Novel fusion protein specific for CD137 and GPC3

Abstract

The present invention provides fusion proteins specific for CD137 and GPC3, wherein the fusion proteins can be used to co-stimulate lymphocyte activation in a GPC3 target-dependent manner. Such fusion proteins can be used in many pharmaceutical applications, for example as anticancer agents and / or immunomodulators for treating or preventing human diseases such as various tumors. The present invention also relates to methods for preparing the fusion proteins described herein and compositions comprising such fusion proteins. The present invention further relates to nucleic acid molecules encoding such fusion proteins and methods for producing such fusion proteins and nucleic acid molecules. In addition, the present application discloses therapeutic and / or diagnostic uses of these fusion proteins and compositions comprising one or more such fusion proteins.

Description

I. Background Technology

[0001] Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS and SGBS1) is a carcinoembryonic antigen belonging to the glypican family of glycosyl-phosphatidylinositol-anchored heparan sulfate proteoglycans. GPC3 is expressed in various tissues during development and plays an important role in morphogenesis and growth through signaling pathways such as fibroblast growth factor (FGF), non-classical Wnt or insulin-like growth factor (Cheng et al., Carcinogenesis, 2008; Song et al., J Biol Chem, 2005; Song et al., J Biol Chem, 1997). However, in most normal adult tissues, GPC3 expression is downregulated or silent. In addition, depending on the cell type, GPC3 can positively and negatively regulate cell growth. Loss-of-function mutations in GPC3 are responsible for Simpson-Golabi-Behmel or Simpson malformation syndrome (SGBS), a rare X-linked overgrowth disorder in which patients have an increased risk of embryonal tumors, including Wilms' tumor (Pilia et al., Nat Genet, 1996).

[0002] GPC3 is expressed in various types of cancer including liver cancer, gastric cancer, melanoma, high-grade urothelial carcinoma, testicular cancer, and some uterine and vaginal cancers (Aydin et al., Diagn Pathol, 2015; Ushiku et al., Cancer Sci, 2009; Gailey and Bellizzi, Am J Clin Pathol, 2013; Yamanaka et al., Oncology, 2007; Nakatsura et al., Clin Cancer Res, 2004; Zynger et al., Am J Surg Pathol, 2006; Montalbano et al., Int J Oncol, 2016; Midorikawa et al., Int J Cancer, 2003). In particular, GPC3 is highly expressed in hepatocellular carcinoma (HCC) (Capurro et al., Gastroenterology, 2003; Nakatsura et al., Biochem Biophys Res Commun, 2003; Sung et al., Cancer Sci, 2003; Zhu et al., Gut, 2001), which is the main form of liver cancer, accounting for 90% of all liver cancers and causing at least 500,000 deaths each year (Jelic et al., Ann Oncol, 2010). GPC3 has been extensively studied as a diagnostic biomarker and therapeutic target for HCC and other cancer types. Several antibodies targeting GPC3 have been obtained, including humanized mouse antibodies YP7 and GC33 and human antibodies HN3 and MDX-1414, however, these antibodies have not shown the ability to inhibit HCC cell proliferation or induce apoptosis (Feng and Ho, FEBS Lett, 2014). In particular, GC33 is currently being evaluated in clinical trials for HCC. The mechanism of GC33 function includes antibody-dependent cellular cytotoxicity (ADCC), but GC33 does not directly inhibit the proliferation of GPC3-positive tumor cells (Takai et al., Cancer Biol Ther, 2009; Nakano et al., Biochem Biophys Res Commun, 2009; Ishiguro et al., Cancer Res, 2008). However, although GC33 has been shown to exhibit tumor growth inhibition in vivo, it has not provided satisfactory clinical efficacy. At the same time, the results of knockdown and siRNA experiments indicate that GPC3 is not a lethal gene for HCC cells, which makes it impossible to determine whether anti-GPC3 antibodies can effectively cause tumor regression.

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

[0004] CD137L is a trimeric protein present in membrane-bound form and soluble variants. However, the ability of soluble CD137L to activate CD137 on lymphocytes such as CD137 is limited, and high concentrations are required to induce effects (Wyzgol et al., J Immunol, 2009). The natural method of activating CD137 is by the engagement of CD137-positive cells with CD137L positive cells. It is believed that the activation of CD137 is then induced by the aggregation of CD137L on relative cells, resulting in signal transduction via TRAF1, 2, and 3 (Yao et al., Nat Rev Drug Discov, 2013; Snell et al., Immunol Rev, 2011) and further in CD137-positive T cells with downstream effects. In the case of activating T cells by the identification of their corresponding homologous targets, the effect caused by costimulation CD137 is further enhanced activation, enhanced survival and proliferation, the production of proinflammatory cytokines and improved killing ability.

