WO2018087108A1

WO2018087108A1 - Proteins specific for cd137

Info

Publication number: WO2018087108A1
Application number: PCT/EP2017/078522
Authority: WO
Inventors: Marlon HINNER; Shane Olwill; Timo EICHNER
Original assignee: Pieris Pharmaceuticals Gmbh
Priority date: 2016-11-09
Filing date: 2017-11-08
Publication date: 2018-05-17

Abstract

The present disclosure provides human lipocalin muteins that bind CD137 and can be used in pharmaceutical applications, for example, as anti-cancer agents and/or immune modulators for the treatment or prevention of human diseases such as cancer, infectiousdiseases, and autoimmune diseases. The present discloser further shows the human lipocalin muteins can compete with CD137L for CD137 binding. The present disclosure also concerns methods of making CD137 binding lipocalin muteins described herein as well as compositions comprising such lipocalin muteins. The present disclosure further relates to nucleic acid molecules encoding such lipocalin muteins and to methods for generation of such lipocalin muteins and nucleic acid molecules. In addition, the application discloses therapeutic and/or diagnostic uses of these lipocalin muteins as well as compositions comprising one or more of such lipocalin muteins.

Description

PROTEINS SPECIFIC FOR CD137

I. BACKGROUND

[0001] CD137 is a costimulatory immune receptor and a member of the tumor necrosis factor receptor (TNFR) superfamily. It is mainly expressed on activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells but can also be found on resting monocytes and dendritic cells (Li and Liu, 2013), or endothelial cells (Snell et al., 201 1 ). CD137 plays an important role in the regulation of immune responses and thus is a target for cancer immunotherapy. CD137 ligand (CD137L) is the only known natural ligand of CD137 and is constitutively expressed on several types of antigen presenting cells (APC), such as activated B cells, monocytes, and splenic dendritic cells, and it can be induced on T lymphocytes.

[0002] As a costimulatory immune receptor, CD137 targeting may be therapeutically relevant both for cancer and autoimmune disease:

[0003] Regarding CD137^'s role as therapeutic cancer target, the benefit of CD137 costimulation for the elimination of cancer cells has been demonstrated in a number of preclinical in-vivo models. The forced expression of CD137L on a tumor, for example, leads to tumor rejection (Melero et al., 1998). Likewise, the forced expression of an anti-CD137 scFv on a tumor leads to a CD4⁺ T-cell and NK-cell dependent elimination of the tumor (Ye et al., 2002, Zhang et al., 2006, Yang et al., 2007). A systemically administered anti-CD137 antibody has also been demonstrated to lead to retardation of tumor growth (Martinet et al., 2002). It has been shown that CD137 is an excellent marker for naturally occurring tumor- reactive T cells in human tumors (Ye et al., 2014), and that anti-CD137 antibodies can be employed to improve the expansion and activity of CD8+ melanoma tumor-infiltrating lymphocytes for the application in adoptive T-cell therapy (Chacon et al., 2013). The preclinical demonstration of the potential therapeutic benefit of CD137 costimulation has spurred the development of therapeutic antibodies targeting CD137, BMS-663513 (Jure- Kunkel, M. et al., US patent 7288638) and PF-05082566 (Fisher et al., 2012); both are currently in early clinical trials. [0004] Regarding CD137^'s role in autoimmunity, there exists a significant body of preclinical work showing the relevance of this target in animal models of disease (reviewed in (Vinay and Kwon, 2016)). To highlight one example: a CD137 ligand double knockout-mouse showed reduced severity of disease in an animal model of multiple sclerosis, EAE (Martinez Gomez et al., 2012). Somewhat surprisingly, the treatment with CD137-agonistic antibodies has been found to ameliorate autoimmune symptoms in animal models of autoimmune disease instead of worsening them (reviewed in (Sun et al., 2003). However, while these results may be explained by a skewing of the T cell repertoire away from the autoreactive Th17 type, the data may be confounded both by antibody-directed cellular cytotoxicity and direct interference with the CD137 / CD137 ligand interaction.

[0005] Due to the roles of CD137 in modulating immune responses, there is a long- felt unmet need for compounds that bind human CD137 and increase a CD137-mediated response, and thereby provide a potential therapeutic for treatment or prevention of various diseases and conditions including cancer and infectious disease. Further, there is an unmet need for compounds that bind human CD137, but reduce a CD137-mediated response by interfering with the interaction of CD137 with its ligand CD137. Such, so-called "competitive" CD137 binders are desired to suppress strong inflammatory or autoimmune reactions, where CD137-positive cells inappropriately interact with CD137 ligand-expressing cells. A particularly unmet need is the provision of competitive CD137 binders that are completely devoid of any agonistic activity, which is best accomplished with a monomeric therapeutic polypeptide and/or non-FcgR-interacting polypeptides easily accessible with the CD137 binders described herein. To be therapeutically relevant, the affinity of such competitive binders should lie in a range that is comparable to that of the CD137 ligand, i.e., at least in the nanomolar range. To be developable as a drug in autoimmune indications, such competitive binders are further desirable to be crossreactive to a relevant toxicology species.

[0006] In addition, it has only recently been appreciated that a bivalent CD137-binder like an antibody may by itself not be sufficient to efficiently cluster CD137 on T-cells or NK- cells and lead to strong activation, in analogy to the lack of activity of the trivalent soluble CD137L (Wyzgol et al., 2009). It is thus both feasible and valuable to design CD137 binders that stimulate CD137-dependent T-cell activation when CD137 is clustered by a secondary means, such as a tumor target (Hinner et al., 2015). For example, when designing a bispecific or multispecific tumor-targeting molecule comprising such a CD137 binder, the tumor-specific T cells are bridged with tumor cells by the CD137/tumor target-bispecific, resulting in the clustering and concomitant activation of CD137, thus providing a local co- activating signal to the T cell. Healthy tissue is spared by the tumor-costimulated T cells due to the lack of target-mediated CD137 clustering. Further, bispecific or multispecific constructs specific for CD137 and other targets may be useful in a number of therapeutic areas, including but not limited to immune activation for cancer therapy.

[0007] It is an object of the present invention to provide for novel lipocalin muteins specific for CD137 that are competitive CD137 binders, which - alone or as fusion polypeptides to, e.g., an antibody - may be applied to the therapy of autoimmune disease, cancer or infectious disease. The compounds are muteins derived from lipocalins. Muteins of various lipocalins are a rapidly expanding class of therapeutics. They can be constructed through highly sophisticated artificial engineering to exhibit a high affinity and specificity against a target that is different than a natural ligand of wild-type lipocalins (see, e.g., WO 1999/16873, WO 2000/75308, WO 2003/029463, WO 2003/029471 and WO 2005/19256).

II. DEFINITIONS

[0008] The following list defines terms, phrases, and abbreviations used throughout the instant specification. All terms listed and defined herein are intended to encompass all grammatical forms.

[0009] As used herein, unless otherwise specified, "CD137" means human CD137.

CD137 is also known as "4-1 BB" or "tumor necrosis factor receptor superfamily member 9 (TNFRSF9)" or "induced by lymphocyte activation (I LA)". Human CD137 means a full-length protein defined by UniProt Q0701 1 , a fragment thereof, or a variant thereof.

[0010] As used herein, "detectable affinity" means the ability to bind to a selected target with an affinity, generally measured by K_d or EC₅₀, of at most about 10^"5 M or below (a lower _d or EC₅₀ value reflects better binding activity). Lower affinities are generally no longer measurable with common methods such as ELISA (enzyme-linked immunosorbent assay) and therefore of secondary importance.

[0011] As used herein, "binding affinity" of a protein of the disclosure (e.g., a mutein of a lipocalin) or a fusion polypeptide thereof to a selected target (in the present case, CD137), can be measured (and thereby K_d values of a mutein-ligand complex be determined) by a multitude of methods known to those skilled in the art. Such methods include, but are not limited to, fluorescence titration, competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Such methods are well established in the art and examples thereof are also detailed below.

[0012] It is also noted that the complex formation between the respective binder and its ligand is influenced by many different factors such as the concentrations of the respective binding partners, the presence of competitors, pH and the ionic strength of the buffer system used, and the experimental method used for determination of the dissociation constant K_d (for example fluorescence titration, competition ELISA or surface plasmon resonance, just to name a few) or even the mathematical algorithm which is used for evaluation of the experimental data.

[0013] Therefore, it is also clear to the skilled person that the K_d values (dissociation constant of the complex formed between the respective binder and its target ligand) may vary within a certain experimental range, depending on the method and experimental setup that is used for determining the affinity of a particular lipocalin mutein for a given ligand. This means that there may be a slight deviation in the measured K_d values or a tolerance range depending, for example, on whether the K_D value was determined by surface plasmon resonance (SPR), by competitive ELISA, or by direct ELISA.

[0014] As used herein, a "mutein," a "mutated" entity (whether protein or nucleic acid), or "mutant" refers to the exchange, deletion, or insertion of one or more nucleotides or amino acids, compared to the naturally occurring (wild-type) nucleic acid or protein "reference" scaffold. Said term also includes fragments of a mutein and variants as described herein. Lipocalin muteins of the present invention, fragments or variants thereof preferably have the function of binding to CD137 as described herein.

[0015] The term "fragment" as used herein in connection with the muteins of the disclosure relates to proteins or peptides derived from full-length mature human neutrophil gelatinase-associated lipocalin (hNGAL) that are N-terminally and/or C-terminally shortened, i.e., lacking at least one of the N-terminal and/or C-terminal amino acids. Such fragments may include at least 10, more such as 20 or 30 or more consecutive amino acids of the primary sequence of mature hNGAL and are usually detectable in an immunoassay of mature hNGAL. 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) of the N-terminal and/or C-terminal amino acids. It is understood that the fragment is preferably a functional fragment of the full-length mature hNGAL (mutein), which means that it preferably comprises the binding pocket of the full length mature hNGAL (mutein) it is derived from. As an illustrative example, such a functional fragment may comprise at least amino acids 13-157 of the linear polypeptide sequence of the full length mature hNGAL.

[0016] In general, the term "fragment," as used herein with respect to the corresponding protein ligand CD137 of a lipocalin mutein of the disclosure or of the combination according to the disclosure or of a fusion polypeptide described herein, relates to N-terminally and/or C-terminally shortened protein or peptide ligands, which retain the capability of the full-length ligand to be recognized and/or bound by a mutein according to the disclosure. As an illustrative example, the fragment may be an extracellular fragment of CD137. Such an extracellular fragment may comprise amino acids of the extracellular subdomains of CD137, such as the individual or combined amino acid sequences of domain 1 (residues 24-45 of Uniprot Protein ID Q0701 1 ), domain 2 (residues 46-86), domain 3 (87- 1 18) and domain 4 (residues 1 19-159).

[0017] The term "mutagenesis" as used herein means that the experimental conditions are chosen such that the amino acid naturally occurring at a given sequence position of the mature lipocalin can be substituted by at least one amino acid that is not present at this specific position in the respective natural polypeptide sequence. The term "mutagenesis" also includes the (additional) modification of the length of sequence segments by deletion or insertion of one or more amino acids. Thus, it is within the scope of the disclosure that, for example, one amino acid at a chosen sequence position is replaced by a stretch of three random mutations, leading to an insertion of two amino acid residues compared to the length of the respective segment of the wild-type protein. Such an insertion or deletion may be introduced independently from each other in any of the peptide segments that can be subjected to mutagenesis in the disclosure. In one exemplary embodiment of the disclosure, an insertion of several mutations may be introduced into the loop AB of the chosen lipocalin scaffold (cf. International Patent Publication No. WO 2005/019256 which is incorporated by reference its entirety herein).

[0018] The term "random mutagenesis" means that no predetermined single amino acid (mutation) is present at a certain sequence position but that at least two amino acids can be incorporated with a certain probability at a predefined sequence position during mutagenesis.

[0019] "Identity" is a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present disclosure means the percentage of pair-wise identical residues— following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question— with respect to the number of residues in the longer of these two sequences. 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.

[0020] The term "homology" is used herein in its usual meaning and includes identical amino acids as well as amino acids which are regarded to be conservative substitutions (for example, exchange of a glutamate residue by an aspartate residue) at equivalent positions in the linear amino acid sequence of a polypeptide of the disclosure (e.g., any lipocalin muteins of the disclosure).

[0021] The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5 (November 16, 2002; (Altschul et al., 1997). In this embodiment, the percentage of homology is based on the alignment of the entire polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cutoff value set to 10^"3) including the propeptide sequences, preferably using the wild-type protein scaffold as reference in a pairwise comparison. It is calculated as the percentage of numbers of "positives" (homologous amino acids) indicated as result in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0022] Specifically, in order to determine whether an amino acid residue of the amino acid sequence of a lipocalin (mutein) is different from a wild-type lipocalin corresponding to a certain position in the amino acid sequence of a wild-type lipocalin, a skilled artisan can use means and methods well-known in the art, e.g., alignments, either manually or by using computer programs such as BLAST 2.0, which stands for Basic Local Alignment Search Tool, or ClustalW, or any other suitable program which is suitable to generate sequence alignments. Accordingly, a wild-type sequence of lipocalin can serve as "subject sequence" or "reference sequence," while the amino acid sequence of a lipocalin different from the wild- type lipocalin described herein serves as "query sequence." The terms "wild-type sequence" and "reference sequence" and "subject sequence" are used interchangeably herein. A preferred wild-type sequence of lipocalin is the sequence of hNGAL as shown in SEQ ID NO: 2.

[0023] "Gaps" are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of sequence identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity using standard parameters, for example BLAST (Altschul et al., 1997, Altschul et al., 1990, Smith and Waterman, 1981 )

[0024] The term "variant" as used in the present disclosure relates to derivatives of a protein or peptide that include modifications of the amino acid sequence, for example by substitution, deletion, insertion or chemical modification. Such modifications do in some embodiments not reduce the functionality of the protein or peptide. Such variants include proteins, wherein one or more amino acids have been replaced by their respective D- stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline. However, such substitutions may also be conservative, i.e., an amino acid residue is replaced with a chemically similar amino acid residue. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. The term "variant," as used herein with respect to the corresponding protein ligand CD137 of a lipocalin mutein of the disclosure or of the combination according to the disclosure or of a fusion polypeptide described herein, relates to CD137or 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 in comparison to a wild-type CD137 protein, respectively, such as a CD137 reference protein as deposited with SwissProt as described herein. A CD137 variant, respectively, has preferably an amino acid identity of at least 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type human CD137, such as a CD137 reference protein as deposited with SwissProt as described herein.

[0025] By a "native sequence" of a lipocalin is meant that the sequence of a lipocalin that has the same amino acid sequence as the corresponding polypeptide derived from nature. Thus, a native sequence lipocalin can have the amino acid sequence of the respective naturally-occurring lipocalin from any organism, in particular, a mammal. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence" polypeptide specifically encompasses naturally-occurring truncated or secreted forms of the lipocalin, naturally-occurring variant forms such as alternatively spliced forms and naturally-occurring allelic variants of the lipocalin. A polypeptide "variant" means a biologically active polypeptide having at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or at least about 98% amino acid sequence identity with the native sequence polypeptide. Such variants include, for instance, polypeptides in which one or more amino acid residues are added or deleted at the N- or C- terminus of the polypeptide. Generally, a variant has at least about 70%, including at least about 75%, including at least about 80%, such as at least about 85% amino acid sequence identity, including at least about 90% amino acid sequence identity, at least about 95% or at least about 98% amino acid sequence identity with the native sequence polypeptide.

[0026] The term "position" when used in accordance with the disclosure means the position of either an amino acid within an amino acid sequence depicted herein or the position of a nucleotide within a nucleic acid sequence depicted herein. To understand the term "correspond" or "corresponding" as used herein in the context of the amino acid sequence positions of one or more lipocalin muteins, a corresponding position is not only determined by the number of the preceding nucleotides/amino acids. Accordingly, the position of a given amino acid in accordance with the disclosure which may be substituted may vary due to deletion or addition of amino acids elsewhere in a (mutant or wild-type) lipocalin. Similarly, the position of a given nucleotide in accordance with the present disclosure which may be substituted may vary due to deletions or additional nucleotides elsewhere in a mutein or wild-type lipocalin 5'-untranslated region (UTR) including the promoter and/or any other regulatory sequences or gene (including exons and introns).

[0027] Thus, for a "corresponding position" in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides/amino acids may differ in the indicated number than similar neighboring nucleotides/amino acids, but said neighboring nucleotides/amino acids, which may be exchanged, deleted, or added, are also comprised by the one or more "corresponding positions".

[0028] In addition, for a corresponding position in a lipocalin mutein based on a reference scaffold in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides/amino acids are structurally corresponding to the positions elsewhere in a (mutant or wild-type) lipocalin, even if they may differ in the indicated number, as appreciated by the skilled in light of the highly-conserved overall folding pattern among lipocalins.

[0029] The term "albumin" includes all mammal albumins such as human serum albumin or bovine serum albumin or rat serum albumin.

[0030] The term "organic molecule" or "small organic molecule" as used herein for the non-natural target denotes an organic molecule comprising at least two carbon atoms, but preferably not more than 7 or 12 rotatable carbon bonds, having a molecular weight in the range between 100 and 2000 Dalton, preferably between 100 and 1000 Dalton, and optionally including one or two metal atoms.

[0031] The word "detect," "detection," "detectable," or "detecting" as used herein is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative and qualitative measurements of a molecule of interest.

[0032] A "subject" is a vertebrate, 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, domestic and farm animals, and zoo, sports, or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgus monkeys, to name only a few illustrative examples. Preferably, the "mammal" herein is human. [0033] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations.

[0034] A "sample" is defined as a biological sample taken from any subject.

Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue.

III. DESCRIPTIONS OF FIGURES

[0035] Figure 1 : provides typical measurements of on-rate and off-rate by surface plasmon resonance (SPR) for the interaction of various representative lipocalin muteins (SEQ ID NOs indicated in the graph) with human CD137 (Fc-fusion) as the target. The targets were immobilized via an anti-human IgG-Fc antibody, which was in turn immobilized on a sensor chip using standard amine coupling chemistry. The lipocalin muteins were employed as the soluble analyte which was flowed at different concentrations across the chip surface. There are clear SPR binding signals towards the human target, human CD137-Fc fusion protein (huCD137-Fc), for all muteins tested, while the negative controls of SEQ ID NO: 3 and SEQ ID NO: 4 exhibit no binding. The dissociation constants resulting from a fit (1 :1 binding model) of the depicted data for all SEQ ID NOs are provided in Table 1.

[0036] Figure 2: provides representative examples of an SPR-based experiment designed to investigate whether muteins of SEQ ID NO: 5, SEQ ID NO: 12 and SEQ ID NO: 13 interfere with the binding of CD137 ligand (CD137L) to CD137. This is performed by generating a complex of CD137 and CD137L on the SPR sensor chip, and checking whether the tested lipocalin muteins can bind to this complex or not. As a reference, CD137 in the absence of CD137L is incubated with the lipocalin muteins. In the figures, only the relevant segments of the sensorgrams are provided. The SPR trace for the binding of the respective lipocalin mutein to huCD137-Fc alone is marked with an arrow with a solid stem. The SPR trace for the binding of the respective lipocalin mutein to huCD137-Fc that has been saturated with CD137L is marked with an arrow with a broken stem. Figure 2(A) shows SEQ ID NO: 5 cannot bind to huCD137-Fc in the presence of CD137L. Figure 2(B) and Figure 2(C) show SEQ ID NO: 12 and SEQ ID NO: 13 bind to huCD137-Fc with a very similar response both in the absence and presence of CD137L, showing that there is no competition in the binding between the two lipocalin muteins and CD137L.

