monomeric protein content of exemplary fusion proteins after SEC purification is summarized in Table 1, which also provides isoelectric point (pI) values. The pI values of the provided fusion proteins (SEQ ID NO: 87 and 82) were increased compared to the already known CD137/GPC3 bispecific fusion protein SEQ ID NO: 83.

[00334] Table 1. Titers during transient expression

[00335] Example 2: Binding of GPC3 Fusion Proteins Determined by Surface Plasmon Resonance (SPR)

[00336] The binding kinetics and affinity of exemplary recombinant human GPC3 (huGPC3) and cynomolgus GPC3 (cyGPC3) fusion proteins (R&D Systems) were determined by surface plasmon resonance (SPR) using a Biacore T200 instrument (GE Healthcare).

[00337] Anti-human IgG Fc antibody (GE Healthcare) was immobilized onto the CM5 sensor chip using standard amine chemistry: the carboxyl groups on the chip were activated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). After this, a solution of anti-human IgG Fc antibodies (GE Healthcare) at a concentration of 25 μg/ml in 10 mM sodium acetate (pH 5.0) was added at a flow rate of 5 μl/min. until the immobilization level reaches 6000-10000 resonance units (RU). The remaining unreacted NHS ester groups were blocked by passing a 1 M ethanolamine solution over the surface. The comparison channel was processed in a similar way. Thereafter, test fusion proteins (SEQ ID NOs: 87 and 82 and CD137Ac1 fusion protein 1) or anti-GPC3 antibody included in the fusion protein (SEQ ID NOs: 81 and 82) at a concentration of 0.2 μg/ml in HBS- EP+ buffer was captured by anti-human IgG Fc antibody on the chip surface for 180 seconds at a flow rate of 10 μL/min. The needle was washed after each capture step.

[00338] For affinity determination, dilutions (100 nM, 25 nM, 6.25 nM, and 1.56 nM) of huGPC3 or cyGPC3 were prepared in HBS-EP+ buffer (GE Healthcare) and applied to the prepared chip surface. The binding assay was performed with a contact time of 180 s, a dissociation time of 900 s, and a flow rate of 30 μL/min. After each injection, the needle was cleaned with 40 mM NaOH + 20% isopropanol. All measurements were carried out at 25°C. Regeneration of the chip surface was carried out by introducing 3 M MgCl ₂ for 120 s. Before protein measurements, three run cycles were performed for conditioning purposes. Data were evaluated using Biacore T200 Evaluation software (ver. 2.0). A double comparison and a 1:1 linkage model were used to fit the raw data.

[00339] The values determined for k _on , k _off and the resulting equilibrium dissociation constant (K _D ) for exemplary fusion proteins are summarized in Table 2. Fusion Proteins Tested (SEQ ID NOs: 87 and 82 and CD137Ac1 Fusion Protein 1) bound huGPC3 as well as cyGPC3 with subnanomolar affinities comparable to those of the GPC3 fusion protein antibody and a certain already known bispecific CD137/GPC3 fusion protein (SEQ ID NO: 83).

[00340] Table 2. Kinetic constants and affinities of fusion proteins determined by SPR

[00341] Example 3: Binding of Fusion Proteins to GPC3 or CD137 in an Enzyme-Linked Immunosorbent Assay (ELISA)

[00342] An enzyme-linked immunosorbent assay (ELISA) was used to determine the binding efficiency of exemplary GPC3 and CD137 fusion proteins.

[00343] Recombinant huGPC3 (R&D Systems) at a concentration of 1 μg/ml in PBS was applied overnight to microtiter plates at 4°C. After five washes with 100 μl of PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1% T (PBS-0.1%T-2%BSA) for 1 hour at room temperature. After five washes with 100 μl of PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), exemplary fusion proteins (SEQ ID NOs: 87 and 92 and CD137Ac1-fusion protein 1-7 ) or the anti-GPC3 fusion protein antibody (SEQ ID NOs: 81 and 82) were added to the wells at various concentrations and incubated for 1 hour at room temperature, followed by another washing step. Bound molecules of interest were detected by incubation with a 1:5000 dilution of HRP-anti-human IgG Fc conjugate (Jackson Laboratory) in PBS-0.1%T-2%BSA. After an additional washing step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and fluorescence intensity was detected using a fluorescence microplate reader.

[00344] The same ELISA conditions were also used to determine the binding potency of cynomolgus GPC3 and huCD137 fusion proteins, instead recombinant cyGPC3 (R&D Systems) or huCD137-His (recombinant human CD137 with a C-terminal polyhistidine tag) was applied to the microtiter plate , R&D Systems). The agents tested had similar dilution titers, and bound agents were detected using anti-NGAL HRP conjugates.

[00345] Exemplary results are depicted in Figure 2A-2D along with fitting curves obtained from a 1:1 sigmoidal coupling fit where the EC ₅₀ value and maximum signal were free parameters and the slope was fixed to unity. The obtained EC ₅₀ values are presented in Table 3.

[00346] The observed human GPC3 EC ₅₀ values of the present fusion proteins (SEQ ID NOs: 87 and 82 and CD137Ac1 fusion protein 1-7) were very similar or comparable to those of the already known bispecific CD137/GPC3 fusion protein ( SEQ ID NO: 83) and/or anti-GPC3 antibodies included in fusion proteins (SEQ ID NOs: 81 and 82). Some of the fusion proteins tested (SEQ ID NOs: 87 and 82, CD137Ac1 fusion protein 3 and CD137Ac1 fusion protein 4) retained strong binding affinity to human CD137. In addition, the specific fusion protein tested (SEQ ID NOs: 87 and 82) was also observed to cross-react with cynomolgus GPC3 at a level comparable to that of human GPC3, i.e. it associated cynomolgus GPC3 with EC ₅₀ values in the same range as the corresponding EC ₅₀ values for human GPC3.

[00347] Table 3. ELISA data for GPC3 or CD137 binding

[00348] Example 4: Simultaneous binding of GPC3 and CD137 fusion proteins in ELISA

[00349] A dual binding ELISA format was used to demonstrate simultaneous binding of exemplary fusion proteins to GPC3 and CD137.

[00350] Recombinant huCD137-His (R&D Systems) in PBS (1 μg/ml) was coated overnight onto microtiter plates at 4°C. After each incubation step, the plates were washed five times with 100 μl of PBS-0.05%T. The plates were blocked with PBS-0.1%T-2%BSA for 1 hour at room temperature and then washed again. Various concentrations of the test fusion proteins were added to the wells and incubated for 1 hour at room temperature, followed by a washing step. Then biotinylated huGPC3 was added at a constant concentration of 1 μg/ml in PBS-0.1%T-2%BSA for 1 hour. After washing, a 1:5000 diluted solution of ExtrAvidin-HRP conjugate (Sigma-Aldrich) in PBS-0 was added to the wells .1%T-2%BSA and incubated for 1 hour. After an additional washing step, the fluorogenic substrate HRP (QuantaBlu, Thermo) was added to each well and the fluorescence intensity was detected using a fluorescence microplate reader.

[00351] Double binding of fusion proteins was also tested in a reverse manner where recombinant huGPC3 (R&D Systems) was applied to microtiter plates at a concentration of 1 μg/ml and bound fusion proteins were detected by spiking with 5 μg/ml biotinylated huCD137-His.

[00352] Exemplary double binding data results are presented in Figure 3 along with fitting curves obtained from a 1:1 sigmoidal binding fit where the EC ₅₀ value and maximum signal were free parameters and the slope was fixed to unity. The EC ₅₀ values are summarized in Table 4. The fusion proteins (SEQ ID NOs: 87 and 82 and CD137Ac1 fusion proteins 1-7) showed clear binding signals, indicating that they can simultaneously target GPC3 and CD137. Most of these fusion proteins are capable of simultaneously targeting GPC3 and CD137 at a level comparable to the already known bispecific CD137/GPC3 fusion protein SEQ ID NO: 83.

[00353] Table 4. ELISA Data for Simultaneous Targeting of Both GPC3 and CD37

[00354] Example 5: Off-target binding of fusion proteins as determined by ELISA

[00355] An ELISA-based assay was used to evaluate off-target binding of fusion proteins to 32 different targets, including GPC3, GPC5 and other TNF receptor proteins (Frese et al., MAbs, 2013). Targets at a concentration of 5 μg/ml in PBS were applied overnight to microtiter plates at 4°C. After washing with PBS-0.05%T, the plates were blocked with PBS-0.1%T-2%BSA for 1 hour at room temperature. After five washing cycles with 100 μl of PBS-0.05%T, test fusion proteins were added to the wells at a concentration of 100 nM or 10 nM and incubated for 1 h at room temperature, followed by another washing step. Bound antibodies of interest were detected by incubation with a 1:5000 dilution of HRP goat anti-human IgG Fc conjugate (Jackson Laboratory) in PBS-0.1%T-2%BSA. After an additional washing step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well and incubated for 1 hour. Fluorescence intensity was detected using a fluorescence microplate reader and normalized to the control antibody signal (SEQ ID NOs: 106 and 107). The normalized binding signal values of each test molecule to the 32 targets were summed to obtain the cumulative binding coefficient for the antibody at a given concentration, i.e., 100 nM or 10 nM. The cumulative binding coefficients at 100 nM and 10 nM for each test molecule were further summed to obtain the sum of the cumulative binding coefficients. For example, the control antibody (SEQ ID NOs: 106 and 107), to which the fluorescence intensity was normalized for binding to each of the 32 targets, has a cumulative binding coefficient of 32 when tested at either 100 nM or 10 nM and the sum of the cumulative binding coefficients equal to 64. The results are presented in Table 5. Higher cumulative binding coefficients correlate with stronger off-target binding.