[0005] The benefits of CD137 co-stimulation for the elimination of cancer cells have been demonstrated in many in vivo models. Overexpression of CD137L on tumors leads to, for example, tumor rejection (Melero et al., Eur J Immunol, 1998). Similarly, overexpression of anti-CD137 scFv on tumors leads to CD4 + T cell and NK cell-dependent tumor elimination (Yang et al., Cancer Res, 2007; Zhang et al., Mol Cancer Ther, 2006; Ye et al., Nat Med, 2002). It has also been demonstrated that systemic administration of anti-CD137 antibodies leads to tumor growth retardation (Martinet et al., Gene Ther, 2002).

[0006] Studies have also shown that CD137 is an excellent marker for tumor-reactive T cells naturally present in human tumors (Ye et al., Clin Cancer Res, 2014), and anti-CD137 antibodies can be used to improve CD8 + Expansion and activation of melanoma-infiltrating lymphocytes for adoptive T-cell therapy (Chacon et al., PLoS One, 2013).

[0007] Preclinical demonstration of the potential therapeutic benefits of CD137 co-stimulation has prompted the development of therapeutic antibodies targeting CD137, including BMS-663513 (described in US Pat. No. 7,288,638) and PF-05082566 (Fisher et al., Cancer Immunol Immunother, 2012).

[0008] Among other things, the present invention provides a novel means of simultaneously engaging CD137 and GPC3 via one or more fusion proteins that possess the properties of a binding specificity for CD137 and a binding specificity for GPC3.

[0009] II. Definitions

[0010] The following list defines the terms, phrases and abbreviations used in this specification. All terms listed and defined herein are intended to encompass all grammatical forms.

[0011] As used herein, unless otherwise specified, "CD137" means human CD137 (huCD137). Human CD137 means the full-length protein defined by UniProt Q07011, its fragment or its variant. CD137 is also called 4-1BB or tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is lymphocyte activation-induced (ILA). In some specific embodiments, CD137 of non-human species is used, such as macaque (cynomolgus) CD137 and mouse CD137.

[0012] As used herein, unless otherwise specified, "Glypican-3" or "GPC3" means human GPC3 (huGPC3). Human GPC3 means the full-length protein defined by UniProt P51654, a fragment thereof, or a variant thereof. Human GPC3 is encoded by the GPC3 gene. GPC3 is also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, or SGBS1. In some specific embodiments, GPC3 of non-human species is used, such as macaque GPC3 and mouse GPC3.

[0013] As used herein, "binding affinity" describes the ability of a biomolecule of the invention (e.g., a polypeptide or protein, such as a lipocalin mutein, an antibody, a fusion protein, or any other peptide or protein) to bind to a selected target and form a complex. Binding affinity can be measured by a variety 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 ELISAs), calorimetric methods such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well known in the art, and some examples thereof are further described below. Thus, binding affinity is expressed as the dissociation constant (K) measured using these methods. D ), half maximal effective concentration (EC 50 ) or half maximal inhibitory concentration (IC 50 ) value. The lower K D ,EC 50 or IC 50 The value reflects better (higher) binding ability (affinity). Thus, the binding affinity of two biomolecules to a selected target can be measured and compared. When comparing the binding affinity of two biomolecules to a selected target, the terms "substantially the same", "substantially the same" or "substantially similar" mean that within the experimental variability of the binding affinity measurement, one biomolecule has a K expressed as the same or similar to that of another molecule. D ,EC 50 or IC 50 The experimental variation of the binding affinity measurement depends on the specific method used and is known to those skilled in the art.

[0014] As used herein, the term "substantially" may also refer to the qualitative condition of exhibiting all or nearly all of the range or degree of a target feature or characteristic. One of ordinary skill in the biological field will appreciate that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid an absolute result. Therefore, the term "substantially" as used herein is used to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0015] As used herein, the terms "detect", "detection", "detecting" or "detectable" are understood to be both at a quantitative and qualitative level and combinations thereof. It thus includes quantitative, semi-quantitative and qualitative measurements performed on the biomolecules of the invention.

[0016] As used herein, "detectable affinity" generally means that the binding capacity between a biomolecule and its target is at most about 10 -5 M or lower, which is represented by KD ,EC 50 or IC 50 Values ​​above 10 are generally no longer measurable by conventional methods such as ELISA and SPR -5 The binding affinity of M (expressed as K D ,EC 50 or IC 50 value), and therefore it is secondary.

[0017] It is noted that the formation of a complex between a biomolecule of the present invention and its target is influenced by a number of different factors, such as the concentration of the respective target, the presence of competitors, the pH and ionic strength of the buffer system employed, the experimental method used to determine the binding affinity (e.g., fluorescence titration, competitive ELISA (also known as competition ELISA) and surface plasmon resonance), and even the mathematical algorithm used to evaluate the experimental data. Therefore, it is also clear to those skilled in the art that K D ,EC 50 or IC 50 The binding affinity of a given protein may vary within a certain experimental range, depending on the method and experimental setup. This means that the measured K D ,EC 50 or IC 50 Slight deviations from the values ​​may occur within a tolerance range, for example, depending on whether the value is determined by ELISA (including direct or competitive ELISA), by SPR or another method.