[0037] Figure 3: shows representative examples of fluorescence-activated cell sorting (FACS) studies carried out to assess the specific binding of representative lipocalin muteins (SEQ ID NOs indicated in the graph) to human CD137 expressed on mammalian cells. Mock-transfected cells served as the negative control. [0038] Figure 4: depicts the results of a T-cell activation assay performed to assess the ability of representative CD137-binding lipocalin muteins (SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15) to co-stimulate T-cell responses and regulate downstream signaling, when coated on a plastic dish and enabled clustering. In addition, the activation of T-cells by incubation with soluble lipocalin muteins was tested to investigate whether the respective binders display agonistic activity in the absence of clustering. In Figure 4(A) lipocalin muteins were coated onto a plastic dish together with an anti-human-CD3 antibody and purified T-cells were subsequently incubated on the coated surface in the presence of soluble anti-human CD28. In Figure 4(B) an anti-human-CD3 antibody was coated onto a plastic dish and purified T-cells were subsequently incubated on the coated surface in the presence of soluble anti-human CD28 and the lipocalin muteins in solution. In both cases, supernatant interleukin 2 (IL-2) levels served as the readout. As a negative control, SEQ ID NO: 4 was employed. In the experiment of Figure 4(A), there is a clearly increased IL-2 concentration in the supernatant due to T-cell activation by the lipocalin muteins of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 compared to the negative control of SEQ ID NO: 4. For the experiment utilizing the lipocalin muteins in solution in Figure 4(B), there is no significant increase in IL-2 concentration in the supernatant for any of the lipocalin muteins tested compared to the negative control SEQ ID NO: 4. Taken together, Figure 4(A) and 4(B) show that the tested lipocalin muteins display the desired behavior: clustering of CD137 on the T-cell surface via plastic-coated anti-CD137 muteins leads to the desired T-cell costimulation, while the respective mutein in solution - while binding to CD137 as shown in Example 4 and Figure 3 - does not induce any T-cell costimulation.

[0039] Figure 5: provides the result of a T-cell activation experiment utilizing the

CD137-binding lipocalin mutein of SEQ ID NO: 13 as the test molecule. SEQ ID NO: 4 was used as the negative control. The experiment was performed with additional, suboptimal anti- CD3 and anti-CD28 stimulation of T-cells. The readouts are Figure 5(A) continued proliferation of the T-cells after three-days incubation measured using a 4 h BrdU pulse, Figure 5(C) supernatant IL-2 concentration, and Figure 5(E) supernatant IFN-γ levels. Alternatively, only suboptimal anti-CD3 concentration was utilized and CD28 stimulation was omitted, with readouts being Figure 5(B) continued proliferation, Figure 5(D) supernatant IL- 2 concentration and Figure 5(F) supernatant IFN-γ levels. The experiment demonstrates SEQ ID NO: 13-dose-dependent increases in proliferation, IL-2, and IFN-γ levels both utilizing anti-CD3/anti-CD28 stimulation and anti-CD3 stimulation alone.

[0040] Figure 6: depicts an alignment of amino acid sequences of optimized CD137 specific hNGAL muteins (SEQ ID NOs: 47-56), in comparison with the linear polypeptide sequence of wild-type hNGAL (SEQ ID NO: 2). The negative control mutein based on hNGAL (SEQ ID NO:4) is also listed.

[0041] Figure 7: provides typical measurements of on-rate and off-rate by surface plasmon resonance (SPR) for the interaction of various representative lipocalin muteins (SEQ ID NOs indicated in the graph) with human CD137 (Fc-fusion) as the target. The targets were immobilized via an anti-human IgG-Fc antibody, which was in turn immobilized on a sensor chip using standard amine coupling chemistry. The lipocalin muteins were employed as the soluble analyte which was flowed at different concentrations across the chip surface. There are clear SPR binding signals towards the human target, human CD137-Fc fusion protein (huCD137-Fc), for all muteins tested. The binding parameters resulting from a fit (1 :1 binding model) of the depicted data for all SEQ ID NOs are provided in Table 4.

[0042] Figure 8: provides typical measurements of on-rate and off-rate by surface plasmon resonance (SPR) for the interaction of various representative lipocalin muteins (SEQ ID NOs indicated in the graph) with cynomolgus monkey CD137 (Fc-fusion) as the target. The targets were immobilized via an anti-human IgG-Fc antibody, which was in turn immobilized on a sensor chip using standard amine coupling chemistry. The lipocalin muteins were employed as the soluble analyte which was flowed at different concentrations across the chip surface. There are clear SPR binding signals towards the cynomolgus monkey target, cynomolgus monkey CD137-Fc fusion protein (cyCD137-Fc), for all muteins tested. The binding parameters resulting from a fit (1 :1 binding model) of the depicted data for all SEQ ID NOs are provided in Table 5.

[0043] Figure 9: provides representative examples of an SPR-based experiment designed to investigate whether the mutein of SEQ ID NO: 47 interferes with the binding of CD137 ligand (CD137L) to CD137. This is investigated by generating a complex of CD137 and CD137L on the SPR sensor chip, and checking whether the tested lipocalin muteins can bind to this complex or not. As a reference, CD137 in the absence of CD137L is incubated with the lipocalin mutein of SEQ ID NO: 47. In the figures, only the relevant segments of the sensorgrams are provided. The experiment was performed both with (A) human CD137-Fc and (B) cynomolgus monkey CD137-Fc. The SPR trace for the binding of the respective lipocalin mutein to CD137-Fc alone is marked with an arrow with a solid stem. The SPR trace for the binding of the respective lipocalin mutein to huCD137-Fc that has been saturated with CD137L is marked with an arrow with a broken stem. The experiment shows that SEQ ID NO: 47 cannot bind to CD137-Fc from either human or cynomolgus monkey in the presence of CD137L.

[0044] Figure 10: shows representative plots of the results of fluorescence-activated cell sorting (FACS) studies carried out in order to assess the specific binding of representative lipocalin muteins (SEQ ID NOs indicated in the graph) to (A) human CD137 or (B) cynomolgus monkey CD137 expressed on mammalian cells. SEQ ID NO: 4 served as the negative control.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE

[0045] As used herein, a "lipocalin" is defined as a monomeric protein of approximately 18-20 kDa in weight, having a cylindrical β-pleated sheet supersecondary structural region comprising a plurality of (preferably eight) β-strands connected pair-wise by a plurality of (preferably four) loops at one end to define thereby a binding pocket. It is the diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes among the lipocalin family members, each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g., in Skerra, 2000, Flower et al., 2000, Flower, 1996). Indeed, the lipocalin family of proteins have naturally evolved to bind a wide spectrum of ligands, sharing unusually low levels of overall sequence conservation (often with sequence identities of less than 20%) yet retaining a highly conserved overall folding pattern. The correspondence between positions in various lipocalins is well known to one of skill in the art (see, e.g., U.S. Patent No. 7,250,297).

[0046] As noted above, a lipocalin is a polypeptide defined by its supersecondary structure, namely cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to define thereby a binding pocket. The present disclosure is not limited to lipocalin muteins specifically disclosed herein. In this regard, the disclosure relates to a lipocalin mutein having a cylindrical β-pleated sheet supersecondary structural region comprising eight β-strands connected pair-wise by four loops at one end to define thereby a binding pocket, wherein at least one amino acid of each of at least three of said four loops has been mutated as compared to the reference sequence, and wherein said lipocalin is effective to bind CD137 with detectable affinity.

[0047] In one particular embodiment, a lipocalin mutein disclosed herein is a mutein of human lipocalin 2. The term "human lipocalin 2" or "human Lcn 2" or "human NGAL" as used herein refers to the mature human neutrophil gelatinase-associated lipocalin (NGAL) with the SWISS-PROT/UniProt Data Bank Accession Number P80188. A human lipocalin 2 mutein of the disclosure may also be designated herein as "an hNGAL mutein." The amino acid sequence shown in SWISS-PROT/UniProt Data Bank Accession Number P80188 may be used as a preferred "reference sequence," more preferably the amino acid sequence shown in SEQ ID NO:2 is used as reference sequence.

[0048] In some embodiments, a lipocalin mutein binding CD137 with detectable affinity may include at least one amino acid substitution of a native cysteine residue of the reference sequence by another amino acid, for example, a serine residue. In some other embodiments, a lipocalin mutein binding CD137 with detectable affinity may include one or more non-native cysteine residues substituting one or more amino acids of a wild-type lipocalin. In a further particular embodiment, a lipocalin mutein according to the disclosure includes at least two amino acid substitutions of a native amino acid by a cysteine residue, hereby to form one or more cysteine begins. In some embodiments, said cysteine bridge may connect at least two loop regions. The definition of these regions is used herein in accordance with Flower (2000), Flower (1996) and Breustedt et al. (2005). In a related embodiment, the disclosure teaches one or more lipocalin muteins that are capable of activating downstream signaling pathways of CD137 by binding to CD137.

[0049] Proteins of the disclosure, which are directed against or specific for CD137, include any number of specific-binding protein muteins that are based on a defined protein scaffold, preferably a lipocalin scaffold. Also preferably, the number of nucleotides or amino acids, respectively, that is exchanged, deleted or inserted is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 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, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 being preferred and 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 being even more preferred. However, it is preferred that protein muteins of the disclosure are still capable of binding CD137.

[0050] In one aspect, the present disclosure includes various lipocalin muteins that bind CD137 with at least detectable affinity. In this sense, CD137 can be regarded as a non- natural ligand of the reference wild-type lipocalins, where "non-natural ligand" refers to a compound that does not bind to wild-type lipocalins under physiological conditions. By engineering wild-type lipocalin with one or more mutations at certain sequence positions, the present inventors have demonstrated that high affinity and high specificity for the non-natural ligand, CD137, is possible. In some embodiments, at 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or even more nucleotide triplet(s) encoding certain sequence positions on wild-type lipocalins, a random mutagenesis may be carried out through substitution at these positions by a subset of nucleotide triplets.

[0051] Further, the lipocalin muteins of the disclosure may have a mutated amino acid residue at any one or more, including at least at any 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more of the sequence positions corresponding to certain sequence positions of the linear polypeptide sequence of the reference lipocalin.

[0052] A protein of the disclosure may include the wild-type (natural) amino acid sequence of the "parental" protein scaffold (such as a lipocalin scaffold) outside the mutated amino acid sequence positions. In some embodiments, a lipocalin mutein according to the disclosure may also carry one or more amino acid mutations at one or more sequence position(s) as long as such a mutation does, at least essentially not hamper or not interfere with the binding activity and the folding of the mutein. Such mutations can be accomplished very easily on DNA level using established standard methods (Sambrook and Russell, 2001 , Molecular cloning: a laboratory manual). Illustrative examples of alterations of the amino acid sequence are insertions or deletions as well as amino acid substitutions.

[0053] Such substitutions may be conservative, i.e., an amino acid residue is replaced with an amino acid residue of chemically similar properties, in particular with regard to polarity as well as size. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) iso-leucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. Another example of conservative substitutions are the replacements among members of the following groups: 1 ) glycine, alanine, valine, leucine, and isoleucine 2) phenylalanine, tryptophan, and tyrosine 3) cysteine and methionine 4) serine and threonine 5) arginine, histidine, and lysine 6) aspartic acid, glutamic acid, asparagine, and glutamine. On the other hand, it is also possible to introduce non-conservative alterations in the amino acid sequence. In addition, instead of replacing single amino acid residues, it is also possible to either insert or delete one or more continuous amino acids of the primary structure a parental protein (for example, hNGAL) as long as these deletions or insertion result in a stable folded/functional mutein (for example, hNGAL muteins with truncated N- and C-terminus). In such mutein, for instance, one or more amino acid residues are added or deleted at the N- or C- terminus of the polypeptide. Generally, such a mutein may have about at least 70%, such as at least about 75%, including at least about 80%, such as at least about 85%, at least about 90%, at least about 95% or at least about 98% amino acid sequence identity, with the amino acid sequence of hNGAL. In addition, as another illustrative example, the present disclosure also encompasses hNGAL muteins, in which amino acid residues (Lys-Asp-Pro, positions 46-48) of the linear polypeptide sequence of the mature human lipocalin 2 (hNGAL) have been deleted (SEQ ID NO: 16).

[0054] The amino acid sequence of a lipocalin mutein disclosed herein has a high sequence identity to the reference lipocalin, preferably hNGAL, when compared to sequence identities with other lipocalins. In this general context, the amino acid sequence of a lipocalin mutein of the disclosure is at least substantially similar to the amino acid sequence of the reference lipocalin, with the proviso that possibly there are gaps (as defined below) in an alignment that are the result of additions or deletions of amino acids. A respective sequence of a lipocalin mutein of the disclosure, being substantially similar to the sequences of the reference lipocalin, has, in some embodiments, at least 70% identity or sequence identity, at least 75% identity or sequence identity, at least 80% identity or sequence identity, at least 82% identity or sequence identity, at least 85% identity or sequence identity, at least 87% identity or sequence identity, or at least 90% identity or sequence identity including at least 95% identity or sequence identity, to the sequence of the reference lipocalin, with the proviso that the altered position or sequence is retained and that one or more gaps are possible.

[0055] As used herein, a lipocalin mutein of the disclosure "specifically binds" a target

(for example, CD137) if it is able to discriminate between that target and one or more reference targets, since binding specificity is not an absolute, but a relative property. "Specific binding" can be determined, for example, in accordance with western blots, ELISA, FACS, RIA (radioimmunoassay), ECL (electrochemiluminescence), immunoradiometric assay (IRMA), IHC (ImmunoHistoChemistry), and peptide scans.

[0056] In one embodiment, the lipocalin muteins of the disclosure are fused at its N- terminus and/or its C-terminus to a fusion partner, which is a protein domain that extends the serum half-life of the mutein. In further particular embodiments, the protein domain is an Fc part of an immunoglobulin, a C_H3 domain of an immunoglobulin, a C_H4 domain of an immunoglobulin, an albumin binding peptide, or an albumin binding protein.

[0057] In another embodiment, the lipocalin muteins of the disclosure are chemically conjugated to a compound that extends the serum half-life of the mutein. More preferably, the muteins are conjugated to a compound selected from the group consisting of a polyalkylene glycol molecule, a hydroxyethyl starch, an Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

[0058] In yet another embodiment, the current disclosure relates to a nucleic acid sequences comprising a nucleotide sequence encoding a lipocalin muteins disclosed herein. The disclosure encompasses a host cell containing said nucleic acid molecule.

A. Lipocalin muteins specific for CD137

[0059] In one aspect, the present disclosure provides human lipocalin muteins that bind to human CD137 with high affinity and useful applications of such muteins. The disclosure also provides methods of making CD137 binding proteins described herein as well as compositions comprising such proteins. The disclosed CD137 binding proteins have a similar or comparable binding pattern to both human and cynomolgus CD137, and only costimulate T-cell responses when CD137 clustering is induced. This mechanism may be exploited therapeutically in cancer or infectious disease. By interfering with the binding of CD137 to its ligand, CD137 ligand, the disclosed CD137 binding proteins may also be applied therapeutically in autoimmune indications. Finally, CD137 binding proteins of the disclosure as well as compositions thereof may be used in methods of detecting CD137 protein in a sample or in methods of binding of CD137 in a subject to stimulate or inhibit immune responses. No such human lipocalin muteins having these features attendant to the uses provided by present disclosure have been previously described.

1. Exemplary Lipocalin muteins specific for CD137

[0060] Some embodiments of the current disclosure relate to a lipocalin mutein that is capable of binding CD137, preferably human CD137 (huCD137), with an affinity measured by a K_d of about 700 nM or lower, about 200 nM or lower, about 140 nM or lower, about 50 nM or lower, about 30 nM or lower, about 10 nM or lower, or even lower, such as about 5.5 nM or lower. Such affinity can be determined, for example, by surface plasmon resonance (SPR) analysis, such as essentially described in Examples 4 and 13.

[0061] In other embodiments, the CD137 binding lipocalin mutein may be cross- reactive with cynomolgus CD137 (cyCD137), and in some further embodiments, capable of binding cyCD137 with an affinity measured by a K_d of about 20 nM, 10 nM, 5 nM, 2 nM, 1 .5 nM, or even lower. Such affinity can be determined, for example, by SPR analysis, such as essentially described in Examples 4 and 13.

[0062] In other embodiments, the lipocalin muteins of the disclosure do not competitively bind CD137 in the presence of CD137 ligand (CD137L) as determined by a surface plasmon resonance (SPR) assay as essentially described in Examples 5 and 14.

[0063] In other embodiments, the lipocalin muteins of the disclosure competitively bind CD137 in the presence of CD137 ligand (CD137L) as determined by a surface plasmon resonance (SPR) assay, such as essentially described in Examples 5 and 14.

[0064] In other embodiments, the lipocalin mutein is capable of binding CD137 on

Chinese hamster ovary (CHO) cells transfected with huCD137 with an EC₅₀ value of about 1000 nM, 100 nM, 50 nM, or even lower such as 34 nM. In other embodiments, the lipocalin mutein is capable of binding CD137 on CHO cells transfected with cyCD137 with an EC₅₀ of about 100 nM, 50 nM, 30 nM, or even lower such as 27 nM. The EC₅₀ value can, for example, be determined by a fluorescence-activated cell sorting (FACS), such as essentially described in Examples 6 and 15.

[0065] Another embodiment of the current disclosure provides a lipocalin mutein that is capable of activating downstream signaling pathways of CD137 by binding to CD137. [0066] In some embodiments, a lipocalin mutein of the disclosure is capable of inducing higher IL-2 concentration, for example, when measured in a functional T-cell activation assay and compared to the negative control of SEQ ID NO: 4, such as essentially described in Example 7.

[0067] In some other embodiments, a lipocalin mutein of the disclosure does not lead to higher IL-2 concentration, for example, when measured in a functional T-cell activation assay and compared to the negative control of SEQ ID NO: 4, such as essentially described in Example 8.

[0068] In some embodiments, a lipocalin mutein of the disclosure is capable of inducing higher IL-2 and IFN-γ proliferation, for example, when measured in a functional T- cell activation assay and compared to the negative control of SEQ ID NO: 4, such as essentially described in Example 9.

[0069] In one aspect, the present disclosure provides CD137-binding hNGAL muteins.

[0070] In this regard, the disclosure provides one or more hNGAL muteins that are capable of binding CD137 with an affinity measured by a K_D of about 1000 nM or lower, 300 nM or lower, 100 nM or lower, and even about 50 nM or lower.

[0071] In some embodiments, such hNGAL muteins comprise mutated amino acid residues at two 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/or 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0072] In some particular embodiments, such hNGAL mutein may contain a mutated 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/or 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0073] In further particular embodiments, such hNGAL mutein may further include a mutated amino acid residue at one or more positions corresponding to positions 20, 25, 33, 44, 59, 71 , 78, 80, 82, 87, 92, 98, 101 , and/or 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0074] In some further embodiments, the hNGAL mutein may comprise at least 1 , 2,

3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or even more, mutated amino acid residues at one or more sequence positions corresponding to sequence positions 20, 25, 33, 36, 40, 41 , 44, 49, 52, 59, 68, 70-73, 77-82, 92, 96, 98, 100, 101 , 103, 122, 125, 127, 132, and/or 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) and wherein said polypeptide binds CD137, in particular human CD137.

[0075] In some embodiments, a CD137-binding hNGAL mutein according to the disclosure includes, at one or more positions corresponding to positions 20, 25, 33, 36, 40, 41 , 44, 49, 52, 59, 68, 70-73, 77-82, 92, 96, 98, 100, 101 , 103, 122, 125, 127, 132, and/or 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Gin 20→ Arg; Asn 25→ Tyr or Asp; Val 33→ lie; Leu 36→Met; Ala 40→ Asn; lie 41→ Leu; Glu 44→ Val or Asp; Gin 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→ Gin or His; Tyr 78→ His; Trp 79→ lie; lie 80→ Asn; Arg 81 → Trp or Gin; Thr 82→ Pro; 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→ lie; Tyr 132→ Trp; and/or Lys 134→ Gly. In some embodiments, an hNGAL mutein according to the disclosure includes 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, 25, 26 or all mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2).