[00356] The provided fusion protein of SEQ ID NOs: 87 and 82 showed no or little off-target binding (sum of cumulative binding coefficients <150), as did the anti-GPC3 antibody and the CD137-specific lipocalin mutein (Fc fusion product), included in the fusion protein (SEQ ID NO: 81 and 82 and SEQ ID NO: 98, respectively), while a certain already known bispecific CD137/GPC3 fusion protein (SEQ ID NO: 83) showed unwanted off-target binding (sum of cumulative binding coefficients >250).

[00357] Table 5. Off-target binding of fusion proteins

[00358] Example 6 Flow cytometric analysis of fusion protein binding to cells expressing CD137 or GPC3

[00359] Target specific binding of exemplary fusion proteins to cells expressing human CD137 and cells expressing human GPC3 was assessed by flow cytometry.

[00360] CHO cells were stable transfected with human CD137 or blank control using the Flp-In system (Life technologies) according to the manufacturer's instructions. SK-Hep1 cells were stable transfected with human GPC3 or blank control using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected CHO cells were maintained in Ham's F12 medium (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 500 μg/ml hygromycin B (Roth). Transfected SK-Hep1 cells were cultured in RPMI 1640 + GlutaMAX medium (Gibco) supplemented with 20% fetal bovine serum (Sigma-Aldrich) and 500 μg/ml G418 (Gibco). Cells were cultured in cell culture flasks according to the manufacturer's instructions (37°C, 5% CO ₂ atmosphere).

[00361] For flow cytometry analysis, appropriate cell lines were incubated with an exemplary fusion protein (SEQ ID NOs: 87 and 82), an anti-GPC3 antibody, or a lipocalin mutein specific for CD137 (Fc fusion product) included in the fusion protein (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively), the reference anti-CD137 antibody of SEQ ID NOs: 26 and 27 or isotype control (SEQ ID NOs: 24 and 25) and detected using fluorescently labeled anti-human IgG antibody in FACS analysis as described below:

[00362] 5 x 10 ⁴ cells per well were incubated for 1 hour in ice-cold PBS containing 5% fetal calf serum (PBS-FCS). A series of dilutions of the test molecule were added to the cells and incubated for 1 hour on ice. Cells were washed twice with PBS and then incubated with Alexa647-labeled goat anti-hIgG antibody or Alexa488-labeled goat anti-hIgG antibody for 30 minutes on ice. Cells were then washed and analyzed using an iQue Flow cytometer (Intellicyte Screener). Geometric means of fluorescent signals were plotted and fitted using Graphpad software. Geometric mean fluorescence intensity values were used to calculate EC ₅₀ values using four-parameter logistic regression, with the lowest value set as background.

[00363] The ability of the test molecules to bind human GPC3 and CD137 is depicted in Figure 4. The binding affinity (EC ₅₀ ) values of the fusion protein SEQ ID NOs: 87 and 82 with cells expressing human GPC3 and human CD137 were in the low nanomolar range, comparable with that of the anti-GPC3 antibody (SEQ ID NOs: 81 and 82) and the CD137-specific lipocalin mutein (SEQ ID NO: 98) or antibody (SEQ ID NOs: 26 and 27), respectively (summarized in Table 6) . The already known CD137/GPC3 bispecific fusion protein (SEQ ID NO: 83), however, showed limited dose-dependent binding to GPC3-expressing cells as the binding curve did not reach a plateau at concentrations up to 100 nM (Figure 4B). None of the molecules tested bound to blank control-transfected cells (data not shown).

[00364] Table 6. Binding affinity values of fusion proteins to cells expressing GPC3 or CD137

[00365] Example 7: Binding Affinity of Fusion Proteins to GPC3-Positive Tumor Cells

[00366] The binding of fusion proteins to tumor cells expressing various levels of GPC3 - HepG2, Hep3B, MKN-45 and NCI-N87 - was assessed using flow cytometry.

[00367] The HepG2 cell line was cultured in Dulbecco's modified Eagle's medium (DMSO, Pan Biotech) supplemented with 10% fetal bovine serum (Sigma-Aldrich). The Hep3B cell line was cultured in minimal essential medium (Earl's salts) (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 2 mM L-glutamine (Gibco). Cell lines MKN-45 and NCI-N87 were cultured in RPMI-1640 + GlutaMAX medium (Gibco) supplemented with 20% or 10% fetal bovine serum (Sigma-Aldrich), respectively. All tumor cell lines were cultured in cell culture flasks according to the manufacturer's instructions (37°C, 5% CO ₂ atmosphere).

[00368] For flow cytometry analysis, tumor cell lines with varying levels of GPC3 expression (high to moderate expression: HepG2 > Hep3B > MKN-45) and the GPC3 negative cell line NCI-N87 were incubated with an exemplary fusion protein SEQ ID NO: 87 and 82, an anti-GPC3 antibody or a CD137-specific lipocalin mutein (Fc fusion product) included in the fusion protein (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively), the reference anti-CD137 antibody with SEQ ID NO: 26 and 27 or isotype control (SEQ ID NO: 24 and 25) and detected using fluorescently labeled anti-human IgG antibody as described in Example 6.

[00369] The ability of the fusion protein SEQ ID NO: 87 and 82 to bind GPC3-positive tumor cells is depicted in Figure 5, and the corresponding binding affinity values (EC ₅₀ ) are summarized in Table 7. The binding affinity values of the fusion protein to GPC3-expressing tumor cells are were in the lower nanomolar range, comparable to that of the anti-GPC3 antibody included in the fusion proteins (SEQ ID NOs: 81 and 82). The results further indicated that the fusion protein does not bind to GPC3-negative tumor cells.

[00370] Table 7. Binding affinity of fusion proteins to GPC3-positive tumor cells

[00371] Example 8: GPC3-Dependent T Cell Co-Stimulation Using CD137 Bioassay

[00372] The potential of selected fusion proteins to induce activation of the CD137 signaling pathway in a GPC3-dependent manner was assessed using a commercially available double stable transfection Jurkat cell line expressing CD137 and the luc2 gene (a humanized version of firefly luciferase), with luc2 expression under the control of NFκB -sensitive element. In this bioassay, targeting CD137 results in intracellular CD137 signaling, resulting in NFκB-mediated luminescence.

[00373] Hepatocellular cancer cell lines HepG2 and Hep3B, which have high levels of GPC3 expression, and gastric cancer cell lines MKN-45, which have moderate levels of GPC3 expression, and NCI-N87, which is GPC3 negative, were cultured as described in Example 7. The day before analysis, the appropriate tumor cells were seeded at 6.25 x 10 ³ cells per well and allowed to attach overnight at 37°C in a humidified atmosphere of 5% CO ₂ .

[00374] The next day, 3.75 x 10 ⁴ NF-kB-Luc2/CD137 Jurkat cells were added to each well, followed by the addition of various concentrations, typically ranging from 0.00488 nM to 10 nM, of an exemplary fusion protein (SEQ ID NO: 87 and 82), anti-GPC3 antibody or lipocalin mutein specific for CD137 (Fc fusion product) included in the fusion protein (SEQ ID NO: 81 and 82 and SEQ ID NO: 98, respectively), reference anti-CD137 antibody with SEQ ID NO: 26 and 27 or isotype control (SEQ ID NO: 24 and 25). The plates were covered with a gas-permeable lid and incubated at 37°C in a humidified atmosphere with 5% CO ₂ . After 4 hours, 30 μl of Bio-Glo™ reagent was added to each well and the bioluminescent signal was quantified using a luminometer (PHERAstar). Four-parameter logistic curve analysis was performed using GraphPad Prism® to calculate EC ₅₀ values (with a common lower limit), which are summarized in Table 8. To demonstrate the GPC3 dependence of CD137 fusion proteins, the same experiment was performed in parallel in the absence of tumor cells using the highest concentration of the molecules tested. The analysis was carried out in triplicate.

[00375] The results of a representative experiment are depicted in Figure 6. The data presented in Figure 6A and 6B indicate that the exemplary fusion protein of SEQ ID NOs: 87 and 82 induced strong CD137-mediated co-stimulation of T cells in the presence of a tumor cell line with high level of GPC3 expression. Figures 6C-6E show that CD137 activation by the fusion protein is GPC3 dependent, as NF-κB-Luc2/CD137 activation of Jurkat cells was not detected in the presence of tumor cells with moderate or low GPC3 expression or in the absence of GPC3-expressing tumor cells. In contrast, the reference CD137 mAb (SEQ ID NOs: 26 and 27) demonstrated CD137-mediated co-stimulation of T cells regardless of GPC3 expression level and in the absence of target cells.

[00376] Table 8. Results of T cell activation assessment using CD137 bioassay

[00377] Example 9: Evaluation of GPC3-dependent T cell activation induced by fusion proteins

[00378] GPC3 target-dependent co-stimulation of T cells by fusion proteins was analyzed using a T cell activation assay. The fusion proteins were applied at various concentrations to anti-CD3 T cells stimulated with anti-CD3 T cells cocultured with SK-Hep1 cells transfected with human GPC3 or transfected with a blank control, or with a HepG2 GPC3-positive tumor cell line. IL-2 secretion levels were measured in the supernatants.

[00379] PBMC were isolated from buffy coats from healthy volunteer donors by polysucrose density gradient centrifugation (Biocoll, 1.077 g/ml, Biochrom) according to Biochrom protocols. T lymphocytes were further purified from PBMCs by magnetic cell sorting using a total T cell purification kit (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Purified total T cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen, and stored in liquid nitrogen until further use. For analysis, T cells were thawed and placed in culture medium (RPMI-1640 + GlutaMAX medium, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin mixture (Gibco) overnight at 37°C at humidified atmosphere with 5% CO ₂ . SK-Hep1 and HepG2 cells were cultured as described in Example 6 and Example 7, respectively.