[0018] As used herein, "specific for", "specific binding", "specifically binds", or "binding specificity" refers to the ability of a biomolecule to distinguish a desired target (e.g., CD137 and GPC3) from one or more reference targets (e.g., cellular receptor for neutrophil gelatinase-associated lipocalin). It should be understood that such specificity is not an absolute property, but rather a relative property, and can be determined, for example, by SPR, Western blot, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), immunohistochemistry (IHC), and peptide scanning.

[0019] When used in the context of the fusion protein of the present invention that binds to CD137 and GPC3 herein, the term "specific for", "specific binding", "specifically binds" or "binding specificity" means that the fusion protein as described herein binds to, reacts with or is directed to CD137 and GPC3, but does not substantially bind to another protein. The term "another protein" includes any protein that is not CD137 or GPC3 or a protein that is closely related or homologous to CD137 or GPC3. However, CD137 or GPC3 from species other than humans and fragments and / or variants of CD137 or GPC3 are not excluded by the term "another protein". The term "substantially does not bind" means that the fusion protein of the present invention binds to another protein with a lower binding affinity than CD137 and / or GPC3, i.e., exhibits less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9%, 8%, 7%, 6% or 5% cross-reactivity. Whether the fusion protein as defined above reacts specifically can be easily tested, in particular by comparing the reaction of the fusion protein of the invention with CD137 and / or GPC3 with the reaction of the fusion protein with other (another) protein.

[0020] As used herein, the term "lipocalin" refers to a monomeric protein weighing about 18-20 kDa, which has a cylindrical β-pleated sheet supersecondary structural region, which comprises a plurality of β-strands (preferably 8 β-strands, labeled A to H) connected in pairs by a plurality of (preferably 4) loops at one end to thereby contain a ligand binding pocket and define the entrance to the ligand binding pocket. Preferably, the loops containing the ligand binding pocket used in the present invention are loops connecting the open ends of β-strands A and B, C and D, E and F, and G and H, and are labeled as AB, CD, EF and GH loops. It is well recognized that, among members of the lipocalin family, it is this diversity of loops in the otherwise rigid lipocalin backbone that generates a variety of different binding modes, each capable of accommodating targets of different size, shape, and chemical characteristics (e.g., reviewed in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996). It is understood that the lipocalin family of proteins has evolved naturally to bind a wide range of ligands, with very low levels of overall sequence conservation (typically with less than 20% sequence identity), but retains a highly conserved overall folding pattern. The correspondence between positions in different lipocalins is also well known to those skilled in the art (see, e.g., U.S. Pat. No. 7,250,297). Proteins falling within the definition of "lipocalin" as used herein include, but are not limited to, human lipocalins including tear lipocalin (Tlc, Lcn1), lipocalin-2 (Lcn2), or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, α 1 -Acid glycoprotein 1, alpha 1 -Acid glycoprotein 2, alpha 1 -microglobulin, complement component 8γ, retinol binding protein (RBP), epididymal retinoic acid binding protein, glycodelin, odorant binding protein IIa, odorant binding protein IIb, lipocalin-15 (Lcn15), and prostaglandin D synthase.

[0021] As used herein, unless otherwise specified, "tear lipocalin" refers to human tear lipocalin (hTlc), and further refers to mature human tear lipocalin. When the term "mature" is used to characterize a protein, it means that the protein is substantially free of a signal peptide. "Mature hTlc" of the present invention refers to the mature form of human tear lipocalin, which does not have a signal peptide. Mature hTlc is described as residues 19-176 having a sequence of SWISS-PROT database accession number P31025, and its amino acids are shown in SEQ ID NO: 1.

[0022] As used herein, "lipocalin-2" or "neutrophil gelatinase-associated lipocalin" refers to human lipocalin-2 (hTlc2) or human neutrophil gelatinase-associated lipocalin (hNGAL), and further refers to mature human lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin. When the term "mature" is used to characterize a protein, it means that the protein is substantially free of a signal peptide. "Mature hNGAL" of the present invention refers to the mature form of human neutrophil gelatinase-associated lipocalin, which does not have a signal peptide. Mature hNGAL is described as residues 21-198 of the sequence having SWISS-PROT database accession number P80188, and its amino acids are shown in SEQ ID NO: 2.

[0023] As used herein, "native sequence" refers to a protein or polypeptide having a sequence that exists in nature or having a wild-type sequence, regardless of how the protein or polypeptide is prepared. Such native sequence proteins or polypeptides can be isolated from nature or can be produced by other means such as recombinant or synthetic methods.