[0076] In some embodiments, a CD137-binding hNGAL mutein according to the disclosure may (further) include one or two of the following mutated amino acid residues: Gin 28→ His and Cys 87→ Ser, of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0077] In some additional embodiments, the hNGAL muteins binding CD137 include one of the following sets of amino acid substitutions in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) . Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser 68→ Asp;

Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81→ Trp; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125→ Ser; Ser 127→ lie; Tyr 132 →Trp; Lys 134→ Gly;

(b) . Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser 68→ Asp;

Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; 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→ lie; Tyr 132→ Trp; Lys 134→ Gly; (c) . Asn 25→ Tyr; Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Gly;

Ser 68→ Asp; Leu 70→ Met; Phe 71→ Leu; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81→ Gin; Phe 92→ Ser; Asn 96→ Phe; Tyr 100→ Asp; Leu 103 → His; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134→ Gly;

(d) . Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Gly; Ser 68→ Asp;

Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Tyr 78→ His; Trp 79→ lie; Arg 81→ Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134→ Gly;

(e) . Asn 25→ Asp; Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Gly;

Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81→ Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134→ Gly;

(f) . Val 33→ lie; Leu 36→ Met; Ala 40→ Asn; lie 41 → Leu; Gin 49→ His; Tyr 52→ Gly;

(g) . Gin 20→ Arg; Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Glu 44→ Val; Gin 49→ His;

Tyr 52→ Gly; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81→ Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100→ Asp; Leu 103 → His; Phe 122→ Tyr; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134→ Gly;

(h) . Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser 68→ Asp;

Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; lie 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→ lie; Tyr 132→ Trp; Lys 134→ Gly;

(i) . Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Gly; Lys 59→ Asn;

G). Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Glu 44→ Asp; Gin 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→ lie; Arg 81→ Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100→ Asp; Leu 103 → His; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134→ Gly;

[0078] In the residual region, i.e., the region differing from sequence 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/or 134, an hNGAL mutein of the disclosure may include the wild-type (natural) amino acids of mature hNGAL or mutated amino acids.

[0079] In other embodiments, an hNGAL mutein of the disclosure includes a mutated 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 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0080] In particular embodiments, a lipocalin mutein of the disclosure comprises at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or even more, mutated amino acid residues at one or more sequence 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 linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). Preferably, it is envisaged that the disclosure relates to a lipocalin mutein which comprises, in addition to one or more mutated amino acid residues at sequence positions corresponding to positions 36, 87, and/or 96 of the linear polypeptide sequence of mature human NGAL (SEQ ID NO: 2), one or more mutated amino acid residues at sequence 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 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0081] In some still further embodiments, the disclosure relates to a polypeptide, wherein said polypeptide is an hNGAL mutein, in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), comprising at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , or even more, mutated amino acid residues at the sequence 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, and wherein said polypeptide binds CD137, in particular human CD137.

[0082] In some embodiments, a CD137-binding hNGAL mutein of the disclosure includes, at any one or more of the sequence positions 36, 40-41 , 49, 52, 65, 68, 70, 72-73, 77, 79, 81 , 83, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Leu 36→ Gin; Ala 40→ lie; lie 41→ Arg or Lys; Gin 49→ Val, lie, 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; 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. [0083] In some embodiments, an hNGAL mutein of the disclosure includes 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, 25 or all mutated amino acid residues at the sequence positions of mature hNGAL (SEQ ID NO: 2).

[0084] In some additional embodiments, an hNGAL mutein of the disclosure, which binds to CD137 includes the following amino acid replacements in comparison with the linear polypeptide sequence of mature hNGAL:

(a) Leu 36→ Gin; Ala 40→ lie; lie 41 → Lys; Gin 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; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr;

(b) Leu 36→ Gin; Ala 40→ lie; lie 41→ Arg; Gin 49→ lie; 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; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr;

(c) Leu 36→ Gin; Ala 40→ lie; lie 41 → Arg; Gin 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; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr;

(d) Leu 36→ Gin; Ala 40→ lie; lie 41 → Lys; Gin 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; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr;

(e) Leu 36→ Gin; Ala 40→ lie; lie 41 → Lys; Gin 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; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr;

(f) Leu 36→ Gin; Ala 40→ lie; lie 41 → Lys; Gin 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; Leu 94→ Phe; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr; (g) Leu 36→ Gin; Ala 40→ lie; lie 41 → Arg; Gin 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; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr;

(h) Leu 36→ Gin; Ala 40→ lie; lie 41 → Lys; Gin 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; Leu 94→ Phe; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr; or

(i) Leu 36→ Gin; Ala 40→ lie; lie 41→ Arg; Gin 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; Asn 96→ Lys; Tyr 100→ Phe; Leu 103→ His; Tyr 106→ Ser; Lys 125→ Phe; Ser 127→ Phe; Tyr 132→ Glu; Lys 134→ Tyr.

[0085] In the residual region, i.e., the region differing from sequence 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, an hNGAL mutein of the disclosure may include the wild-type (natural) amino acids of mature hNGAL or mutated amino acids.

[0086] In still further embodiments, an hNGAL mutein according to the current disclosure has at least 70% sequence identity to the sequence of mature hNGAL (SEQ ID NO: 2). As an illustrative example, the mutein of the SEQ ID NO: 47 has an amino acid sequence identity of 88.2% with the amino acid sequence of mature hNGAL (SEQ ID NO: 2).

[0087] In further particular embodiments, an hNGAL mutein of the disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 12-20 and 47-56 or a fragment or variant thereof.

[0088] In further particular embodiments, an hNGAL mutein of the disclosure has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-20 and 47-56.

[0089] The disclosure also includes structural homologues of an hNGAL mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-20 and 47-56, which structural homologues have an amino acid sequence identity of more than about 60%, preferably more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 92%, and most preferably more than 95% in relation to said hNGAL mutein.

[0090] An hNGAL mutein according to the present disclosure can be obtained by means of mutagenesis of a naturally occurring form of mature hNGAL (SEQ ID NO: 2). In some embodiments of the mutagenesis, a substitution (or replacement) is a conservative substitution. Nevertheless, any substitution - including non-conservative substitution or one or more from the exemplary substitutions below - is envisaged as long as the lipocalin mutein retains its capability to bind to CD137, and/or it has a sequence identity to the then substituted sequence in that it is at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or higher sequence identity to the amino acid sequence of mature hNGAL (SEQ ID NO: 2).

[0091] In some additional embodiments, an hNGAL mutein of the disclosure is capable of interfering with the binding of CD137L to CD137, for example, as measured in a surface plasmon resonance (SPR) assay essentially described in Examples 5 and 14.

[0092] In some particular embodiments, the present disclosure provides a lipocalin mutein that binds human CD137 with an affinity measured by a K_D of about 30 nM or lower, wherein the lipocalin mutein has at least 90% or higher, such as 95%, identity to the amino acid sequence of SEQ ID NO: 13.

2. Applications of Lipocalin muteins specific for CD137

[0093] CD137 is a T-cell costimulatory receptor induced on T-cell receptor (TCR) activation (Nam et al., 2005, Watts, 2005). In addition to its expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells, activated B cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, has been described on antigen-presenting cells including B cells, monocyte/macrophages, and dendritic cells (Watts, 2005). On interaction with its ligand, CD137 leads to increased TCR-induced T-cell proliferation, cytokine production, functional maturation, and prolonged CD8+ T-cell survival (Nam et al., 2005, Watts, 2005).

[0094] The CD137/CD137L interaction is involved in various aspects of an immune response. It appears to be important in inhibiting activation-induced cell death in T cells (Hurtado et al., 1997), but abrogates anti-apoptotic effects of other cytokines in neutrophils (Heinisch et al., 2001 ). CD137 thus may play a role in immune function homeostasis (Ebata et al., 2001 ) and may represent a target costimulatory system that can be targeted in treatment of cancer or the inflammatory response (Blazar et al., 2001 , Takahashi et al., 2001 , Kim and Broxmeyer, 2001 , Kim et al., 2001 ). [0095] Numerous possible applications for the CD137-binding lipocalin muteins of the disclosure, therefore, exist in medicine. In one further aspect, the disclosure relates to the use of a CD137-binding lipocalin mutein disclosed herein for detecting CD137 in a sample as well as a respective method of diagnosis.

[0096] The present disclosure also involves the use of one or more CD137-binding lipocalin muteins as described for complex formation with CD137.

[0097] Therefore, in another aspect of the disclosure, the disclosed lipocalin muteins are used for the detection of CD137. Such use may include the steps of contacting one or more said muteins, under suitable conditions, with a sample suspected of containing CD137, thereby allowing formation of a complex between the muteins and CD137, and detecting the complex by a suitable signal.

[0098] The detectable signal can be caused by a label, as explained above, or by a change of physical properties due to the binding, i.e., the complex formation, itself. One example is surface plasmon resonance, the value of which is changed during binding of binding partners from which one is immobilized on a surface such as a gold foil.

[0099] The CD137-binding lipocalin muteins disclosed herein may also be used for the separation of CD137. Such use may include the steps of contacting one or more said muteins, under suitable conditions, with a sample supposed to contain CD137, thereby allowing formation of a complex between the muteins and CD137, and separating the complex from the sample.

[00100] In the use of the disclosed muteins for the detection of CD137 as well as the separation of CD137, the muteins and/or CD137 or a domain or fragment thereof may be immobilized on a suitable solid phase.

[00101] In still another aspect, the present disclosure features a diagnostic or analytical kit comprising a CD137-binding lipocalin mutein according to the disclosure.

[00102] In addition to their use in diagnostics, in yet another aspect, the disclosure contemplates a pharmaceutical composition comprising a mutein of the disclosure and a pharmaceutically acceptable excipient.

[00103] Furthermore, the present disclosure provides human lipocalin muteins that bind CD137 for use as anti-cancer agents and/or immune modulators. As such the lipocalin muteins of the present disclosure that bind CD137 are envisaged to be used in a method of treatment or prevention of human diseases such as cancer, infectious diseases, and autoimmune diseases. Accordingly, also provided are methods of treatment or prevention of human diseases such as cancer, infectious diseases, and autoimmune diseases in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a lipocalin mutein of the present invention that bind CD137. For the treatment of autoimmune disease, a human lipocalin mutein directly interfering with the interaction of CD137 with its ligand CD137L is highly preferred.

[00104] In T cells, CD137- mediated signaling leads to the recruitment of TRAF family members and activation of several kinases, including ASK-1 , MKK, MAPK3/MAPK4, p38, and JNK/SAPK. Kinase activation is then followed by the activation and nuclear translocation of several transcription factors, including ATF-2, Jun, and NF-κΒ. In addition to augmenting suboptimal TCR-induced proliferation, CD137-mediated signaling protects T cells, and in particular, CD8+T cells from activation-induced cell death (AICD).

[00105] The present disclosure encompasses the use of a CD137-binding lipocalin mutein of the disclosure or a composition comprising such lipocalin mutein for the binding of CD137, costimulating T-cells, and/or activating downstream signaling pathways of CD137 by binding to CD137, including enhancing IL-2 secretion and producing IFN-γ.

[00106] The present disclosure also features a method of binding CD137 or costimulating T-cells, comprising applying one or more CD137-binding lipocalin muteins of the disclosure or of one or more compositions comprising such lipocalin muteins.

[00107] Furthermore, the present disclosure involves a method of activating downstream signaling pathways of CD137, including enhancing the secretion of proinflammatory cytokines including but not limited to IL-2 and IFN-γ, comprising applying one or more CD137-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

[00108] The present disclosure also contemplates a method of inducing T lymphocyte proliferation, comprising applying one or more CD137-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

[00109] Moreover, absence of CD137/CD137L interactions prevents the development of certain autoimmune diseases (Seo et al., 2004, Seo et al., 2003). CD137/CD137L interactions are involved in the network of hematopoietic and nonhematopoietic cells in addition to the well characterized antigen-presenting cell (APC)-T cell interactions. Signaling through CD137L plays a critical role in the differentiation of myeloid cells and their cellular activities, suggesting that CD137L signals trigger and sustain inflammation (Kwon, 2009).

[00110] The present disclosure encompasses the use of a CD137-binding lipocalin mutein of the disclosure that is capable of binding to CD137 competitively with CD137L or a composition comprising such lipocalin mutein that is caplable of binding to CD137 competitively with CD137L.

[00111] The present disclosure also features a method of interfering with the binding of CD137L to CD137, comprising applying one or more CD137-competitive-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

[00112] Furthermore, the present disclosure involves a method of interfering with natural signaling of CD137L, comprising applying one or more CD137-competitive-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

[00113] The present disclosure also contemplates a method of reducing production of proinflammatory cytokines and chemokines, comprising applying one or more CD137- competitive-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

B. Lipocalin muteins of the disclosure

[00114] Lipocalins are proteinaceous binding molecules that have naturally evolved to bind ligands. Lipocalins occur in many organisms, including vertebrates, insects, plants and bacteria. The members of the lipocalin protein family (Pervaiz and Brew, 1987) are typically small, secreted proteins and have a single polypeptide chain. They are characterized by a range of different molecular-recognition properties: their binding to various, principally hydrophobic small molecules (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants, and odorants), and their binding to specific cell-surface receptors and their formation of macromolecular complexes. Although they have, in the past, been classified primarily as transport proteins, it is now clear that the lipocalins fulfill a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and the synthesis of prostaglandins. Lipocalins have also been implicated in the regulation of the immune response and the mediation of cell homoeostasis (reviewed, e.g., in Flower et al., 2000, Flower, 1996).

[00115] Lipocalins share unusually low levels of overall sequence conservation, often with sequence identities of less than 20%. In strong contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight- stranded anti-parallel β-sheet closed back on itself to form a continuously hydrogen-bonded β-barrel. This β-barrel forms a central cavity. One end of the barrel is sterically blocked by the N-terminal peptide segment that runs across its bottom as well as three peptide loops connecting the β-strands. The other end of the β-barrel is open to the solvent and encompasses a target-binding site, which is formed by four flexible peptide loops. It is this diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g., in Skerra, 2000, Flower et al., 2000, Flower, 1996).

[00116] A lipocalin mutein according to the present disclosure may be a mutein of any chosen lipocalin. Examples of suitable lipocalins (also sometimes designated as "protein 'reference' scaffolds" or simply "scaffolds") of which a mutein may be used include, but are not limited to, tear lipocalin (lipocalin-1 , von Ebner gland protein), retinol binding protein, neutrophil lipocalin-type prostaglandin D-synthase, β-lactoglobulin, bilin-binding protein (BBP), apolipoprotein D (APO D), neutrophil gelatinase associated lipocalin (NGAL), tear lipocalin (Tic), including human tear lipocalim (hTIc), a2-microglobulin-related protein (A2m), 24p3/uterocalin (24p3), von Ebners gland protein 1 (VEGP 1 ), von Ebners gland protein 2 (VEGP 2), and Major allergen Can f1 precursor (ALL-1 ). In related embodiments, the lipocalin mutein is selected from the group consisting of human neutrophil gelatinase associated lipocalin (NGAL), human apolipoprotein D (APO D) and the bilin-binding protein of Pieris brassicae.

[00117] When used herein in the context of the lipocalin muteins of the present disclosure that bind to CD137, the term "specific for" includes that the lipocalin mutein is directed against, binds to, or reacts with CD137, respectively. Thus, being directed to, binding to or reacting with includes that the lipocalin mutein specifically binds to CD137, respectively. The term "specifically" in this context means that the lipocalin mutein reacts with a CD137 protein, as described herein, but essentially not with another protein. The term "another protein" includes any non-CD137 protein, respectively, including proteins closely related to or being homologous to CD137 against which the lipocalins disclosed herein are directed to. However, CD137 proteins, fragments and/or variants from species other than human such as those described in the context of the definition "subject" are not excluded by the term "another protein." The term "does not essentially bind" means that the lipocalin mutein of the present disclosure does not bind another protein, i.e., shows a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6, or 5%. Whether the lipocalin specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of a lipocalin mutein of the present disclosure with CD137 and the reaction of said lipocalin with (an)other protein(s). "Specific binding" can also be determined, for example, with Western blots, ELISA, RIA, ECL, IRMA, FACS, IHC and peptide scans. [00118] The amino acid sequence of a lipocalin mutein according to the disclosure has a high sequence identity to the reference lipocalin, for example hNGAL, as compared to such mutein's sequence identity with another lipocalin (see also above). In this general context, the amino acid sequence of a lipocalin mutein of the combination according to the disclosure is at least substantially similar to the amino acid sequence of the corresponding wild-type or reference lipocalin. A respective sequence of a lipocalin mutein of the combination according to the disclosure, being substantially similar to the sequences of the corresponding reference lipocalin, has 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 to the sequence of the corresponding lipocalin. In this regard, a lipocalin mutein of the disclosure of course may contain, in comparison substitutions as described herein which renders the lipocalin mutein capable of binding to CD137. Typically, a mutein of a lipocalin includes one or more mutations - relative to the sequence of the reference lipocalin - of amino acids in the four loops at the open end of the ligand binding site of the lipocalin (cf. above). As explained above, these regions are essential in determining the binding specificity of a lipocalin mutein for a desired target.

[00119] A mutein of the present disclosure may also contain mutations in regions outside of the four flexible peptide loops that form the target binding site of the lipocalin. For example, a mutein of the present invention may contain one or more mutations in one or more of the three peptide loops (designated BC, DE, and FG) connecting the β-strands at the closed end of the lipocalin. As an illustrative example, a mutein derived from a polypeptide of hNGAL or a homologue 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 arranged at the end of the β-barrel structure that is located opposite to the natural lipocalin binding pocket. As a further illustrative example, a mutein derived from a polypeptide of hNGAL or a homologue thereof, may have no mutated amino acid residues in peptide loop DE arranged at the end of the β-barrel structure, compared to wild-type sequence of hNGAL.

[00120] A lipocalin mutein according to the disclosure includes one or more, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or even 20 substitutions in comparison to the corresponding native lipocalin, provided that such a lipocalin mutein should be capable of binding to CD137. For example, a lipocalin mutein can have a substitution at a position corresponding to a distinct position (i.e., at a corresponding position) of the wild-type lipocalin having the wild-type sequence of, for example, hNGAL. In some embodiments, a lipocalin mutein of the combination according to the disclosure includes at least two amino acid substitutions, including 2, 3, 4, 5, or even more, amino acid substitutions of a native amino acid by an arginine residue. Accordingly, the nucleic acid of a protein "reference protein" scaffold as described herein is subject to mutagenesis with the aim of generating a lipocalin mutein which is capable of binding to CD137.

[00121] Also, a lipocalin mutein of the present disclosure can comprise a heterologous amino acid sequence, such as a Strep-tag II sequence (SEQ ID NO: 23), at its N-or C- Terminus, preferably C-terminus, without affecting the biological activity (binding to its target e.g., CD137) of the lipocalin mutein.

[00122] Likewise, a lipocalin mutein of the present disclosure may lack 1 , 2, 3, 4 or more amino acids at its N-terminal end and/or 1 , 2 or more amino acids at its C-terminal end, in comparison to the respective wild-type hNGAL

[00123] In some embodiments, a substitution (or replacement) is a conservative substitution. Nevertheless, any substitution— including non-conservative substitution or one or more from the exemplary substitutions listed below— is envisaged as long as the lipocalin mutein retains its capability to bind to CD137, and/or it has an identity to the then substituted sequence in that it is at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85 % or higher identical to the "reference sequence".