[00380] Using triplicates for each experimental condition, the following procedure was performed: flat-bottomed tissue culture plates were pre-coated with 0.25 μg/ml anti-CD3 antibody for 2 hours at 37°C and then washed twice with PBS. SK-Hep1 cells transfected with human GPC3 or transfected with blank control and GPC3-positive HepG2 tumor cells were treated for 30 minutes with 30 μg/ml mitomycin C (Sigma Aldrich) to block proliferation. Mitomycin-treated cells were then washed twice with PBS and seeded at 1.0 x 10 ⁴ cells per well in culture medium and allowed to attach overnight at 37°C in a humidified 5% CO ₂ atmosphere. Target-expressing cells were grown under standard conditions, separated using Accutase (PAA Laboratories), and resuspended in culture medium.

[00381] On subsequent days, after washing the plates twice with PBS, 2.5 x 10 ⁴ T cells were added to each well. A dilution series of fusion proteins (SEQ ID NOs: 87 and 82), an anti-GPC3 antibody included in the fusion protein (SEQ ID NOs: 81 and 82), already known to be bispecific for the CD137/GPC3 fusion protein (SEQ ID NO: 83) , a GPC3-specific lipocalin mutein (Fc fusion product) (SEQ ID NO: 97), a reference CD137 antibody (SEQ ID NOs: 26 and 27), or an isotype control (SEQ ID NOs: 24 and 25), typically in the range from 0.003 nM to 10 nM were added to the corresponding wells. The plates were covered with a gas-permeable lid and incubated at 37°C in a humidified atmosphere with 5% CO ₂ for 3 days. IL-2 levels in the supernatant were assessed using the Human IL-2 DuoSet (R&D Systems) as described in the following procedures.

[00382] In 384-well plates, 1 μg/ml “human IL-2 capture antibody” in PBS was applied for 2 hours at room temperature. After this, the wells were washed 5 times with 80 μl of PBS-0.05%T. After 1 hour blocking in PBS-0.05%T containing 1% casein (w/w), assay supernatants and a series of concentrations of IL-2 standard diluted in culture medium were transferred to appropriate wells and incubated overnight at 4 °C. The next day, a mixture of 100 ng/ml goat anti-hIL-2-Bio detection antibody (R&D Systems) and 1 μg/ml sulfomethane-labeled streptavidin (Mesoscale Discovery) in PBS-0.05%T containing 0.5% casein was added. and incubated at room temperature for 1 hour. After washing, 25 μl of reading buffer (Mesoscale Discovery) was added to each well and the resulting electrochemiluminescent signal (ECL) was detected using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[00383] A T cell activation assay was also used in a similar manner to evaluate the ability of additional already known CD137/GPC3 bispecific fusion proteins SEQ ID NOs: 99 and 27, SEQ ID NO: 83 and SEQ ID NO: 84 to co-stimulate T cells answers. In the experiment, tissue culture plates were pre-coated with 200 μl of 0.25 μg/ml anti-CD3 antibody for 1 hour at 37°C and washed twice with PBS. 1.25 x 10 ⁴ HepG2 tumor cells per well were seeded, allowed to attach overnight at 37°C in a humidified 5% CO ₂ atmosphere, and treated for 2 hours at 37°C with 10 μg/ml mitomycin C. The plates were washed twice with PBS and 5×10 ⁴ T cells and test molecules were added to each well at a concentration of 1 μg/ml. The plates were covered with a gas-permeable lid and incubated at 37°C in a humidified atmosphere with 5% CO ₂ for 3 days. After this, the concentration of IL-2 in the supernatant was assessed.

[00384] Exemplary data is presented in Figure 7. Coculture of total T cells with SK-Hep1 cells transfected with human GPC3 (Figure 7B) or GPC-3-expressing HepG2 tumor cells (Figure 7C) in the presence of a fusion protein (SEQ ID NOs: 87 and 82) resulted in a strong dose-dependent secretion of IL-2 compared to isotype control and much stronger than that of the anti-GPC3 antibody (SEQ ID NOs: 81 and 82, GPC3-specific lipocalin mutein (SEQ ID NO : 129) or already known as a bispecific CD137/GPC3 fusion protein (SEQ ID NO: 83), where no increase in IL-2 or a slight increase in IL-2 secretion was observed at high dose levels. When co-cultured with blank control transfected cells SK-Hep1 (GPC3 negative), none of the tested molecules caused a dose-dependent increase in IL-2 secretion.The results show that the activation of T cells by the present fusion proteins was GPC3-dependent.

[00385] Example 10: Assessing T Cell Activation in the Presence of Tumor Cells Expressing Different Levels of GPC3

[00386] An additional T cell assay was used to evaluate the ability of typical fusion proteins to co-promote T cell activation in a GPC3-targeting dependent manner. The fusion proteins were applied at various concentrations to T cells stimulated with anti-CD3 antibodies in the presence of tumor cell lines with different levels of GPC3 expression. Tumor cell lines tested included HepG2, Hep3B, MKN-45, and NCI-N87 (high to moderate expression: HepG2 > Hep3B > MKN-45, GPC3 negative control: NCI-N87). IL-2 secretion levels were measured in the supernatants.

[00387] PBMC from healthy volunteer donors were isolated from buffy coats, and lymphocytes were purified from PBMC as described in Example 9.

[00388] For analysis, T cells were thawed and placed in culture medium (RPMI-1640 + GlutaMAX medium, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin mixture (Gibco) for 16 hours. at 37°C in a humidified atmosphere with 5% CO ₂ .

[00389] Using triplicate for each experimental condition, the following procedure was performed: Flat-bottomed tissue culture plates were pre-coated with 0.25 μg/ml anti-CD3 antibody for 2 hours at 37°C and then washed twice with PBS. To block proliferation, tumor cell lines HepG2, Hep3B, MKN-45, or NIC-N87 were treated for 30 min. with 30 μg/ml mitomycin C (Sigma Aldrich). Mitomycin-treated tumor cells were then washed twice with PBS and seeded at 8.3 x 10 ³ cells per well in culture medium and allowed to attach overnight at 37°C in a humidified 5% CO ₂ atmosphere. Target cells were grown under standard conditions, separated using Accutase (PAA Laboratories), and resuspended in culture medium.

[00390] In the following days, after washing the plates twice with PBS, 2.5 x 10 ⁴ T cells were added to each well of the tumor cells. A dilution series of an exemplary fusion protein (SEQ ID NOs: 87 and 82), an anti-GPC3 antibody, or a CD137-specific lipocalin mutein (Fc fusion product) included in the fusion protein (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively), reference CD137 antibody (SEQ ID NOs: 26 and 27) or isotype control (SEQ ID NOs: 24 and 25), typically ranging from 0.26 nM to 10 nM, was added to the appropriate wells. The plates were covered with a gas-permeable lid and incubated at 37°C in a humidified atmosphere with 5% CO ₂ for 3 days.

[00391] After 3 days of co-culture, the level of IL-2 in the supernatant was assessed as described in Example 9.

[00392] Exemplary data is presented in Figure 8. Co-culture of total T cells with HepG2 and Hep3B cells having high levels of GPC3 expression in the presence of the fusion protein SEQ ID NO: 87 and 82 resulted in a clear increase in IL-2 secretion compared to hIgG4 isotype control. In addition, co-culture with MKN-45 (moderate GPC3) or NIC-N87 (negative GPC3) did not increase the levels of IL-2 secretion from the fusion protein. The data show that the functional activity of the fusion proteins, as measured by their ability to activate T cells or increase IL-2 secretion, was GPC3-dependent. In contrast, T cell activation or IL-2 secretion induced by the reference CD137 antibody (SEQ ID NOs: 26 and 27) was not always GPC3 dependent and was difficult to predict.

[00393] Example 11: Evaluation of T cell-mediated cytolysis of GPC3-expressing tumor cells induced by fusion proteins

[00394] An impedance T cell killing assay was used to evaluate the ability of the fusion proteins to activate the CD137 co-stimulatory signaling pathway and induce T cell-mediated cytolysis of GPC3-expressing tumor cells. For this purpose, adherent tumor cells were seeded into the wells of an electronic microtiter plate (E-plate). Cell adhesion on gold microelectrodes prevents the passage of electrical current between the electrodes. This impedance is measured as a dimensionless parameter “cell coefficient”. Cellular coefficient (CI) increases as cells attach and then proliferate over time. Nonadherent CD8+ T cells, which produced anti-CD3 antibodies that independently activated T-cell antigens, and test molecules at various concentrations were added to HepG2 cells. Direct addition did not cause impedance changes. If the test molecule has a co-stimulatory effect on cytotoxic T cells by activating the CD137 signaling pathway, T cell-mediated cytolysis of target cells will be increased, and destruction of adherent tumor cells by effector cells can be detected as a decrease in CI, making it possible to construct a killing curve in real time.

[00395] PBMC from healthy volunteer donors were isolated from buffy coats. CD8+ T cells were isolated from these PBMCs and stored in liquid nitrogen until further use. For analysis, CD8+ T cells were thawed and placed in assay medium consisting of RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin for 24 hours at 37°C in a humidified atmosphere with 5% CO ₂ .

[00396] The procedure described below was performed using three replicates for each experimental condition. Tumor cell lines HepG2 (a GPC3-expressing hepatocellular carcinoma cell line) or NCI-N87 (a GPC3-negative gastric carcinoma cell line) were seeded into an E-plate in the analytical medium and incubated for 24 hours at 37°C in a humidified atmosphere with 5 % CO ₂ to ensure cell adhesion and proliferation.