[0024] "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 may have the amino acid sequence of a corresponding naturally occurring (wild-type) lipocalin from any organism, in particular a mammal. When used in the context of a lipocalin, the term "native sequence" specifically encompasses naturally occurring truncated or secreted forms, naturally occurring variant forms (such as alternatively spliced ​​forms and naturally occurring allelic variants of a lipocalin). The term "native sequence lipocalin" is used interchangeably with "wild-type lipocalin" herein.

[0025] As used herein, "mutant", "mutated" entity (protein or nucleic acid) or "mutant" refers to an exchange, deletion or insertion of one or more amino acids or nucleotides compared to a naturally occurring (wild type) protein or nucleic acid. The term also includes fragments of mutant proteins as described herein. The present invention explicitly encompasses such lipocalin mutants as described herein, which have a cylindrical β-pleated sheet supersecondary structure region, the structural region comprising 8 β-strands connected in pairs by 4 loops at one end to thereby contain a ligand binding pocket and an entrance defining the ligand binding pocket, wherein at least one amino acid in each of at least three of the 4 loops has been mutated compared to the native sequence lipocalin. Preferably, the lipocalin mutant protein of the present invention has the function of binding CD137 as described herein.

[0026] As used herein, the term "fragment" in connection with the lipocalin mutein of the invention refers to a protein or polypeptide derived from full-length mature hTlc or hNGAL or lipocalin mutein that is N-terminally and / or C-terminally truncated (i.e., lacks at least one of the N-terminal and / or C-terminal amino acids). Such a fragment may include at least 10 or more, such as 20 or 30 or more consecutive amino acids of the primary sequence of the mature hTlc or hNGAL or lipocalin mutein from which it is derived, and is typically detectable in an immunoassay for mature hTlc 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 1, 2, 3 or 4 N-terminal amino acids (His-His-Leu-Leu) and / or 1 or 2 C-terminal amino acids (Ser-Asp) of mature hTlc. It should be understood that the fragment is preferably a functional fragment of the mature hTlc or hNGAL or lipocalin mutant protein from which it is derived, which means that the fragment preferably retains the preferred binding specificity for CD137 of the mature hTlc / hNGAL or lipocalin mutant protein from which it is derived. As an illustrative example, such a functional fragment may at least contain amino acids at positions 5-153, 5-150, 9-148, 12-140, 20-135 or 26-133 corresponding to the linear polypeptide sequence of mature hTlc. As another illustrative example, such a functional fragment may comprise at least the amino acids at positions 13-157, 15-150, 18-141, 20-134, 25-134 or 28-134 corresponding to the linear polypeptide sequence of mature hNGAL.

[0027] The term "fragment" with respect to the corresponding target CD137 or GPC3 of the fusion protein of the present invention refers to CD137 or GPC3 or the protein domain of CD137 or GPC3 truncated at the N-terminus and / or C-terminus. The fragment of CD137 or the fragment of GPC3 described herein retains the ability of full-length CD137 or GPC3 to be recognized and / or bound by the fusion protein of the present invention. As an illustrative example, the fragment can be the extracellular domain of CD137 or GPC3. As an illustrative example, such an extracellular domain can include amino acids of the extracellular subdomain of CD137, such as domain 1 (residues 24-45 of UniProt Q07011), domain 2 (residues 46-86), domain 3 (87-118) and domain 4 (residues 119-159) alone or in combination.

[0028] As used herein, the term "variant" refers to a derivative of a protein or polypeptide comprising a mutation, for example, by substitution, deletion, insertion and / or chemical modification of an amino acid sequence or a nucleotide sequence. In some embodiments, such mutation and / or chemical modification does not reduce the function 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 substitutions between members of the following groups: 1) alanine, serine, threonine and valine; 2) aspartic acid, glutamic acid, glutamine, asparagine and histidine;

[0029] 3) arginine, lysine, glutamine, asparagine and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine and proline; and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Such variants include proteins or polypeptides in which one or more amino acids are replaced by their respective D-stereoisomers or amino acids other than the 20 naturally occurring amino acids (such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, valerine). Such variants also include, for example, proteins or polypeptides in which one or more amino acid residues are added or deleted at the N- and / or C-terminus. Generally, variants have 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. Variants preferably retain biological activity, such as binding to the same target as the protein or polypeptide from which they are derived.

[0030] The term "variant" as used herein in relation to the corresponding protein ligand CD137 or GPC3 of the fusion protein of the invention relates to CD137 or GPC3 or fragments thereof having one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 60, 70, 80 or more) amino acid substitutions, deletions and / or insertions compared to the native sequence of CD137 or GPC3 (wild-type CD137 or GPC3, such as CD137 deposited as UniProt Q07011 as described herein or GPC3 deposited as UniProt P51654). CD137 variants or GPC3 variants have preferably at least 50%, 60%, 70%, 80%, 85%, 90% or 95% amino acid identity with wild-type CD137 or GPC3, respectively. The CD137 variants or GPC3 variants described herein retain the ability to bind to the fusion proteins disclosed in the present invention that are specific for CD137 and GPC3.

