WO2022243341A1 - Lipocalin muteins with binding affinity for ox40 - Google Patents

Abstract

The present disclosure provides human lipocalin muteins that specifically bind to OX40, which 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, infectious diseases, and autoimmune diseases. The present disclosure also concerns methods of making OX40-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 generating such nucleic acid molecules. In addition, the present disclosure provides therapeutic and/or diagnostic uses of these lipocalin muteins as well as of compositions comprising one or more of such lipocalin muteins.

Description

Lipocalin muteins with binding affinity for 0X40

I. BACKGROUND

[0001] 0X40, also known as CD134 or tumor necrosis factor receptor superfamily member 4 (TNFRSF4), is a tumor necrosis factor (TNF) receptor impacting many aspects of immune function and is one of the most prominent co-stimulatory receptors to control T cells (Croft et al., 2009). 0X40 is predominantly expressed on activated T cells, preferentially on regulatory T cells (Tregs) and conventional CD4⁺ T cells while also on CD8⁺ cells at lower level (Croft, 2010), but not on naive cells. Lower level of 0X40 expression is also seen on natural killer T (NKT) cells (Zaini et al., 2007), natural killer (NK) cells (Melero et al., 2013) and neutrophils (Baumann et al., 2004). The only known ligand of 0X40, 0X40 ligand (OX40L), also known as CD252 or TNFSF4, is also not constitutively expressed. It is mainly found on activated antigen-presenting cells (e.g., dendritic cells, B cells, macrophages, and endothelial cells) as well as on activated T cells (Flynn et al., 1998, Ohshima et al., 1997, Stuber et al., 1995, Chen et al., 1999).

[0002] Expression of both 0X40 and OX40L is increased after antigen presentation to T cells. While T cell receptor ligation alone can drive 0X40 expression on CD4⁺T cells, such expression is augmented and sustained by co-stimulatory ligation of CD28, as well as 0X40- OX40L ligation (Rogers et al., 2001 , Walker et al., 1999). Meanwhile, 0X40 expression on previously unstimulated CD4⁺ T cells peak from 24 hours to 4-5 days after initial stimulation (Baumann et al., 2004, Karulf et al., 2010).

[0003] Co-stimulatory signals from 0X40 engagement by OX40L or anti-OX40 agonistic antibodies increase proliferation, effector function and survival of T cells (Weinberg et al., 2011 , Croft et al., 2009). 0X40 signaling also influences Treg function, resulting in either impaired suppressing ability of Tregs or Treg expansion depending on the microenvironment (Aspeslagh et al., 2016). In addition, 0X40 and OX40L regulate cytokine production from T cells leading to generation and maintenance of memory CD4⁺ and memory CD8⁺ T cells.

[0004] The capability of T cell activity regulation makes 0X40 a promising candidate for anti-tumor therapy. 0X40 is expressed on tumor infiltrating T cells in different tumor indications including breast cancer, melanoma, B-cell lymphoma and head and neck cancer (Marabelle et al., 2013, Morris et al., 2009, Vetto et al., 1997, Xie et al., 2010, Sarff et al. , 2008). Studies have shown that the use of 0X40 agonists, including OX40L, 0X40 fusion proteins, 0X40 antibodies, and RNA aptamers, leads to tumor regression in several preclinical models (AN et al., 2004, Kjaergaard et al., 2000, Morris et al., 2001 , Piconese et al., 2008, Gough et al., 2010, Redmond et al., 2009). As a result, some anti-OX40 agonistic monoclonal antibodies (e.g., MEDI0562, Medlmmune LLC) are currently undergoing early phase cancer clinical trials in monotherapy or in combination. Almost all reported effects of 0X40 agonists involve directly regulating CD8⁺T cell survival (Vetto et al., 1997), promoting CD4⁺T cell help for CD8⁺T cells (Lee et al., 2004), and/or suppressive action on Tregs (Aspeslagh et al., 2016). Of note, the efficacy of 0X40 agonists may be affected by many factors, including tumor microenvironment and tumor burden.

[0005] In autoimmunity, on the other hand, 0X40 and OX40L are typically upregulated at sites of inflammation or autoimmunity. Blockade of 0X40 and/or OX40L have been examined for most of the major animal models and showed significantly attenuated symptoms for diseases, including allergic asthma (Arestides et al., 2002, Jember et al., 2001), experimental allergic encephalomyelitis (EAE) (Weinberg et al., 1999), multiple sclerosis (MS) (Weinberg et al., 1996), colitis (Higgins et al., 1999), diabetes (Pakala et al., 2004), arthritis (Yoshioka et al., 2000), atherosclerosis (van Wanrooij et al., 2007), graft versus host disease (Tsukada et al., 2000), and allograft rejection (Curry et al., 2004). In most cases, reduced disease has been associated with suppressing T cells (weak CD4⁺ or CD8⁺ T cell responses) or T cell-derived cytokines, although it is highly likely that decreased activities of other cell types including NK cells, NKT cells, mast cells, and neutrophils, which can all express OX40L and/or 0X40, might have partially accounted for reduced clinical symptoms (Croft et al., 2009).

[0006] Due to the roles of 0X40 in modulating the immune response, there is a need for compounds that bind human 0X40 and increase an OX40-mediated response, providing potential therapeutics for various diseases, including cancer. Further, there is an unmet need for compounds that bind human 0X40 but reduce an OX40-stimulation-mediated response by disrupting OX40-OX40L interaction, so that to suppress strong inflammatory or autoimmune reactions. Such “competitive” 0X40 binders are desired to bind 0X40 with an affinity comparable to that of OX40L, i.e. , at least in the nanomolar range, and are further desired to be cross-reactive to a relevant toxicology species. [0007] In addition, despite all the positive data supporting the use of 0X40 binders in cancer, it is unlikely that anti-OX40 alone will be sufficient to cure all patients or all tumor types. However, combination immunotherapy with both 0X40 and checkpoint inhibition may be able to do what single agents alone cannot. For instance, in an ovarian cancer model, combined anti- PD-1/OX40 mAb treatment induced a synergistic anti-tumor effect, remarkedly increasing survival in that over half of the mice were tumor free at day 90 (Guo et al., 2014). Further, bispecific or multispecific constructs specific for 0X40 and other targets may also be useful in various therapeutic implications, including but not limited to immune activation for cancer therapy.

[0008] It is an object of the present disclosure to provide for novel lipocalin muteins specific for 0X40, some of which are competitive 0X40 binders. The provided OX40-specific lipocalin muteins are preferably reactive with both human and cynomolgus 0X40 with a comparable binding pattern. These lipocalin muteins alone, as fusion polypeptides, or in combination with other therapeutic composition(s) may be applied to the therapy of autoimmune diseases, cancer or infectious diseases.

[0009] The provided 0X40 binders are muteins derived from lipocalins. Muteins of various lipocalins are a rapidly expanding class of therapeutics and 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

[0010] 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.

[0011] As used herein, unless otherwise specified, ΌC40” means human 0X40

(huOX40). Human 0X40 means a full-length protein defined by UniProt P43489 (version 177 of 7 April 2021), a fragment thereof, or a variant thereof. Human 0X40 is encoded by the gene TNFRSF4. 0X40 is also known as cluster of differentiation 134 (CD134) or tumor necrosis factor receptor superfamily member 4 (TNFRSF4), which terms are used interchangeably. Cynomolgus 0X40 (cyOX40) refers to the 0X40 of cynomolgus monkeys. In some particular embodiments, 0X40 of non-human species, e.g., cynomolgus 0X40 and mouse 0X40, is used. [0012] As used herein, “binding affinity” describes the ability of a biomolecule (e.g., a polypeptide or a protein) of the disclosure (e.g., a lipocalin mutein, an antibody, a fusion protein, or any other peptide or protein) to bind a selected target (and form a complex). Binding affinity is measured by a number of methods known to those skilled in the art, including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays, including direct and competitive ELISA, calorimetric methods, such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well-established in the art and some examples of such methods are further described herein. Binding affinity is thereby reported as a value of the dissociation constant (K_D), half maximal effective concentration (EC₅₀), or half maximal inhibitory concentration (IC₅₀) measured using such methods. A lower K_D, EC₅₀, or IC₅₀ value reflects better (higher) binding ability (affinity). Accordingly, the binding affinities of two biomolecules toward a selected target can be measured and compared. When comparing the binding affinities of two biomolecules toward the selected target, the term “comparable to”, “about the same,” “substantially the same” or “substantially similar” means one biomolecule has a binding affinity reported as a K_D, an EC₅₀, or an IC₅₀ value that is identical or similar to that of another molecule within the experimental variability of the binding affinity measurement. Preferably, “comparable to”, “about the same,” “substantially the same” or “substantially similar” relate to a value that is within 50% deviation to a given reference value, more preferably within 20% deviation, most preferably within 10% deviation. The experimental variability of the binding affinity measurement is dependent upon the specific method used and is known to those skilled in the art.

[0013] As used herein, the term “substantially” may also refer to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0014] As used herein, the term “detect,” “detection,” “detectable,” or “detecting” 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 performed on a biomolecule of the disclosure. [0015] As used herein, “detectable affinity” generally means the binding ability between a biomolecule and its target, reported by a K_D, EC₅₀, or IC₅₀ value, is at most about 10⁵ M or lower. A binding affinity, reported by a K_D, EC₅₀, or IC₅₀ value, higher than 10⁵ M is generally no longer measurable with common methods such as ELISA and SPR and is therefore of secondary importance. Thus, “detectable affinity” may refer to a K_D value of about 10⁵ M or lower as determined by ELISA or SPR, preferably SPR.

[0016] It is noted that the complex formation between a biomolecule of the disclosure and its target is influenced by many different factors, such as the concentrations of the respective target, the presence of competitors, pH and the ionic strength of the buffer system used, the experimental method used for determination of the binding affinity (e.g., fluorescence titration, competitive ELISA (also called competition ELISA), and surface plasmon resonance), and even the mathematical algorithm used for evaluation of the experimental data. Therefore, it is clear to the skilled person that binding affinity reported by a K_D, EC₅o, or IC₅o value may vary within a certain experimental range, depending on the method and experimental setup. This means that there may be a slight deviation in the measured K_D, EC₅₀, or IC₅₀ values or a tolerance range depending, for example, on whether such values were determined by ELISA (including direct or competition ELISA), by SPR, or by another method.

[0017] As used herein, “specific for,” “specific binding,” “specifically bind,” or “binding specificity” relates to the ability of a biomolecule to discriminate between the desired target (for example, 0X40) and one or more reference targets (for example, physiological ligands of human tear lipocalin). It is understood that such specificity is not an absolute but a relative property and can be determined, for example, by means of SPR, western blots, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), ImmunoHistoChemistry (IHC), and peptide scans.

[0018] When used herein in the context of the lipocalin muteins of the present disclosure that bind to 0X40, the term “specific for,” “specific binding,” “specifically bind,” or “binding specificity” means that the lipocalin mutein binds to, reacts with, or is directed against 0X40, as described herein, but does not essentially bind another protein. The term “another protein” includes any proteins that are not 0X40 or proteins closely related to or being homologous to 0X40. However, 0X40 from species other than human and fragments and/or variants of 0X40 are not excluded by the term “another protein.” The term “does not essentially bind” means that the lipocalin muteins of the present disclosure bind another protein with lower binding affinity than 0X40, i.e., show 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 mutein 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 0X40 and the reaction of said lipocalin with (an)other protein(s).

[0019] As used herein, the term “lipocalin” refers to a monomeric protein of approximately 18-20 kDa in weight, having a cylindrical b-pleated sheet supersecondary structural region comprising a plurality of b-strands (preferably eight b-strands designated A to H) connected pair-wise by a plurality of (preferably four) loops at one end to thereby comprise a ligand-binding pocket and define the entrance to the ligand-binding pocket. Preferably, the loops comprising the ligand-binding pocket used in the present disclosure are loops connecting the open ends of b-strands A and B, C and D, E and F, and G and H, and are designated loops AB, CD, EF, and GH. It is well-established that the diversity of said loops in the otherwise rigid lipocalin scaffold 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). It is understood that the lipocalin family of proteins has 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 also well-known to one of skill in the art (see, e.g., U.S. Patent No. 7,250,297). Proteins falling in the definition of “lipocalin”, as used herein, include, but are not limited to, human lipocalins including human tear lipocalin (Tic, Lcn1), Lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, cq-acid glycoprotein 1 , cq-acid glycoprotein 2, cq-microglobulin, complement component 8y, retinol-binding protein (RBP), the epididymal retinoic acid-binding protein, glycodelin, odorant binding protein lla, odorant-binding protein lib, lipocalin-15 (Lcn15), and prostaglandin D synthase.

[0020] As used herein, unless otherwise specified, “tear lipocalin” refers to human tear lipocalin (hTIc) and further refers to mature human tear lipocalin. The term “mature” when used to characterize a protein means a protein essentially free from the signal peptide. A “mature hTIc” of the instant disclosure refers to the mature form of human tear lipocalin, which is free from the signal peptide. Mature hTIc is described by residues 19-176 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P31025, the amino acid sequence of which is indicated in SEQ ID NO: 1. An exemplary DNA sequence encoding human tear lipocalin is shown in SEQ ID NO: 62.

[0021] As used herein, “Lipocalin-2” or “neutrophil gelatinase-associated lipocalin” refers to human Lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL) and further refers to the mature human Lipocalin-2 or mature human neutrophil gelatinase- associated lipocalin. The term “mature” when used to characterize a protein means a protein essentially free from the signal peptide. A “mature hNGAL” of the instant disclosure refers to the mature form of human neutrophil gelatinase-associated lipocalin, which is free from the signal peptide. Mature hNGAL is described by residues 21-198 of the sequence deposited with the SWISS-PROT Data Bank under Accession Number P80188, the amino acid sequence of which is indicated in SEQ ID NO: 2. An exemplary DNA sequence encoding hNGAL is shown in SEQ ID NO: 63.

[0022] As used herein, a “native sequence” refers to a protein or a polypeptide having a sequence that occurs in nature or having a wild-type sequence, regardless of its mode of preparation. Such native sequence protein or polypeptide can be isolated from nature or can be produced by other means, such as by recombinant or synthetic methods.

[0023] The term “native sequence lipocalin” refers to a lipocalin having 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 (wild-type) lipocalin from any organism, in particular, a mammal. The term “native sequence”, when used in the context of a lipocalin 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. The terms “native sequence lipocalin” and “wild-type lipocalin” are used interchangeably herein.

[0024] 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 amino acids or nucleotides, compared to the naturally occurring (wild-type) protein or nucleic acid. Said term also includes fragments of a mutein as described herein. The present disclosure explicitly encompasses lipocalin muteins, as described herein, having a cylindrical b-pleated sheet supersecondary structural region comprising eight b-strands connected pair-wise by four loops at one end to thereby comprise a ligand-binding pocket and define the entrance of the ligand binding pocket, wherein at least one amino acid located within said four loops has been mutated as compared to the native sequence lipocalin. Lipocalin muteins of the present disclosure preferably have the function of binding 0X40 as described herein.

[0025] As used herein, the term “fragment,” in connection with the lipocalin muteins of the disclosure, refers to proteins or polypeptides derived from full-length mature hTIc or hNGAL or lipocalin muteins that are N-terminally and/or C-terminally truncated, i.e., lacking at least one of the N-terminal and/or C-terminal amino acids. Such fragments may include at least 10 or more, such as 20 or 30 or more consecutive amino acids of the primary sequence of mature hTIc or hNGAL or the lipocalin mutein it is derived from and are usually detectable in an immunoassay of mature hTIc or 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. As an illustrative example, such a fragment may lack the one, two, three, or four N-terminal (His-His-Leu-Leu) and/or one or two C-terminal amino acids (Ser-Asp) of mature hTIc. It is understood that the fragment is preferably a functional fragment of mature hTIc or hNGAL or the lipocalin mutein from which it is derived, which means that it preferably retains the binding specificity, preferably to 0X40, of mature hTIc/hNGAL or the lipocalin mutein it is derived from. As an illustrative example, such a functional fragment may comprise at least amino acids at positions 5-153, 5-150, 9-148, 12-140, 20-135, or 26-133 corresponding to the linear polypeptide sequence of mature hTIc. As another illustrative example, such a functional fragment may comprise at least amino acids at positions 13-157, IS- 150, 18-141 , 20-134, 25-134, or 28-134 corresponding to the linear polypeptide sequence of mature hNGAL.