[00397] The next day, an anti-CD3 antibody was added to the tumor cells, followed by a series of dilutions of an exemplary fusion protein (SEQ ID NOs: 87 and 82), an anti-GPC3 antibody, or a CD137-specific lipocalin mutein (Fc fusion product) included in fusion protein (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively), a reference anti-CD137 antibody (SEQ ID NOs: 26 and 27), already identified as a bispecific CD137/GPC3 fusion protein (SEQ ID NO: 83), GPC3-specific lipocalin mutein (Fc fusion product) (SEQ ID NO: 97) or isotype control (SEQ ID NO: 24 and 25). Resting CD8+ T cells were then added to the tumor cells in a 5:1 ratio. The plates were covered with a gas-permeable lid and incubated for 3 days, during which time the impedance was measured periodically.

[00398] CI values were plotted against time using RTCA HT Software V 1.0.1 (ACEA Biosciences). Normalized CI was calculated as the CI at a given time point (20 h, 30 h, 40 h, 50 h, 60 h, 70 h after addition of effector cells) divided by the CI at the normalization time point (first measurement after addition of effector cells). For every three replicates, the mean normalized CI value and corresponding SD value were used to calculate the specific killing value using the following formula: 100 - (normalized mean CI of test molecule/normalized mean CI of target and effector cells) x 100. Specific killing values were exported to GraphPad Prism v7 and plotted with corresponding SD values against time intervals of interest on the X- and Y-axis plot.

[00399] The results of illustrative experiments are presented in Figure 9. The data shows that the fusion protein of SEQ ID NOs: 87 and 82 induced dose-dependent T cell-mediated lysis of GPC3-expressing HepG2 cells (Figure 9A-9C), while specific lysis of target negative no NCI-N87 cells were observed (Figure 9D), indicating that the fusion protein-induced activation of the CD137 pathway leading to T cell-mediated killing was GPC3-dependent. The provided fusion protein SEQ ID NO: 87 and 82 induced a much higher level of killing of GPC3-positive target cells by cytotoxic T cells compared to an equimolar amount of the already known bispecific CD137/GPC3 fusion protein (SEQ ID NO: 83), antibody to GPC3 (SEQ ID NOs: 81 and 82) or the GPC3-specific lipocalin mutein (Fc fusion product) (SEQ ID NO: 97) (Figure 9C). In addition, the CD137-specific lipocalin mutein (Fc fusion product) (SEQ ID NO: 98), the reference CD137 antibody (SEQ ID NOs: 26 and 27), and the hIgG4 isotype control (SEQ ID NOs: 24 and 25) do not induced CD8+ T cell-mediated killing (Figure 9C).

[00400] An additional experiment was performed to demonstrate that the potent T cell-mediated lysis of HepG2 cells by the CD137/GPC3 bispecific fusion protein (SEQ ID NOs: 87 and 82) was dependent on the structure of the bispecific protein and not just on the antibody combination to GPC3 and antibodies to CD137. The experiment was performed essentially as described above, but all constructs were added at a concentration of 10 nM and compared with a mixture of antibodies to GPC3 (SEQ ID NO: 81 and 82) and the reference antibody to CD137 (SEQ ID NO: 26 and 27), and a mixture of an anti-GPC3 antibody (SEQ ID NOs: 81 and 82) and a CD137-specific lipocalin mutein (Fc fusion product) (SEQ ID NO: 98). Although strong T cell-mediated lysis was again detected for the fusion protein SEQ ID NO: 87 and 82, specific lysis mediated by both mixtures was only moderate, indicating the advantage of this bispecific format for achieving T-cell-mediated lysis .

[00401] Example 12: Evaluation of in vivo functional activity in a xenograft mouse model inoculated with human PBMCs

[00402] To study the in vivo activity of the presented fusion proteins, immunodeficient NOG mice engrafted with human HepG2 tumor cells and human PBMCs were used.

[00403] 4-6 week old NOG mice were injected subcutaneously (sc) with 5 x 10 ⁶ HepG2 cells in a Matrigel/PBS solution (1:1). The tumors were allowed to grow to a size of 80-100 mm ³ , a time point that was defined as day 0 of the experiment. On day 0, mice were randomized into treatment groups (or control group) according to tumor size and animal weight. Mice were given 5×10 ⁶ fresh human PBMC intravenously by tail vein injection (iv). Mice were administered the treatment or control (PBS) by intraperitoneal (ip) injection after iv injection of PBMC on day 1 and were again administered the treatment or control on day 8 and day 15. The molecules tested included the exemplary fusion protein SEQ ID NOs: 87 and 82 (0.5, 5 or 20 mg/kg), anti-GPC3 antibody included in fusion protein (SEQ ID NOs: 81 and 82) (0.39 or 3.9 mg/kg, concentration equimolar to 0.5 or 5 mg/kg fusion protein treatment), and reference anti-CD137 antibody (SEQ ID NOs: 26 and 27) (3.9 mg/kg, concentration equimolar to 5 mg/kg fusion protein treatment). Tumor growth was recorded every 3-4 days. Animals that were not treated with the drug were excluded from data analysis.

[00404] At the end of the study (day 16), mice were sacrificed and tumors were formalin fixed and paraffin embedded (FFPE). Histological and immunohistochemical analyzes of tumors were performed in the BioSiteHisto laboratory. FFPE xenograft tumor tissue was sectioned and stained with H&E or for the T cell markers CD3, CD4, or CD8. Stained slides were photographed and digitized using 3D Histech Pannoramic MIDI instrumentation with a 20× objective lens. CaseViewer 2.2 was used for microscopic examination of digital images, and Image J was used for tumor area analysis. The percentage of antitumor effector lymphocytes (TILs) per tumor area minus necrotic area was calculated.

[00405] Figure 10 depicts the change in tumor volume as measured on day 2, day 6, day 9, day 13, and day 16 of the study. Tumor growth inhibition was achieved by the fusion protein SEQ ID NOs: 87 and 82 and the anti-GPC3 antibody included in the fusion protein SEQ ID NOs: 81 and 82. Both the fusion protein and the anti-GPC3 antibody (at a concentration of 5 or 3.9 mg/kg respectively) completely inhibited tumor growth during the study. The effects of the fusion protein and anti-GPC3 antibody were dose-dependent, with lower concentrations (0.5 or 0.39 mg/kg) having little effect on tumor growth. Only limited effects on tumor growth were achieved when treated with the reference CD137 antibody (SEQ ID NOs: 26 and 27).

[00406] Table 9 summarizes the data obtained from the histological and immunohistochemical analysis of antitumor effector lymphocytes. Intratumoral infiltration of CD3, CD4, or CD8 T cells is presented as % TIL per (tumor area - necrotic area). Treatment with the fusion protein resulted in T cell infiltration of up to 10% (tumor area - necrotic area) (all treatment groups). Intratumoral T cell infiltration was not induced by treatment with anti-GPC3 antibody or reference anti-CD137 antibody compared with control media.

[00407] Table 9. Percentage of antitumor effector lymphocytes (TIL) per tumor area minus necrotic area

[00408] Example 13: Pharmacokinetics of Fusion Proteins in Mice

[00409] Pharmacokinetics analyzes of a representative fusion protein (SEQ ID NOs: 87 and 82) and an anti-GPC3 antibody included in the fusion protein (SEQ ID NOs: 81 and 82) were performed in mice and compared with the results of the analysis of two already known bispecific for CD137 /GPC3 fusion proteins (SEQ ID NO: 83 and SEQ ID NO: 84). Male CD-1 mice approximately 5 weeks old (2 mice per time point; Charles River Laboratories) were treated with the appropriate construct at a dose of 2 mg/kg (SEQ ID NOs: 87 and 82, SEQ ID NOs: 81 and 82) into the tail vein. or 10 mg/kg (SEQ ID NO: 83, SEQ ID NO: 84). Mouse plasma samples were collected at time points of 5 min, 24 h, 168 h, and 336 h for antibody constructs and 5 min, 2 h, 4 h, 8 h, 24 h, 2 days, 3 days, 4 days, 7 days, 9 days, 11 days, 14 days and 21 days. Sufficient whole blood was collected under isoflurane anesthesia to obtain at least 30-50 μl of Li- and heparin-supplemented plasma per animal and time point. Plasma drug levels were then analyzed by ELISA.

[00410] For SEQ ID NOs: 87 and 82 or SEQ ID NOs: 81 and 82, the following protocol was used: human GPC3 was dissolved in PBS (1 μg/ml) and coated overnight on microtiter plates at 4°C. After each incubation step, the plates were washed five times with 80 μl of PBS-0.05%T. The plates were blocked with PBS-0.1%T-2%BSA for 1 hour at room temperature and then washed. Plasma samples were diluted in PBS-0.1%T-2%BSA to a plasma concentration of 20%, added to wells, and incubated for 1 h at room temperature. After this there was another washing stage. Bound agents of interest were detected after 1 h of incubation with sulfonated anti-human antibody (Mesoscale Discovery) or polyclonal antiserum purified on an NGAL affinity chromatography column at a concentration of 1 μg/ml diluted in PBS-0.1%T-2 %BSA. After an additional washing step, 25 μl of reading buffer was added to each well and the electrochemiluminescence (ECL) signal was read from each well using a Mesoscale Discovery reader.