[0031] The term "variant" as used herein with respect to lipocalin mutants relates to lipocalin mutants of the invention or fragments thereof, wherein the sequence thereof has mutations and / or chemical modifications including substitutions, deletions and insertions. The variants of the lipocalin mutants described herein retain the biological activity of the lipocalin mutants from which the variants are derived, such as the biological activity of binding to CD137. In general, the variants of the lipocalin mutants have at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% amino acid sequence identity with the lipocalin mutants from which they are derived.

[0032] As used herein, the term "mutagenesis" refers to the introduction of mutations into a polynucleotide or amino acid sequence. Preferably, mutations are introduced under experimental conditions so that the naturally occurring amino acid at a given position of a protein or polypeptide sequence can be altered, for example, replaced by at least one amino acid. The term "mutagenesis" also includes (additionally) changing the length of a sequence segment by deleting or inserting one or more amino acids. Thus, the following is within the scope of the present invention: for example, an amino acid at a selected sequence position is replaced by a stretch of three amino acids, resulting in the addition of two amino acid residues compared to the length of the corresponding segment of the natural protein or polypeptide amino acid sequence. Such insertions or deletions can be introduced independently of each other into any sequence segment that can be mutagenized in the present invention. In an exemplary embodiment of the present invention, an insertion can be introduced into the amino acid sequence segment corresponding to the AB loop of a native sequence lipocalin (see International Patent Publication No. WO 2005 / 019256, which is incorporated herein by reference in its entirety).

[0033] As used herein, the term "random mutagenesis" means that no predetermined mutation (change of amino acid) exists at a certain sequence position, but at least two amino acids can be incorporated at a predefined sequence position with a certain probability during mutagenesis.

[0034] As used herein, the term "sequence identity" or "identity" is a sequence property that measures the similarity or relationship of sequences. As used herein, the term "sequence identity" or "identity" means the percentage of paired identical residues relative to the number of residues in the longer of the two sequences after alignment of a protein or polypeptide sequence of the invention with the sequence in question (homology). Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the result by 100.

[0035] As used herein, the term "sequence homology" or "homology" is its general meaning, and homologous amino acids include identical amino acids as well as amino acids that are considered to be conservative substitutions at equivalent positions in the linear amino acid sequence of a protein or polypeptide of the invention (such as any fusion protein or lipocalin mutein of the invention).

[0036] The skilled person will recognize available computer programs such as BLAST (Altschul et al., Nucleic Acids Res, 1997), BLAST2 (Altschul et al., J Mol Biol, 1990) and Smith-Waterman (Smith and Waterman, J Mol Biol, 1981) for determining sequence homology or sequence identity using standard parameters. Sequence homology or sequence identity percentages may be determined herein using, for example, the program BLASTP, version 2.2.5 (November 16, 2002) (Altschul et al., Nucleic Acids Res, 1997). In this embodiment, the homology percentages are based on alignments of the complete protein or polypeptide sequence including the propeptide sequence (matrix: BLOSUM 62; gap penalty: 11.1; cutoff value set to 10 -3 ), preferably using the wild-type protein backbone as a reference in the pairwise comparison. It is calculated as the percentage of the number of "positives" (homologous amino acids) represented as the result of the BLASTP program output divided by the total number of amino acids selected by the program for comparison.

[0037] Specifically, in order to determine whether the amino acid residues of the amino acid sequence of the lipocalin mutant protein are different from the wild-type lipocalin corresponding to a certain position in the wild-type lipocalin amino acid sequence, the skilled person can use means and methods well known in the art, such as manually or by using a computer program such as BLAST 2.0 (which stands for Basic Local Alignment Search Tool) or ClustalW or any other suitable program for generating sequence alignments. Therefore, the wild-type sequence of the lipocalin protein can be used as a "subject sequence" or "reference sequence", and the amino acid sequence of the lipocalin mutant protein described herein that is different from the wild-type lipocalin protein is used as a "query sequence". The terms "wild-type sequence", "reference sequence" and "subject sequence" are used interchangeably herein. Preferably, the wild-type sequence of the lipocalin protein is the hTlc sequence shown in SEQ ID NO: 1 or the hNGAL sequence shown in SEQ ID NO: 2.

[0038] A "gap" is a space in an alignment caused by the addition or deletion of an amino acid. Thus, two identical sequences have 100% identity, but sequences that are less highly conserved and have deletions, additions or substitutions may have a lower degree of sequence identity.