[0026] A “fragment” with respect to the corresponding target 0X40 of a lipocalin mutein of the disclosure, refers to N-terminally and/or C-terminally truncated 0X40 or protein domains of 0X40. Fragments of 0X40 as described herein retain the capability of the full-length 0X40 to be recognized and/or bound by a lipocalin mutein of the disclosure. As an illustrative example, the fragment may be an extracellular domain of 0X40. Such an extracellular domain of human 0X40 may comprise residues 29-214 of UniProt P43489 or residues 1-186 of SEQ ID NO: 7. Such an extracellular domain may comprise, consist essentially of, or consist of amino acids of the extracellular subdomains of 0X40, such as the individual or combined amino acid sequences of domain 1 (residues 30-65 of UniProt P43489), domain 2 (residues 66-107 of UniProt P43489), domain 3 (residues 108-126 of UniProt P43489) and domain 4 (residues 127- 167 of UniProt P43489). Such an extracellular domain of cynomolgus 0X40 may comprise residues 1-186 of SEQ ID NO: 9. [0027] As used herein, the term “variant” relates to derivatives of a protein or polypeptide that include mutations, for example by substitutions, deletions, insertions, and/or chemical modifications of an amino acid sequence or nucleotide sequence. In some embodiments, such mutations and/or chemical modifications do not reduce the functionality of the protein or peptide. Such substitutions may be conservative, i.e. , 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, threonine, and valine; 2) aspartic acid, glutamic acid, glutamine, asparagine, and histidine; 3) arginine, lysine, glutamine, asparagine, and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine, and proline; and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Such variants include proteins or polypeptides, wherein one or more amino acids have been substituted 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. Such variants also include, for instance, proteins or polypeptides in which one or more amino acid residues are added or deleted at the N- and/or C-terminus. Generally, a variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95% or at least about 98% amino acid sequence identity with the native sequence protein or polypeptide. A variant preferably retains the biological activity, e.g., binding the same target, of the protein or polypeptide it is derived from.

[0028] The term “variant”, as used herein with respect to the corresponding protein ligand 0X40 of a lipocalin mutein of the disclosure, relates to 0X40 or a fragment thereof 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 the native sequence of 0X40 (wild-type 0X40), such as 0X40 as deposited with UniProt P43489 as described herein. An 0X40 variant has preferably an amino acid sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90% or 95% with a wild-type 0X40, such as 0X40 as deposited with UniProt P43489 as described herein. An 0X40 variant as described herein retains the ability to bind lipocalin muteins specific to 0X40 disclosed herein.

[0029] The term “variant”, as used herein with respect to a lipocalin mutein, relates to a lipocalin mutein or fragment thereof of the disclosure, wherein the sequence has mutations, including substitutions, deletions, and insertions, and/or chemical modifications. A variant of a lipocalin mutein as described herein retains the biological activity, e.g., binding to 0X40, of the lipocalin mutein from which it is derived. Generally, a lipocalin mutein variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% amino acid sequence identity with the lipocalin mutein from which it is derived.

[0030] As used herein, the term “mutagenesis” refers to the introduction of mutations into a polynucleotide or amino acid sequence. Mutations are preferably introduced under experimental conditions such that the amino acid naturally occurring at a given position of the protein or polypeptide sequence can be altered, for example substituted by at least one amino acid. 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 amino acids, leading to an addition of two amino acid residues compared to the length of the respective segment of the native protein or polypeptide amino acid sequence. Such an insertion or deletion may be introduced independently from each other in any of the sequence segments that can be subjected to mutagenesis in the disclosure. In one exemplary embodiment of the disclosure, an insertion may be introduced into an amino acid sequence segment corresponding to the loop AB of the native sequence lipocalin (of. International Patent Publication No. WO 2005/019256, which is incorporated herein by reference in its entirety).

[0031] As used herein, the term “random mutagenesis” means that no predetermined mutation (alteration of an amino acid) 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.

[0032] As used herein, the term “sequence identity” or “identity” denotes 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.

[0033] As used herein, the term “sequence homology” or “homology” has its usual meaning, and a homologous amino acid includes identical amino acids as well as amino acids which are regarded to be conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or polypeptide of the disclosure (e.g., any lipocalin muteins of the disclosure).

[0034] A skilled artisan will recognize available computer programs, for example BLAST

(Altschul et al., 1997), BLAST2 (Altschul et al., 1990), and Smith- Waterman (Smith and Waterman, 1981), for determining sequence homology or sequence identity using standard parameters. 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 some embodiments, the percentage of homology is based on the alignment of the entire protein or polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1 ; cutoff value set to 10³) 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.

[0035] Specifically, in order to determine whether the amino acid sequence of a lipocalin

(mutein) is different from that of a reference (wild-type) lipocalin with regard to a certain position in the amino acid sequence of the reference (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, the amino acid sequence of a reference (wild-type) lipocalin can serve as “subject sequence” or “reference sequence”, while the amino acid sequence of a lipocalin mutein serves as “query sequence.” The terms “wild-type sequence,” “reference sequence,” and “subject sequence” are used interchangeably herein. A preferred wild-type sequence of a lipocalin is the sequence of hTLc as shown in SEQ ID NO: 1 or hNGAL as shown in SEQ ID NO: 2.

[0036] “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.

[0037] As used herein, the term “position” means the position of either an amino acid within an amino acid sequence disclosed herein or the position of a nucleotide within a nucleic acid sequence disclosed herein. It is to be understood that when the term “correspond” or “corresponding” is 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 or amino acids. Accordingly, the absolute position of a given amino acid in accordance with the disclosure may vary from the corresponding position due to deletion or addition of amino acids elsewhere in a (mutant or wild-type) lipocalin. Similarly, the absolute position of a given nucleotide in accordance with the present disclosure may vary from the corresponding position 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 regions (including exons and introns).

[0038] A “corresponding position” in accordance with the disclosure may be the sequence position that aligns to the sequence position it corresponds to in a pairwise or multiple sequence alignment according to the present disclosure. It is preferably to be understood that for a “corresponding position” in accordance with the disclosure, the absolute positions of nucleotides or amino acids may differ from adjacent nucleotides or amino acids but said adjacent nucleotides or amino acids which may have been exchanged, deleted, or added may be comprised by the same one or more “corresponding positions”.

[0039] In addition, for a corresponding position in a lipocalin mutein based on a reference sequence in accordance with the disclosure, it is preferably to be understood that the positions of nucleotides or amino acids of a lipocalin mutein can structurally correspond to the positions elsewhere in a reference lipocalin (wild-type lipocalin) or another lipocalin mutein, even if they may differ in the absolute position numbers, as appreciated by the skilled person in light of the highly-conserved overall folding pattern among lipocalins.

[0040] As used interchangeably herein, the terms “conjugate,” “conjugation,” “fuse,”

“fusion,” or “linked” refer to the joining together of two or more subunits, through all forms of covalent or non-covalent linkage, by means including, but not limited to, genetic fusion, chemical conjugation, coupling through a linker or a cross-linking agent, and non-covalent association.

[0041] The term “fusion polypeptide” or “fusion protein”, as used herein, refers to a polypeptide or protein comprising two or more subunits. In some embodiments, a fusion protein as described herein comprises two or more subunits, wherein at least one of these subunits binds to 0X40. In some embodiments, at least two subunits bind to 0X40. Within the fusion protein, the subunits may be linked by covalent or non-covalent linkage. Preferably, the fusion protein is a translational fusion between the two or more subunits. The translational fusion may be generated by genetically engineering the coding sequence for one subunit in a reading frame with the coding sequence of a further subunit. Both subunits may be interspersed by a nucleotide sequence encoding a linker. However, the subunits of a fusion protein of the present disclosure may also be linked through chemical conjugation. The subunits forming the fusion protein are typically linked to each other as follows: C-terminus of one subunit to N-terminus of another subunit, or C-terminus of one subunit to C-terminus of another subunit, or N-terminus of one subunit to N-terminus of another subunit, or N-terminus of one subunit to C-terminus of another subunit. The subunits of the fusion protein can be linked in any order and may include more than one of any of the constituent subunits. If one or more of the subunits is part of a protein (complex) that consists of more than one polypeptide chain, the term “fusion protein” may also refer to the protein comprising the fused sequences and all other polypeptide chain(s) of the protein (complex). As an illustrative example, where a full-length immunoglobulin is fused to a lipocalin mutein via a heavy or light chain of the immunoglobulin, the term “fusion protein” may refer to the single polypeptide chain comprising the lipocalin mutein and the heavy or light chain of the immunoglobulin. The term “fusion protein” may also refer to the entire immunoglobulin (both light and heavy chains) and the lipocalin mutein fused to one or both of its heavy and/or light chains.

[0042] As used herein, the term “subunit” of a fusion protein/polypeptide disclosed herein refers to a single protein or a separate polypeptide chain, which can form a stable folded structure by itself and may define a unique function of providing a binding motif towards a target. In some embodiments, a preferred subunit of the disclosure is a lipocalin mutein. In some other embodiments, a preferred subunit of the disclosure is a full-length immunoglobulin or an antigen-binding domain or fragment thereof.

[0043] A “linker” that may be comprised by a fusion protein or polypeptide of the present disclosure joins together two or more subunits of a fusion protein as described herein. The linkage can be covalent or non-covalent. A preferred covalent linkage is via a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. Accordingly, in a preferred embodiment, said linker comprises one or more amino acids, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids. Preferred peptide linkers are described herein, including glycine-serine (GS) linkers, glycosylated GS linkers, and proline-alanine-serine polymer (PAS) linkers. In some preferred embodiments, a GS linker, such as a (G₄S)₃ linker as described in SEQ ID NO: 13, is used to join together the subunits of a fusion protein or polypeptide. Other preferred linkers include chemical linkers. [0044] As used herein, the term “albumin” includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat serum albumin.

[0045] As used herein, the term “organic molecule” or “small organic molecule” 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 2,000 daltons, preferably between 100 and 1,000 daltons, and optionally including one or two metal atoms.

[0046] 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, including tumor tissue.

[0047] 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” used herein is a human.

[0048] An “effective amount” is an amount sufficient to yield beneficial or desired results.

An effective amount can be administered in one or more individual administrations or doses.

[0049] As used herein, “antibody” includes whole antibodies or any antigen binding fragment (i.e. , “antigen-binding portion” or “antigen-binding domain”) or single chain thereof. A whole antibody refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable domain (V_H or HCVR) and a heavy chain constant region (C_H). The heavy chain constant region is comprised of three domains, C_Hi, C and C_H . Each light chain is comprised of a light chain variable domain (V_L or LCVR) and a light chain constant region (C_L). The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each V_H and V_L is composed of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may optionally mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0050] As used herein, “antigen binding fragment” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody include (i) a Fab fragment consisting of the V_H, V_L, C_L and C_Hi domains; (ii) a F(ab')₂ fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment consisting of the V_H, V_L, C_L and C_Hi domains and the region between the C_Hi and C_H2 domains; (iv) an Fd fragment consisting of the V_H and C_Hi domains; (v) a single-chain Fv fragment consisting of the V_H and V_L domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al. , Nature, 1989) consisting of a V_H domain; (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs which may optionally be joined by a synthetic linker; (viii) a “diabody” comprising the V_H and V_L connected in the same polypeptide chain using a short linker (see, e.g., patent documents EP 404,097; WO 93/11161 ; and Holliger et al., Proc Natl Acad Sci U S A, 1993); and (ix) a “domain antibody fragment” containing only the V_H or V_L, where in some instances two or more V_H regions are covalently joined.

[0051] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies may also be fully human.

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

[0053] “Fragment crystallizable region” or “Fc region” refers to the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof (numbering according to EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000)). The C-terminal lysine (residue 447 according to EU index of Kabat) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue. Suitable native-sequence Fc regions for use in the antibodies of the disclosure include human lgG1 , lgG2 (lgG2A, lgG2B), lgG3, and lgG4.

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

[0055] As used herein, “isolated antibody” refers to an antibody that is substantially free of its natural environment. For instance, an isolated antibody is substantially free of cellular material and other proteins from the cell or tissue source from which it is derived. An “isolated antibody” further refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In an illustrative example, an isolated antibody that specifically binds 0X40 is substantially free of antibodies that specifically bind antigens other than 0X40. However, an isolated antibody that specifically binds 0X40 may have cross-reactivity with other antigens, such as 0X40 molecules from other species.

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

[0057] As used herein, “humanized antibody” refers to an antibody that consists of the

CDRs of antibodies derived from mammals other than human, and the FR region and the constant region of a human antibody or derived from a human antibody. A humanized antibody may comprise a variable domain that has a variable region amino acid sequence which, analyzed as a whole, is closer to human than to other species as assessed using the Immunogenetics Information System (IMGT) DomainGapAlign tool, as described by Ehrenmann et al. (2010). A humanized antibody may be useful as an effective component in a therapeutic agent due to the reduced antigenicity. The term “therapeutic agent” or “therapeutically active agent”, as used herein, refers to an agent which is therapeutically useful. A therapeutic agent may be any agent for the prevention, amelioration, or treatment of a disease, a physiological condition, a symptom, or for the evaluation or diagnosis thereof. A humanized antibody is useful as an effective component in a therapeutic agent due to the reduced antigenicity.

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

III. DESCRIPTIONS OF FIGURES

[0059] Figure 1 : depicts an alignment of amino acid sequences of OX40-specific lipocalin muteins (SEQ ID NOs: 36-61 and 73-75), in comparison with the linear polypeptide sequence of wild-type mature hTIc (SEQ ID NO: 1) and mature hNGAL (SEQ ID NO: 2) (Figure 1A) and provides the design of the lipocalin mutein Fc fusions generated by fusing one or more OX40-specific lipocalin muteins to the C-terminus of the Fc region of an antibody via a peptide linker (Figure 1B).

[0060] Figure 2: depicts the results of fluorescence-activated cell sorting (FACS) studies carried out in order to assess the specific binding of lipocalin muteins (SEQ ID NOs: 37- 44 and 47-60) to human 0X40 (Figure 2A) or cynomolgus 0X40 (Figure 2B), respectively, expressed on mammalian cells as described in Example 5. Chinese hamster ovary (CHO) cells stably transfected with human or cynomolgus 0X40 were incubated with lipocalin muteins, and the bound muteins were detected using a fluorescently labeled anti-hTIc or anti-hNGAL antibody. All lipocalin muteins show binding to 0X40 expressed on CHO cells. The negative control lipocalin muteins of SEQ ID NOs: 3 and 4 showed no binding. The geometric means of the fluorescence intensity were normalized to maximal mean and fit with a 1 :1 binding model. The resulting EC₅₀ values are provided in Table 4.

[0061] Figure 3: shows that exemplary lipocalin muteins (SEQ ID NOs: 36, 38, 39, 41-

44, 47, 49, 52-55, and 57-61) compete with human 0X40 ligand (huOX40L, OX40’s natural ligand) for the binding to 0X40 in an ELISA assay as described in Example 6. A huOX40L coated assay plate was incubated with lipocalin mutein and huOX40-Fc (human 0X40 extracellular domain fused to human lgG1 Fc fragment), and bound huOX40-Fc was detected using a goat anti-human IgG antibody conjugated with horseradish peroxidase. A dose dependent inhibition of huOX40-Fc binding to huOX40L molecules by OX40-specific lipocalin muteins was shown. The optimized lipocalin muteins (SEQ ID NOs: 38, 39, 41-44, 49, 54, 55, and 57-61) showed improved inhibitory effect on OX40/OX40L interaction compared to the parental mutein (SEQ ID NO: 36 or 47). The negative control lipocalin mutein (SEQ ID NO: 3 or 4) did not lead to measurable inhibition of huOX40 binding to huOX40L coated on the assay plate.

[0062] Figure 4: shows that the OX40-specific hTIc muteins (SEQ ID NOs: 43 and 44) and hNGAL muteins (SEQ ID NOs: 52, 53, 55, and 58) bind overlapping epitopes, as shown in the competitive ELISA studies described in Example 8. A constant concentration of hTIc mutein SEQ ID NO: 43 or SEQ ID NO: 44 was coated on a microtiter plate, followed by adding a mixture of hNGAL muteins (SEQ ID NO: 52, 53, 55, or 58) at different concentrations and the tracer of biotinylated human 0X40 with a C-terminal polyhistidine tag (huOX40-His-bio) at a fixed concentration. Bound tracer was detected via ExtrAvidin-Peroxidase.