[00124] Conservative substitutions are generally the following substitutions, listed according to the amino acid to be mutated, each followed by one or more replacement(s) that can be taken to be conservative: Ala→ Gly, Ser, Val; Arg→ Lys; Asn→ Gin, His; Asp→ Glu; Cys→ Ser; Gin→ Asn; Glu→ Asp; Gly→ Ala; His→ Arg, Asn, Gin; lie→ Leu, Val; Leu→ lie, Val; Lys→ Arg, Gin, Glu; Met→ Leu, Tyr, lie; Phe→ Met, Leu, Tyr; Ser→ Thr; Thr → Ser; Trp → Tyr; Tyr → Trp, Phe; Val → lie, Leu. Other substitutions are also permissible and can be determined empirically or in accord with other known conservative or non-conservative substitutions. As a further orientation, the following eight groups each contain amino acids that can typically be taken to define conservative substitutions for one another: a. Alanine (Ala), Glycine (Gly); b. Aspartic acid (Asp), Glutamic acid (Glu); c. Asparagine (Asn), Glutamine (Gin); d. Arginine (Arg), Lysine (Lys); e. Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val); f. Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp); g. Serine (Ser), Threonine (Thr); and h. Cysteine (Cys), Methionine (Met)

[00125] If such substitutions result in a change in biological activity, then more substantial changes, such as the following, or as further described below in reference to amino acid classes, may be introduced and the products screened for a desired characteristic. Examples of such more substantial changes are: Ala→ Leu, lie; Arg→ Gin; Asn→ Asp, Lys, Arg, His; Asp→ Asn; Cys→ Ala; Gin→ Glu; Glu→ Gin; His→ Lys; lie→ Met, Ala, Phe; Leu→ Ala, Met; Lys→ Asn; Met→ Phe; Phe→ Val, lie, Ala; Trp→ Phe; Tyr → Thr, Ser; Val→ Met, Phe, Ala.

[00126] Substantial modifications in the biological properties of the lipocalin are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1 ) hydrophobic: methionine, alanine, valine, leucine, iso-leucine; (2) neutral hydrophilic: cysteine, serine, threonine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues that influence chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine.

[00127] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformation of the respective lipocalin also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond (s) may be added to the lipocalin to improve its stability.

[00128] Any mutation, including an insertion as discussed above, can be accomplished very easily on the nucleic acid, e.g., DNA level using established standard methods. Illustrative examples of alterations of the amino acid sequence are insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e., an amino acid residue is replaced with an amino acid residue of chemically similar properties, in particular with regard to polarity as well as size. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) iso-leucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. On the other hand, it is also possible to introduce non-conservative alterations in the amino acid sequence. In addition, instead of replacing single amino acid residues, it is also possible to either insert or delete one or more continuous amino acids of the primary structure of tear lipocalin as long as these deletions or insertion result in a stable folded/functional mutein.

[00129] Modifications of the amino acid sequence include directed mutagenesis of single amino acid positions in order to simplify sub-cloning of the mutated lipocalin gene or its parts by incorporating cleavage sites for certain restriction enzymes. In addition, these mutations can also be incorporated to further improve the affinity of a lipocalin mutein for a given target such as CD137. Furthermore, mutations can be introduced in order to modulate certain characteristics of the mutein such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary. For example, naturally occurring cysteine residues may be mutated to other amino acids to prevent disulphide bridge formation.

[00130] It is also possible to deliberately mutate other amino acid sequence positions to cysteine in order to introduce new reactive groups, for example for the conjugation to other compounds, such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for the formation of non-naturally occurring disulphide linkages. The generated thiol moiety may be used to PEGylate or HESylate the mutein, for example, in order to increase the serum half-life of a respective lipocalin mutein.

[00131] With respect to a mutein of human lipocalin 2, exemplary possibilities of such a mutation to introduce a cysteine residue into the amino acid sequence of a lipocalin including human lipocalin 2 mutein to include the introduction of a cysteine (Cys) residue at least at one of the sequence positions that correspond to sequence positions 14, 21 , 60, 84, 88, 1 16, 141 , 145, 143, 146 or 158 of the wild type sequence of human NGAL. In some embodiments where a human lipocalin 2 mutein of the disclosure has a sequence in which, in comparison to the sequence of the SWISS-PROT/UniProt Data Bank Accession Number P80188, a cysteine has been replaced by another amino acid residue, the corresponding cysteine may be reintroduced into the sequence. As an illustrative example, a cysteine residue at amino acid position 87 may be introduced in such a case by reverting to a cysteine as originally present in the sequence of SWISS-PROT accession No. P80188. The generated thiol moiety at the side of any of the amino acid positions 14, 21 , 60, 84, 88, 1 16, 141 , 145, 143, 146 and/or 158 may be used to PEGylate or HESylate the mutein, for example, in order to increase the serum half-life of a respective human lipocalin 2 mutein.

[00132] A lipocalin mutein disclosed herein may also be a mutein of human tear lipocalin (TLPC or Tic), also termed lipocalin-1 , tear pre-albumin or von Ebner gland protein. The term "human tear lipocalin" or "Tic" or "lipocalin-1 " as used herein refers to the mature human tear lipocalin with the SWISS-PROT/UniProt Data Bank Accession Number P31025 (Isoform 1 ). The amino acid sequence is shown in shown in SEQ ID NO: 1.

[00133] A mutein of human tear lipocalin may include the wild-type (natural) amino acid sequence of the "parental" wild-type human tear lipocalin outside the mutated amino acid sequence positions. A mutein of human tear lipocalin according to the disclosure may also carry one or more amino acid mutations at a sequence position/ positions as long as such a mutation does, at least essentially not hamper or not interfere with the binding activity and the folding of the mutein. Such mutations can be accomplished very easily on DNA level using established standard methods (Sambrook, J. et al. (2001 ) Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Illustrative examples of alterations of the amino acid sequence are insertions or deletions as well as amino acid substitutions. Such substitutions may be conservative, i.e. an amino acid residue is replaced with an amino acid residue of chemically similar properties, in particular with regard to polarity as well as size. Examples of conservative substitutions are the replacements among the members of the following groups: 1 ) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) iso-leucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan. On the other hand, it is also possible to introduce non-conservative alterations in the amino acid sequence. In addition, instead of replacing single amino acid residues, it is also possible to either insert or delete one or more continuous amino acids of the primary structure of the human tear lipocalin as long as these deletions or insertion result in a stable folded/functional mutein (for example, Tic muteins with truncated N- and C-terminus). In such mutein, for instance, one or more amino acid residues are added or deleted at the N- or C- terminus of the polypeptide. As an illustrative example, the first four N-terminal amino acid residues of the sequence of mature human tear lipocalin (His-His-Leu-Leu; positions 1 -4) and/or the last two C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of the mature human tear lipocalin can been deleted. Generally such a mutein may have about at least 70%, including at least about 80%, such as at least about 85% amino acid sequence identity, with the amino acid sequence of the mature human tear lipocalin. As an illustrative example, the present disclosure also encompasses Tic muteins as defined above, in which the first four N-terminal amino acid residues of the sequence of mature human tear lipocalin (His-His-Leu-Leu; positions 1 -4) and/or the last two C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of the mature human tear lipocalin have been deleted (SEQ ID NOs: 5-1 1 ). [00134] In some embodiments, if one of the above moieties is conjugated to a lipocalin mutein of the disclosure, conjugation to an amino acid side chain can be advantageous. Suitable amino acid side chains may occur naturally in the amino acid sequence of a human lipocalin or may be introduced by mutagenesis. In case a suitable binding site is introduced via mutagenesis, one possibility is the replacement of an amino acid at the appropriate position by a cysteine residue.

[00135] In another embodiment, in order to provide suitable amino acid side chains for conjugating one of the above compounds to a lipocalin mutein according to the present disclosure, artificial amino acids may be introduced by mutagenesis. Generally, such artificial amino acids are designed to be more reactive and thus to facilitate the conjugation to the desired compound. One example of such an artificial amino acid that may be introduced via an artificial tRNA is para-acetyl-phenylalanine.

[00136] For several applications of the muteins disclosed herein it may be advantageous to use them in the form of fusion polypeptides. In some embodiments, a lipocalin mutein of the disclosure is fused at its N-terminus or its C-terminus to a protein, a protein domain or a peptide, for instance, an antibody, a signal sequence and/or an affinity tag.

[00137] Affinity tags such as the Strep-tag or Strep-tag II (Schmidt et al., 1996), the c- myc-tag, the FLAG-tag, the His-tag or the HA-tag or proteins such as glutathione-S- transferase, which allow easy detection and/or purification of recombinant proteins, are further examples of suitable fusion partners. Finally, proteins with chromogenic or fluorescent properties such as the green fluorescent protein (GFP) or the yellow fluorescent protein (YFP) are suitable fusion partners for lipocalin muteins of the disclosure as well.

[00138] In general, it is possible to label the lipocalin muteins of the disclosure with any appropriate chemical substance or enzyme, which directly or indirectly generates a detectable compound or signal in a chemical, physical, optical, or enzymatic reaction. An example for a physical reaction and at the same time optical reaction/marker is the emission of fluorescence upon irradiation or the emission of x-rays when using a radioactive label. Alkaline phosphatase, horseradish peroxidase and β-galactosidase are examples of enzyme labels (and at the same time optical labels) which catalyze the formation of chromogenic reaction products. In general, all labels commonly used for antibodies (except those exclusively used with the sugar moiety in the Fc part of immunoglobulins) can also be used for conjugation to the lipocalin muteins of the disclosure. The lipocalin muteins of the disclosure may also be conjugated with any suitable therapeutically active agent, e.g., for the targeted delivery of such agents to a given cell, tissue or organ or for the selective targeting of cells (e.g., tumor cells) without affecting the surrounding normal cells. Examples of such therapeutically active agents include radionuclides, toxins, small organic molecules, and therapeutic peptides (such as peptides acting as agonists/antagonists of a cell surface receptor or peptides competing for a protein binding site on a given cellular target). The lipocalin muteins of the disclosure may, however, also be conjugated with therapeutically active nucleic acids such as antisense nucleic acid molecules, small interfering RNAs, micro RNAs or ribozymes. Such conjugates can be produced by methods well known in the art.

[00139] As indicated above, a lipocalin mutein of the disclosure may in some embodiments be conjugated to a moiety that extends the serum half-life of the mutein (in this regard see also International Patent Publication No. WO 2006/056464, where such conjugation strategies are described with reference to muteins of human neutrophil gelatinase-associated lipocalin (hNGAL) with binding affinity for CTLA-4). The moiety that extends the serum half-life may be a polyalkylene glycol molecule, hydroxyethyl starch, fatty acid molecules, such as palmitic acid (Vajo and Duckworth, 2000), an Fc part of an immunoglobulin, a C_H3 domain of an immunoglobulin, a C_H4 domain of an immunoglobulin, , an albumin binding peptide, or an albumin binding protein, transferrin to name only a few. The albumin binding protein may be a bacterial albumin binding protein, an antibody, an antibody fragment including domain antibodies (e.g., US Patent No. 6,696,245), or a lipocalin mutein with binding activity for albumin. Accordingly, suitable conjugation partners for extending the half-life of a lipocalin mutein of the disclosure include an albumin binding protein, for example, a bacterial albumin binding domain, such as the one of streptococcal protein G (Konig and Skerra, 1998). Other examples of albumin binding peptides that can be used as conjugation partner are, for instance, those having a Cys-Xaai-Xaa2-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaai is Asp, Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gin, His, lie, Leu, or Lys; Xaa₃ is Ala, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thr as described in U.S. Patent Publication No. 20030069395 or Dennis et al. (2002).

[00140] In other embodiments, albumin itself (Osborn et al., 2002), or a biologically fragment of albumin can be used as conjugation partner of a lipocalin mutein of the disclosure. The term "albumin" includes all mammal albumins such as human serum albumin or bovine serum albumin or rat albumin. The albumin or fragment thereof can be recombinantly produced as described in U.S. Patent No. 5,728,553 or European Patent Publication Nos. EP0330451 and EP0361991. Recombinant human albumin (e.g., Recombumin® from Novozymes Delta Ltd., Nottingham, UK) can be conjugated or fused to a lipocalin mutein of the disclosure in order to extend the half-life of the mutein.

[00141] If the albumin-binding protein is an antibody fragment it may be a domain antibody. Domain Antibodies (dAbs) are engineered to allow precise control over biophysical properties and in vivo half-life to create the optimal safety and efficacy product profile. Domain Antibodies are for example commercially available from Domantis Ltd. (Cambridge, UK and MA, USA).

[00142] If a transferrin is used as a moiety to extend the serum half-life of the lipocalin muteins of the disclosure, the muteins can be genetically fused to the N or C terminus, or both, 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 provides high bioavailability, biodistribution, and circulating stability. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, 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).

[00143] If an Fc part of an immunoglobulin is used for the purpose to prolong the serum half-life of the lipocalin muteins of the disclosure, the SynFusion™ technology, commercially available from Syntonix Pharmaceuticals, Inc (MA, USA), may be used. The use of this Fc-fusion technology allows the creation of longer-acting biopharmaceuticals and may for example consist of two copies of the mutein linked to the Fc region of an antibody to improve pharmacokinetics, solubility, and production efficiency.

[00144] Yet another alternative to prolong the half-life of the lipocalin muteins of the disclosure is to fuse to the N-or C-terminus of a mutein a long, unstructured, flexible glycine- rich sequences (for example poly-glycine with about 20 to 80 consecutive glycine residues). This approach disclosed in International Patent Publication No. WO2007/038619, for example, has also been term "rPEG" (recombinant PEG).

[00145] If PEG is used as conjugation partner, the polyalkylene glycol can be substituted, unsubstituted, linear, or branched. It can also be an activated polyalkylene derivative. Examples of suitable compounds are PEG molecules as described in International Patent Publication No. WO 1999/64016, in U.S. Patent No. 6,177,074 or in U.S. Patent No. 6,403,564 in relation to interferon, or as described for other proteins such as PEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (Fuertges and Abuchowski, 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, of about 20,000, of about 30,000 or of about 40,000 Daltons. Moreover, as e.g., described in U.S. Patents No. 6,500,930 or 6,620,413, carbohydrate oligomers and polymers such as starch or hydroxyethyl starch (HES) can be conjugated to a mutein of the disclosure for the purpose of serum half-life extension.

[00146] In addition, a lipocalin mutein disclosed herein may be fused to a moiety may confer new characteristics to the lipocalin muteins of the disclosure such as enzymatic activity or binding affinity for other targets. Examples of suitable fusion partners are alkaline phosphatase, horseradish peroxidase, glutathione-S-transferase, the albumin-binding domain of protein G, protein A, antibodies fragments, oligomerization domains, or toxins.

[00147] In particular, it may be possible to fuse a lipocalin mutein disclosed herein with a separate enzyme active site such that both "components" of the resulting fusion polypeptide together act on a given therapeutic target. The binding domain of the lipocalin mutein attaches to the disease-causing target, allowing the enzyme domain to abolish the biological function of the target.

[00148] The present disclosure also relates to nucleic acid molecules (DNA and RNA) that include nucleotide sequences encoding the lipocalin muteins of the disclosure. Since the degeneracy of the genetic code permits substitutions of certain codons by other codons specifying the same amino acid, the disclosure is not limited to a specific nucleic acid molecule encoding a lipocalin mutein as described herein but encompasses all nucleic acid molecules that include nucleotide sequences encoding a functional mutein. In this regard, the present disclosure provides nucleotide sequences encoding some lipocalin muteins of the disclosure as shown in SEQ ID NOs: 24-39 and 57-66.

[00149] In one embodiment of the disclosure, the method includes subjecting the nucleic acid molecule to mutagenesis at nucleotide triplets coding for at least one, or even more, of the sequence positions corresponding to the sequence 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/or 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00150] In one embodiment of the disclosure, the method includes subjecting the nucleic acid molecule to mutagenesis at nucleotide triplets coding for at least one, or even more, of the sequence positions corresponding to the sequence 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 polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00151] The disclosure also includes nucleic acid molecules encoding the lipocalin muteins of the disclosure, which include additional mutations outside the indicated sequence positions of experimental mutagenesis. Such mutations are often tolerated or can even prove to be advantageous, for example if they contribute to an improved folding efficiency, serum stability, thermal stability, formulation stability, or ligand binding affinity of the muteins.

[00152] A nucleic acid molecule disclosed in this application may be "operably linked" to one or more regulatory sequence(s) to allow expression of this nucleic acid molecule.

[00153] A nucleic acid molecule, such as DNA, is referred to as "capable of expressing a nucleic acid molecule" or capable "to allow expression of a nucleotide sequence" if it includes sequence elements which contain information regarding to transcriptional and/or translational regulation, and such sequences are "operably linked" to the nucleotide sequence encoding the polypeptide. An operable linkage is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions include a promoter, which, in prokaryotes, contains both the promoter per se, i.e., DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35/-10 boxes and the Shine- Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.

[00154] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.

[00155] Therefore, a nucleic acid molecule of the disclosure can include a regulatory sequence, such as a promoter sequence. In some embodiments, a nucleic acid molecule of the disclosure includes a promoter sequence and a transcriptional termination sequence. Suitable prokaryotic promoters are, for example, the tet promoter, the /acUV5 promoter or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

[00156] The nucleic acid molecules of the disclosure can also be part of a vector or any other kind of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid or an artificial chromosome.

[00157] In one embodiment, the nucleic acid molecule is included in a phasmid. A phasmid vector denotes a vector encoding the intergenic region of a temperent phage, such as M 13 or f1 , or a functional part thereof fused to the cDNA of interest. After superinfection of the bacterial host cells with such an phagemid vector and an appropriate helper phage (e.g., M13K07, VCS-M13 or R408) intact phage particles are produced, thereby enabling physical coupling of the encoded heterologous cDNA to its corresponding polypeptide displayed on the phage surface (Lowman, 1997, Rodi and Makowski, 1999).

[00158] Such cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a lipocalin mutein as described herein, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art, and are commercially available.

[00159] The DNA molecule encoding a lipocalin mutein as described herein, and in particular a cloning vector containing the coding sequence of such a mutein can be transformed into a host cell capable of expressing the gene. Transformation can be performed using standard techniques. Thus, the disclosure is also directed to a host cell containing a nucleic acid molecule as disclosed herein.

[00160] The transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a fusion polypeptide of the disclosure. Suitable host cells can be prokaryotic, such as Escherichia coii (£. coii) 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.

[00161] The disclosure also relates to a method for the production of a lipocalin mutein as described herein, wherein the mutein, a fragment of the mutein or a fusion polypeptide of the mutein and another polypeptide (e.g., another lipocalin mutein or antibody or antibody fragment) is produced starting from the nucleic acid coding for the mutein by means of genetic engineering methods. The method can be carried out in vivo, the lipocalin mutein can for example be produced in a bacterial or eukaryotic host organism and then isolated from this host organism or its culture. It is also possible to produce a protein in vitro, for example by use of an in vitro translation system.

[00162] When producing the lipocalin mutein in vivo a nucleic acid encoding such mutein is introduced into a suitable bacterial or eukaryotic host organism by means of recombinant DNA technology (as already outlined above). For this purpose, the host cell is first transformed with a cloning vector that includes a nucleic acid molecule encoding a lipocalin mutein as described herein using established standard methods. The host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or from the cultivation medium.

[00163] In some embodiments, a nucleic acid molecule, such as DNA, disclosed in this application may be "operably linked" to another nucleic acid molecule of the disclosure to allow expression of a fusion polypeptide of the disclosure. In this regard, an operable linkage is a linkage in which the sequence elements of the first nucleic acid molecule and the sequence elements of the second nucleic acid molecule are connected in a way that enables expression of the fusion polypeptide as a single polypeptide.

[00164] In addition, in some embodiments, the naturally occurring disulfide bond between Cys 76 and Cys 175 may be removed in hNGAL muteins of the disclosure. Accordingly, such muteins can be produced in a cell compartment having a reducing redox milieu, for example, in the cytoplasm of Gram-negative bacteria.