[0039] As used herein, the term "position" means the position of an amino acid in an amino acid sequence described herein or the position of a nucleotide in a nucleic acid sequence described herein. It is to be understood that, as used herein in the context of one or more lipocalin mutant amino acid sequence positions, the corresponding position is not only determined by the number of preceding nucleotides or amino acids. Thus, the absolute position of a given amino acid according to the present invention may differ from the corresponding position due to deletions or additions of amino acids at other positions of the (mutant or wild-type) lipocalin. Similarly, the absolute position of a given nucleotide according to the present invention may differ from the corresponding position due to deletions or additions of nucleotides at other positions of the mutant or wild-type lipocalin 5'-untranslated region (UTR) (including promoters and / or any other regulatory sequences or genes (including exons and introns)).

[0040] A "corresponding position" according to the present invention may be a sequence position that matches the sequence position to which it corresponds in a pairing or multiple sequence alignment according to the present invention. For a "corresponding position" according to the present invention, it is preferably understood that the absolute position of a nucleotide or amino acid may be different from an adjacent nucleotide or amino acid, but the same one or more "corresponding positions" may still contain the adjacent nucleotide or amino acid that may have been exchanged, deleted or added.

[0041] Furthermore, for corresponding positions in a lipocalin mutein based on a reference sequence according to the invention, it is preferably understood that the position of the nucleotide or amino acid of the lipocalin mutein may structurally correspond to positions at other positions in a reference lipocalin (wild-type lipocalin) or another lipocalin mutein, although these positions may differ in absolute position numbers, as the skilled person understands in view of the highly conserved overall folding form between lipocalins.

[0042] As used interchangeably herein, the terms "conjugate", "conjugation", "fuse", "fusion" or "linking" refer to the joining together of two or more subunits via all forms of covalent or non-covalent linkage by means including but not limited to genetic fusion, chemical conjugation, coupling through linkers or cross-linking reagents, and non-covalent association.

[0043] As used herein, "fusion polypeptide" or "fusion protein" refers to a polypeptide or protein comprising two or more subunits. In some embodiments, the fusion protein described herein comprises two or more subunits, at least one of which can specifically bind to CD137, and another subunit can specifically bind to GPC3. In the fusion protein, these subunits can be connected by covalent or non-covalent bonds. Preferably, the fusion protein is a translational fusion between two or more subunits. The translational fusion can be produced by genetic engineering the coding sequence of one subunit in the reading frame with the coding sequence of another subunit. Both subunits can be interspersed with the nucleotide sequence encoding the linker. However, the subunits of the fusion protein of the present invention can also be connected by chemical conjugation. Typically, the subunits forming the fusion protein are connected to each other, the C-terminus of one subunit is connected to the N-terminus of another subunit, or the C-terminus of one subunit is connected to the C-terminus of another subunit, or the N-terminus of one subunit is connected to the N-terminus of another subunit, or the N-terminus of one subunit is connected to the C-terminus of another subunit. The subunits of a fusion protein may be linked in any order and may include more than one of any of the constituent subunits. If one or more subunits are part of a protein (complex) consisting of more than one polypeptide chain, the term "fusion protein" may also refer to a protein comprising the fused sequence and all other polypeptide chains of the protein (complex). As an illustrative example, in the case where a full-length immunoglobulin is fused to a lipocalin mutant protein via the heavy or light chain of the immunoglobulin, the term "fusion protein" may refer to a single polypeptide chain comprising a lipocalin mutant protein and a heavy or light chain of the immunoglobulin. The term "fusion protein" may also refer to a complete immunoglobulin (light and heavy chains) and a lipocalin mutant protein fused to one or both of its heavy and / or light chains.

[0044] As used herein, the term "subunit" of the fusion proteins disclosed herein refers to a single protein or independent polypeptide chain that can form a stable folded structure by itself and define a unique function that provides a binding motif for a target. In some embodiments, the preferred subunits of the present invention are lipocalin mutant proteins. In some other embodiments, the preferred subunits of the present invention are full-length immunoglobulins or antigen binding domains thereof.

[0045] The "linker" that can be included in the fusion protein of the present invention connects two or more subunits of the fusion protein as described herein together. The linkage can be covalent or non-covalent. A preferred covalent linkage is through a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. Therefore, in a preferred embodiment, the 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, the GS linker is (G) as described in SEQ ID NO: 13. 4 S) 3 , which is used to connect the subunits of the fusion protein together. Other preferred linkers include chemical linkers.

[0046] As used herein, the term "albumin" includes all mammalian albumins, such as human serum albumin or bovine serum albumin or rat serum albumin.

[0047] As used herein, the term "organic molecule" or "small organic molecule" refers to an organic molecule comprising at least two carbon atoms, but preferably comprising no more than 7 or 12 rotatable carbon bonds, having a molecular weight ranging from 100 to 2,000 Daltons, preferably 100 to 1,000 Daltons, and optionally comprising one or two metal atoms.