[0063] Figure 5: shows that the OX40-specific muteins (SEQ ID NOs: 43, 44, 52, 53,

55, and 58) bind epitopes that overlap with those of certain reference 0X40 antibodies, as shown in the competitive ELISA studies described in Example 9. A constant concentration of an 0X40 antibody (SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 30 and 31) was coated on a microtiter plate, followed by adding a mixture of molecules to be tested at different concentrations and the tracer of huOX40-His-bio at a fixed concentration. Bound tracer was detected via ExtrAvidin-Peroxidase. The hTIc muteins (SEQ ID NOs: 43 and 44) compete with all tested 0X40 antibodies for 0X40 binding. The hNGAL muteins (SEQ ID NOs: 52, 53, 55, and 58) compete with SEQ ID NOs: 28 and 29 and SEQ ID NOs: 30 and 31 , but not with SEQ ID NOs: 26 and 27, for 0X40 binding.

[0064] Figure 6: depicts the results of a T cell activation assay evaluating the ability of a set of representative OX40-specific lipocalin muteins (SEQ ID NOs: 41-45 and 52-56) to co stimulate T cell responses as described in Example 10. The lipocalin muteins were captured via an anti-lipocalin scaffold antibody coated directly on a flat bottom tissue culture plate 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. Supernatant interleukin 2 (IL-2) levels served as readout for T cell activation. There is a clear increase in IL-2 concentration in the supernatant due to T cell activation induced by the lipocalin muteins of SEQ ID NOs: 41-45 and 52-56, while the negative controls of SEQ ID NO: 3 and SEQ ID NO: 4, as well as the lgG1 isotype do not induce measurable T cell co-stimulation.

IV. DETAILED DESCRIPTION OF THE DISCLOSURE [0065] In one aspect, the present disclosure provides human lipocalin muteins that bind

0X40 and useful applications therefor. The disclosure also provides methods of making 0X40- binding lipocalin muteins described herein, as well as compositions comprising such lipocalin muteins. Provided OX40-binding lipocalin muteins as well as compositions thereof may be used in methods of detecting or binding 0X40 in a sample or in methods of modulating immune responses. No such human lipocalin muteins having these features attendant to the uses provided by the present disclosure have been previously described.

A. Exemplary lipocalin muteins of the disclosure.

[0066] 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 homeostasis (reviewed, e.g., in Flower et al. , 2000, Flower, 1996).

[0067] 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 b-sheet closed back on itself to form a continuously hydrogen-bonded b-barrel. This b-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 b- strands. The other end of the b-barrel is open to the solvent and encompasses a target-binding site, which is formed by four flexible peptide loops (AB, CD, EF, and GH). It is the 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). [0068] A lipocalin mutein according to the present disclosure may be a mutein of any lipocalin. Examples of suitable lipocalins (also sometimes designated as “reference lipocalin,” “wild-type lipocalin,” “reference protein scaffolds,” or simply “scaffolds”) of which a mutein may be used include, but are not limited to, tear lipocalin (lipocalin-1 , Tic, or von Ebner’s gland protein), retinol binding protein, neutrophil lipocalin-type prostaglandin D-synthase, b- lactoglobulin, bilin-binding protein (BBP), apolipoprotein D (APOD), neutrophil gelatinase- associated lipocalin (NGAL), a2-microglobulin-related protein (A2m), 24p3/uterocalin (24p3), von Ebner’s gland protein 1 (VEGP 1), von Ebner’s gland protein 2 (VEGP 2), and Major allergen Can f 1 (ALL-1). In particular embodiments, a lipocalin mutein is derived from the lipocalin group consisting of human tear lipocalin (hTIc), human neutrophil gelatinase- associated lipocalin (hNGAL), human apolipoprotein D (hAPOD) and the bilin-binding protein of Pieris brassicae.

[0069] The amino acid sequence of a lipocalin mutein according to the disclosure may have a high sequence identity to the reference (or wild-type) lipocalin from which it is derived, for example, hTIc or hNGAL, when compared to sequence identities with another lipocalin (see also above). In this general context, the amino acid sequence of a lipocalin mutein according to the disclosure is at least substantially similar to the amino acid sequence of the corresponding reference (wild-type) lipocalin, with the proviso that there may be gaps (as defined herein) 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 corresponding reference (wild-type) lipocalin, has, in some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, or 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 with the wild-type lipocalin, substitutions as described herein, which renders the lipocalin mutein capable of binding to 0X40.

[0070] Typically, a lipocalin mutein of the disclosure contains one or more mutated amino acid residues - relative to the amino acid sequence of the wild-type or reference lipocalin, for example, hTIc and hNGAL - in the four loops at the open end that comprise a ligand-binding pocket and define the entrance of the ligand-binding pocket (cf. above). As explained above, these regions are essential in determining the binding specificity of a lipocalin mutein for the desired target. In some embodiments, a lipocalin mutein of the disclosure may also contain mutated amino acid residues in regions outside of the four loops. A lipocalin mutein of the disclosure may contain one or more mutated amino acid residues in one or more of the three peptide loops (designated BC, DE, and FG) connecting the b-strands at the closed end of the lipocalin. In some embodiments, a mutein derived from of tear lipocalin, NGAL 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 b-barrel structure that is located opposite to the natural lipocalin binding pocket. In some embodiments, a mutein derived from tear lipocalin, NGAL or a homologue thereof, may have no mutated amino acid residues in peptide loop DE arranged at the end of the b-barrel structure, compared to the wild-type sequence of tear lipocalin, NGAL or a homologue thereof.

[0071] A lipocalin mutein according to the disclosure includes one or more, such as 2, 3,

4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more mutated amino acid residues in comparison to the amino acid sequence of a corresponding reference (wild-type) lipocalin, provided that such a lipocalin mutein should be capable of binding to 0X40. In some embodiments, a lipocalin mutein of the disclosure includes at least two, including 2, 3, 4, 5, or even more, mutated amino acid residues, where a native amino acid residue of the corresponding reference (wild-type) lipocalin is substituted by an arginine residue.

[0072] Any types and numbers of mutations, including substitutions, deletions, and insertions, are envisaged as long as the lipocalin mutein retains its capability to bind 0X40, and/or it has a sequence identity of at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or higher identity to the amino acid sequence of the reference (wild-type) lipocalin, for example, mature hTIc or mature hNGAL.

[0073] In some embodiments, a substitution is a conservative substitution. In some embodiments, a substitution is a non-conservative substitution or one or more from the exemplary substitutions below.

[0074] 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 Ser, Thr, or Val; Arg Lys, Gin, Asn, or His; Asn Gin, Glu, Asp, or His; Asp Glu, Gin, Asn, or His; Gin Asn, Asp, Glu, or His; Glu Asp, Asn, Gin, or His; His Arg, Lys, Asn, Gin, Asp, or Glu; lie Thr, Leu, Met, Phe, Val, Trp, Tyr, Ala, or Pro; Leu Thr, lie, Val, Met, Ala, Phe, Pro, Tyr, or Trp; Lys Arg, His, Gin, or Asn; Met ® Thr, Leu, Tyr, lie, Phe, Val, Ala, Pro, or Trp; Phe Thr, Met, Leu, Tyr, lie, Pro, Trp, Val, or Ala; Ser ® Thr, Ala, or Val; Thr ® Ser, Ala, Val, lie, Met, Val, Phe, Pro, or Leu; Trp ® Tyr, Phe, Met, lie, or Leu; Tyr ® Trp, Phe, lie, Leu, or Met; Val ® Thr, lie, Leu, Met, Phe, Ala, Ser, or Pro. Other substitutions are also permissible and can be determined empirically or in accordance with other known conservative or non-conservative substitutions. As a further orientation, the following groups each contain amino acids that can typically be taken to define conservative substitutions for one another: a. Alanine (Ala), Serine (Ser), Threonine (Thr), Valine (Val); b. Aspartic acid (Asp), Glutamic acid (Glu), Glutamine (Gin), Asparagine (Asn), Histidine (His); c. Arginine (Arg), Lysine (Lys), Glutamine (Gin), Asparagine (Asn), Histidine (His); d. Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val), Alanine (Ala), Phenylalanine (Phe), Threonine (Thr), Proline (Pro); e. Isoleucine (lie), Leucine (Leu), Methionine (Met), Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp).

[0075] If such conservative 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 be screened for a desired characteristic. Examples of such more substantial changes are: Ala ® Leu or Phe; Arg ® Glu; Asn ® lie, Val, or Trp; Asp ® Met; Cys ® Pro; Gin ® Phe; Glu ® Arg; His ® Gly; lie ® Lys, Glu, or Gin; Leu ® Lys or Ser; Lys ® Tyr; Met ® Glu; Phe ® Glu, Gin, or Asp; Trp ® Cys; Tyr ® Glu or Asp; Val ® Lys, Arg, His.

[0076] In some embodiments, substantial modifications in the physical and biological properties of the lipocalin (mutein) 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.

[0077] 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, asparagine, glutamine; (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. In some embodiments, substitutions may entail exchanging a member of one of these classes for a member of another class.

[0078] 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.

B. Exemplary OX40-specific lipocalin muteins of the disclosure.

[0079] As noted above, a lipocalin is a polypeptide defined by its supersecondary structure, namely a cylindrical b-pleated sheet supersecondary structural region comprising eight b-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 b-pleated sheet supersecondary structural region comprising eight b-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 and wherein said lipocalin is effective to bind 0X40 with detectable affinity.

[0080] In some embodiments, lipocalin muteins disclosed herein may be or comprise a mutein of mature human tear lipocalin (hTIc). A mutein of mature hTIc may be designated herein as an “hTIc mutein”. In some other embodiments, a lipocalin mutein disclosed herein is a mutein of mature human neutrophil gelatinase-associated lipocalin (hNGAL). A mutein of mature hNGAL may be designated herein as an “hNGAL mutein”.

[0081] In one aspect, the present disclosure includes any number of lipocalin muteins derived from a reference (wild-type) lipocalin, preferably derived from mature hTIc or mature hNGAL, that bind 0X40 with detectable affinity. In a related aspect, the disclosure includes various lipocalin muteins that are capable of activating the downstream signaling pathways of 0X40 by binding to 0X40. In this sense, 0X40 can be regarded as a non-natural target of the reference (wild-type) lipocalin, preferably hTIc or hNGAL, where “non-natural target” refers to a substance that does not bind to the reference (wild-type) lipocalins under physiological conditions. By engineering reference (wild-type) lipocalins with one or more mutations at certain sequence positions, the present inventors have demonstrated that high affinity and high specificity for the non-natural target, 0X40, is possible. In some embodiments, at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 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, with the aim of generating a lipocalin mutein which is capable of binding 0X40.

[0082] The lipocalin muteins of the disclosure may have mutated, including substituted, deleted and inserted, amino acid residue(s) at one or more sequence positions corresponding to sequence positions in the linear polypeptide sequence of a reference lipocalin, preferably hTIc or hNGAL. Preferably, the number of amino acid residues of a lipocalin mutein of the disclosure that is mutated in comparison with the amino acid sequence of the reference lipocalin, preferably hTIc or hNGAL, is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more such as 25, 30, 35, 40, 45 or 50, with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 being preferred and 9, 10 or 11 being even more preferred. Even more preferred is that the number of amino acid residues of a lipocalin mutein of the disclosure that is mutated in comparison with the amino acid sequence of the reference lipocalin, preferably hTIc or hNGAL, is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, such as 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 40, 45 or 50, with 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, or 33 being preferred and 29, 30, or 31 being even more preferred. However, it is preferred that a lipocalin mutein of the disclosure is still capable of binding 0X40.

[0083] In some embodiments, 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 reference (wild-type) lipocalin; for example, SEQ ID NOs: 36-61 and 73-75. In some embodiments, the present disclosure encompasses hTIc muteins as defined above, in which the first one, two, three, or four N-terminal amino acid residues of the sequence of mature hTIc (His-His-Leu-Leu; positions 1-4) and/or the last one or two C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of the mature hTIc have been deleted (e.g., SEQ ID NOs: 36-46). Further, a lipocalin mutein of the disclosure may include the wild-type (natural) amino acid sequence of the reference (wild-type) lipocalin, preferably hTIc or hNGAL, outside the mutated amino acid sequence positions.

[0084] In some embodiments, one or more mutated amino acid residues incorporated into a lipocalin mutein of the disclosure do, at least essentially, not hamper or not interfere with or not influence the binding activity to the designated target and/or the folding of the mutein. Such mutations, including substitution, deletion and insertion, can be accomplished at the DNA level using established standard methods (Sambrook and Russell, 2001, Molecular cloning: a laboratory manual). In some embodiments, (a) mutated amino acid residue(s) at one or more sequence positions corresponding to the linear polypeptide sequence of the reference (wild- type) lipocalin, preferably hTIc or hNGAL, is/are introduced through random mutagenesis by substituting the nucleotide triplet(s) encoding the corresponding sequence positions of the reference lipocalin with a subset of nucleotide triplets.

[0085] In some embodiments, a lipocalin mutein that binds 0X40 with detectable affinity may include at least one amino acid substitution of a native cysteine residue by another amino acid, for example, a serine residue. In some embodiments, a lipocalin mutein that binds 0X40 with detectable affinity may include one or more non-native cysteine residues substituting one or more amino acids of a reference (wild-type) lipocalin, preferably hTIc or hNGAL. In some embodiments, 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 bridges. In some embodiments, said cysteine bridge may connect at least two loop regions. The definition of these regions is used herein in accordance with (2000), Flower (1996) and Breustedt et al. (2005).

[0086] Generally, a lipocalin mutein of the disclosure may have at least about 70%, including at least about 80%, such as at least about 85% amino acid sequence identity, with the amino acid sequence of the mature hTIc (SEQ ID NO: 1) or mature hNGAL (SEQ ID NO: 2).

[0087] In some embodiments, the present disclosure provides OX40-binding lipocalin muteins. In this regard, the disclosure provides one or more lipocalin muteins that are capable of binding 0X40 with a detectable affinity, preferably with an affinity measured by a K_D of about 10⁵ M or lower. The preferred lipocalin muteins are capable of binding 0X40 with an affinity measured by a K_D of about 500 nM or lower, about 400 nM or lower, about 300 nM or lower, about 200 nM or lower, about 150 nM or lower, about 100 nM or lower, about 70 nM or lower, about 50 nM or lower, about 30 nM or lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, about 2 nM or lower, about 1 nM or lower, or about 0.5 nM or even lower. In some embodiments, the OX40-binding lipocalin mutein may be cross-reactive with cynomolgus 0X40 (cyOX40), and in some embodiments, capable of binding cyOX40 with an affinity measured by a K_D of about 500 nM or lower, about 400 nM or lower, about 300 nM or lower, about 200 nM or lower, about 150 nM or lower, about 100 nM or lower, about 70 nM or lower, about 50 nM or lower, about 30 nM or lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, about 2 nM or lower, about 1 nM or lower, or about 0.5 nM or even lower. The K_D values can be, for example, measured in a surface-plasmon-resonance (SPR) assay, such as the SPR assay as essentially described in Example 4.

[0088] In some embodiments, a lipocalin mutein of the disclosure may be capable of binding 0X40 expressed on a cell. In some embodiments, a lipocalin mutein of the disclosure may be capable of binding 0X40 expressed on a cell with an EC₅₀ value of about 250 nM or lower, about 200 nM or lower, about 150 nM or lower, about 100 nM or lower, about 70 nM or lower, about 50 nM or lower, about 30 nM or lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 7 nM or lower, about 5 nM or lower, about 3 nM or lower, about 2 nM or lower, or about 1 nM or even lower. The EC₅₀ of a mutein may be measured, for example, in a fluorescence activated cell sorting (FACS) assay, such as the FACS assay as essentially described in Example 5. A cell expressing 0X40 may, for example, be a Chinese hamster ovary (CHO) cell transfected with human 0X40 or cynomolgus 0X40. In some embodiments, a lipocalin mutein of the disclosure does not essentially bind cells that do not express 0X40.

[0089] In some embodiments, a lipocalin mutein of the disclosure may be capable of competing with 0X40 ligand (OX40L) for binding to 0X40. In some further embodiments, a provided lipocalin mutein may be capable of competing with OX40L for binding to 0X40 with an IC₅₀ value of about 500 nM or lower, about 400 nM or lower, about 300 nM or lower, about 250 nM or lower, about 200 nM or lower, about 170 nM or lower, about 150 nM or lower, about 120 nM or lower, about 100 nM or lower, about 70 nM or lower, about 50 nM or lower, about 30 nM or lower, about 20 nM or lower, about 15 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.2 nM or lower, about 0.1 nM or lower, or about 0.05 nM or even lower. In some embodiments, an hTIc mutein of the disclosure may be able to inhibit the binding of 0X40 to OX40L. In some embodiments, a provided hTIc mutein may be able to inhibit the binding of 0X40 to OX40L with an IC₅₀ value of about 170 nM or lower, about 15 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.2 nM or lower, about 0.1 nM or lower, or about 0.05 nM or even lower. The inhibitory mode of action can, for example, be determined by an enzyme- linked immunosorbent assay (ELISA), such as the ELISA assay as essentially described in Example 6.