[00165] In case a lipocalin mutein of the disclosure includes intramolecular disulfide bonds, it may be preferred to direct the nascent polypeptide to a cell compartment having an oxidizing redox milieu using an appropriate signal sequence. Such an oxidizing environment may be provided by the periplasm of Gram-negative bacteria such as E. coli, in the extracellular milieu of Gram-positive bacteria or in the lumen of the endoplasmic reticulum of eukaryotic cells and usually favors the formation of structural disulfide bonds.

[00166] It is, however, also possible to produce a mutein of the disclosure in the cytosol of a host cell, preferably E. coli. In this case, the polypeptide can either be directly obtained in a soluble and folded state or recovered in form of inclusion bodies, followed by renaturation in vitro. A further option is the use of specific host strains having an oxidizing intracellular milieu, which may thus allow the formation of disulfide bonds in the cytosol (Venturi et al., 2002).

[00167] However, a lipocalin mutein as described herein may not necessarily be generated or produced only by use of genetic engineering. Rather, such a mutein can also be obtained by chemical synthesis such as Merrifield solid phase polypeptide synthesis or by in vitro transcription and translation. It is for example possible that promising mutations are identified using molecular modeling, polypeptides continuing such mutations synthesized in vitro, and investigated for binding activity with respect to CD137 and other desirable properties (such as stability). Methods for the solid phase and/or solution phase synthesis of polypeptides/proteins are well known in the art (see e.g., Bruckdorfer et al., 2004). [00168] In another embodiment, the lipocalin muteins of the disclosure may be produced by in vitro transcription/translation employing well-established methods known to those skilled in the art.

[00169] The skilled worker will appreciate methods useful to prepare lipocalin muteins contemplated by the present disclosure but whose protein or nucleic acid sequences are not explicitly disclosed herein. As an overview, such modifications of the amino acid sequence include, e.g., directed mutagenesis of single amino acid positions in order to simplify sub- cloning of a mutated lipocalin gene or its parts by incorporating cleavage sites for certain restriction enzymes. In addition, these mutations can also be incorporated to further improve the affinity of a lipocalin mutein for its target (e.g., CD137). Furthermore, mutations can be introduced to modulate certain characteristics of the mutein such as to improve folding stability, serum stability, protein resistance or water solubility or to reduce aggregation tendency, if necessary. For example, naturally occurring cysteine residues may be mutated to other amino acids to prevent disulphide bridge formation.

[00170] The lipocalin muteins disclosed herein and its derivatives can be used in many fields similar to antibodies or fragments thereof. For example, the lipocalin muteins can be used for labelling with an enzyme, an antibody, a radioactive substance or any other group having biochemical activity or defined binding characteristics. By doing so, their respective targets or conjugates or fusion polypeptides thereof can be detected or brought in contact with them. In addition, lipocalin muteins of the disclosure can serve to detect chemical structures by means of established analytical methods (e.g., ELISA or Western Blot) or by microscopy or immunosensorics. In this regard, the detection signal can either be generated directly by use of a suitable mutein conjugate or fusion polypeptide or indirectly by immunochemical detection of the bound mutein via an antibody.

[00171] Additional objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present disclosure is specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure.

[00172] The invention is further characterized by following items.

[00173] Item 1. A lipocalin mutein capable of binding human CD137 with an affinity by a _d of about 700 nM or lower and cynomolgus monkey CD137 with an affinity by a _d of about 20 nM or lower, wherein said mutein is capable of interfering with the binding of CD137 to CD137 ligand.

[00174] Item 2. The mutein of item 1 , wherein the capability of interfering with the binding of the CD137 ligand to CD137 is analyzed by surface plasmon resonance or any other applicable method, such as FACS or ELISA.

[00175] Item 3. The lipocalin mutein of item 1 , wherein the mutein is capable of binding human CD137 with an affinity by a K_d of about 30 nM or lower.

[00176] Item 4. The lipocalin mutein of item 1 , wherein the mutein is capable of binding human CD137 with an affinity by a K_d of about 8 nM or lower.

[00177] Item 5. The lipocalin mutein of item 1 , wherein the mutein is capable of binding cynomolgus monkey CD137 with an affinity by a Κ_ά of about 5 nM or lower.

[00178] Item 6. The lipocalin mutein of item 1 , wherein the mutein is capable of binding cynomolgus monkey CD137 with an affinity by a K_d of about 2 nM or lower.

[00179] Item 7. A lipocalin mutein as in one of items 1 to 6, wherein the said K_d values are determined by surface plasmon resonance.

[00180] Item 8. The lipocalin mutein of item 1 , wherein the mutein is capable of binding human CD137 with an EC₅₀ value of about 1000 nM or lower.

[00181] Item 9. The lipocalin mutein of item 1 , wherein the mutein is capable of binding cynomolgus monkey CD137 with an EC₅₀ value of about 90 nM or lower.

[00182] Item 10. The lipocalin mutein of item 8 or 9, wherein the said EC₅₀ values are determined by fluorescence-activated cell sorting analysis.

[00183] Item 1 1. A lipocalin mutein as in any of the preceding itemscomprising at least two or more mutated amino acid residues at sequence 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/or 134 in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00184] Item 12. The lipocalin mutein of item 1 1 , comprising at least one or more mutated amino acid residues at sequence positions 36, 40, 41 , 49, 52, 68, 70, 72, 73, 77, 79, 81 , 96, 100, 103, 125, 127, 132, and 134 in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00185] Item 13. The lipocalin mutein of item 1 1 , further comprising at least one or more mutated amino acid residues at sequence positions 20, 25, 33, 44, 59, 71 , 78, 80, 82, 87, 92, 98, 101 , and 122 in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[00186] Item 14. A lipocalin mutein as in any of the preceding items, wherein the amino acid sequence of the mutein comprises at least one or more of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) Gin 20→ Arg; Asn 25→ Tyr or Asp; Val 33→ lie; Leu 36→Met; Ala 40→ Asn; lie 41→ Leu; Glu 44→ Val or Asp; Gin 49→ His; Tyr 52→Ser or Giy; Lys 59→ Asn; Ser 68→ Asp; Leu 70→ Met; Phe 71→ Leu; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin or His; Tyr 78→ His; Trp 79→ lie; lie 80→ Asn; Arg 81 → Trp or Gin; Thr 82→ Pro; 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→ lie; Tyr 132→ Trp; and Lys 134 → Gly.

[00187] Item 15. A lipocalin mutein as in any of the preceding items, wherein the amino acid sequence of the mutein comprises at least one or more of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Leu 36→Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→Ser or Gly; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin or His; Trp 79→ lie; Arg 81 → Trp or Gin; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His or Pro; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; and Lys 134→ Gly.

[00188] Item 16. A lipocalin mutein as in any of the preceding items, wherein the amino acid sequence of the mutein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Leu 36→Met; Ala 40→ Asn; lie 41 → Leu; Gin 49→ His; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Trp 79→ lie; Asn 96→ Phe; Tyr 100→ Asp; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; and Lys 134 → Gly.

[00189] Item 17. A lipocalin mutein as in any of the preceding items, wherein the amino acid sequence of the mutein comprises at least 1 , 2, 3, or 4 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Tyr 52→ Ser or Gly; Asp 77→ Gin or His; Arg 81→ Trp or Gin; Leu 103 — > His or Pro;

[00190] Item 18. A lipocalin mutein as in any of the preceding items, wherein the amino acid sequence of the mutein comprises at least one or more of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gin 20→ Arg; Asn 25→ Tyr or Asp; Val 33→ lie; Glu 44→ Val or Asp; Lys 59→ Asn; Phe 71→ Leu; Tyr 78→ His; lie 80→ Asn; Thr 82→ Pro; Phe 92→ Leu or Ser; Lys 98→ Arg; Pro 101→ Leu; and Phe 122→ Tyr.

[00191] Item 19. A lipocalin mutein as in any of the preceding items, wherein the amino acid sequence of the mutein comprises one of the following sets of amino acid substitutions in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID

NO: 2):

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

(b) . Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; 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→ lie; Tyr 132→ Trp; Lys 134→ Gly;

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

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

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

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

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

(h) . Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser 68→ Asp; Leu 70→ Met; Arg 72 Leu; Lys 73 Asp; Asp 77 Gin; Trp 79→ lie; lie 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 lie; Tyr 132→ Trp; Lys 134 Gly;

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

(j). Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Glu 44→ Asp; Gin 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→ lie; Arg 81→ Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125→ Ser; Ser 127 lie; Tyr 132→ Trp; Lys 134→ Gly.

[00192] Item 20. A lipocalin mutein as in any of the preceding items, wherein the amino acid sequence of the mutein comprises 1 or 2 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gin 28→ His and Cys 87→ Ser.

[00193] Item 21 . A lipocalin mutein as in any of the preceding items, wherein the mutein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-56 and a fragment or variant thereof.

[00194] Item 22. A lipocalin mutein as in any of the preceding items, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-56 and a fragment or variant thereof.

[00195] Item 23. A lipocalin mutein as in any of the preceding items, wherein the mutein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[00196] Item 24. A lipocalin mutein as in any of the preceding items, wherein the mutein is conjugated to a compound selected from the group consisting of an organic molecule, an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a cytostatic agent, a toxin, a metal complex, a metal, and colloidal gold.

[00197] Item 25. A lipocalin mutein as in any of the preceding items, wherein the mutein is fused at its N- terminus and/or its C-terminus to a fusion partner which is a protein, a protein domain, or a peptide.

[00198] Item 26. A lipocalin mutein as in any of the preceding items, wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein.

[00199] Item 27. The lipocalin mutein of 26, wherein the compound that extends the serum half-life is selected from the group consisting of a polyalkylene glycol molecule, hydroethylstarch, a Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein. [00200] Item 28. The lipocalin mutein of item 27, wherein the polyalkylene glycol is polyethylene (PEG) or an activated derivative thereof.

[00201] Item 29. A nucleic acid molecule comprising a nucleotide sequence encoding a lipocalin mutein as in any of the preceding items.

[00202] Item 30. The nucleic acid molecule of item 29, wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of said nucleic acid molecule.

[00203] Item 31 . An expression vector comprising the nucleic acid molecule of item 29 or 30.

[00204] Item 32. A host cell containing a nucleic acid molecule of item 29 or 30.

[00205] Item 33. A method of producing a lipocalin mutein according to any one of items 1 to 28, wherein the mutein is produced starting from the nucleic acid coding for the mutein or fragment thereof by means of genetic engineering methods.

[00206] Item 34. The method of item 33, wherein the mutein is produced in a bacterial or eukaryotic host organism and is isolated from this host organism or its culture.

[00207] Item 35. A method for treating a subject having cancer, inflammatory diseases or autoimmune diseases, comprising administering an effective amount of one or more lipocalin muteins of any one of items 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, or one or more compositions comprising such muteins.

[00208] Item 36. The method for treating a subject having cancers or inflammatory diseases or autoimmune diseases of item 35, wherein the lipocalin muteins are applied to bind CD137 and/or activate the downstream signaling pathway of CD137.

[00209] Item 37. A method of stimulating immune response in a subject, comprising administering an effective amount of one or more lipocalin muteins of any one of items 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, or one or more compositions comprising such muteins.

[00210] Item 38. The method of stimulating immune response in a subject of item 37, wherein the immune response comprises T lymphocyte proliferation and/or reduced production of proinflammatory cytokines and chemokines.

[00211] Item 39. A method of antagonizing CD137 in a subject, comprising administering an effective amount of one or more lipocalin muteins of any one of items 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, or one or more compositions comprising such muteins.

[00212] Item 40. The method of antagonizing CD137 in a subject of item 39, wherein the lipocalin muteins are applied to interfere with the binding of CD137 ligand to CD137.

[00213] Item 41 . A method of detecting the presence of CD137, comprising:

(a) , contacting a lipocalin mutein according to any one of the items 1 -28 with a test sample suspected to contain CD137 under suitable conditions, thereby allowing the formation of a complex between the mutein and CD137;

(b) . detecting the complex of the mutein and CD137 by a suitable signal.

[00214] Item 42. A method for the separation of CD137, comprising:

(a) , contacting a lipocalin mutein according to any one of the items 1 -28 with a test sample under suitable conditions, thereby allowing the formation of a complex between the mutein and CD137;

(b) . separating the complex of the mutein and CD137 from tested sample.

[00215] Item 43. The method of item 41 or 42, wherein the test sample 7 is a biological sample.

[00216] Item 44. The method of any one of items 43, wherein the biological sample is isolated from a human.

[00217] Item 45. The method of any one of items 43, wherein the biological sample comprises body fluid.

[00218] Item 46. A pharmaceutical composition comprising a lipocalin mutein of any one of items 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, and pharmaceutically acceptable excipient.

[00219] Item 47. A diagnostic or analytical kit comprising a lipocalin mutein of any one of items 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof.

[00220] Item 48. The method for treating cancers or inflammatory diseases or autoimmune diseases of item 35, wherein the cancers to be treated are selected from the group consisting of ovarian, colon, breast, lung cancers, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias, B-cell derived leukemias, T-cell derived leukemias, B-cell derived lymphomas, T-cell derived lymphomas, mast cell derived tumors, and combinations thereof.

[00221] Item 49. The method for treating cancers or inflammatory diseases or autoimmune diseases of item 35, wherein the autoimmune diseases or inflammatory diseases are selected from the group consisting of intestinal mucosal inflammation, wasting disease associated with colitis, multiple sclerosis, systemic lupus erythematosus, viral infections, rheumatoid arthritis, osteoarthritis, psoriasis, Conn's disease, and inflammatory bowel disease.

[00222] Item 50. The use of a lipocalin mutein according to any one of items 1 -28 for the treatment of autoimmune disease.

V. EXAMPLES

[00223] Example 1 : Selection and optimization of muteins specifically binding to CD137

[00224] The representative CD137-specific lipocalin muteins disclosed in this application were selected from naive mutant libraries based on hNGAL. Different strategies and targets were employed to obtain CD137-binding muteins. Recombinant targets utilized were the commercially available Fc-fusion of the full extracellular domain of CD137 from human (huCD137-Fc, R&D Systems 838-4B) and individual subdomains of human CD137, all generated as fusions to the human Fc fragment. As an alternative non-Fc fused target we employed the His-tagged human CD137 extracellular domain (Invitrogen, 10041 -H08H-250). Alternatively, a cell-based panning using CHO cells transfected with the full cDNA of human CD137 was employed. Protein- and cell-based pannings were performed using standard procedures. The clones obtained after selection were subjected to a screening process as described in Example 2. For optimization of these CD137-specific muteins, so-called maturation libraries were generated using either a parsimonious randomization of selected positions or error prone polymerase chain reaction (PCR) based methods.

[00225] Example 2: Identification of muteins specifically binding to CD137 using high-throughput ELISA screening

[00226] Individual lipocalin muteins fused to a C-terminal Strep-tag (SEQ ID NO: 23, cf. Example 3) were used to inoculate 2xYT/Amp medium and grown overnight (14-18 h) to stationary phase. Subsequently, 50 μΙ_ 2xYT/Amp were inoculated from the stationary phase cultures and incubated for 3 h at 37°C and then shifted to 22°C until an OD₅₉₅ of 0.6-0.8 was reached. Production of muteins was induced by addition of 10 μΙ_ 2xYT/Amp supplemented with 1.2 μg ml anhydrotetracyclin. Cultures were incubated at 22°C until the next day. After addition of 40 μΙ_ of 5% (w/v) BSA in PBS/T and incubation for 1 h at 25°C cultures were ready for use in screening assays.

[00227] Binding of the isolated muteins to human CD137 was tested by coating huCD137-Fc (5 μg ml in PBS) of the relevant species overnight at 4°C on microtiterplates. After blocking the plate with PBST containing 2% BSA, 20 μΙ_ of BSA-blocked cultures were added to the microtiter plates and incubated for 1 h at 25°C. Bound muteins were detected with anti-StrepTag antibody conjugated with horseradish peroxidase (1 h incubation; IBA, Goettingen). For quantification, 20 μΙ_ of QuantaBlu fluorogenic peroxidase substrate were added and the resulting fluorescence was determined at an excitation wavelength of 330 nM and an emission wavelength of 420 nM.

[00228] To select for muteins with increased temperature resistance, BSA-blocked cultures were incubated for 1 h at 60°C and then allowed to cool down to room temperature before adding them to CD137 coated and BSA-blocked microtiterplates as described in the previous paragraph. The muteins were subsequently processed as described in the previous paragraph and were selected for bacterial expression, purification, and further characterization.

[00229] Example 3: Expression of muteins

[00230] Unique muteins were expressed with C-terminal tag SAWSHPQFEK (SEQ ID NO: 21 ) or PSAWSHPQFEK (SEQ ID NO: 22); including an SA or PSA linker and Strep-tag® II, WSHPQFEK (SEQ ID NO: 23) in E. coli in 2YT-Amp medium to purify the muteins after expression using Streptactin affinity chromatography and preparative size exclusion chromatography. Finally, lipocalin muteins were subjected to an endotoxin depletion step utilizing Mustang E columns. Purified lipocalin muteins were then characterized as detailed in all following examples.

[00231] Example 4: Affinity of muteins binding to human CD137-Fc fusion protein determined by surface plasmon resonance (SPR)

[00232] Surface plasmon resonance (SPR) was used to measure binding kinetics and affinity of the representative lipocalin muteins disclosed herein.

[00233] SPR analysis of the binding of the representative muteins to human CD137- Fc fusion protein (huCD137-Fc) was performed at 37°C on a Biacore T200 instrument (GE Healthcare) using HBS-EP+ (1 x; BR-1006-69; GE Healthcare) as running buffer.

[00234] Prior to the protein measurements three regeneration cycles were performed for conditioning purposes. Regeneration of the derivatized chip surface was achieved by applying 3M MgCI₂ for 60 s followed by 10 mM glycine, pH 1 .7 for 180 s. Anti-human IgG-Fc antibody was utilized to immobilize huCD137-Fc in a subsequent step and taken from the human antibody capture kit (GE Healthcare, BR-1008-39). It was immobilized on a CM5 sensor chip using standard amine coupling chemistry and the immobilization buffer included in the kit (10 mM sodium acetate pH 5.0), resulting in a ligand density of about 13000 resonance units (RU). The reference channel was treated accordingly.

[00235] HuCD137-Fc at a concentration of 0.5 μg mL was captured on this surface for 180 s at a flow rate of 10 μΙ_Ληίη in HBS-EP+ buffer. No target protein was applied to the reference channel. Subsequently, the lipocalin muteins were applied in an appropriate dilution series in HBS-EP+ buffer at a flow rate of 30 μΙ_/η"ΐίη. Regeneration of the derivatized chip surface was achieved as described above. Data were evaluated with Biacore T200 Evaluation software (V 2.0). Double referencing was used and the 1 :1 Binding model was used to fit the raw data.

[00236] Figure 1 shows the SPR traces and fit curves determined for the lipocalin muteins tested, with the corresponding SEQ ID NOs provided in the graphs. The data is depicted for the binding to huCD137-Fc. There are clear SPR binding signals towards the human target, while the negative controls SEQ ID NO: 3 and SEQ ID NO: 4 exhibit no binding. The affinities resulting from a fit of this data are provided in Table 1 below.

[00237] Table 1 :

K_d huCD137

SEQ ID AA [nM]

SEQ ID NO 5 162

SEQ ID NO 6 1 12

SEQ ID NO 7 1 10

SEQ ID NO 8 151

SEQ ID NO 9 209

SEQ ID NO 10 1 12

SEQ ID NO 1 1 269

SEQ ID NO 12 36

SEQ ID NO 13 2

SEQ ID NO 14 9

SEQ ID NO 15 23

SEQ ID NO 16 30

SEQ ID NO 17 50

SEQ ID NO 18 77

SEQ ID NO 19 98

SEQ ID NO 20 138

SEQ ID NO 3 (Ctrl) not binding

SEQ ID NO 4 (Ctrl) not binding

[00238] Example 5: Surface plasmon resonance (SPR) assay to determine competition between human CD137L and muteins in binding to human CD137-Fc fusion protein [00239] With respect to a lipocalin mutein described in this application that binds CD137, generally, two modes of binding are possible: in the first case, the mutein's binding site overlaps with the binding site of human CD137 ligand (CD137L) to CD137. When such lipocalin mutein binds to CD137, this interferes with binding of CD137L to CD137 and concomitantly leads to interference with natural CD137L signaling ("competitive binding"); in the second case, the mutein's binding site does not overlap with the CD137L's binding site and such lipocalin mutein can bind to CD137 without interfering with CD137L binding and natural CD137L signaling ("non-competitive binding").