[00411] For SEQ ID NO: 83 or SEQ ID NO: 84, the following protocol was used: human CD137 was dissolved in PBS (1 μg/ml) and coated overnight onto microtiter plates at 4°C. After each incubation step, the plates were washed with 80 μl of PBS-0.05%T. The plates were blocked with PBS-0.1%T-2%BSA for 1 hour at room temperature and then washed. Plasma samples were diluted in PBS-0.1%T-2%BSA to a plasma concentration of 20%, added to wells, and incubated for 1 hour at room temperature. After this there was another washing stage. Test bound agents were detected after 1 hour incubation with sulfonated human Glypican-bio and 1 μg/ml streptavidin, each diluted in PBS-0.1%T-2%BSA. After an additional washing step, 25 μl of reading buffer was added to each well and the ECL signal from each well was read using a Mesoscale Discovery reader.

[00412] A calibration curve using dilutions of standard proteins was also generated to analyze and quantify the data. Plasma concentrations over time for the test molecules in the illustrative experiment are plotted in Figure 12. The data were subjected to compartment-neutral analysis using Phoenix WinNonlin version 8.1 and the results are presented in Table 10.

[00413] From the data it is clear that the provided fusion protein SEQ ID NO: 87 and 82 had typical antibody pharmacokinetics, while the already known CD137/GPC3 bispecific fusion protein SEQ ID NO: 83 and SEQ ID NO: 84 had significantly worse pharmacokinetics profiles.

[00414] Table 10. Pharmacokinetics in mice

[00415] Example 14: Evaluation of Thermal Stability of Fusion Proteins

[00416] To determine the melting temperatures (T _m ) of the fusion proteins, which is a general indicator of overall stability, test molecules with a protein concentration of 1 mg/ml in PBS (Gibco) were subjected to scanning (25-100°C) in 1°C/step increments. min using a nanoDSC capillary instrument (CSC 6300, TA Instruments). T _m values were calculated based on the obtained thermograms using the built-in Nano Analyze software.

[00417] The resulting maximum melting temperatures as well as melting onset temperatures for the exemplary fusion proteins are presented in Table 11 below. The fusion proteins SEQ ID NOs: 72 and 82, as well as CD137Ac1-fusion protein 2, had improved thermal stability compared to certain previously known CD137/GPC3 bispecific fusion proteins SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 26 and 102.

[00418] Table 11. T _m and melting temperature determined using nanoDSC

[00419] The embodiments described herein by way of illustration may, as appropriate, be practiced in the absence of any element or elements, limitation or restrictions not specifically disclosed herein. So, for example, the terms “comprising”, “including”, “containing”, etc. should be read in its broadest sense and without limitation. In addition, the terms and expressions used herein have been used as terms of description and not limitation, and the use of these terms and expressions is not intended to exclude any equivalents of the features shown and described, or portions thereof, but instead is intended to Within the scope of the claimed invention, various modifications are possible. Thus, it should be understood that while the present embodiments have been specifically disclosed by way of preferred embodiments and additional features, modifications and variations thereof may be made by those skilled in the art, and that such modifications and variations are considered to fall within the scope of the present invention. . All patents, patent applications, scientific textbooks and peer-reviewed publications described herein are incorporated herein by reference in their entirety. In addition, in the event that the definition or application of a term in the literature that is incorporated by reference herein is inconsistent or inconsistent with the definition of that term given herein, the definition of that term given in this document and the definition of that term in literature source not applicable. Each of the narrower species and each of the narrower subgeneric groups falling within the general disclosure also forms part of the present invention. This includes a general description of the present invention with a condition or negative limitation excluding any subject matter, regardless of whether cut-out material is specifically mentioned herein. Moreover, if features are described in terms of Markush groups, those skilled in the art will understand that the present invention is also thus described in terms of any individual member or subset of members of a Markush group. Additional embodiments will become apparent from the following claims.

[00420] Equivalents: Those skilled in the art will recognize, or through simple experimentation will be able to determine, many equivalents to the specific embodiments of the present invention that are described herein. Such equivalents are intended to be covered by the following claims. All publications, patents and patent applications mentioned herein are hereby incorporated herein by reference to the same extent as if each individual publication, patent or patent application were specifically and separately identified as being incorporated herein by reference.

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--->

LIST OF SEQUENCES

<110> Pieris Pharmaceuticals GmbH

<120> New fusion proteins specific for CD137

and GPC3

<130>PIE16678PCT

<150>EP 19000100.8

<151> 2019-02-26

<160> 129

<170> PatentIn version 3.5

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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> 2

<211> 178

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<213> people

<400> 2

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> 3

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<213> Artificial sequence

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<223> lipocalin mutein

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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 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 Ser 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> 4

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 4

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 Arg Glu Cys Pro Glu Met Asn Leu 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 Leu Ile Ser Gly Arg Ser Gln Glu Val 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 Glu Cys His Gly Lys Pro Val Pro Gly Val Trp Leu Val

100 105 110

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

115 120 125

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

130 135 140

Gln Ser Glu Thr Ser Ser Pro Gly

145 150

<210> 5

<211> 4

<212> Protein

<213> people

<400> 5

His His Leu Leu

1

<210> 6

<211> 535

<212> Protein

<213> people

<400> 6

Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser

1 5 10 15

Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro

20 25 30

Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys

35 40 45

Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn

50 55 60

Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile

65 70 75 80

Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg

85 90 95

His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser

100 105 110

Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val

115 120 125

Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn

130 135 140

Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn

145 150 155 160

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

165 170 175

Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met

180 185 190

Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala

195 200 205

Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe

210 215 220

Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr

225 230 235 240

Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val

245 250 255

Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp

260 265 270

Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg

275 280 285

Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His

290 295 300

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

305 310 315 320

Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala

325 330 335

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

340 345 350

His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile

355 360 365

Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr

370 375 380

Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn

385 390 395 400

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

405 410 415

Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu

420 425 430

Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu

435 440 445

Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu

450 455 460

Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu

465 470 475 480

Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu

485 490 495

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

500 505 510

Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe

515 520 525

His Asn Leu Gly Asn Val His

530 535

<210> 7

<211> 232

<212> Protein

<213> people

<400> 7

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

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

130 135 140

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu

165 170 175

Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg

180 185 190

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

195 200 205

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

210 215 220

Glu Glu Glu Gly Gly Cys Glu Leu

225 230

<210> 8

<211> 535

<212> Protein

<213> Macaca fascicularis

<400> 8

Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser

1 5 10 15

Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro

20 25 30

Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys

35 40 45

Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn

50 55 60

Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile

65 70 75 80

Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg

85 90 95

His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser

100 105 110

Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val

115 120 125

Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn

130 135 140

Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn

145 150 155 160

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

165 170 175

Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met

180 185 190

Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala

195 200 205

Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe

210 215 220

Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr

225 230 235 240

Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val

245 250 255

Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp

260 265 270

Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg

275 280 285

Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His

290 295 300

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

305 310 315 320

Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala

325 330 335

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

340 345 350

His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile

355 360 365

Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr

370 375 380

Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn

385 390 395 400

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

405 410 415

Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu

420 425 430

Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu

435 440 445

Leu Arg Thr Met Ser Val Pro Lys Gly Arg Val Leu Asp Lys Asn Leu

450 455 460

Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu

465 470 475 480

Cys Ile Gly Gly Ser Gly Asp Gly Met Met Lys Val Lys Asn Gln Leu

485 490 495

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

500 505 510

Gly Asn Asn Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe

515 520 525

His Asn Leu Gly Asn Val His

530 535

<210> 9

<211> 231

<212> Protein

<213> Macaca fascicularis

<400> 9

Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

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

130 135 140

Gly Ala Ser Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln Ile Ile Phe Phe Leu Ala Leu Thr Ser Thr Val Val Leu

165 170 175

Phe Leu Leu Phe Phe Leu Val Leu Arg Phe Ser Val Val Lys Arg Ser

180 185 190

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val

195 200 205

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

210 215 220

Glu Glu Gly Gly Cys Glu Leu

225 230

<210> 10

<211> 4

<212> Protein

<213> Artificial sequence

<220>

<223> cleavage site

<400> 10

Ile Glu Gly Arg

1

<210> 11

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> fusion peptide

<400> 11

Ser Ala Trp Ser His Pro Gln Phe Glu Lys

1 5 10

<210> 12

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> streptavidin-tagged peptide II

<400> 12

Trp Ser His Pro Gln Phe Glu Lys

15

<210> 13

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 13

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

1 5 10 15

<210> 14

<211> 18

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 14

Pro Ser Thr Pro Pro Thr Asn Ser Ser Ser Thr Pro Pro Thr Pro Ser

1 5 10 15

Pro Ser

<210> 15

<211> 14

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 15

Gly Gly Ser Gly Asn Ser Ser Gly Ser Gly Gly Ser Pro Val

1 5 10

<210> 16

<211> 20

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 16

Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala Ala Pro Ala Pro

1 5 10 15

Ser Ala Pro Ala

20

<210> 17

<211> 66

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 17

Ala Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Pro Val Pro Ser

1 5 10 15

Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser

20 25 30

Gly Gly Ser Gly Asn Ser Ser Gly Ser Gly Gly Ser Pro Val Pro Ser

35 40 45

Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser

50 55 60

Ala Ser

65

<210> 18

<211> 32

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 18

Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser

1 5 10 15

Pro Ser Gly Gly Ser Gly Asn Ser Ser Gly Ser Gly Gly Ser Pro Val

20 25 30

<210> 19

<211> 74

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 19

Ala Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Pro Val Pro Ser

1 5 10 15

Thr Pro Pro Thr Asn Ser Ser Ser Thr Pro Pro Thr Pro Ser Pro Ser

20 25 30

Pro Val Pro Ser Thr Pro Pro Thr Asn Ser Ser Ser Thr Pro Pro Thr

35 40 45

Pro Ser Pro Ser Pro Val Pro Ser Thr Pro Pro Thr Asn Ser Ser Ser

50 55 60

Thr Pro Pro Thr Pro Ser Pro Ser Ala Ser

65 70

<210> 20

<211> 40

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 20

Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala Ala Pro Ala Pro

1 5 10 15

Ser Ala Pro Ala Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala

20 25 30

Ala Pro Ala Pro Ser Ala Pro Ala

35 40

<210> 21

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 21

Val Asp Asp Ile Glu Gly Arg Met Asp Glu

1 5 10

<210> 22

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 22

Glu Asn Leu Tyr Phe Gln Gly Arg Met Asp Glu

1 5 10

<210> 23

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> linker

<400> 23

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 24

<211> 327

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain

<400> 24

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

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

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 25

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain

<400> 25

Glu Ile Val Leu Thr Gln Ser Pro Ala 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 Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