[0048] A "sample" is defined as a biological sample collected from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissue, including tumor tissue.

[0049] A "subject" is a vertebrate, preferably a mammal, more preferably a human. As used herein, the term "mammal" refers to any animal classified as a mammal, including but not limited to humans, livestock and agricultural animals, and zoo, sports or pet animals, such as sheep, dogs, horses, cats, cows, rats, pigs, apes such as cynomolgous monkeys, to name a few illustrative examples. Preferably, a "mammal" as used herein is a human.

[0050] An "effective amount" is an amount sufficient to produce beneficial or desired results. An effective amount can be administered in one or more individual administrations or dosings.

[0051] As used herein, "antibody" includes whole antibodies or any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. Whole antibodies refer to glycoproteins comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable domain (V H or HCVR) and heavy chain constant region (C H ). The heavy chain constant region consists of three domains C H1 , C H2 and C H3 Each light chain consists of a light chain variable domain (V L or LCVR) and the light chain constant region (C L ). The light chain constant region consists of a domain C L Composition. H With V L The V regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens (e.g., GPC3). The constant region of an antibody can mediate the binding of immunoglobulins 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.

[0052] As used herein, an "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 (such as GPC3). Studies have shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) H 、V L , C L , and C H1 (ii) a F(ab') fragment consisting of two Fab fragments linked by a disulfide bridge in the hinge region 2 Fragment; (iii) by V H 、V L , C L , and C H1 Domain and C H1 and C H2 The Fab' fragment consists of the region between the domains; (iv) the V H and C H1 The Fd fragment consists of a single-arm V H and VL A single-chain Fv fragment consisting of a V H and (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker; (viii) a dAb fragment comprising a V domain linked by a short linker in the same polypeptide chain. H and V L "diabodies" (see, e.g., patent documents EP404,097; WO 93 / 11161; and Holliger et al., Proc Natl Acad Sci USA, 1993); (ix) containing only V H or V L "Domain antibody fragments" wherein, in some cases, two or more V H The regions are covalently linked.

[0053] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic or homologous; or modified forms thereof (eg, humanized, chimeric or multispecific). Antibodies can also be fully human.

[0054] As used herein, "framework" or "FR" refers to the variable domain residues rather than the hypervariable region (CDR) residues.

[0055] "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 may change, the human IgG heavy chain Fc region is generally defined as a segment from Cys226 or from an amino acid residue at the position of Pro230 (numbered according to the EU index of Kabat) to its carboxyl terminus (Johnson and Wu, Nucleic Acids Res, 2000). The C-terminal lysine (numbered according to the EU index of Kabat, residue 447) of the Fc region can be removed, for example, during the production or purification of the antibody or by recombinant engineering of nucleic acids encoding the heavy chain of the antibody. Therefore, the composition of the complete antibody can include antibody groups with all K447 residues removed, antibody groups without K447 residues removed, and antibody groups with and without K447 residues. Suitable native sequence Fc regions used in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0056] "Fc" receptor or "FcR" refers to a receptor that binds the Fc region of an antibody.

[0057] As used herein, an "isolated antibody" refers to an antibody that is substantially free of its natural environment. For example, 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. Herein, an isolated antibody that specifically binds GPC3 is substantially free of antibodies that specifically bind antigens other than GPC3. However, an isolated antibody that specifically binds GPC3 may have cross-reactivity with other antigens, such as GPC3 molecules from other species.

[0058] 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.

[0059] As used herein, "humanized antibody" refers to an antibody composed of antibody CDRs derived from mammals other than humans and human antibodies or FR regions and constant regions derived from human antibodies. In some embodiments, humanized antibodies include variable domains with variable region amino acid sequences, such as the Immunogenetics Information System (IMGT) Domain Gap Align tool assessment as described in Ehrenmann et al., (2010), and overall analysis shows that the variable region amino acid sequences are closer to humans rather than other species. In some embodiments, due to reduced antigenicity, humanized antibodies can be used as active ingredients in therapeutic agents. As used herein, the term "therapeutic agent" or "therapeutically active agent" refers to a therapeutically useful agent. The therapeutic agent can be any agent used to prevent, improve or treat a disease, a physiological state, a symptom, or for the assessment or diagnosis of a disease, a physiological state, a symptom.