[00240] Clustering of CD137 via its ligand activates the downstream signaling pathways of CD137. In the case of T-cells, CD137 activation leads to costimulation of the T- cell^'s activatory responses, such as proliferation and the production of proinflammatory cytokines.

[00241] Another way to induce CD137 clustering is to use immobilized CD137-binding agents. When coated on the plate (e.g., on a plastic culture dish and incubating the T-cells in the dish), both competitive and non-competitive CD137 binders achieve CD137 clustering and thereby activate downstream signaling.

[00242] Therefore, on one hand, both competitive and non-competitive CD137 binders, when applied as described above, can activate the downstream signaling pathways of CD137.

[00243] A competitive CD137 binder, on the other hand, can be employed to inhibit the natural CD137/CD137L interaction, and thereby suppress the natural signaling induced by the encounter of CD137-positive cells with CD137L-expressing cells, for example, antigen- presenting cells. Such a mode of action is desirable in the cases where it is desired to suppress an inappropriately strong inflammatory or autoimmune reaction.

[00244] To demonstrate that this application provides both the competitive-type muteins and the non-competitive-type muteins, we employed a surface plasmon resonance (SPR) experiment. We used it to investigate the competition between CD137L and three representative lipocalin muteins disclosed herein in binding to the human CD137-Fc fusion protein (huCD137-Fc). In this assay, it is investigated whether a lipocalin mutein can bind to the preformed complex of CD137 and CD137L. If this is not the case, then this is evidence that the lipocalin mutein binding epitope on CD137 overlaps with the CD137L binding epitope on CD137. The respective mutein therefore binds to CD137 competitively with respect to the CD137/CD137L interaction. If both CD137L and the lipocalin mutein can bind at the same time, then the binding is non-competitive with respect to the CD137/CD137L interaction. [00245] The competition assay was performed at 37°C on a Biacore T200 instrument (GE Healthcare) using HBS-EP+ (1 x; BR-1006-69; GE Healthcare) as running buffer. The Biotin CAPture Kit (GE Healthcare) was used to immobilize biotinylated huCD137-Fc to a chip surface. CD137-Fc proteins were biotinylated using standard NHS chemistry. Undiluted Biotin CAPture Reagent (streptavidin conjugated with ss-DNA oligo) was captured on a sensor chip CAP with the pre-immobilized complementary ss-DNA oligo. Thereafter, biotinylated CD137-Fc protein at 2 μg mL was applied for 300 s at a flow rate of 5 μΙ_Ληίη. Regeneration of the chip surface was achieved by applying 6M guanidinium-HCI in 250 mM NaOH for 120 s at a flow rate of 10 μΙ_Ληίη.

[00246] In the two first measurement cycles, successful binding of CD137L to CD137 under the experimental conditions was ascertained, and the reference level for the individual binding of a tested lipocalin mutein in the absence of ligand was obtained. In the third cycle, CD137 was saturated with CD137L before the lipocalin mutein was added as described in detail as follows.

[00247] The human CD137 ligand-Fc (R&D Systems 2295-4 L-025/CF) ligand was applied to the immobilized CD137-Fc protein at a concentration of 500 nM and a flow rate of 30 μΙ_Ληίη for 30s. After regeneration, the lipocalin muteins were applied at a concentration of 5 μΜ at a flow rate of 30 μΙ_/η"ΐίη for 30 s. Finally, after another regeneration cycle the human CD137 ligand-Fc was applied to the immobilized CD137-Fc proteins at a concentration of 500 nM for 30 s directly followed by the muteins at a concentration of 5 μΜ for 30 s both at a flow rate of 30 μΙ_Ληίη. Regeneration of the chip surface was achieved by applying 6M guanidinium-HCI in 250 mM NaOH for 120 s at a flow rate of 10 μί/ιηϊη. The resulting sensorgrams were analyzed visually and it was determined whether bound CD137 ligand-Fc had an impact on the interaction of the muteins with the immobilized CD137-Fc proteins. The sensorgrams of the cycles were CD137 ligand-Fc interaction or muteins were applied alone served as controls.

[00248] Representative examples for the relevant segment of the resulting sensorgrams are provided in Figure 2 for the muteins of SEQ ID NO: 5, SEQ ID NO: 12, and SEQ ID NO: 13. The SPR trace for the binding of the respective lipocalin mutein to huCD137-Fc alone is marked with an arrow with a solid stem. The SPR trace for the binding of the lipocalin mutein to huCD137-Fc that has been saturated with CD137L is marked with an arrow with a broken stem. The data shows that the mutein of SEQ ID NO: 5 for example cannot bind to huCD137-Fc in the presence of CD137L (Figure 2A). In contrast, both the mutein of SEQ ID NO: 12 and the mutein of SEQ ID NO: 13 bind to huCD137-Fc with a very similar response both in the absence and presence of CD137L, showing that there is no competition in the binding between the lipocalin muteins and CD137L. This data is summarized in Table 2.

Table 2:

Mode of

SEQ ID AA binding

SEQ ID NO: 5 competitive

SEQ ID NO: 12 non-competitive

SEQ ID NO: 13 non-competitive

[00249] Example 6: FACS analysis of lipocalin muteins binding to cells expressing human CD137

[00250] We employed FACS studies in order to assess the specific binding of lipocalin muteins and negative controls to Chinese hamster ovary (CHO) cells stably transfected with human CD137 (CHO-huCD137). The cell line was generated using the Flp-ln system (Invitrogen) according to the manufacturer^'s instructions. Mock-transfected Flp-ln CHO cells served as the negative control.

[00251] Transfected CHO cells were maintained in Ham^'s F12 medium (Invitrogen) supplemented with 10% Fetal Calf Serum (FCS, Biochrom) and 500 μg ml Hygromycin B (Roth). Cells were cultured in cell culture flasks under standard conditions according to manufacturer's instruction (37°C, 5% C0₂ atmosphere). In order to dissociate the adherent cells for subculture or FACS experiments, Accutase (PAA) was employed according to the manufacturer^'s instructions.

[00252] To perform the experiment, CD137-positive and negative Flp-ln CHO cells were incubated with lipocalin muteins, and bound mutein was labeled using anti-lipocalin primary antibodies and fluorescently labeled secondary antibodies, which were detected by FACS analysis as described in the following.

[00253] 1 x 10⁵ cells per well were pre-incubated for 1 h in ice-cold PBS containing 5% fetal calf serum (PBS-FCS). Subsequently, a dilution series of lipocalin muteins and negative controls typically ranging from 10 μΜ to 1 nM was added to the cells and incubation was continued on ice for 1 h. Cells were washed twice in ice-cold PBS using centrifugation at 300 g and then incubated with a rabbit anti-lipocalin primary antibody (Pieris, (polyclonal rabbit anti-hNGAL; Pieris) for 30 min on ice. Cells were washed twice in ice-cold PBS, re- suspended in PBS-FCS and incubated 30 min on ice with a secondary anti-rabbit antibody labelled with the fluorescent dye Alexa488 (Life Technologies). Cells were subsequently washed and analyzed using a Guava easyCyte HT Flow cytometer. Typically, a gate was set to exclude non-viable cells and 5.000 events were recorded. Numerical results are expressed as the geometric mean of the fluorescence intensity.

[00254] FACS histograms for all clones tested are provided in Figure 3. In the respective plots, the SEQ ID NOs of the respective lipocalin muteins are depicted. In line with the SPR data (Figure 1 , Table 1 ), all muteins show a clear binding to cell-expressed CD137. The EC₅₀ resulting from a fit of this data are provided in Table 3 below.

[00255] Table 3:

SEQ ID AA ECso CHO::hCD137 [nM]

SEQ ID NO 6 61 .1

SEQ ID NO 7 67.6

SEQ ID NO 8 234.6

SEQ ID NO 9 1 13.3

SEQ ID NO 1 1 53

SEQ ID NO 13 4.3

SEQ ID NO 14 4.5

SEQ ID NO 15 7.8

SEQ ID NO 17 17.9

SEQ ID NO 18 13.7

SEQ ID NO 20 18

SEQ ID NO 3 (Ctrl) no binding

SEQ ID NO 4 (Ctrl) no binding

[00256] Example 7: Functional T-cell activation assay using coated lipocalin muteins

[00257] We employed a T-cell activation assay to assess the ability of a set of representative CD137-binding lipocalin muteins to co-stimulate T-cell responses. The tested muteins (SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15) span an SPR affinity ranging from 2 nM to >23 nM in Example 4 (cf. Table 1 ). As discussed in Example 5, there are several ways to induce CD137 clustering and in this experiment, we applied immobilized CD137-binding agents. In this experiment, the lipocalin muteins were coated onto a plastic dish together with an anti-human CD3 antibody (Muronomab, Janssen-Cilag) and purified T- cells were subsequently incubated on the coated surface in the presence of soluble anti- human CD28 antibody (Clone 28.2; eBioscience). Anti-CD3 and anti-CD28 antibodies were used to provide a sub-threshold stimulus to the T-cells that could be costimulated by CD137 costimulation. As a readout, we measured supernatant interleukin 2 (IL-2) levels. An increased IL-2 production is one of the hallmarks of T-cell activation, and the increase in IL-2 levels by costimulation with an anti-CD137 antibody has been described in the literature (Fisher et al., 2012). As a negative control, SEQ ID NO: 4 was utilized. In the following, we provide a detailed description of the experiment.

[00258] Human peripheral blood mononuclear cells (PBMC) from healthy volunteer donors were isolated from buffy coats by centrifugation through a Polysucrose density gradient (Biocoll 1 .077 g/mL from Biochrom), following Biochrom^'s protocols. The T lymphocytes were isolated from the resulting PBMC using a Pan T-cell purification Kit (Miltenyi Biotec GmbH) and the manufacturer^'s protocols, Purified T-cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down using liquid nitrogen and stored in liquid nitrogen until further use. For the assay, T cells were thawed for 16 h and cultivated in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1 % Penicillin-Streptomycin (Life Technologies).

[00259] The following procedure was performed using triplicates for each experimental condition. Flat-bottom tissue culture plates were coated overnight at 4°C using 200 μί of a mixture of 0.5 μg mL anti-CD3 antibody and 25 μg mL rabbit anti-lipocalin antibodies (polyclonal rabbit anti-hNGAL, Pieris). The latter was employed to allow for immobilization of lipocalin muteins by affinity capturing. The following day, wells were washed twice with PBS, and 50 μί of CD137-binding lipocalin muteins of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, all at a concentration of 25μg mL, were captured on the precoated plates for 1 h at 37°C. SEQ ID NO: 4 was employed likewise and served as the negative control. After again washing twice with PBS, 100 μί of the T-cell suspension (corresponding to 5x10⁴ T cells) in culture media supplemented with 2 μg mL hCD28 antibody was added to each well. Plates were covered with a gas permeable seal (4titude) and incubated at 37°C in a humidified 5% C02 atmosphere for 3 days. Subsequently, IL-2 in the supernatant was assessed.

[00260] Human IL-2 levels in the pooled cell culture supernatants were quantified using the IL-2 DuoSet kit from R&D Systems. In the first step, a 384 well plate was coated at room temperature for 2 h with 1 μg mL "Human IL-2 Capture Antibody" (R&D System) diluted in PBS. Subsequently, wells were washed 5 times with 80 μί PBS-T (PBS containing 0.05% Tween20) using a Biotek EL405 select CW washer (Biotek). After 1 h blocking in PBS-T additionally containing 1 % casein (w/w), pooled supernatant and a concentration series of an IL-2 standard diluted in culture medium were incubated in the 384-well plate overnight at 4°C. To allow for detection and quantitation of captured IL-2, a mixture of 100 ng/mL biotinylated goat anti-hlL-2-Bio detection antibody (R&D System) and ^g mL Sulfotag-labelled streptavidin (Mesoscale Discovery) were added in PBS-T containing 0.5% casein and incubated at room temperature for 1 h. After washing, 25 μί reading buffer was added to each well and the electrochemiluminescence (ECL) signal of every well was read using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[00261] The resulting data is plotted in Figure 4A. There is a clearly increased IL-2 concentration in the supernatant due to T-cell activation for the lipocalin muteins of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 compared to the negative control of SEQ ID NO: 4. The experiment indicates that all muteins tested are able to costimulate a T-cell response when coated on a plastic culture dish.

[00262] Example 8: Functional T-cell activation assay using lipocalin muteins in solution

[00263] To test whether representative lipocalin muteins also activate CD137 by simple binding without clustering, the assay of Example 7 was carried out in an analogous fashion to Example 5, but using soluble instead of captured lipocalin muteins. In this assay, flat-bottom tissue culture plates were coated as described above, but using anti-CD3 antibody only. Plates were processed as described above until after the T-cell addition step (including 2 μg mL hCD28), which was followed by the addition of 50 μΙ_ of the lipocalin muteins in solution at a concentration of 25 μg mL.

[00264] The resulting data is plotted in Figure 4B. There is no significant increase in IL-2 concentration in the supernatant due to T-cell activation for any of the lipocalin muteins tested compared to the negative control of SEQ ID NO: 4. The experiment indicates that monomeric lipocalin muteins in solution, at a concentration that is sufficient to saturate all CD137 receptors, do not costimulate T-cells.

[00265] Example 9: Functional T-cell activation assay using coated lipocalin muteins

[00266] To investigate in more detail the ability of mutein of SEQ ID NO: 13 to costimulate T-cell responses, we employed a T-cell activation assay as in Example 7. As readouts, we assessed continued proliferation of the T-cells after three days' incubation using a 4 h BrdU pulse, and measured supernatant IL-2 and Interferon gamma (IFN-γ levels. Beside proliferation and IL-2 production, an increased IFN-γ production is a further hallmark of T-cell activation, and the increase in IFN-γ levels by costimulation with an anti-CD137 antibody has been described in the literature (Jure-Kunkel, M. et al., US patent 7288638).

[00267] As a negative control, the wild-type like lipocalin mutein SEQ ID NO: 4 was utilized. This experiment was, in some aspects, carried out in an identical manner to the experiment described in Example 8. In the following, we provide a detailed description of the experiment.

[00268] Human peripheral blood mononuclear cells (PBMC) from healthy volunteer donors were isolated from buffy coats by centrifugation through a Polysucrose density gradient (Biocoll 1 .077 g/mL from Biochrom), following Biochrom^'s protocols. The T lymphocytes were isolated from the resulting PBMC using a Pan T-cell purification Kit (Miltenyi Biotec GmbH) and the manufacturer^'s protocols. Purified T-cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen down using liquid nitrogen and stored in liquid nitrogen until further use. For the assay, T-cells were thawed for 16 h and cultivated in culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1 % Penicillin-Streptomycin (Life Technologies).

[00269] The following procedure was performed using triplicates for each experimental condition. Flat-bottom tissue culture plates were coated overnight at 4°C using 200 μί of a mixture of 5 μg mL anti-CD3 antibody and 25 μg mL rabbit anti-lipocalin-scaffold antibody (polyclonal rabbit anti-hNGAL, Pieris). The latter was employed to allow for immobilization of SEQ ID NO: 13 by affinity capturing. As a negative control, an lgG1 isotype control was coated at 5 μg mL instead of the anti-CD3 antibody, together with the 25 μg mL rabbit anti- lipocalin-scaffold antibody. The following day, wells were washed twice with PBS, and 50 μί of a dilution series of SEQ I D NO: 13 ranging from 50 μg mL to 0.8 μg mL in seven steps was captured on the precoated plates for 1 h at 37°C. As a negative control, SEQ ID NO: 4 was captured at three concentrations (50 μg mL, 25 μg mL, 12.5 μg mL). As a further negative control, SEQ ID NO: 13 was captured at 50 μg mL to the wells that had been coated with lgG1 isotype and the anti-hNGAL capture antibody (see above). After again washing twice with PBS, 100 L of the T-cell suspension (corresponding to 5 x 10⁴ T cells) in culture media was added to the wells. This was performed either in the presence or absence of hCD28 antibody at a concentration of 2 μg mL. Plates were covered with a gas permeable seal (4titude) and incubated at 37°C in a humidified 5% C02 atmosphere for 3 days. Subsequently, IL-2 and IFN-γ concentration in the supernatant, as well as cell proliferation were assessed.

[00270] In order to quantify T-cell proliferation, the chemiluminescent cell proliferation ELISA kit based on BrdU incorporation (Roche) was used according to the manufacturer^'s instructions. Briefly, on day 3, 10 μί of BrdU labeling solution were added to each well and proliferation was allowed to proceed for a further 4 h at 37°C under a humidified 5% C02 atmosphere. Plates were centrifuged at 300 g for 10 min and supernatants of the triplicates were pooled and immediately stored at -20°C for later IL-2 and IFN-γ quantification. Plates were subsequently dried at 60°C for 1 hour. 200 μΙ_ of "FixDenat" solution was added to each well and the plates were incubated at room temperature for 30 min. Incorporated BRDU was labeled with a peroxidase-labelled anti-BrdU antibody by 2 h incubation at room temperature. BrdU levels were assessed by quantifying a chemiluminescent peroxidase-catalyzed reaction in a PheraStar FS reader.

[00271 ] Human IL-2 and IFN-γ levels in the pooled cell culture supernatants were quantified using the I L-2 DuoSet and IFN-γ DuoSet kits from R&D Systems. The procedure is carried out analogously for both cytokines, and described only for I L-2 in the following. In the first step, a 384 well plate was coated at room temperature for 2 h with 1 μg mL "Human I L-2 Capture Antibody" (R&D System) diluted in PBS. Subsequently, wells were washed 5 times with 80 μί PBS-T (PBS containing 0.05% Tween20) using a Biotek EL405 select CW washer (Biotek). After 1 h blocking in PBS-T additionally containing 1 % casein (w/w), pooled supernatant and a concentration series of an IL-2 standard diluted in culture medium were incubated in the 384-well plate overnight at 4°C. To allow for detection and quantitation of captured IL-2, a mixture of 100 ng/mL biotinylated goat anti-hl L-2-Bio detection antibody (R&D System) and ^g mL Sulfotag-labelled streptavidin (Mesoscale Discovery) were added in PBS-T containing 0.5% casein and incubated at room temperature for 1 h. After washing, 25 L reading buffer was added to each well and the electrochemiluminescence (ECL) signal of every well was read using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[00272] The result of the experiment is depicted in Figure 5. Readouts of proliferation, IL-2 and IFN-γ in the supernatant for the experiment using both anti-CD3 and anti-CD28 antibodies are provided in Figure 5A, 5C, and 5E, respectively. The same readouts for the experiment performed with anti-CD3 antibody only are provided in Figures 5B, 5D, and 5F.

[00273] In the experiment employing stimulation by anti-CD3 and anti-CD28 antibodies, there is a clear dose-dependent increase in proliferation rate (Figure 5A), which is up to 14-fold higher than for the negative control of SEQ ID NO: 4. Proliferation in the absence of anti-CD3 mAb (column labeled as "lgG1 ) is negligible. Regarding IL-2 production (Figure 5C), there is also a clear dose-dependent increase that levels at a maximum response at a coating concentration of SEQ ID NO: 13 of 6.25 μg mL and at higher concentrations remains constantly at levels of up to around 6fold compared to the negative control. Regarding IFN-γ production (Figure 5E), the pattern is very similar, with maximum IFN-γ levels reaching up to 2.5-fold values compared to the negative control.