[0090] In some embodiments, an hTIc mutein of the disclosure may be able to compete with an anti-OX40 antibody comprising the amino acid sequences shown in SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 30 and 31 for binding to 0X40. Such competition may be assessed by an ELISA assay as essentially described in Example 9. In some embodiments, an hTIc mutein of the disclosure may have an epitope that overlaps with the epitope of an anti-OX40 antibody comprising the amino acid sequences shown in SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 30 and 31 .

[0091] In some embodiments, an hTIc mutein of the disclosure may be able to induce increased IL-2 secretion. In some embodiments, a provided hTIc mutein may be able to induce a concentration-dependent IL-2 secretion and/or demonstrate a tendency to induce enhanced IL-2 secretion at higher concentrations. IL-2 secretion may be measured, for example, in a functional T cell activation assay as essentially described in Example 10.

[0092] In some embodiments, an hTIc mutein of the disclosure may be able to costimulate T cell responses and/or T cell activation. The stimulated T cell response or T cell activation may be measured, for example, in a functional T cell activation assay as essentially described in Example 10.

[0093] In some embodiments, provided hTIc muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 5, 6, 8, 11 , 19, 23, 26-34, 36, 37, 40, 52, 55-56, 58, 60-61 , 65, 79, 86, 101 , 104-106, 108, 111, 113-114, 116, 121, 124, 137, 140, 148, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTIc muteins are capable of binding 0X40, in particular human 0X40.

[0094] In some embodiments, provided hTIc muteins may comprise a mutated amino acid residue at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or more positions corresponding to positions 5, 6, 8, 11, 19, 23, 26-34, 36, 37, 40, 52, 55-56, 58, 60-61 , 65, 79, 86, 101 , 104-106, 108, 111, 113-114, 116, 121 , 124, 137, 140,

148, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 20 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTIc muteins are capable of binding 0X40, in particular human 0X40.

[0095] In some embodiments, provided hTIc muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 26-34, 55-56, 60, 101 , 104- 105, 108, 111 , and 114 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTIc muteins are capable of binding 0X40, in particular human 0X40.

[0096] In some embodiments, provided hTIc muteins may comprise a mutated amino acid residue at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18 positions corresponding to positions 26-34, 55-56, 60, 101 , 104-105, 108, 111 , and 114 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above- mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTIc muteins are capable of binding 0X40, in particular human 0X40.

[0097] In some embodiments, provided hTIc muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 23, 26-34, 55-56, 58, 60-61 , 101 , 104-106, 108, 111 , 114, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above- mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 20 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTIc muteins are capable of binding 0X40, in particular human 0X40.

[0098] In some embodiments, provided hTIc muteins may comprise a mutated amino acid residue at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 positions corresponding to positions 23, 26-34, 55-56, 58, 60-61 , 101 , 104-106, 108, 111 , 114, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 20 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTIc muteins are capable of binding 0X40, in particular human 0X40.

[0099] In some embodiments, provided hTIc muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 5, 6, 8, 11 , 19, 36, 37, 40, 52, 65, 79, 86, 113, 116, 121 , 124, 137, 140, and 148 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above- mentioned positions of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTLc muteins are capable of binding 0X40, in particular human 0X40.

[0100] In other embodiments, provided hTIc muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 23, 26-34, 55-56, 58, 60-61 , 101 , 104-106, 108, 111 , 114, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1) and at one or more positions corresponding to positions 5, 6, 8, 11 , 19, 36, 37, 40, 52, 65, 79, 86, 113, 116, 121 , 124, 137, 140, and 148 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). In some preferred embodiments, the provided hTIc muteins are capable of binding 0X40, in particular human 0X40. [0101] In some embodiments, a lipocalin mutein according to the disclosure may include at least one amino acid substitution of a native cysteine residue by, e.g., a serine residue. In some embodiments, an hTIc mutein according to the disclosure includes an amino acid substitution of a native cysteine residue at positions corresponding to positions 61 and/or 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO:1) by another amino acid, such as a serine residue. In this context it is noted that it has been found that removal of the structural disulfide bond (on the level of a respective naive nucleic acid library) of wild-type hTIc that is formed by the cysteine residues 61 and 153 (of. Breustedt et al. , 2005) may provide hTIc muteins that are not only stably folded but are also able to bind a given non-natural target with high affinity. In some embodiments, the elimination of the structural disulfide bond may provide the further advantage of allowing for the generation or deliberate introduction of non-natural disulfide bonds into muteins of the disclosure, thereby, increasing the stability of the muteins. However, hTIc muteins that bind 0X40 and that have the disulfide bridge formed between Cys 61 and Cys 153 are also part of the present disclosure.

[0102] In some particular embodiments, an hTIc mutein of the disclosure may include one or more of the amino acid substitutions Cys 61 Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gin, Asp, Leu, Tyr, Met, Ser, Pro or Trp and/or Cys 153 Ser or Ala, at positions corresponding to positions 61 and/or 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO:1).

[0103] In some embodiments, either two or all three of the cysteine codons at positions corresponding to positions 61 , 101 , and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO:1) are replaced by a codon of another amino acid. Further, in some embodiments, an hTIc mutein according to the disclosure includes an amino acid substitution of a native cysteine residue at the position corresponding to position 101 of the linear polypeptide sequence of mature hTIc (SEQ ID NO:1) by a serine residue or a histidine residue.

[0104] In some embodiments, a mutein according to the disclosure comprises an amino acid substitution of a native amino acid by a cysteine residue at positions corresponding to positions 28 or 105 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1). Further, in some embodiments, a mutein according to the disclosure comprises an amino acid substitution of a native arginine residue at a position corresponding to position 111 of the linear polypeptide sequence of mature hTIc (SEQ ID NO:1) by a proline residue. Further, in some embodiments, a mutein according to the disclosure comprises an amino acid substitution of a native lysine residue at a position corresponding to position 114 of the linear polypeptide sequence of mature hTIc (SEQ ID NO:1) by a tryptophan residue or a glutamic acid residue.

[0105] In some embodiments, provided OX40-binding hTIc muteins may comprise, at one or more positions corresponding to positions 5, 6, 8, 11, 19, 23, 26-34, 36, 37, 40, 52, 55- 56, 58, 60-61 , 65, 79, 86, 101 , 104-106, 108, 111 , 113-114, 116, 121, 124, 137, 140, 148, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Ala 5 Thr; Ser 6 Thr; Glu 8 Lys; Gin 11 Arg; Leu 19 Met or Gin; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Thr 37 Ala; Thr 40 lie; Lys 52 Glu; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Lys 65 lie; Ala 79 Thr; Ala 86 Thr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met or Leu; Lys 114 Trp; Val 116 Ala; Lys 121 Met; Leu 124 Lys; Arg 137 His; Ser 140 Arg; Arg 148 Ser or Trp; and Cys 153 Ser. In some embodiments, an hTIc mutein of the disclosure comprises two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28 or more, or even all of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 15 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 20 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1).

[0106] In some embodiments, provided OX40-binding hTIc muteins may comprise, at one or more positions corresponding to positions 26-34, 55-56, 60, 101 , 104-105, 108, 111 , and 114 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Arg 60 Lys; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; Lys 108 lie; Arg 111 Pro; and Lys 114 Trp. In some embodiments, an hTIc mutein of the disclosure comprises two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1).

[0107] In some embodiments, provided OX40-binding hTIc muteins may comprise, at one or more positions corresponding to positions 23, 26-34, 55-56, 58, 60-61 , 101 , 104-106, 108, 111 , 114, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; and Cys 153 Ser. In some embodiments, an hTIc mutein of the disclosure comprises two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1). In some embodiments, an hTIc mutein of the disclosure comprises 10 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hTIc (SEQ ID NO: 1).

[0108] In some embodiments, provided OX40-binding hTIc muteins may comprise, at one or more positions corresponding to positions 5, 6, 8, 11 , 19, 36, 37, 40, 52, 65, 79, 86, 113, 116, 121 , 124, 137, 140, 148, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Ala 5 Thr; Ser 6 Thr; Glu 8 Lys; Gin 11 Arg; Leu 19 Met or Gin; Val 36 Asp; Thr 37 Ala; Thr 40 lie; Lys 52 Glu; Lys 65 lie; Ala 79 Thr; Ala 86 Thr; Val 113 Met or Leu; Val 116 Ala; Lys 121 Met; Leu 124 Lys; Arg 137 His; Ser 140 Arg; Arg 148 Ser orTrp; and Cys 153 Ser.

[0109] In some embodiments, provided OX40-binding hTIc muteins may comprise one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1):

(a) Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met

31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; and Cys 153 Ser; (b) Leu 19 Gin; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro

29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Thr 40 lie; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Ala 86 Thr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met; Lys 114 Trp; and Cys 153 Ser ;

(c) Leu 19 Met; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro

29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met; Lys 114 Trp; and Cys 153 Ser;

(d) Ala 5 Thr; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Lys 52

Glu; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met; Lys 114 Trp; Arg 137 His; and Cys 153 Ser;

(e) Ser 6 Thr; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Thr 37 Ala; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Lys 65 lie; Ala 79 Thr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; Val 116 Ala; and Cys 153 Ser;

(f) Ala 5 Thr; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36

Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; Arg 148 Ser; and Cys 153 Ser;

(g) Glu 8 Lys; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36

Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Leu; Lys 114 Trp; Lys 121 Met; and Cys 153 Ser; (h) Gin 11 Arg; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro

29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; Ser 140 Arg; Arg 148 Trp; and Cys 153 Ser;

(i) Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu

30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; and Cys 153 Ser;

(j) Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu

30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; Leu 124 Lys; and Cys 153 Ser; or

(k) Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu

30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; and Lys 114 Trp.

In some embodiments, an OX40-binding hTIc mutein includes all but three, all but two, or all but one mutated amino acid residues of one of the aforementioned sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1).

[0110] In some embodiments, the residual region, i.e. , the region differing from positions corresponding to positions 5, 6, 8, 11, 19, 23, 26-34, 36, 37, 40, 52, 55-56, 58, 60-61 , 65, 79, 86, 101 , 104-106, 108, 111, 113-114, 116, 121, 124, 137, 140, 148, and 153 of the linear polypeptide sequence of mature hTIc (SEQ ID NO: 1), of an hTIc mutein of the disclosure may comprise the wild-type (natural) amino acid sequence of the linear polypeptide sequence of mature hTIc outside the mutated amino acid sequence positions.

[0111] In some embodiments, an hTIc mutein of the disclosure has at least 70% sequence identity or at least 70% sequence homology to the sequence of mature hTIc (SEQ ID NO: 1). As an illustrative example, the mutein of SEQ ID NO: 44 has an amino acid sequence identity or a sequence homology of approximately 84% with the amino acid sequence of the mature hTIc.

[0112] In some embodiments, an hTIc mutein of the disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 36-46 or a fragment or variant thereof.

[0113] In some embodiments, an hTIc mutein of the disclosure has at least 75%, at least

80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-46.

[0114] The present disclosure also includes structural homologues of an hTIc mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-46, which structural homologues have an amino acid sequence homology or sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% in relation to said hTIc mutein.

[0115] In some embodiments, the present disclosure provides OX40-binding hNGAL muteins. In this regard, the disclosure provides one or more hNGAL muteins that are capable of binding 0X40 with a detectable affinity, preferably with an affinity measured by a K_D of about 10⁵ M or lower. The preferred hNGAL muteins are capable of binding 0X40 with an affinity measured by a K_D of about 400 nM or lower, about 135 nM or lower, about 50 nM or lower, about 30 nM or lower, about 20 nM or lower, about 10 nM or lower, about 5 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.3 nM or lower, about 0.2 nM or lower, about 0.1 nM or lower, or about 0.05 nM or even lower. In some embodiments, the OX40-binding hNGAL mutein may be cross-reactive with cynomolgus 0X40 (cyOX40) and, in some embodiments, capable of binding cyOX40 with an affinity measured by a K_D of about 290 nM or lower, about 115 nM or lower, about 55 nM or lower, about 10 nM or lower, about 5 nM or lower, about 4 nM or lower, about 2 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.4 nM or lower, about 0.3 nM or lower, or about 0.2 nM or even lower. The K_D values can be, for example, measured in an SPR assay, such as the SPR assay as essentially described in Example 4.

[0116] In some embodiments, an hNGAL mutein of the disclosure may be capable of binding 0X40 expressed on a cell. In some embodiments, an hNGAL mutein of the disclosure may be capable of binding 0X40 expressed on a cell with an EC₅₀ value of about 35 nM or lower, about 25 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 4 nM or lower, about 3 nM or lower, about 2 nM or lower, about 1 nM or lower, about 0.8 nM or lower, or about 0.5 nM or even lower. The EC₅₀ of a mutein may be measured, for example, in a FACS assay, such as the FACS assay as essentially described in Example 5. A cell expressing 0X40 may, for example, be a Chinese hamster ovary (CHO) cell transfected with human 0X40 or cynomolgus 0X40. In some embodiments, an hNGAL mutein of the disclosure does not essentially bind cells that do not express 0X40.

[0117] In some embodiments, an hNGAL mutein of the disclosure may be able to compete with 0X40 ligand (OX40L) for binding to 0X40. In some further embodiments, a provided hNGAL mutein may be able to compete with OX40L for binding to 0X40 with an IC₅₀ value of about 150 nM or lower, about 25 nM or lower, about 15 nM or lower, about 5 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.2 nM or lower, about 0.1 nM or lower, about 0.05 nM or lower, about 0.02 nM or lower, or about 0.01 nM or even lower. In some embodiments, an hNGAL mutein of the disclosure may be able to inhibit the binding of 0X40 to OX40L. In some embodiments, a provided hNGAL mutein may be able to inhibit the binding of 0X40 to OX40L with an IC₅₀ value of about 150 nM or lower, about 25 nM or lower, about 15 nM or lower, about 5 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.2 nM or lower, about 0.1 nM or lower, about 0.05 nM or lower, about 0.02 nM or lower, or about 0.01 nM or even lower. The inhibitory mode of action can, for example, be determined by an ELISA, such as the ELISA assay as essentially described in Example 6.

[0118] In some embodiments, an hNGAL mutein of the disclosure may be able to compete with an anti-OX40 antibody comprising the amino acid sequences shown in SEQ ID NOs: 28 and 29 or SEQ ID NOs: 30 and 31 for binding to 0X40. Such competition may be assessed by an ELISA assay as essentially described in Example 9. In some embodiments, an hNGAL mutein of the disclosure may have an epitope that overlaps with the epitope of an anti- 0X40 antibody comprising the amino acid sequences shown in SEQ ID NOs: 28 and 29 or SEQ ID NOs: 30 and 31.

[0119] In some embodiments, an OX40-binding hNGAL mutein of the disclosure may be able to compete with an OX40-binding hTIc mutein of the disclosure, such as the mutein shown in SEQ ID NO: 43 or SEQ ID NO: 44, for binding to 0X40. Such competition may be assessed by an ELISA assay as essentially described in Example 8. In some embodiments, an hNGAL mutein of the disclosure may have an epitope that overlaps with the epitope of an hTIc mutein of the disclosure, such as the mutein shown in SEQ ID NO: 43 or SEQ ID NO: 44.

[0120] In some embodiments, an hNGAL mutein of the disclosure may be able to induce increased IL-2 secretion. In some embodiments, a provided hNGAL mutein may be able to induce a concentration-dependent IL-2 secretion and/or demonstrate a tendency to induce enhanced IL-2 secretion at higher concentrations. IL-2 secretion may be measured, for example, in a functional T cell activation assay as essentially described in Example 10.

[0121] In some embodiments, an hNGAL mutein of the disclosure may be able to co stimulate T cell responses and/or T cell activation. The stimulated T cell response or T cell activation may be measured, for example, in a functional T cell activation assay as essentially described in Example 10.

[0122] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 3, 21 , 25-26, 28, 36, 40-41 , 44, 49-50, 52, 55, 59, 60, 62-63, 65, 68, 70, 72-73, 75, 77-83, 87, 93, 96, 98, 100, 103, 106, 108, 114, 118, 125, 127, 129, 132, 134, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 20 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding 0X40, in particular human 0X40.