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> 26

<211> 448

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain

<400> 26

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

1 5 10 15

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

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

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

115 120 125

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 27

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain

<400> 27

Glu Ile Val Leu Thr Gln Ser Pro Ala 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 Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 28

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody Fc region

<400> 28

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

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

20 25 30

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

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly

225

<210> 29

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody Fc region

<400> 29

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

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

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly

225

<210> 30

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody Fc region

<400> 30

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

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

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly

225

<210> 31

<211> 228

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody Fc region

<400> 31

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

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

195 200 205

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

210 215 220

Leu Ser Leu Gly

225

<210> 32

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 32

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 Glu Gly Cys Arg Pro Trp Asn Ile Phe 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 Ala Ile Asp Gly Pro Ala Gln Glu Val 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 Val Cys Asp Gly Ser Pro Val Pro Gly Val Trp Leu Val

100 105 110

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

115 120 125

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

130 135 140

Gln Ser Glu Thr Ser Ser Pro Gly

145 150

<210> 33

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 33

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

1 5 10 15

Ala Met Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe 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 Ala Ile Asp Gly Pro Ala Gln Glu Val Arg 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

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Gln Ser Glu Thr Ser Ser Pro Gly

145 150

<210> 34

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 34

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 Glu Gly Cys Arg Pro Trp Asn Ile Phe 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 Ala Ile Asp Gly Pro Ala Gln Glu Val Asn 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 Arg Asp His Tyr Ile Phe Tyr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Gln Ser Glu Thr Ser Ser Pro Gly

145 150

<210> 35

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 35

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

1 5 10 15

Ala Met Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe 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 Ala Ile Asp Gly Pro Ala Gln Glu Val Arg 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 Glu Asp His Tyr Ile Phe Tyr

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Gln Ser Glu Thr Ser Ser Pro Gly

145 150

<210> 36

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 36

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 Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val

20 25 30

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

35 40 45

Val Thr Met Ala Ile Asp Gly Pro Ala Gln Glu Val 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 Val Cys Asp Gly Ser Pro Val Pro Gly Val Trp Leu Val

100 105 110

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

115 120 125

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

130 135 140

Gln Ile Glu Thr Ser Ser Pro Gly

145 150

<210> 37

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 37

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 Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

Gln Ile Glu Thr Ser Ser Pro Gly

145 150

<210> 38

<211> 152

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 38

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

1 5 10 15

Ala Met Thr Val Asp Glu Gly Cys Arg Pro Trp Asn Ile Phe Ser Val

20 25 30

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

35 40 45

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

50 55 60

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

100 105 110

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

115 120 125

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

130 135 140

Gln Ile Glu Thr Ser Ser Pro Gly

145 150

<210> 39

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 39

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

35 40 45

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

50 55 60

Asn Val Thr Gly Val Thr Phe Asp Asp Lys Lys Cys Thr Tyr Ala Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 40

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 40

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

35 40 45

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

50 55 60

Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 41

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 41

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

35 40 45

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

50 55 60

Asp Val Thr Ala Val Ala Phe Asp Asp Lys Lys Cys Thr Tyr Asp Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 42

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 42

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

35 40 45

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

50 55 60

Asp Val Thr Ala Val Ala Phe Asp Asp Lys Lys Cys Thr Tyr Asp Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 43

<211> 175

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<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 His Gly Lys Trp Tyr

20 25 30

Val Val Gly Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Ser Lys Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Phe Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser Thr Asn Tyr

100 105 110

Asn Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu

115 120 125

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

130 135 140

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

145 150 155 160

Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

165 170 175

<210> 44

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<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 Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 45

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<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 Gln Ala Gly Asn Ile Arg Leu Arg Glu Asp Lys Asp Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 46

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<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 Gln Ala Gly Asn Ile Lys Leu Arg Glu Asp Lys Asp Pro

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 47

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 47

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

35 40 45

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

50 55 60

Asp Val Thr Ala Val Thr Phe Asp Asp Lys Lys Cys Asn Tyr Ala Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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> 48

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 48

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

35 40 45

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

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 49

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 49

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

35 40 45

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

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

Ile Arg Ser Asp Leu Gly His Thr Ser Tyr Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 50

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 50

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 Tyr Gln Phe Gln Gly Lys Trp Tyr

20 25 30

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

35 40 45

His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Asp Val Met Leu Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 51

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 51

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

35 40 45

His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln His Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 52

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 52

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 Asp Gln Phe Gln Gly Lys Trp Tyr

20 25 30

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

35 40 45

His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 53

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 53

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

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

35 40 45

His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 54

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 54

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

1 5 10 15

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

20 25 30

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

35 40 45

His Lys Met Gly Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 55

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 55

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

35 40 45

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

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Asn

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 56

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 56

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

35 40 45

His Lys Met Gly Ala Thr Ile Tyr Glu Leu Asn Glu Asp Lys Ser Tyr

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 57

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 57

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 Met Ala Gly Asn Asn Leu Leu Arg Asp Asp Lys Asp Pro

35 40 45

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

50 55 60

Asn Val Thr Asp Val Met Leu Leu Asp Lys Lys Cys His Tyr Ile Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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> 58

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 58

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

35 40 45

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

50 55 60

Asn Val Thr Gly Val Ser Phe Trp Arg Lys Lys Cys His Tyr Lys Ile

65 70 75 80

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

85 90 95

Ile Lys Ser Gly Pro Gly Gln Thr Ser Asn Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Ala 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> 59

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 59

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

35 40 45

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

50 55 60

Asn Val Thr Asn Val Arg Phe Ala Met Lys Lys Cys Met Tyr Ser Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe 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> 60

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 60

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

35 40 45

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

50 55 60

Asp Val Thr Val Val Ser Phe Trp Arg Lys Lys Cys His Tyr Lys Ile

65 70 75 80

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

85 90 95

Ile Lys Ser Gly Pro Gly Gln Thr Ser Asn Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Ala 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> 61

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 61

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

35 40 45

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

50 55 60

Asp Val Thr Gly Val Ser Phe Arg Gly Lys Lys Cys His Tyr Lys Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Phe 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> 62

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 62

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

35 40 45

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

50 55 60

Asp Val Thr Ser Val Ala Phe Arg Asn Lys Lys Cys His Tyr Lys Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Phe 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> 63

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 63

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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> 64

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 64

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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> 65

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 65

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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> 66

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 66

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Lys Ser Glu Pro Gly Tyr Thr Ala Asn Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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> 67

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 67

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Lys Ser Pro Pro Gly Asn Thr Ala Asn Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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> 68

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 68

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

Asp Val Thr Asn Val Arg Phe Ala Arg Lys Lys Cys Met Tyr Ser Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe 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> 69

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 69

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe 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> 70

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 70

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Lys Ser Gln Pro Gly Asp Thr Ala Asn Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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> 71

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> lipocalin mutein

<400> 71

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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> 72

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody heavy chain CDR1

<400> 72

Gly Tyr Thr Phe Thr Asp Tyr Glu

15

<210> 73

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 of antibody heavy chain

<400> 73

Leu Asp Pro Lys Thr Gly Asp Thr

15

<210> 74

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 of antibody heavy chain

<400> 74

Thr Arg Phe Tyr Ser Tyr Thr Tyr

15

<210> 75

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain CDR1

<400> 75

Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr

1 5 10

<210> 76

<211> 11

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain CDR1

<400> 76

Gln Ser Leu Val His Ser Asn Arg Asn Thr Tyr

1 5 10

<210> 77

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 antibody light chain

<400> 77

Ser Gln Asn Thr His Val Pro Pro Thr

15

<210> 78

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain variable domain

<400> 78

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Leu Val Ser Ser

115

<210> 79

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain variable domain

<400> 79

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr 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 Ser 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 Asn

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 80

<211> 445

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain

<400> 80

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

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

165 170 175

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

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 81

<211> 442

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain

<400> 81

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

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

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

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

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

435 440

<210> 82

<211> 219

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain

<400> 82

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr 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 Ser 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 Asn

85 90 95

Thr His Val Pro Pro 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> 83

<211> 615

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 83

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

195 200 205

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

210 215 220

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

225 230 235 240

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

His Gly Lys Trp Tyr Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg

465 470 475 480

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

485 490 495

Glu Asp Lys Ser Tyr Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys

500 505 510

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

515 520 525

Phe Thr Leu Gly Lys Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Ile Asp Gln Cys Ile Asp Gly

610 615

<210> 84

<211> 610

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 84

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr

180 185 190

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

195 200 205

Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr Val Val Gly Gln

210 215 220

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

225 230 235 240

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

245 250 255

Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr Phe Val

260 265 270

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

275 280 285

Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser Thr Asn Tyr Asn

290 295 300

Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu Glu

305 310 315 320

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

325 330 335

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

340 345 350

His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser Lys

370 375 380

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

385 390 395 400

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

405 410 415

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

420 425 430

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

580 585 590

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

595 600 605

Gly Lys

610

<210> 85

<211> 366

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 85

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

35 40 45

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

50 55 60

Asp Val Thr Ser Val Ala Phe Arg Asn Lys Lys Cys His Tyr Lys Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Phe 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr

180 185 190

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

195 200 205

Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr Val Val Gly Gln

210 215 220

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

225 230 235 240

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

245 250 255

Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr Phe Val

260 265 270

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

275 280 285

Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser Thr Asn Tyr Asn

290 295 300

Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu Glu

305 310 315 320

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

325 330 335

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

340 345 350

His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

355 360 365

<210> 86

<211> 554

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 86

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

35 40 45

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

50 55 60

Asp Val Thr Ser Val Ala Phe Arg Asn Lys Lys Cys His Tyr Lys Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Phe 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr

180 185 190

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

195 200 205

Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr Val Val Gly Gln

210 215 220

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

225 230 235 240

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

245 250 255

Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr Phe Val

260 265 270

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

275 280 285

Pro Gly His Thr Ser Ser Leu Val Arg Val Val Ser Thr Asn Tyr Asn

290 295 300

Gln His Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu Glu

305 310 315 320

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

325 330 335

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

340 345 350

His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp Leu Ile

370 375 380

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

385 390 395 400

Asn Gln Phe His Gly Lys Trp Tyr Val Val Gly Gln Ala Gly Asn Ile

405 410 415

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

420 425 430

Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr Met Val Lys Phe Asp

435 440 445

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

450 455 460

Pro Gly Glu Phe Thr Leu Gly Lys Ile Lys Ser Phe Pro Gly His Thr

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

545 550

<210> 87

<211> 635

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 87

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

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

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

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

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp Leu

450 455 460

Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln

465 470 475 480

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

485 490 495

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

500 505 510

Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr Met Val Lys Phe

515 520 525

Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr Phe Val Pro Gly Ser

530 535 540

Gln Pro Gly Glu Phe Thr Leu Gly Lys Ile Lys Ser Phe Pro Gly His

545 550 555 560

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

565 570 575

Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu Glu Phe Tyr Ile

580 585 590

Thr Leu Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn

595 600 605

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

610 615 620

Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

625 630 635

<210> 88

<211> 828

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 88

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

35 40 45

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

50 55 60

Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys

245 250 255

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

260 265 270

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

275 280 285

Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

595 600 605

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

610 615 620

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser

625 630 635 640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp

645 650 655

Leu Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn Phe

660 665 670

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

675 680 685

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

690 695 700

Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr Met Val Lys

705 710 715 720

Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr Phe Val Pro Gly

725 730 735

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

740 745 750

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

755 760 765

Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu Glu Phe Tyr

770 775 780

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

785 790 795 800

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

805 810 815

Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

820 825

<210> 89

<211> 412

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 89

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr 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 Ser 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 Asn

85 90 95

Thr His Val Pro Pro 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 Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp

225 230 235 240

Leu Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn Phe

245 250 255

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

260 265 270

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

275 280 285

Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr Met Val Lys

290 295 300

Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile Trp Thr Phe Val Pro Gly

305 310 315 320

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

325 330 335

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

340 345 350

Ala Met Val Phe Phe Lys Phe Val Phe Gln Asn Arg Glu Glu Phe Tyr

355 360 365

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

370 375 380

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

385 390 395 400

Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

405 410

<210> 90

<211> 412

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 90

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

35 40 45

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

50 55 60

Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys Met Tyr Asp Ile

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Glu Phe Tyr 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

195 200 205

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His

210 215 220

Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

225 230 235 240

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

245 250 255

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

260 265 270

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln

275 280 285

Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

290 295 300

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

305 310 315 320

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

325 330 335

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

340 345 350

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

355 360 365

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

370 375 380

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

385 390 395 400

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

405 410

<210> 91

<211> 635

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 91

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

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

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

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

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp Leu

450 455 460

Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln

465 470 475 480

Asp Asn Gln Phe Gln Gly Lys Trp Tyr Val Val Gly Met Ala Gly Asn

485 490 495

Asn Leu Leu Arg Glu Asp Lys Asp Pro His Lys Met Ser Ala Thr Ile

500 505 510

Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr Asp Val Met Phe

515 520 525

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

530 535 540

Gln Pro Gly Glu Leu Thr Leu Gly Phe Ile Arg Ser Asp Leu Gly His

545 550 555 560

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

565 570 575

Met Val Phe Phe Lys Ser Val Ile Gln Asn Arg Glu Trp Phe Gly Ile

580 585 590

Thr Leu Tyr Gly Arg Thr Lys Glu Leu Thr Ser Glu Leu Lys Glu Asn

595 600 605

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

610 615 620

Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

625 630 635

<210> 92

<211> 635

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 92

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

35 40 45

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

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

Ile Arg Ser Asp Leu Gly His Thr Ser Tyr Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys

245 250 255

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

260 265 270

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

275 280 285

Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

595 600 605

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

610 615 620

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

625 630 635

<210> 93

<211> 412

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 93

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Arg Asn Thr Tyr 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 Ser 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 Asn

85 90 95

Thr His Val Pro Pro 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 Gly Gly Gly Gly Ser

210 215 220

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp

225 230 235 240

Leu Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn Phe

245 250 255

Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr Val Val Gly Met Ala Gly

260 265 270

Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro His Lys Met Ser Ala Thr

275 280 285

Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asn Val Thr Asp Val Met

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Val Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

405 410

<210> 94

<211> 412

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 94

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

35 40 45

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

50 55 60

Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile

65 70 75 80

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

85 90 95

Ile Arg Ser Asp Leu Gly His Thr Ser Tyr Leu Val Arg Val Val Ser

100 105 110

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

115 120 125

Asn Arg Glu Trp Phe Gly 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

195 200 205

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His

210 215 220

Ser Asn Arg Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

225 230 235 240

Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

245 250 255

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

260 265 270

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln

275 280 285

Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

290 295 300

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

305 310 315 320

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

325 330 335

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

340 345 350

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

355 360 365

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

370 375 380

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

385 390 395 400

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

405 410

<210> 95

<211> 640

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 95

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

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

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

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

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp

450 455 460

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

465 470 475 480

Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr Val Val

485 490 495

Gly Met Ala Gly Asn Asn Leu Leu Arg Glu Asp Lys Asp Pro His Lys

500 505 510

Met Ser Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asn Val

515 520 525

Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln Tyr Ile Ile Trp Thr

530 535 540

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

545 550 555 560

Ser Asp Leu Gly His Thr Ser Tyr Leu Val Arg Val Val Ser Thr Asn

565 570 575

Tyr Asn Gln His Ala Met Val Phe Phe Lys Ser Val Ile Gln Asn Arg

580 585 590

Glu Trp Phe Gly Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu Thr Ser

595 600 605

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

610 615 620

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

625 630 635 640

<210> 96

<211> 645

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 96

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

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

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

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

275 280 285

Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

405 410 415

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

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Lys Asp Pro His Lys Met Ser Ala Thr Ile Tyr Glu Leu Lys Glu Asp

515 520 525

Lys Ser Tyr Asn Val Thr Asp Val Met Phe Leu Asp Lys Lys Cys Gln

530 535 540

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

545 550 555 560

Leu Gly Phe Ile Arg Ser Asp Leu Gly His Thr Ser Tyr Leu Val Arg

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Gln Cys Ile Asp Gly

645

<210> 97

<211> 421

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 97

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

195 200 205

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

210 215 220

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

225 230 235 240

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Ser Leu Ser Leu Gly

420

<210> 98

<211> 422

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 98

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

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

20 25 30

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

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gly Lys Trp Tyr Val Val Gly Gln Ala Gly Asn Ile Arg Leu Arg Glu

275 280 285

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

290 295 300

Asp Lys Ser Tyr Asp Val Thr Met Val Lys Phe Asp Asp Lys Lys Cys

305 310 315 320

Met Tyr Asp Ile Trp Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe

325 330 335

Thr Leu Gly Lys Ile Lys Ser Phe Pro Gly His Thr Ser Ser Leu Val

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asp Gln Cys Ile Asp Gly

420

<210> 99

<211> 641

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 99

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

1 5 10 15

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

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

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

65 70 75 80

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

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

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

115 120 125

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

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

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

Lys Ser Glu Pro Gly Gly Thr Ala Asn Leu Val Arg Val Val Ser Thr

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

Gly

<210> 100

<211> 641

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 100

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

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

195 200 205

Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly

210 215 220

Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp

225 230 235 240

Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu

245 250 255

Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

260 265 270

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

275 280 285

Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro

420 425 430

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

435 440 445

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

450 455 460

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

465 470 475 480

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

485 490 495

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

580 585 590

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

595 600 605

Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu

610 615 620

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

625 630 635 640

Lys

<210> 101

<211> 409

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 101

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

35 40 45

Pro Lys Met Trp Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Asn Arg Glu Ile Phe Phe Ile Ile 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro

275 280 285

Pro Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Thr Lys Ser Phe Asn Arg Gly Glu Cys

405

<210> 102

<211> 408

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 102

Glu Ile Val Leu Thr Gln Ser Pro Ala 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 Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly

210 215 220

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

225 230 235 240

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

245 250 255

Gln Phe Gln Gly Lys Trp Tyr Val Val Gly Arg Ala Gly Asn Val Gly

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Val Pro Ile Asp Gln Cys Ile Asp