[00274] In the experiment employing stimulation by anti-CD3 mAb only, we again find a clear dose-dependent increase in proliferation rate (Figure 5A), which is up to 4-fold higher than for the negative control of SEQ ID NO: 4. There appears to be a maximum response at 6.25 μg mL coating concentration of SEQ ID NO: 13, which reaches a 15-fold value compared to the negative control. At both higher and lower concentrations, the response is less pronounced. Regarding IFN-γ production, there is a dose-dependent increase that levels at a maximum response at a coating concentration of SEQ ID NO: 13 of 6.25 μg mL and at higher concentrations remains constantly at levels of up to around 2.5-fold compared to the negative control.

[00275] Overall, the experiment shown in this Example 9 clearly demonstrates a significant costimulation of T-cell response by the mutein of SEQ ID NO: 13 with regard to proliferation, IL-2 production and IFN-γ production, both in the presence and in the absence of CD28 stimulation.

[00276] Example 10: Generation and selection of optimized muteins specifically binding to CD137

[00277] SEQ ID NO: 47 was found in a selection process using a naive NGAL mutein library as described in Example 1. For optimization of this CD137-specific mutein, so-called maturation libraries were generated based on the mutein sequence using either parsimonious randomization of selected positions or error prone polymerase chain reaction (PCR) based methods.

[00278] The generated lipocalin muteins were cloned with high efficiency into phagemid vector essentially as described (Kim et al., 2009). Phage display was employed to select for optimized muteins with improved heat stability and binding affinity. The phagemid selection was conducted with increased stringency compared to the initial mutein selections and involved preincubation steps at elevated temperature and limiting target concentration amongst other strategies.

[00279] Example 11 : Identification of muteins specifically binding to CD137 using high-throughput ELISA screening

[00280] Individual colonies were used to inoculate 2x Yeast Extract Trypton (2XYT)/Amp medium and grown overnight (14-18 h) to stationary phase. Subsequently, 50 μΙ_ 2xYT/Amp were inoculated from the stationary phase cultures and incubated for 3 h at 37°C and then shifted to 22°C until an OD₅₉₅ of 0.6-0.8 was reached. Production of muteins was induced by addition of 10 μΙ_ 2xYT/Amp supplemented with 1 .2 μg mL anhydrotetracycline. Cultures were incubated at 22°C until the next day. After addition of 40 μΙ_ of 5% (w/v) BSA in PBS/T and incubation for 1 h at 25°C, cultures were ready for use in screening assays. [00281] Binding of the isolated muteins to CD137 was tested by direct coating of Fc- fusion of the full extracellular domain of CD137 from human (huCD137) at 5 μg mL in PBS overnight at 4°C on microtiter plates. After blocking the plate with PBST containing 2% BSA, 20 μΙ_ of BSA-blocked cultures (with or without previous heat incubation) were added to the microtiter plates and incubated for 1 h at 25°C. Bound muteins were detected with anti-Strep- Tag antibody conjugated with horseradish peroxidase (IBA) after 1 h incubation. For quantification, 20 μΙ_ of QuantaBlu fluorogenic peroxidase substrate was added and the fluorescence was determined at an excitation wavelength of 330 nm and an emission wavelength of 420 nm.

[00282] To select for muteins with increased temperature resistance, BSA-blocked cultures were incubated for 1 h at 60°C and then allowed to cool down to room temperature before adding them to CD137 coated and BSA-blocked microtiterplates as described in the previous paragraph. The muteins were subsequently processed as described in the previous paragraph and were selected for bacterial expression, purification, and further characterization.

[00283] Example 12: Expression of muteins

[00284] Selected muteins were expressed with C-terminal sequence SAWSHPQFEK (SEQ ID NO: 21 ) of SA linker or PSAWSHPQFEK (SEQ ID NO: 22) of PSA linker and the Strep-tag II peptide (WSHPQFEK, SEQ ID NO: 23) in E. coli in 2xYT-Amp medium to purify the muteins after expression using Strep-Tactin affinity chromatography and preparative size exclusion chromatography (SEC). Finally, lipocalin muteins were subjected to an endotoxin depletion step utilizing Mustang E columns. Purified lipocalin muteins were then characterized as detailed in all following examples.

[00285] Example 13: Affinity of muteins binding to human and cynomolgus monkey CD137-Fc fusion protein determined by surface plasmon resonance (SPR)

[00286] Surface plasmon resonance (SPR) was used to measure binding kinetics and affinity of the representative lipocalin muteins disclosed herein.

[00287] SPR analysis of the binding of the representative muteins to human CD137- Fc fusion protein (huCD137-Fc) or cynomolgus monkey CD137- Fc fusion protein (cyCD137- Fc), respectively, was performed at 37°C on a Biacore T200 instrument (GE Healthcare) using HBS-EP+ (1 x; BR-1006-69; GE Healthcare) as running buffer.

[00288] Prior to the protein measurements three regeneration cycles were performed for conditioning purposes. Regeneration of the derivatized chip surface was achieved by applying 3M MgCI₂ for 60 s followed by 10 mM glycine, pH 1 .7 for 180 s. Anti-human IgG-Fc antibody was utilized to immobilize huCD137-Fc in a subsequent step and taken from the human antibody capture kit (GE Healthcare, BR-1008-39). It was immobilized on a CM5 sensor chip using standard amine coupling chemistry and the immobilization buffer included in the kit (10 mM sodium acetate pH 5.0), resulting in a ligand density of about 9000 resonance units (RU). The reference channel was treated accordingly.

[00289] HuCD137-Fc at a concentration of 0.5 μg mL was captured on this surface for 180 s at a flow rate of 10 μί/ιηίη in HBS-EP+ buffer. cyCD137-Fc was captured in an identical manner, but using a concentration of 1 μg mL protein. No target protein was applied to the reference channel. Subsequently, the lipocalin muteins were applied in an appropriate dilution series in HBS-EP+ buffer at a flow rate of 30 μΙ_/η"ΐίη. Regeneration of the derivatized chip surface was achieved as described above. Data were evaluated with Biacore T200 Evaluation software (V 2.0). Double referencing was used and the 1 :1 Binding model was used to fit the raw data.

[00290] Figure 7 shows the SPR traces and fit curves determined for the lipocalin muteins tested on huCD137, with the corresponding SEQ ID NOs provided in the graphs. Figure 8 shows the respective data for the target cyCD137. There are clear SPR binding signals towards both targets. The on- and off-rates and the resulting affinities resulting from a fit of the data are provided in Table 4 (huCD137) and Table 5 (cyCD137) below.

[00291] Table 4: Kinetic constants and affinities of CD137-specific muteins toward huCD137 determined by surface-plasmon-resonance (SPR).

SEQ ID AA [MV] [s ¹] [nM]

SEQ ID NO: 47 8.78E+03 6.10E-03 694.8

SEQ ID NO: 48 2.06E+03 1.41E-04 68.6

SEQ ID NO: 49 1.95E+04 2.07E-04 10.6

SEQ ID NO: 50 1.71E+04 9.43E-05 5.5

SEQ ID NO: 51 1.63E+04 2.68E-04 16.4

SEQ ID NO: 52 1.47E+04 2.31E-04 15.7

SEQ ID NO: 53 7.67E+03 8.04E-05 10.5

SEQ ID NO: 54 6.41E+03 1.61E-04 25.1

SEQ ID NO: 55 1.29E+04 2.37E-04 18.4

SEQ ID NO: 56 1.25E+04 3.58E-04 28.6

[00292] Table 5: Kinetic constants and affinities of CD137-specific muteins toward cyCD137 determined by surface-plasmon-resonance (SPR).

SEQ ID AA [MV] [s ¹] [nM]

SEQ ID NO: 47 6.51E+03 1.12E-04 17.2

SEQ ID NO: 48 1.94E+03 1.97E-05 10.1

SEQ ID NO: 49 1.82E+04 3.50E-05 1.9

SEQ ID NO: 50 1.48E+04 2.20E-05 1.5

SEQ ID NO: 51 1.36E+04 4.99E-05 3.7

SEQ ID NO: 52 1.32E+04 4.05E-05 3.1

SEQ ID NO: 53 7.26E+03 3.28E-05 4.5

SEQ ID NO: 54 5.64E+03 4.22E-05 7.5

SEQ ID NO: 55 1.15E+04 4.33E-05 3.8

SEQ ID NO: 56 1.07E+04 4.09E-05 3.8

[00293] Example 14: Surface plasmon resonance (SPR) assay to determine competition between human CD137L and SEQ ID NO: 47 in binding to CD137-Fc fusion protein

[00294] We employed a surface plasmon resonance (SPR) experiment in analogy to investigate the competition between CD137L and the lipocalin mutein of SEQ ID NO: 47 in binding to the human CD137-Fc fusion protein (huCD137-Fc) or cynomolgus monkey CD137-Fc fusion protein (cyCD317-Fc).

[00295] The competition assay was performed at 37°C on a Biacore T200 instrument (GE Healthcare) using HBS-EP+ (1 x; BR-1006-69; GE Healthcare) as running buffer. The Biotin CAPture Kit (GE Healthcare) was used to immobilize biotinylated huCD137-Fc to a chip surface. CD137-Fc proteins were biotinylated using standard NHS chemistry. Undiluted Biotin CAPture Reagent (streptavidin conjugated with ss-DNA oligo) was captured on a sensor chip CAP with the pre-immobilized complementary ss-DNA oligo. Thereafter, biotinylated CD137-Fc protein at 2 μg mL was applied for 300 s at a flow rate of 5 L/min. Regeneration of the chip surface was achieved by applying 6M guanidinium-HCI in 250 mM NaOH for 120 s at a flow rate of 10 μΙ_Ληίη.

[00296] In the two first measurement cycles, successful binding of CD137L to CD137 under the experimental conditions was ascertained, and the reference level for the individual binding of a tested lipocalin mutein in the absence of ligand was obtained. In the third cycle, CD137 was saturated with CD137L before the lipocalin mutein was added as described in detail below. [00297] The human CD137 ligand-Fc (R&D Systems 2295-4 L-025/CF) ligand was applied to the immobilized CD137-Fc protein at a concentration of 500 nM and a flow rate of 30 μΙ_Ληίη for 30s. After regeneration, the lipocalin muteins were applied at a concentration of 5 μΜ at a flow rate of 30 μΙ_/η"ΐίη for 30 s. Finally, after another regeneration cycle the human CD137 ligand-Fc was applied to the immobilized CD137-Fc proteins at a concentration of 500 nM for 30 s directly followed by the muteins at a concentration of 5 μΜ for 30 s both at a flow rate of 30 μΙ_Ληίη. Regeneration of the chip surface was achieved by applying 6M guanidinium-HCI in 250 mM NaOH for 120 s at a flow rate of 10 μί/ιηϊη. The resulting sensorgrams were analyzed visually and it was determined whether bound CD137 ligand-Fc had an impact on the interaction of the muteins with the immobilized CD137-Fc proteins. The sensorgrams of the cycles were CD137 ligand-Fc interaction or muteins were applied alone served as controls.

[00298] Representative examples for the relevant segment of the resulting sensorgrams are provided in Figure 9. The SPR trace for the binding of SEQ ID NO: 47 to huCD137-Fc or cyCD137-Fc alone is marked with an arrow with a solid stem. The SPR trace for the binding of the lipocalin mutein to huCD137-Fc or cyCD137-Fc that has been saturated with CD137L is marked with an arrow with a broken stem. The data shows that the mutein of SEQ ID NO: 47 cannot bind to huCD137-Fc (Figure 9A) or cyCD137-Fc (Figure 9B) in the presence of CD137L, and that therefore SEQ ID NO: 47 is a competitive binder to CD137 with regard to the CD137L/CD137 interaction.

[00299] Example 15: FACS analysis of lipocalin muteins binding to cells expressing CD137

[00300] We employed FACS studies in order to assess the specific binding of lipocalin muteins and negative controls to Chinese hamster ovary (CHO) cells stably transfected with human CD137 (CHO-huCD137) or cynomolgus monkey CD137 (CHO-cyCD137). The cell line was generated using the Flp-ln system (Invitrogen) according to the manufacturer^'s instructions. Mock-transfected Flp-ln CHO cells served as the negative control.

[00301] Transfected CHO cells were maintained in Ham^'s F12 medium (Invitrogen) supplemented with 10% Fetal Calf Serum (FCS, Biochrom) and 500 μg ml Hygromycin B (Roth). Cells were cultured in cell culture flasks under standard conditions according to manufacturer's instruction (37°C, 5% C02 atmosphere). In order to dissociate the adherent cells for subculture or FACS experiments, Accutase (PAA) was employed according to the manufacturer^'s instructions. [00302] To perform the experiment, human or cynomolgus monkey CD137-positive cells as well as mock-transfected Flp-ln CHO cells were incubated with lipocalin muteins, and bound mutein was labeled using anti-lipocalin primary antibodies and fluorescently labeled secondary antibodies, which were detected by FACS analysis in analogy to the description in the following.

[00303] 5 x 10⁴ cells per well were pre-incubated for 1 h in ice-cold PBS containing 5% fetal calf serum (PBS-FCS). Subsequently, a dilution series of lipocalin muteins and negative control ranging from 500 nM 8 pM was added to the cells and incubation was continued on ice for 1 h. Cells were washed twice in ice-cold PBS using centrifugation at 300 g and then incubated with a rabbit anti-lipocalin primary antibody (Pieris, polyclonal rabbit anti-hNGAL; Pieris) for 30 min on ice. Cells were washed twice in ice-cold PBS, re- suspended in PBS-FCS and incubated 30 min on ice with a secondary anti-rabbit antibody labelled with the fluorescent dye Alexa488 (Life Technologies). Cells were subsequently washed and analyzed using an Intellicyt Flow cytometer. Numerical results were determined as the geometric mean of the fluorescence intensity. The data were fitted using GraphPad Prism with a 1 :1 binding model using shared maximum and minimum fluorescence values and a slope fixed to unity.

[00304] The mean fluorescence resulting from the FACS experiment is plotted versus the employed mutein concentrations in Figure 5. The result for huCD137-positive cells is shown in Figure 5A, while the result for cyCD137-positive CHO cells is shown in Figure 5B. In the respective plots, the SEQ ID NOs of the respective lipocalin muteins are depicted. In line with the SPR data (Figures 7 and 8, Tables 4 and 5), all muteins show a clear binding to cell-expressed CD137 both for human CD137 and cynomolgus monkey CD137- expressing cells. The results of a 1 :1 fit for all binding curves is provided in Table 6.

[00305] Table 6. Binding of CD137 specific lipocalin muteins to CHO cells transfected with huCD137or cyCD137.

EC₅₀ (hCD137::CHO) EC₅₀ (cyCD137::CHO)

SEQ ID AA ± STDERR [nM] ± STDERR [nM]

SEQ ID NO: 47 938 ± 162 60 ± 4

SEQ ID NO: 48 33 ± 4 27 ± 2

SEQ ID NO: 49 35 ± 5 27 ± 2

SEQ ID NO: 50 43 ± 6 42 ± 3

SEQ ID NO: 51 92± 12 65 ± 5

SEQ ID NO: 52 74 ± 10 52 ± 4

SEQ ID NO: 53 71 ± 9 79 ± 6

SEQ ID NO: 54 80 ± 10 86 ± 6 [00306] Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variations thereof may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. All patents, patent applications, textbooks and peer-reviewed publications described herein are hereby incorporated by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Further embodiments will become apparent from the following claims.

Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference NON-PATENT REFERENCES

1. ALTSCHUL, S. F., GISH, W., MILLER, W., MYERS, E. W. & LIPMAN, D. J. 1990. Basic local alignment search tool. J Mol Biol, 215, 403-10.

2. ALTSCHUL, S. F., MADDEN, T. L, SCHAFFER, A. A., ZHANG, J., ZHANG, Z., MILLER, W. & LIPMAN, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25, 3389-402.

3. BLAZAR, B. R., KWON, B. S., PANOSKALTSIS-MORTARI, A., KWAK, K. B., PESCHON, J. J. & TAYLOR, P. A. 2001. Ligation of 4-1BB (CDwl37) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients. J Immunol, 166, 3174-83.

4. BREUSTEDT, D. A., KORNDORFER, I. P., REDL, B. & SKERRA, A. 2005. The 1.8-A crystal structure of human tear lipocalin reveals an extended branched cavity with capacity for multiple ligands. J Biol Chem, 280, 484-93.

5. BRUCKDORFER, T., MARDER, O. & ALBERICIO, F. 2004. From production of peptides in milligram amounts for research to multi-tons quantities for drugs of the future. Curr Pharm Biotech nol, 5, 29-43.

6. CHACON, J. A., WU, R. C, SUKHUMALCHANDRA, P., MOLLDREM, J. J., SARNAIK, A.,

PILON-THOMAS, S., WEBER, J., HWU, P. & RADVANYI, L. 2013. Co-stimulation through 4- 1BB/CD137 improves the expansion and function of CD8(+) melanoma tumor-infiltrating lymphocytes for adoptive T-cell therapy. PLoS One, 8, e60031.

7. DENNIS, M. S., ZHANG, M., MENG, Y. G., KADKHODAYAN, M., KIRCHHOFER, D., COMBS, D.

& DAMICO, L. A. 2002. Albumin binding as a general strategy for improving the

pharmacokinetics of proteins. J Biol Chem, 277, 35035-43.

8. EBATA, T., MOGI, S., HATA, Y., FUJIMOTO, J. I., YAGITA, H., OKUMURA, K. & AZUMA, M.

2001. Rapid induction of CD95 ligand and CD4+ T cell-mediated apoptosis by CD137 (4- 1BB) costimulation. Eur J Immunol, 31, 1410-6.

9. FISHER, T. S., KAMPERSCHROER, C, OLIPHANT, T., LOVE, V. A., LIRA, P. D., DOYONNAS, R., BERGOVIST, S., BAXI, S. M., ROHNER, A., SHEN, A. C, HUANG, C, SOKOLOWSKI, S. A. & SHARP, L. L. 2012. Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T- cell function and promotes anti-tumor activity. Cancer Immunol Immunother, 61, 1721- 33.

10. FLOWER, D. R. 1996. The lipocalin protein family: structure and function. Biochem J, 318 ( Pt 1), 1-14.

11. FLOWER, D. R. 2000. Beyond the superfamily: the lipocalin receptors. Biochim Biophys Acta, 1482, 327-36.

12. FLOWER, D. R., NORTH, A. C. & SANSOM, C. E. 2000. The lipocalin protein family:

structural and sequence overview. Biochim Biophys Acta, 1482, 9-24.

13. FUERTGES, F. & ABUCHOWSKI, A. 1990. The clinical efficacy of polyethylene glycol)- modified proteins. Journal of Controlled Release, 11, 139-148.

14. HEINISCH, I. V., BIZER, C, VOLGGER, W. & SIMON, H. U. 2001. Functional CD137

receptors are expressed by eosinophils from patients with IgE-mediated allergic responses but not by eosinophils from patients with non-lgE-mediated eosinophilic disorders. J Allergy Clin Immunol, 108, 21-8.

15. HINNER, M. J., AIBA, R.-S. B., WIEDENMANN, A., SCHLOSSER, C, ALLERSDORFER, A.,

MATSCHINER, G., ROTHE, C, MOEBIUS, U., KOHRT, H. E. & OLWILL, S. A. 2015.

Costimulatory T cell engagement via a novel bispecific anti-CD137 /anti-HER2 protein. J Immunother Cancer, 3, P187-P187. HURTADO, J. C, KIM, Y. J. & KWON, B. S. 1997. Signals through 4-lBB are costimulatory to previously activated splenic T cells and inhibit activation-induced cell death. J Immunol, 158, 2600-9.