[0123] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,

25, 26, 27, 28, 29, 30, 31 , 32, 33 or even more positions corresponding to positions 3, 21 , 25-

26, 28, 36, 40-41 , 44, 49-50, 52, 55, 59, 60, 62-63, 65, 68, 70, 72-73, 75, 77-83, 87, 93, 96, 98, 100, 103, 106, 108, 114, 118, 125, 127, 129, 132, 134, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 20 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding 0X40, in particular human 0X40.

[0124] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 36, 40-41 , 49, 52, 68, 72-73, 77, 79, 81 , 87, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 20 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding 0X40, in particular human 0X40.

[0125] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 positions corresponding to positions 36, 40-41 , 49, 52, 68, 72-73, 77, 79, 81 , 87, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 10 or more mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 15 or more mutated amino acid residues at one or more of the above- mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 20 mutated amino acid residues at one or more of the above-mentioned positions of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding 0X40, in particular human 0X40. [0126] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 3, 21 , 25-26, 28, 44, 50, 55, 59-60, 62-63, 65, 70, 75, 78, 80, 82-83, 93, 98, 108, 114, 118, 129, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding 0X40, in particular human 0X40.

[0127] In some embodiments, provided hNGAL muteins may comprise a mutated amino acid residue at one or more positions corresponding to positions 3, 21 , 25-26, 28, 44, 50, 55, 59-60, 62-63, 65, 70, 75, 78, 80, 82-83, 93, 98, 108, 114, 118, 129, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) and at one or more positions corresponding to positions 3, 21 , 25, 26, 28, 44, 50, 55, 59, 60, 62, 63, 65, 70, 75, 78, 80, 82, 83, 93, 98, 108, 114, 118, 129, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the provided hNGAL muteins are capable of binding 0X40, in particular human 0X40.

[0128] In some embodiments, a lipocalin mutein according to the disclosure may comprise at least one amino acid substitution of a native cysteine residue by, e.g., a serine residue. In some embodiments, an hNGAL mutein according to the disclosure may comprise an amino acid substitution of a native cysteine residue at positions corresponding to positions 76 and/or 175 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) by another amino acid, such as a serine residue. In this context, it is noted that it has been found that removal of the structural disulfide bond (on the level of a respective naive nucleic acid library) of wild-type hNGAL that is formed by the cysteine residues 76 and 175 (of. Breustedt et al., 2005) may provide hNGAL muteins that are not only stably folded but are also able to bind a given non-natural target with high affinity. In some embodiments, the elimination of the structural disulfide bond may provide the further advantage of allowing for the generation or deliberate introduction of non-natural disulfide bonds into muteins of the disclosure, thereby, increasing the stability of the muteins. However, hNGAL muteins that bind 0X40 and that have the disulfide bridge formed between Cys 76 and Cys 175 are also part of the present disclosure.

[0129] In some embodiments, provided OX40-binding hNGAL muteins may comprise, at one or more positions corresponding to positions 3, 21 , 25-26, 28, 36, 40-41 , 44, 49-50, 52, 55, 59, 60, 62-63, 65, 68, 70, 72-73, 75, 77-83, 87, 93, 96, 98, 100, 103, 106, 108, 114, 118, 125, 127, 129, 132, 134, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Ser 3 Phe or Pro; Asn 21 Asp; Asn 25 Ser; Gin 26 Arg; Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp or Arg; Glu 44 Gly; Gin 49 Gly; Lys 50 Glu or Thr; Tyr 52 Gin; lie 55 Val; Lys 59 Arg; Glu 60 Lys; Tyr 62 Arg; Ser 63 Thr or Ala; Asn 65 Gin or Arg; Ser 68 Gly; Leu 70 Pro or Arg; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Tyr 78 Asp or His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie or Val; Phe 83 Leu; Cys 87 lie, Ser, or Arg; Thr 93 lie; Asn 96 Trp; Lys 98 Arg; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Val 108 Ala; Asn 114 Asp; His 118 Tyr; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; Lys 134 Tyr; Glu 143 Ala; Glu 150 Gly; Gin 164 Asp; and Val 170 Ala. In some embodiments, an hNGAL mutein of the disclosure comprises two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, or all mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 10 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 15 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 20 or more of the above-mentioned mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2).

[0130] In some embodiments, provided OX40-binding hNGAL muteins may comprise, at one or more positions corresponding to positions 36, 40-41 , 49, 52, 68, 72-73, 77, 79, 81 , 87, 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 Phe; Ala 40 Tyr; lie 41 Trp or Arg; Gin 49 Gly; Tyr 52 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Cys 87 lie, Ser, or Arg; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr. In some embodiments, an hNGAL mutein of the disclosure comprises two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2). In some embodiments, an hNGAL mutein of the disclosure comprises 10 or more of the above- mentioned mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2). [0131] In some embodiments, provided hNGAL muteins may comprise, at one or more positions corresponding to positions 3, 21 , 25-26, 28, 44, 50, 55, 59-60, 62-63, 65, 70, 75, 78, 80, 82-83, 93, 98, 108, 114, 118, 129, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Ser 3 Phe or Pro; Asn 21 Asp; Asn 25 Ser; Gin 26 Arg; Gin 28 His; Glu 44 Gly; Lys 50 Glu or Thr; lie 55 Val; Lys 59 Arg; Glu 60 Lys; Tyr 62 Arg; Ser 63 Thr or Ala; Asn 65 Gin or Arg; Leu 70 Pro or Arg; Lys 75 Glu; Tyr 78 Asp or His; lie 80 Thr; Thr 82 lie or Val; Phe 83 Leu; Thr 93 lie; Lys 98 Arg; Val 108 Ala; Asn 114 Asp; His 118 Tyr; Asn 129 Asp; Glu 143 Ala; Glu 150 Gly; Gin 164 Asp; and Val 170 Ala.

[0132] In some embodiments, provided OX40-binding hNGAL muteins may comprise one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Cys 87 Ser; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(b) Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52 Gin; Glu 60 Lys; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; Arg 81 Val; Phe 83 Leu; Cys 87 lie; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(c) Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52 Gin; Ser 63 Thr; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(d) Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Cys 87 Arg; Thr 93 lie; Asn 96 Trp; Lys 98 Arg; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr; (e) Ser 3 Phe; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52 Gin; lie 55 Val; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Phe 83 Leu; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Lys 98 Arg; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; His 118 Tyr; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Glu 150 Gly ;

(f) Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Glu 44 Gly; Gin 49 Gly; Lys 50 Thr; Tyr 52 Gin; Tyr 62 Arg; Asn 65 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Phe 83 Leu; Cys 87 Ser; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(g) Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Asn 65 Gin; Ser 68 Gly; Leu 70 Arg; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; His 118 Tyr; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; and Lys 134 Tyr;

(h) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(i) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; Glu 143 Ala; and Gin 164 Asp;

Q) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; Glu 143 Ala; and Gin 164 Asp;

(k) Ser 3 Pro; Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(L) Asn 25 Ser; Gin 26 Arg; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp;

(m) Asn 21 Asp; Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp;

(n) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; and Lys 134 Tyr;

(o) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Arg; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; Arg 81 Val; Thr 82 Val; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Val 108 Ala; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; Lys 134 Tyr; Gin 164 Asp; and Val 170 Ala;

(p) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 T rp; Lys 134 Tyr; and Gin 164 Asp;

(q) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp; or

(r) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp.

In some embodiments, an OX40-binding hNGAL mutein includes all but three, all but two, or all but one mutated amino acid residues of one of the aforementioned sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0133] In some further embodiments, in the residual region, i.e. , the region differing from positions 3, 21 , 25-26, 28, 36, 40-41 , 44, 49-50, 52, 55, 59, 60, 62-63, 65, 68, 70, 72-73, 75, 77- 83, 87, 93, 96, 98, 100, 103, 106, 108, 114, 118, 125, 127, 129, 132, 134, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), an hNGAL mutein of the disclosure may include the wild-type (natural) amino acid sequence of mature hNGAL outside the mutated amino acid sequence positions.

[0134] In some embodiments, an hNGAL mutein of the disclosure has at least 70% sequence identity or at least 70% sequence homology to the sequence of mature hNGAL (SEQ ID NO: 2). As an illustrative example, the mutein of SEQ ID NO: 56 has an amino acid sequence identity or a sequence homology of approximately 83% with the amino acid sequence of the mature hNGAL.

[0135] In some embodiments, an hNGAL mutein of the disclosure comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47-61 and 73-75 or a fragment or variant thereof.

[0136] In some embodiments, an hNGAL mutein of the disclosure has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-61 and 73-75.

[0137] The present disclosure also includes structural homologues of an hNGAL mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-61 and 73-75, which structural homologues have an amino acid sequence homology or sequence identity of at least 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 98%, or at least 99% in relation to said hNGAL mutein.

[0138] In some embodiments, a provided lipocalin mutein that binds 0X40 may be fused at its N-terminus and/or its C-terminus to a fusion partner. In some preferred embodiments, a lipocalin mutein of the disclosure may be fused at its N-terminus and/or its C-terminus to a fusion partner that is an antibody or antibody fragment, for example, an antibody Fc region.

C. Modifications of the lipocalin muteins of the disclosure.

[0139] In some embodiments, a lipocalin mutein of the present disclosure can comprise a heterologous amino acid sequence at its N- or C-terminus, preferably C-terminus, such as a Strep-tag II (SEQ ID NO: 12) or a cleavage site sequence for certain restriction enzymes, without affecting the biological activity (e.g., binding to its target, e.g., 0X40) of the lipocalin mutein.

[0140] In some embodiments, further modifications of a lipocalin mutein may 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, or to introduce new characteristics to the mutein.

[0141] For example, it is possible to mutate one or more amino acid sequence positions of a lipocalin mutein 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 disulfide linkages. The conjugated compound, for example, PEG or HES, can in some cases increase the serum half-life of the corresponding lipocalin mutein.

[0142] In some embodiments, a reactive group of a lipocalin mutein may occur naturally in its amino acid sequence, such as naturally occurring cysteine residues in said amino acid sequence. In some other embodiments, such reactive group may be introduced via mutagenesis. In case a reactive group is introduced via mutagenesis, one possibility is the mutation of an amino acid at the appropriate position by a cysteine residue. Exemplary possibilities of such a mutation to introduce a cysteine residue into the amino acid sequence of an hTIc mutein include the substitutions Thr 40 Cys, Glu 73 Cys, Arg 90 Cys, Asp 95 Cys, and Glu 131 Cys of the wild-type sequence of mature hTIc (SEQ ID NO: 1). Exemplary possibilities of such a mutation to introduce a cysteine residue into the amino acid sequence of an hNGAL mutein include the introduction of a cysteine residue at one or more of the sequence positions that correspond to sequence positions 14, 21 , 60, 84, 88, 116, 141 , 145, 143, 146 or 158 of the wild-type sequence of mature hNGAL (SEQ ID NO: 2). 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.

[0143] In some embodiments, in order to provide suitable amino acid side chains as new reactive groups for conjugating one of the above compounds to a lipocalin mutein, artificial amino acids may be introduced to the amino acid sequence of a lipocalin mutein. Generally, such artificial amino acids are designed to be more reactive and thus to facilitate the conjugation to the desired compound. Such artificial amino acids (e.g., para-acetyl-phenylalanine) may be introduced by mutagenesis, for example, using an artificial tRNA.

[0144] For several applications of the lipocalin muteins disclosed herein it may be advantageous to use them in the form of fusion proteins. 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, an antibody fragment or variant thereof, a lipocalin (mutein), a signal sequence and/or an affinity tag. In some other embodiments, a lipocalin mutein of the disclosure is conjugated at its N-terminus or its C-terminus to a partner, which is a protein, a protein domain or a peptide; for instance, an antibody, an antibody fragment or variant thereof, a lipocalin (mutein), a signal sequence and/or an affinity tag.

[0145] 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 examples of suitable fusion partners. 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.

[0146] 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. For example, a fluorescent or radioactive label can be conjugated to a lipocalin mutein to generate fluorescence or x-rays as detectable signal. Alkaline phosphatase, horseradish peroxidase and b- 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.

[0147] 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.

[0148] In some embodiments, a lipocalin mutein of the disclosure may be fused or 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 strategies are described with reference to hNGAL muteins with binding affinity for CTLA-4). The moiety that extends the serum half-life may be a PEG molecule, a HES molecule, a fatty acid molecule, 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, an albumin binding protein, or a transferrin, to name only a few. Exemplary fusion proteins are shown in SEQ ID NOs: 64-72.

[0149] If PEG is used as a conjugation partner, the PEG molecule can be substituted, unsubstituted, linear, or branched. It can also be an activated polyethylene 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. Patent No. 6,500,930 or 6,620,413, carbohydrate oligomers and polymers such as HES can be conjugated to a mutein of the disclosure for the purpose of serum half-life extension.

[0150] 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.

[0151] Examples of albumin binding peptides that can be used to extend the serum half- life of a lipocalin mutein are, for instance, those having a Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa_! 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. 2003/0069395 or Dennis et al. (2002). The albumin binding protein fused or conjugated to a lipocalin mutein to extend serum half-life may be a bacterial albumin binding protein, an antibody, an antibody fragment including domain antibodies (see U.S. Patent 6,696,245, for example), or a lipocalin mutein with binding activity for albumin. Examples of bacterial albumin binding proteins include streptococcal protein G (Konig and Skerra, 1998).

[0152] 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).

[0153] Particularly, albumin itself (Osborn et al. , 2002), or a biologically active fragment of albumin can be used as a partner of a lipocalin mutein of the disclosure to extend serum half- life. The term “albumin” includes all mammalian 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. EP 0 330 451 and EP 0 361 991. Accordingly, recombinant human albumin (e.g., Recombumin® from Novozymes Delta Ltd., Nottingham, UK) can be conjugated or fused to a lipocalin mutein of the disclosure.

[0154] If a transferrin is used as a partner 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).

[0155] 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 sequence (for example poly-glycine with about 20 to 80 consecutive glycine residues). This approach disclosed in International Patent Publication No. WO 2007/038619, for example, has also been term “rPEG” (recombinant PEG).

[0156] A lipocalin mutein disclosed herein may be fused or conjugated, at its N-terminus and/or its C-terminus, to a moiety that 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 or antibody fragments, oligomerization domains, other lipocalin muteins, or toxins. [0157] In particular, it is possible to fuse a lipocalin mutein disclosed herein with a separate enzyme active site such that both “subunits” of the resulting fusion protein act together 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.

[0158] It is also possible to fuse a lipocalin mutein disclosed herein with a second

“subunit,” which is an antibody, an antibody active fragment, or another lipocalin mutein, such that the resulting fusion protein acts on both the target of the lipocalin mutein and one other given therapeutic target.

D. Production of exemplary lipocalin muteins of the disclosure.

[0159] In one aspect, the disclosure provides methods of making OX40-binding proteins described herein. The present disclosure also relates to nucleic acid molecules (DNA and RNA) that include nucleotide sequences encoding the lipocalin muteins of the disclosure. In yet another embodiment, the disclosure encompasses a host cell containing said nucleic acid molecule. 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 exemplary nucleotide sequences encoding some lipocalin muteins of the disclosure as shown in SEQ ID NOs: 36-61 and 73-75.

[0160] In some examples, the method of the disclosure includes subjecting the nucleic acid molecule encoding mature hTIc to mutagenesis at nucleotide triplets coding for one or more of the sequence positions 5, 6, 8, 11 , 19, 23, 26-34, 36, 37, 40, 52, 55-56, 58, 60-61 , 65, 79, 86, 101 , 104-106, 108, 111 , 113-114, 116, 121 , 124, 137, 140, 148, and 153 of the linear polypeptide sequence of hTIc (SEQ ID NO: 1). In some examples, the method of the disclosure includes subjecting the nucleic acid molecule encoding mature hNGAL to mutagenesis at nucleotide triplets coding for one or more of the sequence positions 3, 21 , 25-26, 28, 36, 40-41 , 44, 49-50, 52, 55, 59, 60, 62-63, 65, 68, 70, 72-73, 75, 77-83, 87, 93, 96, 98, 100, 103, 106, 108, 114, 118, 125, 127, 129, 132, 134, 143, 150, 164, and 170 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 2).

[0161] 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.

[0162] 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 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 the 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.

[0163] 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 satisfactorily functional in a particular host cell, then they may be substituted with signals functional in that cell.

[0164] Therefore, a nucleic acid molecule of the disclosure may be “operably linked” to a regulatory sequence (or regulatory sequences), such as a promoter sequence, to allow expression of this nucleic acid molecule. 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.

[0165] The nucleic acid molecules of the disclosure can 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.