405

<210> 103

<211> 635

<212> Protein

<213> Artificial sequence

<220>

<223> fusion protein

<400> 103

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

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe

180 185 190

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

195 200 205

Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

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

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Asp Ser Thr Ser Asp Leu

450 455 460

Ile Pro Ala Pro Pro Leu Ser Lys Val Pro Leu Gln Gln Asn Phe Gln

465 470 475 480

Asp Asn Gln Phe Gln Gly Lys Trp Tyr Val Val Gly Arg Ala Gly Asn

485 490 495

Val Gly Leu Arg Glu Asp Lys Asp Pro Pro Lys Met Trp Ala Thr Ile

500 505 510

Tyr Glu Leu Lys Glu Asp Lys Ser Tyr Asp Val Thr Asn Val Arg Phe

515 520 525

Ala Arg Lys Lys Cys Thr Tyr Ser Ile Gly Thr Phe Val Pro Gly Ser

530 535 540

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

545 550 555 560

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

565 570 575

Met Val Phe Phe Lys Glu Val Tyr Gln Asn Arg Glu Ile Phe Phe Ile

580 585 590

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

595 600 605

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

610 615 620

Phe Pro Val Pro Ile Asp Gln Cys Ile Asp Gly

625 630 635

<210> 104

<211> 442

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain

<400> 104

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

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Thr Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe

180 185 190

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

195 200 205

Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro

210 215 220

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

225 230 235 240

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

245 250 255

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp

260 265 270

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

275 280 285

Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe

385 390 395 400

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

405 410 415

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

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210> 105

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain

<400> 105

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

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Asn Ile Gly Asp Gln Tyr Ala

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Lys Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Tyr Thr Gly Phe Gly Ser Leu

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

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

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 106

<211> 439

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain

<400> 106

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

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

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

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

225 230 235 240

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

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Ser Leu Ser Leu Ser Leu Gly

435

<210> 107

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain

<400> 107

Glu Ile Val Leu Thr Gln Ser Pro Ala 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 Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

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> 108

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody heavy chain CDR1

<400> 108

Gly Phe Thr Phe Asn Lys Asn Ala

15

<210> 109

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 of antibody heavy chain

<400> 109

Ile Arg Asn Lys Thr Asn Asn Tyr Ala Thr

1 5 10

<210> 110

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 of antibody heavy chain

<400> 110

Val Ala Gly Asn Ser Phe Ala Tyr

15

<210> 111

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain CDR1

<400> 111

Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr

1 5 10

<210> 112

<211> 12

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain CDR1

<400> 112

Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr

1 5 10

<210> 113

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain CDR

<400> 113

Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr

15

<210> 114

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain variable domain

<400> 114

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 Asn Lys Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Cys Val Ala Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser

115

<210> 115

<211> 113

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain variable domain

<400> 115

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ser Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

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

85 90 95

Tyr Tyr Asn Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 116

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody heavy chain CDR1

<400> 116

Tyr Phe Asp Phe Asp Ser Tyr Glu

15

<210> 117

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 of antibody heavy chain

<400> 117

Ile Tyr His Ser Gly Ser Thr

15

<210> 118

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 of antibody heavy chain

<400> 118

Ala Arg Val Asn Met Asp Arg Phe Asp Tyr

1 5 10

<210> 119

<211> 117

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain variable domain

<400> 119

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe Asp Ser Tyr

20 25 30

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

35 40 45

Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

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

85 90 95

Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser Ser

115

<210> 120

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody heavy chain CDR1

<400> 120

Gly Phe Thr Phe Ser Ser Tyr Ala

15

<210> 121

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> CDR2 of antibody heavy chain

<400> 121

Ile Gln Lys Gln Gly Leu Pro Thr

15

<210> 122

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 of antibody heavy chain

<400> 122

Ala Lys Asn Arg Ala Lys Phe Asp Tyr

15

<210> 123

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain CDR1

<400> 123

Gln Ser Ile Ser Ser Tyr

15

<210> 124

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Antibody light chain CDR1

<400> 124

Gln Ser Ile Ser Ser Tyr

15

<210> 125

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> CDR3 antibody light chain

<400> 125

Gln Gln Asn Arg Gly Phe Pro Leu Thr

15

<210> 126

<211> 116

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain variable domain

<400> 126

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Asn Arg Ala Lys Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 127

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain variable domain

<400> 127

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 Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asn Ala Ser Met 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 Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Arg Gly Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 128

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> antibody light chain variable domain

<400> 128

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 Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Met 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 Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Arg Gly Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 129

<211> 115

<212> Protein

<213> Artificial sequence

<220>

<223> antibody heavy chain variable domain

<400> 129

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 Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<---

Claims (26)

1. A fusion protein that specifically binds both cluster of differentiation 137 (CD137) and glypican-3 (GPC3), wherein the fusion protein comprises two subunits, where the first subunit is a full-length immunoglobulin and specifically binds to GPC3, and where the second subunit is lipocalin mutein and specifically binds to CD137, wherein the second subunit is connected at the N-terminus to the C-terminus of the first subunit via a peptide linker, and wherein the fusion protein contains SEQ ID NOs: 87 and 82. 2. Fusion protein according to claim 1, (a) wherein the fusion protein is capable of binding GPC3 with a K _D value of at most 1 nM or comparable to or lower than the K _D value of an immunoglobulin that is included exclusively in the first subunit; (b) wherein the fusion protein is capable of binding CD137 with an EC ₅₀ value of at most 3 nM or comparable to or lower than the EC ₅₀ value of a CD137-specific lipocalin mutein that is included exclusively in the second subunit; (c) where the fusion protein cross-reacts with cynomolgus GPC3; (d) wherein the fusion protein is capable of simultaneously binding CD137 and GPC3 with EC ₅₀ values of up to 10 nM maximum when the fusion protein is measured in an ELISA assay; (e) wherein the fusion protein is capable of binding GPC3-expressing tumor cells; (f) wherein the fusion protein is capable of inducing increased secretion of IL-2; (g) wherein the fusion protein is capable of inducing increased secretion of IL-2 to a level greater than SEQ ID NO: 83 and/or with better potency than SEQ ID NO: 83; (h) wherein the fusion protein is capable of inducing lymphocyte-mediated cytotoxicity; (i) wherein the fusion protein is capable of inducing enhanced T cell-mediated killing of GPC3-expressing tumor cells compared to SEQ ID NO: 83 and/or inducing activation of cytotoxic T cells with better efficacy compared to SEQ ID NO: 83; (j) wherein the fusion protein is capable of co-stimulating T cell responses in a GPC3-dependent manner; or (k) wherein the fusion protein is capable of co-stimulating T cell responses in the tumor microenvironment. 3. The fusion protein of claim 1 or 2, wherein the fusion protein has a half-life in mice of at least 50 hours, at least 75 hours, at least 100 hours, at least 125 hours, at least 150 hours , at least 175 hours, at least 200 hours, at least 250 hours or even longer, and/or where the fusion protein has a half-life in mice that is longer than SEQ ID NO: 83. 4. Fusion protein according to any one of paragraphs. 1-3, wherein the fusion protein has an isoelectric point of at least 6.5, at least 6.8, at least 7.1, at least 7.4, at least 7.5, at least 7.7 or higher, and/or wherein the fusion protein has an isoelectric point that is higher than SEQ ID NO: 83. 5. Fusion protein according to any one of paragraphs. 1-4, where the second subunit is connected at the N-terminus by a linker to the C-terminus of the heavy chain constant region (CH) of the first subunit. 6. Fusion protein according to any one of paragraphs. 1-5, where the first subunit contains the following set of CDR sequences: GYTFTDYE HCDR1, SEQ ID NO: 72, LDPKTGDT HCDR2, SEQ ID NO: 73, TRFYSYTY HCDR3; SEQ ID NO: 74, and the following set of CDR sequences: QSLVHSNRNTY LCDR1, SEQ ID NO: 75, KVS LCDR2, SQNTHVPPT LCDR3; SEQ ID NO: 77. 7. Fusion protein according to any one of paragraphs. 1-6, where the immunoglobulin has an IgG4 backbone. 8. A nucleic acid molecule intended for expression of a fusion protein that specifically binds both CD137 and GPC3, containing a nucleotide sequence encoding the fusion protein according to any one of claims. 1-7, wherein said sequence is operably linked to a regulatory sequence. 9. A method for producing a fusion protein according to any one of claims. 1-7, where the method includes culturing a host cell to obtain a fusion protein and transforming the host cell with the nucleic acid molecule of claim 8. 10. Pharmaceutical composition for the prevention, reduction or treatment of GPC3-positive forms of cancer, containing an effective amount of the fusion protein according to any one of paragraphs. 1-7 and a pharmaceutically acceptable carrier. 11. A method for simultaneous activation of subsequent CD137 signaling pathways and effects on GPC3-positive tumor cells, involving the use of a fusion protein according to any one of paragraphs in relation to tissue containing a tumor. 1-7 or the pharmaceutical composition according to claim 10. 12. A method for simultaneous co-stimulation of T cells and exposure to GPC3-positive tumor cells, involving the use of a fusion protein according to any one of claims in relation to the tissue containing the tumor. 1-7 or the pharmaceutical composition according to claim 10. 13. A method for inducing a localized lymphocytic response in the immediate vicinity of GPC3-positive tumor cells, involving the use of a fusion protein according to any one of claims in relation to the tissue containing the tumor. 1-7 or the pharmaceutical composition according to claim 10. 14. A method for inducing increased lymphocyte-mediated cytolysis of GPC3-positive tumor cells, comprising the use of a fusion protein according to any one of claims in relation to tissue containing a tumor. 1-7 or the pharmaceutical composition according to claim 10. 15. A method for preventing, reducing the intensity or treating GPC3-positive forms of cancer, involving the use of a fusion protein according to any one of paragraphs in relation to the tissue containing the tumor. 1-7 or the pharmaceutical composition according to claim 10.