KIM, H. J., EICHINGER, A. & SKERRA, A. 2009. High-affinity recognition of lanthanide(lll) chelate complexes by a reprogrammed human lipocalin 2. J Am Chem Soc, 131, 3565-76. KIM, Y. J. & BROXMEYER, H. E. 2001. Therapeutic potential of 4-lBB (CD137) as a regulator for effector CD8(+) T cells. J Hematother Stem Cell Res, 10, 441-9.

KIM, Y. S., KONOPLEV, S. N., MONTEMURRO, F., HOY, E., SMITH, T. L, RONDON, G., CHAMPLIN, R. E., SAHIN, A. A. & UENO, N. T. 2001. HER-2/neu overexpression as a poor prognostic factor for patients with metastatic breast cancer undergoing high-dose chemotherapy with autologous stem cell transplantation. Clin Cancer Res, 7, 4008-12. KONIG, T. & SKERRA, A. 1998. Use of an albumin-binding domain for the selective immobilisation of recombinant capture antibody fragments on ELISA plates. J Immunol Methods, 218, 73-83.

KWON, B. 2009. CD137-CD137 Ligand Interactions in Inflammation. Immune Netw, 9, 84- 9.

LI, S. Y. & LIU, Y. 2013. Immunotherapy of melanoma with the immune costimulatory monoclonal antibodies targeting CD137. Clin Pharmacol, 5, 47-53.

LOWMAN, H. B. 1997. Bacteriophage display and discovery of peptide leads for drug development. Annu Rev Biophys Biomol Struct, 26, 401-24.

MARTINET, O., DIVINO, C. M., ZANG, Y., GAN, Y., MANDELI, J., THUNG, S., PAN, P. Y. & CHEN, S. H. 2002. T cell activation with systemic agonistic antibody versus local 4-lBB ligand gene delivery combined with interleukin-12 eradicate liver metastases of breast cancer. Gene Ther, 9, 786-92.

MARTINEZ GOMEZ, J. M., CROXFORD, J. L, YEO, K. P., ANGELI, V., SCHWARZ, H. &

GASSER, S. 2012. Development of experimental autoimmune encephalomyelitis critically depends on CD137 ligand signaling. J Neurosci, 32, 18246-52.

MELERO, I., BACH, N., HELLSTROM, K. E., ARUFFO, A., MITTLER, R. S. & CHEN, L. 1998. Amplification of tumor immunity by gene transfer of the co-stimulatory 4-lBB ligand: synergy with the CD28 co-stimulatory pathway. Eur J Immunol, 28, 1116-21.

NAM, K. O., KANG, W. J., KWON, B. S., KIM, S. J. & LEE, H. W. 2005. The therapeutic potential of 4-lBB (CD137) in cancer. Curr Cancer Drug Targets, 5, 357-63.

OSBORN, B. L, OLSEN, H. S., NARDELLI, B., MURRAY, J. H., ZHOU, J. X., GARCIA, A., MOODY, G., ZARITSKAYA, L. S. & SUNG, C. 2002. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferon-alpha fusion protein in cynomolgus monkeys. J Pharmacol Exp Ther, 303, 540-8.

PERVAIZ, S. & BREW, K. 1987. Homology and structure-function correlations between alpha 1-acid glycoprotein and serum retinol-binding protein and its relatives. FASEB J, 1, 209-14.

RODI, D. J. & MAKOWSKI, L. 1999. Phage-display technology-finding a needle in a vast molecular haystack. Curr Opin Biotechnol, 10, 87-93.

SAMBROOK, J. & RUSSELL, D. W. 2001. Molecular cloning : a laboratory manual, Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press.

SCHMIDT, T. G., KOEPKE, J., FRANK, R. & SKERRA, A. 1996. Molecular interaction between the Strep-tag affinity peptide and its cognate target, streptavidin. J Mol Biol, 255, 753-66. SEO, S. K., CHOI, J. H., KIM, Y. H., KANG, W. J., PARK, H. Y., SUH, J. H., CHOI, B. K., VINAY, D. S. & KWON, B. S. 2004. 4-lBB-mediated immunotherapy of rheumatoid arthritis. Nat Med, 10, 1088-94.

SEO, S. K., PARK, H. Y., CHOI, J. H., KIM, W. Y., KIM, Y. H., JUNG, H. W., KWON, B., LEE, H. W. & KWON, B. S. 2003. Blocking 4-1BB/4-1BB ligand interactions prevents herpetic stromal keratitis. J Immunol, 111, 576-83.

SKERRA, A. 2000. Lipocalins as a scaffold. Biochim Biophys Acta, 1482, 337-50.

SMITH, T. F. & WATERMAN, M. S. 1981. Identification of common molecular

subsequences. J Mol Biol, 147, 195-7.

SNELL, L. M., LIN, G. H., MCPHERSON, A. J., MORAES, T. J. & WATTS, T. H. 2011. T-cell intrinsic effects of GITR and 4-1BB during viral infection and cancer immunotherapy. Immunol Rev, 244, 197-217.

SUN, Y., SUBUDHI, S. K. & FU, Y. X. 2003. Co-stimulation agonists as a new

immunotherapy for autoimmune diseases. Trends Mol Med, 9, 483-9.

TAKAHASHI, C, MITTLER, R. S. & VELLA, A. T. 2001. Differential clonal expansion of CD4 and CD8 T cells in response to 4-1BB ligation: contribution of 4-1BB during inflammatory responses. Immunol Lett, 76, 183-91.

VAJO, Z. & DUCKWORTH, W. C. 2000. Genetically engineered insulin analogs: diabetes in the new millenium. Pharmacol Rev, 52, 1-9.

VENTURI, M., SEIFERT, C. & HUNTE, C. 2002. High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm. J Mol Biol, 315, 1-8.

VINAY, D. S. & KWON, B. S. 2016. Therapeutic potential of anti-CD137 (4-1BB)

monoclonal antibodies. Expert Opin Ther Targets, 20, 361-73.

WATTS, T. H. 2005. TNF/TNFR family members in costimulation of T cell responses. Annu Rev Immunol, 23, 23-68.

WYZGOL, A., MULLER, N., FICK, A., MUNKEL, S., GRIGOLEIT, G. U., PFIZENMAIER, K. & WAJANT, H. 2009. Trimer stabilization, oligomerization, and antibody-mediated cell surface immobilization improve the activity of soluble trimers of CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptor ligand. J Immunol, 183, 1851-61.

YANG, Y., YANG, S., YE, Z., JAFFAR, J., ZHOU, Y., CUTTER, E., LIEBER, A., HELLSTROM, I. & HELLSTROM, K. E. 2007. Tumor cells expressing anti-CD137 scFv induce a tumor- destructive environment. Cancer Res, 67, 2339-44.

YE, a, SONG, D. G., POUSSIN, M., YAMAMOTO, T., BEST, A., LI, C, COUKOS, G. & POWELL, D. J., JR. 2014. CD137 accurately identifies and enriches for naturally occurring tumor- reactive T cells in tumor. Clin Cancer Res, 20, 44-55.

YE, Z., HELLSTROM, I., HAYDEN-LEDBETTER, M., DAHLIN, A., LEDBETTER, J. A. &

HELLSTROM, K. E. 2002. Gene therapy for cancer using single-chain Fv fragments specific for 4-1BB. Nat Med, 8, 343-8.

ZHANG, H., KNUTSON, K. L, HELLSTROM, K. E., DISIS, M. L. & HELLSTROM, I. 2006.

Antitumor efficacy of CD137 ligation is maximized by the use of a CD137 single-chain Fv- expressing whole-cell tumor vaccine compared with CD137-specific monoclonal antibody infusion. Mol Cancer Ther, 5, 149-55.

Claims

1 . A Iipocaiin mutein capable of binding human CD137 with an affinity by a _d of about 700 nM or lower and cynomolgus monkey CD137 with an affinity by a K_d of about 20 nM or lower, wherein said mutein is capable of interfering with the binding of CD137 to CD137 ligand.

2. The mutein of claim 1 , wherein the capability of interfering with the binding of the CD137 ligand to CD137 is analyzed by surface plasmon resonance or any other applicable method, such as FACS or ELISA.

3. The Iipocaiin mutein of claim 1 , wherein the mutein is capable of binding human CD137 with an affinity by a K_d of about 30 nM or lower.

4. The Iipocaiin mutein of claim 1 , wherein the mutein is capable of binding human CD137 with an affinity by a K_d of about 8 nM or lower.

5. The Iipocaiin mutein of claim 1 , wherein the mutein is caplable of binding cynomolgus monkey CD137 with an affinity by a K_d of about 5 nM or lower.

6. The Iipocaiin mutein of claim 1 , wherein the mutein is capable of binding cynomolgus monkey CD137 with an affinity by a _d of about 2 nM or lower.

7. A Iipocaiin mutein as in one of claims 1 to 6, wherein the said _d values are determined by surface plasmon resonance.

8. The Iipocaiin mutein of claim 1 , wherein the mutein is capable of binding human CD137 with an EC₅₀ value of about 1000 nM or lower.

9. The Iipocaiin mutein of claim 1 , wherein the mutein is capable of binding cynomoguls monkey CD137 with an EC₅₀ value of about 90 nM or lower.

10. A Iipocaiin mutein as in claim 8 or 9, wherein the said EC₅₀ values are determined by fluorescence-activated cell sorting analysis.

1 1 . A Iipocaiin mutein as in any of the preceding claims, comprising at Ieast two or more mutated amino acid residues at sequence 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 in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

12. The Iipocaiin mutein of claim 1 1 , comprising at Ieast one or more mutated amino acid residues at sequence positions 36, 40, 41 , 49, 52, 68, 70, 72, 73, 77, 79, 81 , 96, 100, 103, 125, 127, 132, and 134 in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

13. The Iipocaiin mutein of claim 1 1 , further comprising at Ieast one or more mutated amino acid residues at sequence positions 20, 25, 33, 44, 59, 71 , 78, 80, 82, 87, 92, 98, 101 , and 122 in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

14. A lipocalin mutein as in any of the preceding claims, wherein the amino acid sequence of the mutein comprises at least one or more of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) Gin 20 > Arg; Asn 25 > Tyr or Asp; Val 33 > lie; Leu 36

>Met; Ala 40 > Asn; lie 41→ Leu; Glu 44 > Val or Asp; Gin 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 > Gin or His; Tyr 78→ His; Trp 79 > lie; lie 80 > Asn; Arg

81→ Trp or Gin; Thr 82→ Pro; 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→ lie; Tyr 132→ Trp; and Lys 134→ Gly.

15. A lipocalin mutein as in any of the preceding claims, wherein the amino acid sequence of the mutein comprises at least one or more of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Leu 36 >Met; Ala 40→ Asn; lie 41 > Leu; Gin 49 > His;

Tyr 52 >Ser or Gly; Ser 68 > Asp; Leu 70 > Met; Arg 72 > Leu; Lys 73 > Asp; Asp

77 > Gin or His; Trp 79 > lie; Arg 81 > Trp or Gin; Asn 96 > Phe; Tyr 100→ Asp; Leu 103 > His or Pro; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; and Lys 134→ Gly.

16. A lipocalin mutein as in any of the preceding claims, wherein the amino acid sequence of the mutein comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Leu 36→Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Trp 79→ lie; Asn 96→ Phe; Tyr 100→ Asp; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; and Lys 134→ Gly.

17. A lipocalin mutein as in any of the preceding claims, wherein the amino acid sequence of the mutein comprises at least 1 , 2, 3, or 4 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Tyr 52→ Ser or Gly; Asp 77→ Gin or His; Arg 81 → Trp or Gin; Leu 103→ His or Pro;

18. A lipocalin mutein as in any of the preceding claims, wherein the amino acid sequence of the mutein comprises at least one or more of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gin 20 -> Arg; Asn 25 -> Tyr or Asp; Val 33 -> lie; Glu 44 ->

Val or Asp; Lys 59→ Asn; Phe 71→ Leu; Tyr 78→ His; lie 80→ Asn; Thr 82 > Pro;

Phe 92 > Leu or Ser; Lys 98 > Arg; Pro 101 > Leu; and Phe 122 > Tyr.

19. A lipocalin mutein as in any of the preceding claims, wherein the amino acid sequence of the mutein comprises one of the following sets of amino acid substitutions in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) . Leu 36→ Met; Ala 40 Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser

68 Asp; Leu 70 Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81→ Trp; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125→ Ser; Ser 127→ lie; Tyr 132 Trp; Lys 134→ Gly;

(b) . Leu 36 Met; Ala 40 Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser

68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; 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→ lie; Tyr 132 ► Trp; Lys 134 » Gly;

(c) . Asn 25→ Tyr; Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr

52 Gly; Ser 68→ Asp; Leu 70 Met; Phe 71 → Leu; Arg 72 Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81→ Gin; Phe 92→ Ser; Asn 96 Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125 Ser; Ser 127→ lie; Tyr 132 > Trp; Lys 134 > Gly;

(d) . Leu 36 Met; Ala 40→ Asn; lie 41 Leu; Gin 49→ His; Tyr 52→ Gly; Ser

68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Tyr 78→ His; Trp 79→ lie; Arg 81→ Trp; Phe 92→ Leu; Asn 96 Phe; Tyr 100 → Asp; Leu 103→ His; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134 > Gly;

(e) . Asn 25→ Asp; Leu 36 Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr

52→ Gly; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81 → Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100 Asp; Leu 103→ His; Lys 125→ Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134 > Gly:

(f) . Val 33→ lie; Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Gin 49→ His; Tyr

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

49→ His; Tyr 52→ Gly; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73 → Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81→ Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His; Phe 122 Tyr; Lys 125 Ser; Ser 127 → lie; Tyr 132→ Trp; Lys 134→ Gly;

(h) . Leu 36→ Met; Ala 40 Asn; lie 41→ Leu; Gin 49→ His; Tyr 52→ Ser; Ser

68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; lie 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→ lie; Tyr 132 > Trp; Lys 134 Gly;

(i) . Leu 36 Met; Ala 40 Asn; lie 41→ Leu; Gin 49 His; Tyr 52→ Gly; Lys

59→ Asn; Ser 68→ Asp; Leu 70→ Met; Arg 72→ Leu; Lys 73→ Asp; Asp 77→ Gin; Trp 79→ lie; Arg 81 → Trp; Phe 92→ Leu; Asn 96→ Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125 Ser; Ser 127→ lie; Tyr 132→ Trp; Lys 134→ Gly; and

(j). Leu 36→ Met; Ala 40→ Asn; lie 41→ Leu; Glu 44 Asp; Gin 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→ lie; Arg 81→ Trp; Phe 92 Leu; Asn 96 → Phe; Tyr 100→ Asp; Leu 103→ His; Lys 125→ Ser; Ser 127 lie; Tyr 132→Trp; Lys 134 » Gly.

20. A lipocalin mutein as in any of the preceding claims, wherein the amino acid sequence of the mutein comprises 1 or 2 of the following mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gin 28 His and Cys 87→ Ser.

21 . A lipocalin mutein as in any of the preceding claims, wherein the mutein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-56 and a fragment or variant thereof.

22. A lipocalin mutein as in any of the preceding claims, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-56 and a fragment or variant thereof.

23. A lipocalin mutein as in any of the preceding claims, wherein the mutein has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2.

24. A lipocalin mutein as in any of the preceding claims, wherein the mutein is conjugated to a compound selected from the group consisting of an organic molecule, an enzyme label, a radioactive label, a colored label, a fluorescent label, a chromogenic label, a luminescent label, a hapten, digoxigenin, biotin, a cytostatic agent, a toxin, a metal complex, a metal, and colloidal gold.

25. A lipocalin mutein as in any of the preceding claims, wherein the mutein is fused at its N- terminus and/or its C-terminus to a fusion partner which is a protein, a protein domain, or a peptide.

26. A lipocalin mutein as in any of the preceding claims, wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein.

27. The lipocalin mutein of 26, wherein the compound that extends the serum half-life is selected from the group consisting of a polyalkylene glycol molecule, hydroethylstarch, a Fc part of an immunoglobulin, a CH3 domain of an immunoglobulin, a CH4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

28. The lipocalin mutein of claim 27, wherein the polyalkylene glycol is polyethylene (PEG) or an activated derivative thereof.

29. A nucleic acid molecule comprising a nucleotide sequence encoding a lipocalin mutein as in any of the preceding claims.

30. The nucleic acid molecule of claim 29, wherein the nucleic acid molecule is operably linked to a regulatory sequence to allow expression of said nucleic acid molecule.

31 . An expression vector comprising the nucleic acid molecule of claim 29 or 30.

32. A host cell containing a nucleic acid molecule of claim 29 or 30.

33. A method of producing a lipocalin mutein according to any one of claims 1 to 28, wherein the mutein is produced starting from the nucleic acid coding for the mutein or fragment thereof by means of genetic engineering methods.

34. The method of claim 33, wherein the mutein is produced in a bacterial or eukaryotic host organism and is isolated from this host organism or its culture.

35. A method for treating a subject having cancer, inflammatory diseases, or autoimmune diseases, comprising administering an effective amount of one or more lipocalin muteins of any one of claims 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, or one or more compositions comprising such muteins.

36. The method for treating a subject having cancers or inflammatory diseases or autoimmune diseases of claim 35, wherein the lipocalin muteins are applied to bind CD137 and/or activate the downstream signaling pathway of CD137.

37. A method of stimulating immune response in a subject, comprising administering an effective amount of one or more lipocalin muteins of any one of claims 1 -28 or having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, or one or more compositions comprising such muteins.

38. The method of stimulating immune response in a subject of claim 37, wherein the immune response comprises T lymphocyte proliferation and/or reduced production of proinflammatory cytokines and chemokines.

39. A method of antagonizing CD137 in a subject, comprising administering an effective amount of one or more lipocalin muteins of any one of claims 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, or one or more compositions comprising such muteins.

40. The method of antagonizing CD137 in a subject of claim 39, wherein the lipocalin muteins are applied to interfere with the binding of CD137 ligand to CD137.

41 . A method of detecting the presence of CD137, comprising:

(a) . contacting a lipocalin mutein according to any one of the claims 1 -28 with a test sample suspected to contain CD137 under suitable conditions, thereby allowing the formation of a complex between the mutein and CD137:

(b) . detecting the complex of the mutein and CD137 by a suitable signal.

42. A method for the separation of CD137, comprising:

(a) . contacting a lipocalin mutein according to any one of the claims 1 -28 with a test sample under suitable conditions, thereby allowing the formation of a complex between the mutein and CD137;

(b) . separating the complex of the mutein and CD137 from the tested sample.

43. The method of claim 41 or 42, wherein the test sample is a biological sample.

44. The method of any one of claims 43, wherein the biological sample is isolated from a human.

45. The method of any one of claims 43, wherein the biological sample comprises body fluid.

46. A pharmaceutical composition comprising a lipocalin mutein of any one of claims 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof, and pharmaceutically acceptable excipient.

47. A diagnostic or analytical kit comprising a lipocalin mutein of any one of claims 1 -28 or having at least 85%, at least 90%, at least 95% or at least 98% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-20, or of a fragment or variant thereof.

48. The method for treating cancers or inflammatory diseases or autoimmune diseases of claim 35, wherein the cancers to be treated are selected from the group consisting of ovarian, colon, breast, lung cancers, myelomas, neuroblastic-derived CNS tumors, monocytic leukemias, B-cell derived leukemias, T-cell derived leukemias, B-cell derived lymphomas, T-cell derived lymphomas, mast cell derived tumors, and combinations thereof.

49. The method for treating cancers or inflammatory diseases or autoimmune diseases of claim 35, wherein the autoimmune diseases or inflammatory diseases are selected from the group consisting of intestinal mucosal inflammation, wasting disease associated with colitis, multiple sclerosis, systemic lupus erythematosus, viral infections, rheumatoid arthritis, osteoarthritis, psoriasis, Cohn's disease, and inflammatory bowel disease.

50. The use of a Iipocaiin mutein according to any one of claims 1 -28 for the treatment of autoimmune disease.