[0166] In some embodiments, a nucleic acid molecule is included in a phagemid. A phagemid vector denotes a vector encoding the intergenic region of a temperate phage, such as M13 or f1 , or a functional part thereof fused to the cDNA of interest. After superinfection of the bacterial host cells with such a 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).

[0167] Such cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence encoding a lipocalin mutein or fusion protein 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.

[0168] A DNA molecule encoding a lipocalin mutein or fusion protein as described herein, and in particular a cloning vector containing the coding sequence of such a mutein or fusion protein 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.

[0169] The transformed host cells can be cultured under conditions suitable for expression of a nucleotide sequence encoding a mutein or fusion protein of the disclosure. Suitable host cells can be prokaryotic, such as Escherichia coli ( E . coli) or Bacillus subtilis, or eukaryotic, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.

[0170] In some embodiments, the present disclosure also relates to a method for the production of a lipocalin mutein, fragment of the mutein, or fusion protein as described herein, wherein the mutein, a fragment of the mutein or a fusion protein of the mutein and another polypeptide (e.g., another lipocalin mutein or antibody or antibody fragment) is produced starting from a nucleic acid coding for the mutein, fragment of the mutein, or fusion protein, preferably by means of genetic engineering methods. In some embodiments, provided methods can be carried out in vivo, the lipocalin mutein, fragment of the mutein, or fusion protein, can, for example, be produced in a bacterial or eukaryotic host organism and optionally 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.

[0171] When producing a lipocalin mutein, fragment of the mutein, or fusion protein, in vivo a nucleic acid encoding such mutein, fragment or fusion protein is introduced into a suitable bacterial or eukaryotic host organism, preferably using recombinant DNA technology (as already outlined above). For this purpose, a host cell is first transformed with a cloning vector that includes a nucleic acid molecule encoding a lipocalin mutein, fragment of the mutein, or fusion protein as described herein, for example, 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 protein. Subsequently, the protein is recovered either from the cell or the cultivation medium.

[0172] In addition, with respect to hTIc muteins or hNGAL muteins of the disclosure, in some embodiments, the naturally occurring disulfide bond between Cys 61 and Cys 153 or Cys 76 and Cys 175, respectively, may be removed. Accordingly, such muteins can be produced in a cell compartment having a reducing redox milieu, for example, in the cytoplasm of Gram negative bacteria.

[0173] 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 the lumen of the endoplasmic reticulum of eukaryotic cells and usually favors the formation of structural disulfide bonds.

[0174] 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 the 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).

[0175] However, a lipocalin mutein, fragment of the mutein, or fusion protein 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, and polypeptides containing such mutations are synthesized in vitro, and then investigated for binding activity to 0X40 and other desirable properties (such as stability). Methods for the solid phase and solution phase synthesis of polypeptides/proteins are well known in the art (see, e.g., Bruckdorfer et al. , 2004). In another embodiment, the lipocalin mutein, fragment of the mutein, or fusion protein of the disclosure may be produced by in vitro transcription/translation employing well-established methods known to those skilled in the art.

[0176] In addition, the skilled worker will appreciate methods useful to prepare a lipocalin mutein, fragment of the mutein, or a fusion protein 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 to simplify sub-cloning of a mutated lipocalin gene or its parts by incorporating cleavage sites for certain restriction enzymes.

E. Exemplary uses and applications of lipocalin muteins of the disclosure.

[0177] In general, lipocalin muteins disclosed herein and derivatives thereof can be used in many fields similar to antibodies or fragments thereof. Particularly, the disclosure relates to numerous possible applications for provided OX40-binding lipocalin muteins.

[0178] The present disclosure involves the use of one or more OX40-binding lipocalin muteins as described herein for complex formation with 0X40.

[0179] In one aspect, the disclosure relates to the use of one or more OX40-binding lipocalin mutein disclosed herein for detecting 0X40 in a sample as well as a respective method of diagnosis.

[0180] Accordingly, in another aspect, provided lipocalin muteins are used for the detection of 0X40. Such use may include the steps of contacting one or more of said muteins, under suitable conditions, with a sample suspected of containing 0X40, thereby allowing the formation of a complex between the muteins and 0X40, and detecting the complex by a suitable signal. 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, wherein one of them is immobilized on a surface, such as a gold foil. [0181] The OX40-binding lipocalin muteins disclosed herein may also be used for the separation and/or isolation of 0X40. Such use may include the steps of contacting one or more of said muteins, under suitable conditions, with a sample supposed to contain 0X40, thereby allowing formation of a complex between the muteins and 0X40, and separating the complex from the sample.

[0182] In the use of provided muteins for the detection of 0X40 as well as the separation and/or isolation of 0X40, the muteins and/or 0X40 or a domain or fragment thereof may be immobilized on a suitable solid phase.

[0183] In some embodiments, the present disclosure encompasses diagnostic and/or analytical kits comprising an OX40-binding lipocalin mutein according to the disclosure.

[0184] In addition to their use in diagnostics, in yet another aspect, the present disclosure encompasses the use of OX40-binding lipocalin muteins described herein or a composition comprising such lipocalin muteins, for inhibiting or interfering with the binding of 0X40 ligand (OX40L) to 0X40 and/or natural OX40/OX40L signaling. Accordingly, the present disclosure provides a method of inhibiting or interfering with the binding of OX40L to 0X40, comprising applying one or more OX40-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins. The present disclosure also provides a method of interfering with natural signaling of 0X40 through OX40L, comprising applying one or more OX40-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

[0185] In some aspects, the disclosure contemplates a pharmaceutical composition comprising a provided lipocalin mutein and a pharmaceutically acceptable excipient.

[0186] Furthermore, the present disclosure provides human lipocalin muteins that bind

0X40 for use in therapy, such as for use as anti-cancer agents or immune modulators. As such, lipocalin muteins of the present disclosure that bind 0X40 are, in some embodiments, envisaged to be used in methods 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 disclosure that binds 0X40. [0187] The present disclosure encompasses the use of an OX40-binding lipocalin mutein of the disclosure or a composition comprising such lipocalin mutein for activating the downstream signaling pathways of 0X40, such as enhancing the secretion of proinflammatory cytokines including but not limited to IL-2. The present disclosure also encompasses the use of an OX40-binding lipocalin mutein of the disclosure or a composition comprising such lipocalin mutein for co-stimulating T cells and/or modulating immune responses by binding to 0X40.

[0188] Accordingly, the present disclosure contemplates a method of activating the downstream signaling pathways of 0X40, such as enhancing the secretion of proinflammatory cytokines, including but not limited to IL-2, comprising applying one or more OX40-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins. The present disclosure also contemplates a method of co-stimulating T cells and/or stimulating immune responses, comprising applying one or more OX40-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

[0189] The present disclosure also encompasses the use of an OX40-binding lipocalin mutein of the disclosure or a composition comprising such lipocalin mutein for reducing the production of proinflammatory cytokines and chemokines. The present disclosure contemplates a method of reducing production of proinflammatory cytokines and chemokines, comprising applying one or more OX40-binding lipocalin muteins of the disclosure or one or more compositions comprising such lipocalin muteins.

[0190] 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, modifications and variations of the disclosures 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.

V. EXAMPLES

[0191] Example 1: Selection and optimization of muteins specific for 0X40

[0192] The OX40-specific lipocalin muteins disclosed in this application were selected from naive mutant libraries based on hTIc and hNGAL. The libraries were panned against the biotinylated polyhistidine-tagged full-length extracellular domain of human 0X40 (Sino Biologicals) or full-length extracellular domain of human 0X40 in the format of an Fc-fusion (huOX40-Fc, R&D Systems) alternating with the full-length extracellular domain of cynomolgus monkey 0X40 or individual subdomains of extracellular human 0X40 as Fc fusions. Alternatively, cell-based panning using Chinese hamster ovary (CHO) cells transfected with the full cDNA of human 0X40 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.

[0193] For optimization of OX40-specific muteins, libraries were generated based on mutein SEQ ID NOs: 36 or 47 using either a biased randomization of selected positions or error prone polymerase chain reaction (PCR) based methods. The biased design was made such that for each of the selected positions the amino acid encoded corresponds to the amino acid found in the respective mother clone with a probability of 50-90%, while it can be a different amino acid with a 50-10% probability. With N as the number of targeted positions and B as bias, the most probable number of exchanges per clone is N^c (1 -B). The generated lipocalin muteins were cloned with high efficiency into a phagemid vector essentially as described (Kim et al. , 2009). Phage display was employed to select for optimized muteins with improved heat and disulfide bridge stability and binding affinity. The phagemid selection was conducted against huOX40-Fc alternating with the Fc fusion of cynomolgus 0X40 extracellular domain (cyOX40- Fc), under increased stringency compared to the initial mutein selections and involved preincubation steps at elevated temperature and limiting target concentration amongst other things. Resulting OX40-specific muteins, e.g., the mutein of SEQ ID NO: 54, optionally underwent additional rounds of optimization.

[0194] Example 2: Identification of muteins specifically binding to 0X40 using high-throughput enzyme-linked immunosorbent assay (ELISA) screening

[0195] Individual colonies of lipocalin muteins fused to a C-terminal Strep-tag II (SEQ ID

NO: 12) were used to inoculate 2x Yeast Extract Trypton (2XYT)/Amp medium and grown overnight (14-18 h) to stationary phase. Subsequently, 50 pL 2xYT/Amp were inoculated from the stationary phase cultures and incubated for 3.5 h at 37°C and then shifted to 22°C until an

was reached. Production of muteins was induced by addition of 10 pL 2xYT/Amp supplemented with 1.2 pg/mL anhydrotetracycline. Cultures were incubated at 22°C until the next day. After addition of 40 pL of 5% (w/v) BSA in PBS-T and incubation for 1 h at 25°C, cultures were ready for use in screening assays. [0196] Binding of the isolated muteins to 0X40 was tested by direct coating of huOX40-

Fc or cyOX40-Fc or subdomains of human 0X40 at 2 mg/mL in PBS overnight at 4°C on microtiter plates. After blocking the plate with PBS-T containing 5% BSA, 20 mI_ of BSA-blocked cultures were added to the microtiter plates and incubated for 1 h at room temperate. Bound muteins were detected with anti-Strep-tag antibody conjugated with horseradish peroxidase (HRP) (BioTek) after 1 h incubation. For quantification, 20 mI_ 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.

[0197] To select for muteins with increased affinity and stability, the screening was performed with i) reduced antigen concentration, ii) using reverse screening formats where the muteins were captured via the Strep-tag on microtiter plates coated with anti-Strep-tag antibody and different concentrations of the target were added and detected via either Extravidin-HRP (Sigma), and, partially, iii) incubation of the screening supernatant at 70-75 °C before addition to the target plate.

[0198] Clones were then sequenced based on the screening results, and muteins were selected for further characterization.

[0199] Example 3: Expression of muteins

[0200] Selected muteins with a C-terminal SA linker sequence (SAWSHPQFEK, SEQ ID

NO: 11) and the Strep-tag II peptide (WSHPQFEK, SEQ ID NO: 12) were expressed in E. coli in 2XYT/Amp medium and purified using Strep-Tactin affinity chromatography and preparative size exclusion chromatography (SEC). After SEC purification, the fractions containing monomeric protein were pooled and analyzed again using analytical SEC. The yield of exemplary lipocalin muteins after Strep-Tactin affinity chromatography and preparative SEC as well as the monomer content after Strep-Tactin purification are shown in Table 1.

[0201] Table 1: Expression of muteins.

[0202] In addition, monospecific lipocalin mutein Fc fusions may be generated by fusing one or more of the OX40-specific lipocalin muteins, via a linker, to the C-terminus of the Fc region of an antibody. Exemplary Fc fusions were generated by fusing one OX40-specific lipocalin mutein, via an unstructured (G₄S)₃ linker shown in SEQ ID NO: 13, to the C-terminus of the Fc region of an antibody provided in SEQ ID NO: 32 as depicted in Figure 1B. The resulting constructs are provided in SEQ ID NOs: 64-72.

[0203] The constructs of the Fc fusions were generated by gene synthesis and cloned into a mammalian expression vector. They were then transiently expressed in Expi293FTM cells (Life Technologies). The concentration of fusion proteins in the cell culture medium was measured by HPLC (Agilent Technologies) employing a POROS® protein A affinity column (Applied Biosystems). The titers of exemplary Fc fusions are summarized in Table 2.

[0204] The Fc fusions were purified using Protein A chromatography followed by SEC in phosphate-buffered saline (PBS). After SEC purification, the fractions containing monomeric protein were pooled and analyzed again using analytical SEC.

[0205] Table 2: Transient expression titers of lipocalin mutein Fc fusions. SEQ ID NO Expression titer [mg/mL]

64 0.35

65 0.36

66 0.37

67 0.46

68 0.39

69 0.33

70 0.34

71 0.31

72 0.34

[0206] Example 4: Affinity of muteins binding to human and cynomolgus 0X40 determined by surface plasmon resonance (SPR)

[0207] Surface plasmon resonance (SPR) was used to measure binding kinetics and affinity of representative lipocalin muteins disclosed herein.

[0208] The binding of exemplary lipocalin muteins to huOX40-Fc (R&D Systems) and cyOX40-Fc was determined by SPR using a Biacore instrument (GE Healthcare). Recombinant 0X40 from cynomolgus monkeys (cyOX40-Fc) was produced by fusing the extracellular domain of cynomolgus 0X40 (cyOX40) to a human lgG1 Fc fragment via a Factor Xa cleavage site and a (G₄S)₃ linker.

[0209] The anti-human IgG Fc antibody (GE Healthcare) was immobilized on a CM5 sensor chip using standard amine chemistry: the carboxyl groups on the chip were activated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDO) and N-hydroxysuccinimide (NHS). Subsequently, anti-human IgG Fc antibody solution (GE Healthcare) at a concentration of 25 pg/mL in 10 mM sodium acetate (pH 5) was applied at a flow rate of 5 pL/min until an immobilization level of 5500-14000 resonance units (RU) was achieved. Residual non-reacted NHS-esters were blocked by passing a solution of 1M ethanolamine across the surface. The reference channel was treated in an analogous manner. Subsequently, huOX40-Fc at 0.5 or 0.75 pg/mL or cyOX40-Fc at 2 pg/mL in HBS-EP+ buffer was captured by the anti-human IgG- Fc antibody at the chip surface for 180 s at a flow rate of 10 pL/min.

[0210] For affinity determination of the lipocalin muteins, dilutions of each mutein at various concentrations, typically ranging from 0.43 to 1000 nM, were prepared in HBS-EP+ buffer and applied to the prepared chip surface for affinity measurement to human 0X40 and cynomolgus 0X40. The binding assay was carried out with a contact time of 180 s, a dissociation time of 1200 s and a flow rate of 30 pL/min. All measurements were performed at 25°C. The lipocalin muteins of SEQ ID NOs: 3 and 4 were also tested as a negative control. Regeneration of the chip surface was achieved with injections of 3 M MgCI₂ for 60 s and 10 mM glycine-HCI (pH 1.7) for 180 s at a flow rate of 10 pL/min followed by an extra wash with running buffer (HBS-EP+ buffer) and a stabilization period of 120 s. Prior to the protein measurements, three startup cycles were performed for conditioning purposes. Data were evaluated with Biacore Evaluation software. Double referencing was used, and the 1 :1 binding model was used to fit the raw data.

[0211] Additionally, for affinity determination of lipocalin mutein Fc fusions, tested fusions (SEQ ID NOs: 64-66) at 0.5 pg/mL in HBS-EP+ buffer were captured by the anti-human IgG-Fc antibody at the chip surface for 180 s at a flow rate of 10 pL/min. After each capture step, the needle was washed. Dilutions of huOX40-His (huOX-40 with a C-terminal polyhistidine tag, Sino Biologicals) at 150 nM were prepared in HBS-EP+ buffer and applied to the prepared chip surface. The binding assay was carried out with a contact time of 180 s, a dissociation time of 900 s and a flow rate of 30 pL/min. All measurements were performed at 25°C. Regeneration of the chip surface was achieved with injections of 3 M MgCI₂ for 120 s. Prior to the protein measurements, three startup cycles were performed for conditioning purposes. Data were evaluated with Biacore 8K Evaluation software (V1.1.1). Double referencing was used, and the 1 :1 binding model was used to fit the raw data.

[0212] The values determined for k_on, k_0ff and the resulting equilibrium dissociation constant (K_D) for lipocalin muteins SEQ ID NOs: 36-61 and lipocalin mutein Fc fusions SEQ ID NOs: 64-66 are summarized in Table 3. The binding affinities to human and cynomolgus 0X40 are comparable for most of the tested lipocalin muteins, representing a preferred feature for pharmacokinetic and/or drug-safety studies. Optimized lipocalin muteins of SEQ ID NOs: 48-61 had K_D values in the low nano-molar range, exhibiting up to 1500-fold lower K_D values compared to the parent lipocalin mutein of SEQ ID NO: 47.

[0213] Table 3: Kinetic constants and affinities of OX40-specific muteins determined by surface-plasmon-resonance (SPR).

[0214] Example 5: Fluorescence-activated cell sorting (FACS) analysis of lipocalin muteins binding to cells expressing human orcynomolgus 0X40 [0215] Fluorescence-activated cell sorting (FACS) studies were performed to assess the specific binding of the lipocalin muteins of SEQ ID NOs: 37-44 and 47-60 to Chinese hamster ovary (CHO) cells stably transfected with huOX40 (CHO-huOX40) or cyOX40 (CHO-cyOX40). The muteins of SEQ ID NO: 3 and SEQ ID NO: 4 were tested in parallel as negative control. The cell lines were generated using the Flp-ln system (Invitrogen) according to the manufacturer’s instructions. Mock-transfected Flp-ln CHO cells served as a further negative control.

[0216] Transfected Flp-ln CHO cells were maintained in Ham’s F12 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS, Biochrom) and 500 pg/mL Hygromycin B (Roth). Cells were cultured in cell culture flasks under standard conditions according to the manufacturer’s instructions (37°C, 5% C0₂ atmosphere). In order to detach the adherent cells for subculture or FACS experiments, Accutase (PAA) was employed according to the manufacturer’s instructions.

[0217] To perform the experiment, OX40-positive and -negative Flp-ln CHO cells were incubated with lipocalin muteins, bound mutein was labeled using fluorescently labeled anti-hTIc or anti-hNGAL antibodies, and then the signal was analyzed using FACS as described in this example.

[0218] 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 (SEQ ID NOs: 37-44 and 47-60), and the negative control lipocalin muteins (SEQ ID NOs: 3 and 4), typically ranging from 1 pM to 0.01 nM, was added to the cells, and incubated on ice for 1 h. Cells were washed twice in ice-cold PBS using centrifugation at 500 x g and then incubated with polyclonal rabbit anti-lipocalin antibody conjugated to the fluorescent dye Alexa Fluor 488 or Alexa Fluor 647 (Pieris) for 30 min on ice. Cells were subsequently washed and analyzed using an iQue Flow cytometer (Intellicyt). Fluorescent data generated by lipocalin mutein binding to 0X40 expressing cells were analyzed by gating for OX40-expressing CHO cells using Forecyt® software, and resulting geometric fluorescent mean values were plotted and fitted using Graphpad software. Data generated for SEQ ID NOs: 37-44 and 47-60 are shown in Figure 2 and Table 4. OX40-specific muteins SEQ ID NOs: 37-44, 48-51 , and 54-60 showed clear binding to CHO cells expressing either huOX40 or cyOX40. The negative control lipocalin muteins (SEQ ID NOs: 3 and 4), which do not bind 0X40, did not show any binding. No binding of the lipocalin muteins was detected on mock-transfected Flp-ln CHO cells (data not shown). [0219] Table 4: Binding of OX40-specific lipocalin muteins to Flp-ln CHO cells transfected with human or cynomolgus 0X40.

[0220] Example 6: Competition of lipocalin muteins with OX40L in binding to 0X40 determined using ELISA

[0221] To assess whether the lipocalin muteins interfere with 0X40 binding to OX40L, a competitive ELISA format was used.

[0222] Recombinant human 0X40 ligand with N-terminal His-tag (huOX40L-His, R&D

Systems) in PBS (2 pg/mL) was coated overnight on microtiter plates at 4°C. The plates were washed five times after each incubation step with 100 pL PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20). The plates were blocked with 2% BSA (w/v) in PBS-0.1%T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 h at room temperature and subsequently washed again. Lipocalin muteins at various concentrations ranging from 3 pM to 0.02 nM were mixed with 0.05 nM of huOX40-Fc as a tracer and incubated for 1 h at room temperature. The mixtures of lipocalin muteins and the tracer were added to the huOX40L-His coated wells and incubated for 20 min at room temperature followed by five washing steps with 100 pl_ PBS-0.05%T. Subsequently, a 1 :5000 dilution of goat-anti human IgG-Fc HRP (Jackson ImmunoResearch) was added to the wells and incubated for 1h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[0223] Competition data of exemplary lipocalin muteins (SEQ ID NOs: 36, 38, 39, 41-44,

47, 49, 52-55, and 57-61) are shown in Figure 3, together with the fit curves resulting from a 1:1 sigmoidal binding fit, where the IC₅₀ value and the maximum signal were free parameters, and the slope was fixed to unity. The IC₅₀ values are summarized in Table 5. Certain lipocalin muteins (SEQ ID NOs: 38, 39, 41-44, 49, 54, 55, and 57-61) showed clear inhibition of the OX40/OX40L interaction with substantial improvement in IC₅₀ as compared to the parental muteins (SEQ ID NOs: 36 and 47).

[0224] Table 5: Lipocalin muteins compete with binding of huOX40 to its ligand OX40L in fluorescence-ELISA based assays.

[0225] Example 7: Thermal stability assessment of lipocalin muteins

[0226] To determine the melting temperatures (T_ms) of the lipocalin muteins, which is a general indicator for folding stability, the OX40-specific muteins, at a protein concentration of 1 mg/mL in PBS (Gibco), were scanned (25-100°C) at 1°C/min using a capillary nanoDSC instrument (CSC 6300, TA Instruments). The T_ms were calculated from the displayed thermogram using the integrated Nano Analyze software. Several unfolding events can contribute to the observed thermogram. In cases with multiple transitions, the one with the major unfolding energy best represents the overall protein fold and is reported.

[0227] The resulting maximum melting temperatures as well as the onset temperature of melting for exemplary lipocalin muteins (SEQ ID NOs: 36-44 and 47-60) are listed in Table 6 below.

[0228] Table 6: T_m and onset melting temperature as determined by nanoDSC of 0X40- specific lipocalin muteins.

[0229] Example 8: Epitope analysis of the lipocalin muteins

[0230] Competition ELISA experiments were employed to determine whether the hTIc muteins and hNGAL muteins compete for binding to 0X40.

[0231] Microtiter plates were coated with the OX40-specific hTIc mutein of SEQ ID NO: 43 or SEQ ID NO: 44 (1 pg/mL) at 4°C overnight. The plates were washed five times after each incubation step with 100 pl_ PBS-0.05%T. The plates were blocked with 2% BSA (w/v) in PBS- 0.1 %T for 1 h at room temperature and subsequently washed again. Lipocalin muteins (SEQ ID NOs: 52, 53, 55, and 58) at different concentrations were mixed with 10 nM of biotinylated human 0X40 with a C-terminal polyhistidine tag (huOX40-His-bio, Sino Biological) as a tracer and incubated for 1 h at room temperature. The mixtures of molecules to be tested and the tracer were added to the plates and incubated for 20 min at room temperature followed by five washing steps with 100 pl_ PBS-0.05%T. Subsequently, a 1 :5000 dilution of ExtrAvidin-HRP (Sigma-Aldrich) in PBS-0.1%T-2%BSA was added to the wells and incubated for 1 h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[0232] Competition data for an exemplary experiment are shown in Table 7 and Figure 4, where the x-axis represents tested molecule concentration and the y-axis represents the measured trace molecule concentration. The data were fit to a 1:1 sigmoidal curve, where the IC₅₀ value and the maximum signal were free parameters, and the slope was fixed to unity. The results show competition of exemplary OX40-specific hTIc muteins (SEQ ID NO: 43 or SEQ ID NO: 44) with OX40-specific hNGAL muteins (SEQ ID NO: 52, 53, 55, or 58) for 0X40 binding and indicate that the muteins share overlapping epitopes on 0X40.

[0233] Table 7: Competition of lipocalin muteins.

SEQ ID NO: 43 SEQ ID NO: 44

SEQ ID Competition Competition

NO IC₅₀ [nM] IC₅₀ [nM]

58 3.1 0.16

55 1.4 0.09

53 competition competition

52 competition competition

[0234] Example 9: Epitope analysis of the lipocalin muteins

[0235] A competitive ELISA format was used to determine the competition between the lipocalin muteins and clinically relevant 0X40 antibodies.

[0236] Microtiter plates were coated with the reference 0X40 antibody of SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 30 and 31 in PBS (1 pg/mL) at 4°C overnight. The plates were washed five times after each incubation step with 100 pL PBS-0.05%T. The plates were blocked with 2% BSA (w/v) in PBS-0.1%T for 1 h at room temperature and subsequently washed again. Lipocalin muteins (SEQ ID NOs: 43, 44, 52, 53, 55, and 58) at different concentrations were mixed with 10 nM of huOX40-His-bio as a tracer and incubated for 1 h at room temperature. The mixtures of molecules to be tested and the tracer were added to the plates and incubated for 20 min at room temperature followed by five washing steps with 100 pL PBS-0.05%T. Subsequently, a 1 :5000 dilution of ExtrAvidin-HRP (Sigma-Aldrich) in PBS- 0.1%T-2%BSA was added to the wells and incubated for 1 h. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader.

[0237] Competition data for an exemplary experiment are shown in Table 8 and Figure 5, where the x-axis represents tested molecule concentration, and the y-axis represents the measured trace molecule concentration. The data were fit to a 1:1 sigmoidal curve, where the IC₅₀ value and the maximum signal were free parameters, and the slope was fixed to unity. The results demonstrate that exemplary OX40-specific hTIc muteins (SEQ ID NOs: 43 and 44) compete with all three reference 0X40 antibodies, suggesting the lipocalin muteins bind epitopes that overlap with those of the antibodies. On the other hand, exemplary OX40-specific hNGAL muteins (SEQ ID NOs: 52, 53, 55, and 58) compete with 0X40 antibodies SEQ ID NOs: 28 and 29 and SEQ ID NOs: 30 and 31 , but not with SEQ ID NOs: 26 and 27. The results further suggest that, although epitopes may be overlapping, OX40-specific hTLc muteins and hNGAL muteins do not bind exactly the same epitope.

[0238] Table 8: Competition of lipocalin muteins.

[0239] Example 10: Functional T cell activation assay using coated lipocalin muteins

[0240] To investigate the ability of lipocalin muteins to co-stimulate T cell responses, a T cell activation assay was employed. In this experiment, the lipocalin muteins were coated onto a plastic dish together with an anti-human CD3 antibody (Muronomab, Janssen-Cilag) and anti- lipocalin-scaffold antibody (anti-TIc, Pieris), 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 co-stimulated by 0X40 co-stimulation. T cell proliferation was then assessed by measuring supernatant interleukin 2 (IL-2), which has been suggested to be augmented through 0X40 signaling. A detailed description of the experiment is added below.

[0241] Human peripheral blood mononuclear cells (PBMCs) from healthy volunteer donors were isolated from buffy coats by centrifugation through a Polysucrose density gradient (Ficoll-Paque, density 1.077 g/mL), using SepMate-50 ml. tubes and following the manufacturer’s protocol. The T cells were isolated from the resulting PBMCs using a Pan T cell purification Kit (Miltenyi Biotec GmbH) following the manufacturer ‘s protocols. Purified T cells were resuspended in a freezing buffer consisting of 90% FCS and 10% DMSO, immediately frozen down and stored in liquid nitrogen until further use. For the assay, T cells were thawed in a water bath at 37°C and transferred to an appropriate volume of culture media (RPMI 1640, Life Technologies) supplemented with 10% FCS and 1% Penicillin-Streptomycin (Life Technologies). After centrifugation at 300 x g for 5 min, cells were adjusted to a density of 3E6 cells per mL in culture media and incubated in a cell culture flask at 37°C in a humidified 5% C0₂ atmosphere overnight.

[0242] Flat-bottom tissue culture plates were coated overnight at 4°C using 50 pL of a mixture of 5 pg/mL anti-CD3 antibody and 25 pg/mL rabbit anti-lipocalin-scaffold antibody (Pieris). The latter was employed to allow for immobilization of lipocalin muteins (SEQ ID NOs: 41-45 and 52-56) by affinity capturing. In parallel, a mixture of a dilution series of an IgG isotype ranging from 500 nM to 0.8 nM and 5 pg/mL anti-human-CD3 antibody were directly coated in some wells. The following day, wells were washed twice with PBS, and 50 pL of a dilution series of lipocalin mutein ranging from 500 nM to 0.8 nM in five steps was captured on the precoated plates for 2 h at 37°C. As a negative control, SEQ ID NO: 3 or 4 was captured instead. After two washes with RPMI supplemented with 10% FCS and 1% P/S, 25 pl_ of the T cell suspension (corresponding to 2.5^c 10⁴ T cells) in culture media were added to the wells in the presence of 25 mI_ of anti-hCD28 antibody at 2 pg/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 concentration in the supernatant was assessed.

[0243] Human IL-2 in the pooled cell culture supernatants was quantified using the IL-2

DuoSet (R&D Systems). In the first step, a 384-well plate was coated at room temperature for 1 h with 1 pg/mL “Human IL-2 Capture Antibody” (R&D System) diluted in PBS. Subsequently, wells were washed 5 times with 80 pL PBS-T (PBS containing 0.05% Tween20) using a Biotek 405™ LS Microplate Washer (Biotek). After 1 h blocking in PBS-T additionally containing 1% casein (w/w), assay 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 50 ng/mL biotinylated goat anti-human IL-2-Bio detection antibody (R&D System) and 1 pg/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 pL reading buffer were added to each well, and the electrochemiluminescence (ECL) signal of each well was read using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[0244] Exemplary results are depicted in Figure 6. There is a clear increase in IL-2 production for selected lipocalin muteins SEQ ID NOs: 41-45 and 52-56, demonstrating substantial co-stimulation of T cell responses by these lipocalin muteins.

[0245] Example 11: Determination of binding characteristics of optimized 0X40 binding lipocalin muteins using enzyme-linked immunosorbent assay (ELISA)

[0246] Binding characteristics for the binding to human 0X40 of further OX40-specific hNGAL muteins (SEQ ID NOs: 73-75) were determined by direct ELISA. For this, the lipocalin muteins of SEQ ID NOs: 73-75 were C-terminally fused to a human lgG4 Fc region (SEQ ID NO: 23) via a glycine-serine linker (SEQ ID NO: 13). The amino acid sequences of the corresponding Fc fusions are shown in SEQ ID NOs: 76-78.

[0247] Binding of the hNGAL muteins to 0X40 was tested by direct coating of huOX40-

His at 1 pg/mL in PBS overnight at 4°C on microtiter plates. After washing with PBS-0.05%T (PBS supplemented with 0.05% (v/v) Tween 20), the plates were blocked with 2% BSA (w/v) in PBS-0.1%T (PBS supplemented with 0.1% (v/v) Tween 20) for 1 h at room temperature. After washing with 100 pl_ PBS-0.05%T five times, Fc fusions (SEQ ID NOs: 76-78) at different concentrations were added to the wells and incubated for 1 h at room temperature, followed by another wash step. Bound molecules under study were detected by incubation with 1 :25000 diluted anti-human IgG Fc-HRP (Jackson Laboratory) in PBS-0.1%T-2%BSA. After an additional wash step, fluorogenic HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescence microplate reader. The EC₅₀ values are shown in Table 9.

[0248] Table 9: EC₅₀ values of OX40-specific hNGAL muteins.

SEQ ID NO EC₅₀ [nM]

76 0.51

77 0.58

78 0.45

[0249] 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.

[0250] 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.

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Blockade of CD134 (OX40)-CD134L interaction ameliorates lethal acute graft-versus-host disease in a murine model of allogeneic bone marrow transplantation. Blood, 95, 2434-9. VAJO, Z. & DUCKWORTH, W. C. 2000. Genetically engineered insulin analogs: diabetes in the new millenium. Pharmacol Rev, 52, 1-9. VAN WANROOIJ, E. J., VAN PUIJVELDE, G. H., DE VOS, P., YAGITA, H., VAN BERKEL, T. J. & KUIPER, J. 2007. Interruption of the Tnfrsf4/Tnfsf4 (OX40/OX40L) pathway attenuates atherogenesis in low-density lipoprotein receptor-deficient mic e. Arterioscler Thromb Vase Biol, 27, 204-10. 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. VETTO, J. T., LUM, S., MORRIS, A., SICOTTE, M., DAVIS, J., LEMON, M. & WEINBERG, A. 1997. Presence of the T-cell activation marker OX-40 on tumor infiltrating lymphocytes and draining lymph node cells from patients with melanoma and head and neck cancers. Am J Surg, 174, 258-65. WALKER, L. S., GULBRANSON-JUDGE, A., FLYNN, S., BROCKER, T., RAYKUNDALIA, C., GOODALL, M., FORSTER, R., LIPP, M. & LANE, P. 1999. Compromised 0X40 function in CD28- deficient mice is linked with failure to develop CXC chemokine receptor 5-positive CD4 cells and germinal centers. J Exp Med, 190, 1115-22. WEINBERG, A. D., BOURDETTE, D. N., SULLIVAN, T. J., LEMON, M., WALLIN, J. J., MAZIARZ,

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Claims

1. A lipocalin mutein that is capable of binding 0X40 with a detectable affinity.

2. The mutein of claim 1, wherein the mutein is capable of binding 0X40 with an affinity measured by a K_D of about 500 nM or lower, about 400 nM or lower, about 300 nM or lower, about 200 nM or lower, about 150 nM or lower, about 100 nM or lower, about 50 nM or lower, or about 30 nM or lower.

3. The mutein of claim 1 or 2, wherein the mutein binds 0X40 with an EC₅₀ value of about 250 nM or lower, about 200 nM or lower, about 150 nM or lower, about 100 nM or lower, or about 50 nM or lower.

4. The mutein of any one of claims 1-3, wherein the mutein is cross-reactive with cynomolgus 0X40.

5. The mutein of any one of claims 1-4, wherein the mutein interferes with the binding of 0X40 ligand (OX40L) to 0X40.

6. The mutein of any one of claims 1-5, wherein the mutein competes with OX40L for binding to 0X40.

7. The mutein of any one of claims 1-6, wherein the mutein comprises, at ten or more positions corresponding to positions 5-6, 8, 11, 19, 23, 26-34, 36-37, 40, 52, 55-56, 58, 60-61 , 65, 79, 86, 101 , 104-106, 108, 111, 113-114, 116, 121 , 124, 137, 140, 148, and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1), ten or more of the following mutated amino acid residues: Ala 5 Thr; Ser 6 Thr; Glu 8 Lys; Gin 11 Arg; Leu 19 Met or Gin; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Thr 37 Ala; Thr 40 lie; Lys 52 Glu; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Lys 65 lie; Ala 79 Thr; Ala 86 Thr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met or Leu; Lys 114 Trp; Val 116 Ala; Lys 121 Met; Leu 124 Lys; Arg 137 His; Ser 140 Arg; Arg 148 Ser or Trp; and Cys 153 Ser.

8. The mutein of any one of claims 1-7, wherein the mutein comprises, at ten or more positions corresponding to positions 26-34, 55-56, 60, 101 , 104-105, 108, 111 , and 114 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1), ten or more of the following mutated amino acid residues: Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Arg 60 Lys; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; Lys 108 lie; Arg 111 Pro; and Lys 114 Trp.

9. The mutein of any one of claims 1-8, wherein the mutein comprises, at ten or more positions corresponding to positions 23, 26-34, 55-56, 58, 60-61 , 101 , 104-106, 108, 111 , 114, and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1), ten or more of the following mutated amino acid residues: Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; and Cys 153 Ser.

10. The mutein of any one of claims 1-9, wherein the mutein comprises, at one or more positions corresponding to positions 5, 6, 8, 11 , 19, 36, 37, 40, 52, 65, 79, 86, 113, 116, 121 , 124, 137, 140, 148, and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1), one or more of the following mutated amino acid residues: Ala 5 Thr; Ser 6 Thr; Glu 8 Lys; Gin 11 Arg; Leu 19 Met or Gin; Val 36 Asp; Thr 37 Ala; Thr 40 lie; Lys 52 Glu; Lys 65 lie; Ala 79 Thr; Ala 86 Thr; Val 113 Met or Leu; Val 116 Ala; Lys 121 Met; Leu 124 Lys; Arg 137 His; Ser 140 Arg; Arg 148 Ser or Trp; and Cys 153 Ser.

11 . The mutein of any one of claims 1-10, wherein the mutein comprises one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1):

(a) Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; and Cys 153 Ser;

(b) Leu 19 Gin; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Thr 40 lie; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Ala 86 Thr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met; Lys 114 Trp; and Cys 153 Ser;

(c) Leu 19 Met; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met; Lys 114 Trp; and Cys 153 Ser;

(d) Ala 5 Thr; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Lys 52 Glu; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Met; Lys 114 Trp; Arg 137 His; and Cys 153 Ser;

(e) Ser 6 Thr; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Thr 37 Ala; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Lys 65 lie; Ala 79 Thr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; Val 116 Ala; and Cys 153 Ser;

(f) Ala 5 Thr; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; Arg 148 Ser; and Cys 153 Ser;

(g) Glu 8 Lys; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Val 113 Leu; Lys 114 Trp; Lys 121 Met; and Cys 153 Ser;

(h) Gin 11 Arg; Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; Ser 140 Arg; Arg 148 Trp; and Cys 153 Ser;

(i) Thr 23 Lys; Arg 26 Trp; Glu 27 Asp; Phe 28 Cys; Pro 29 Asn; Glu 30 Gin; Met 31 Pro; Asn 32 lie; Leu 33 Phe; Glu 34 Asp; Val 36 Asp; Met 55 lie; Leu 56 Phe; Ser 58 Asp; Arg 60 Lys; Cys 61 Tyr; Cys 101 Ser; Glu 104 Gin; Leu 105 Cys; His 106 Pro; Lys 108 lie; Arg 111 Pro; Lys 114 Trp; and Cys 153 Ser; (j) Thr 23 ® Lys; Arg 26 ® Trp; Glu 27 ® Asp; Phe 28 ® Cys; Pro 29 ® Asn; Glu 30

® Gin; Met 31 ® Pro; Asn 32 ® lie; Leu 33 ® Phe; Glu 34 ® Asp; Val 36 ® Asp;

Met 55 ® lie; Leu 56 ® Phe; Arg 60 ® Lys; Cys 61 ® Tyr; Cys 101 ® Ser; Glu 104 ® Gin; Leu 105 ® Cys; Lys 108 ® lie; Arg 111 ® Pro; Lys 114 ® Trp; Leu 124 ® Lys; and Cys 153 ® Ser; or

(k) Thr 23 ® Lys; Arg 26 ® Trp; Glu 27 ® Asp; Phe 28 ® Cys; Pro 29 ® Asn; Glu 30

® Gin; Met 31 ® Pro; Asn 32 ® lie; Leu 33 ® Phe; Glu 34 ® Asp; Val 36 ® Asp;

Met 55 ® lie; Leu 56 ® Phe; Ser 58 ® Asp; Arg 60 ® Lys; Cys 101 ® Ser; Glu 104 ® Gin; Leu 105 ® Cys; His 106 ® Pro; Lys 108 ® lie; Arg 111 ® Pro; and Lys 114 ® Trp.

12. The mutein of any one of claims 1-11 , wherein the mutein binds to an epitope on 0X40 that overlaps with the epitope of an antibody comprising the amino acid sequences shown in SEQ ID NOs: 26 and 27, SEQ ID NOs: 28 and 29, or SEQ ID NOs: 30 and 31.

13. The mutein of any one of claims 1-12, wherein the mutein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-46.

14. The mutein of any one of claims 1-13, wherein the mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-46 or a fragment or variant thereof.

15. The mutein of any one of claims 1-6, wherein the mutein comprises, at ten or more positions corresponding to positions 3, 21 , 25-26, 28, 36, 40-41 , 44, 49-50, 52, 55, 59, 60, 62-63, 65, 68, 70, 72-73, 75, 77-83, 87, 93, 96, 98, 100, 103, 106, 108, 114, 118, 125, 127, 129, 132, 134, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), ten or more of the following mutated amino acid residues: Ser 3 Phe or Pro; Asn 21 Asp; Asn 25 Ser; Gin 26 Arg; Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp or Arg; Glu 44 Gly; Gin 49 Gly; Lys 50 Glu or Thr; Tyr 52 Gin; lie 55 Val; Lys 59 Arg; Glu 60 Lys; Tyr 62 Arg; Ser 63 Thr or Ala; Asn 65 Gin or Arg; Ser 68 Gly; Leu 70 Pro or Arg; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Tyr 78 Asp or His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie or Val; Phe 83 Leu; Cys 87 lie, Ser, or Arg; Thr 93 lie; Asn 96 Trp; Lys 98

Arg; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Val 108 Ala; Asn 114 Asp; His 118 Tyr; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; Lys 134 Tyr; Glu

143 Ala; Glu 150 Gly; Gin 164 Asp; and Val 170 Ala.

16. The mutein of any one of claims 1-6 and 15, wherein the mutein comprises, at ten or more positions corresponding to positions 36, 40-41 , 49, 52, 68, 72-73, 77, 79, 81 , 87, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL

(SEQ ID NO: 2), ten or more of the following mutated amino acid residues: Leu 36 Phe; Ala 40 Tyr; lie 41 Trp or Arg; Gin 49 Gly; Tyr 52 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Cys 87 lie, Ser, or Arg; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr.

17. The mutein of any one of claims 1-6 and 15-16, wherein the mutein comprises, at one or more positions corresponding to positions 3, 21 , 25-26, 28, 44, 50, 55, 59-60, 62-63, 65, 70, 75, 78, 80, 82-83, 93, 98, 108, 114, 118, 129, 143, 150, 164, and 170 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), one or more of the following mutated amino acid residues: Ser 3 Phe or Pro; Asn 21 Asp; Asn 25 Ser; Gin 26 Arg; Gin 28 His; Glu 44 Gly; Lys 50 Glu or Thr; lie 55 Val; Lys 59 Arg; Glu 60 Lys; Tyr 62 Arg; Ser 63 Thr or Ala; Asn 65 Gin or Arg; Leu 70 Pro or Arg; Lys 75 Glu; Tyr 78 Asp or His; lie 80 Thr; Thr 82 lie or Val; Phe 83 Leu; Thr 93 lie; Lys 98 Arg; Val 108 Ala; Asn 114 Asp; His 118 Tyr; Asn 129 Asp; Glu 143 Ala; Glu 150 Gly; Gin 164 Asp; and Val 170 Ala.

18. The mutein of any one of claims 1-6 and 15-17, wherein the mutein comprises one of the following sets of mutated amino acid residues in comparison with the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

(a) Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52

Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg

81 Val; Cys 87 Ser; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(b) Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52 Gin; Glu 60

Lys; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp

79 Asp; Arg 81 Val; Phe 83 Leu; Cys 87 lie; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 — > Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(c) Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52 Gin; Ser 63 Thr; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(d) Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Cys 87 Arg; Thr 93 lie; Asn 96 Trp; Lys 98 Arg; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 T rp; and Lys 134 Tyr;

(e) Ser 3 Phe; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Tyr 52 Gin; lie 55 Val; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Phe 83 Leu; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Lys 98 Arg; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; His 118 Tyr; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Glu 150 Gly ;

(f) Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Glu 44 Gly; Gin 49 Gly; Lys 50 Thr; Tyr 52 Gin; Tyr 62 Arg; Asn 65 Gin; Ser 68 Gly; Arg

72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Phe 83 Leu; Cys 87 Ser; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(g) Gin 28 His; Leu 36 Phe; Ala 40 Tyr; lie 41 Trp; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Asn 65 Gin; Ser 68 Gly; Leu 70 Arg; Arg 72 Pro; Lys

73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; His 118 Tyr; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; and Lys 134 Tyr;

(h) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; Arg 81 Val; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr; (i) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg

72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 T rp; Lys 134 Tyr; Glu 143 Ala; and Gin 164 Asp;

0) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Arg 72 Pro; Lys

73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; Glu 143 Ala; and Gin 164 Asp;

(k) Ser 3 Pro; Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; and Lys 134 Tyr;

(I) Asn 25 Ser; Gin 26 Arg; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp;

(m) Asn 21 Asp; Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp;

(n) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; and Lys 134 Tyr;

(o) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Lys 50 Glu; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Arg; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Lys 75 Glu; Asp 77 His; Trp 79 Asp; Arg 81 Val; Thr 82 Val; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Val 108 Ala; Lys 125 Trp; Ser 127 Phe; Asn 129 Asp; Tyr 132 Trp; Lys 134 Tyr; Gin 164 Asp; and Val 170 Ala;

(P) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp;

(q) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp; or

(r) Asn 25 Ser; Leu 36 Phe; Ala 40 Tyr; lie 41 Arg; Gin 49 Gly; Tyr 52 Gin; Lys 59 Arg; Ser 63 Ala; Asn 65 Gin; Ser 68 Gly; Leu 70 Pro; Arg 72 Pro; Lys 73 His; Asp 77 His; Tyr 78 Asp; Trp 79 Asp; lie 80 Thr; Arg 81 Val; Thr 82 lie; Cys 87 Ser; Thr 93 lie; Asn 96 Trp; Tyr 100 Asp; Leu 103 lie; Tyr 106 Asp; Asn 114 Asp; Lys 125 Trp; Ser 127 Phe; Tyr 132 Trp; Lys 134 Tyr; and Gin 164 Asp.

19. The mutein of any one of claims 1-6 and 15-18, wherein the mutein binds to an epitope on 0X40 that overlaps with the epitope of an antibody comprising the amino acid sequences shown in SEQ ID NOs: 28 and 29 or SEQ ID NOs: 30 and 31.

20. The mutein of any one of claims 1-6 and 15-19, wherein the mutein binds to an epitope on 0X40 that overlaps with the epitope of a lipocalin mutein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-46.

21. The mutein of any one of claims 1-6 and 15-20, wherein the mutein has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-61 and 73-75.

22. The mutein of any one of claims 1-6 and 15-21, wherein the mutein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 47-61 and 73-75 or a fragment or variant thereof.

23. The mutein of any one of claims 1-22, 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.

24. The mutein of any one of claims 1-23, wherein the mutein is fused at its N-terminus and/or its C-terminus to a fusion partner that is a protein, a protein domain, a peptide, or a lipocalin mutein.

25. The mutein of any one of claims 1-24, wherein the mutein is fused at its N-terminus and/or its C-terminus to a fusion partner that is an antibody or antibody fragment.

26. The mutein of any one of claims 1-25, wherein the mutein is conjugated to a compound that extends the serum half-life of the mutein.

27. The mutein of claim 26, wherein the compound that extends the serum half-life is selected from the group consisting of a polyethylene glycol (PEG) molecule, hydroxyethyl starch, an Fc part of an immunoglobulin, a C_H3 domain of an immunoglobulin, a C_H4 domain of an immunoglobulin, an albumin binding peptide, and an albumin binding protein.

28. A nucleic acid molecule comprising a nucleotide sequence encoding a mutein of any one of claims 1-27.

29. An expression vector comprising the nucleic acid molecule of claim 28.

30. A host cell containing a nucleic acid molecule of claim 28 or the expression vector of claim 29.

31. A method of producing a mutein of any one of claims 1-27, wherein the mutein is produced starting from the nucleic acid coding for the mutein.

32. A method of binding and/or detecting 0X40 in a subject, comprising applying one or more muteins of any one of claims 1-27 or one or more compositions comprising such muteins.

33. A method of stimulating an immune response in a subject, comprising applying one or more muteins of any one of claims 1-27 or one or more compositions comprising such muteins.

34. A method of interfering with the binding of human 0X40 to OX40L in a subject, comprising applying one or more muteins of any one of claims 1-27 or one or more compositions comprising such muteins.

35. A method of activating downstream signaling pathways of 0X40, comprising applying one or more muteins of any one of claims 1-27 or one or more compositions comprising such muteins.

36. A method of co-stimulating T cells, comprising applying one or more muteins of any one of claims 1-27 or one or more compositions comprising such mutein to a tissue comprising a tumor.

37. A pharmaceutical composition comprising one or more muteins of any one of claims 1-27.

38. A mutein of any one of claims 1-27 or the pharmaceutical composition of claim 37 for use in therapy.

39. The mutein or pharmaceutical composition for the use of claim 38, wherein the use is in the treatment of a cancer, an infectious disease, or an autoimmune disease.

40. Use of a mutein of any one of claims 1-27 for the manufacture of a medicament.

41. The use of claim 40, wherein the medicament is for the treatment of a cancer, an infectious disease, or an autoimmune disease.

42. A method of treating a disease comprising administering to a subject in need thereof an effective amount of a mutein of any one of claims 1-27 or a pharmaceutical composition of claim 37.

43. The method of claim 42, wherein the disease is a cancer, an infectious disease, or an autoimmune disease.