[0060] As used herein, "human antibodies" include antibodies having variable regions in which the framework region and CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from another mammalian species germline (such as mice) are transplanted to human framework sequences. III. Description of the drawings

[0061] Figure 1: Provides an overview of the design of representative fusion proteins described in this application that are bispecific for the targets CD137 and GPC3. Based on an antibody specific for GPC3 (e.g., an antibody whose heavy chain is provided by SEQ ID NO:81, or comprises a heavy chain variable domain of SEQ ID NO:78, or comprises a CDR sequence of GYTFTDYE (HCDR1, SEQ ID NO:72), LDPKTGDT (HCDR2, SEQ ID NO:73) and TRFYSYTY (HCDR3; SEQ ID NO:74), and whose light chain is provided by SEQ ID NO:82, or comprises a light chain variable domain of SEQ ID NO:79, or comprises a CDR sequence of QSLVHSNRNTY (LCDR1, SEQ ID NO:75), KVS (LCDR2, SEQ ID NO:76) and SQNTHVPPT (LCDR3; SEQ ID NO:77)) and one or more lipocalin mutants specific for CD137 (e.g., a lipocalin mutant of SEQ ID NO:40 or a CDR sequence of SEQ ID NO:41). Representative fusion proteins were prepared using a lipocalin mutant protein (NO: 49) with 97% sequence identity and named CD137Ac1. Figure 1 As shown in A-II, one or more lipocalin mutant proteins are genetically fused to the C- and / or N-terminus of the heavy or light chain of a GPC3-specific antibody to obtain fusion proteins, such as SEQ ID NOs: 87 and 82, SEQ ID NOs: 88 and 82, SEQ ID NOs: 81 and 89, SEQ ID NOs: 81 and 90, and CD137Ac1-Fusion 1, CD137Ac1-Fusion 2, CD137Ac1-Fusion 3, CD137Ac1-Fusion 4, CD137Ac1-Fusion 5, CD137Ac1-Fusion 6, and CD137Ac1-Fusion 7 (fusion proteins with 97% sequence identity to SEQ ID NOs: 91 and 82, SEQ ID NOs: 92 and 82, SEQ ID NOs: 81 and 93, SEQ ID NOs: 81 and 94, SEQ ID NOs: 95 and 82, or SEQ ID NOs: 96 and 82). In addition, the resulting fusion protein can be a bivalent protein directed against CD137 (e.g. Figure 1 A-1D), or a tetravalent antibody targeting CD137 (as shown in Figure 1 E-1H) or have an even higher affinity for CD137 (as shown in Figure 1I). Additional monospecific fusion proteins (such as Figure 1 J-1K). The resulting monospecific fusion proteins are provided as SEQ ID NO:97 and SEQ ID NO:98.

[0062] Figure 2 : Shows the results of an ELISA experiment in which representative fusion proteins were assayed for binding to human GPC3 ( Figure 2 A), macaque GPC3 ( Figure 2 B) and human CD137 ( Figure 2 C). GPC3 or C-terminal His-tagged CD137 were coated on microtiter plates and the tested reagents were titrated starting with a maximum concentration of 100 nM. The bound reagents in the study were detected by anti-human IgG Fc-horseradish peroxidase (HRP) or anti-NGAL-HRP, respectively. The data were fitted using a 1:1 binding model, where EC 50 The value and the maximum signal are taken as free parameters, and the slope is fixed to 1. The obtained EC 50 The values ​​are provided in Table 4.

[0063] Figure 3 : shows the results of an exemplary ELISA experiment in which the ability of representative fusion proteins to simultaneously bind to two targets, GPC3 and CD137, was determined as described in Example 4. Recombinant huGPC3 ( Figure 3 A) or huCD137-His( Figure 3 B) coated on a microtiter plate, followed by titration of the fusion protein. Subsequently, constant concentrations of biotinylated huCD137-His ( Figure 3 A) or biotinylated huGPC3 ( Figure 3 B), which was detected by ExtrAvidin-Peroxidase. The data were fitted using a 1:1 binding model, where EC 50 The value and the maximum signal are taken as free parameters, and the slope is fixed to unity. The obtained EC 50 The values ​​are provided in Table 3.

[0064] Figure 4 : shows the results of the evaluation of target binding of the fusion protein, which was performed using CHO cells expressing human CD137 as described in Example 6 ( Figure 4 A) and SK-Hep1 cells expressing human GPC3 ( Figure 4B) By flow cytometry. No binding was observed when mock-transfected cells were used. The geometric mean of the fluorescence intensity was used to calculate the EC 50 value, the EC 50 The values ​​are provided in Table 6.

[0065] Figure 5 : shows the binding of fusion proteins to GPC3-positive tumor cells assessed by flow cytometry. As described in Example 7, different fusion proteins or controls were incubated with cells having different GPC3 expression levels (high to moderate expression: HepG2 ( Figure 5 A)>Hep3B( Figure 5 B)>MKN-45( Figure 5 C)) tumor cell line and GPC3-negative cell line NCI-N87 ( Figure 5 D), and the corresponding binding affinity (EC 50 ) are summarized in Table 7.

[0066] Figure 6 : The potential of representative fusion proteins to costimulate T cell activation in a GPC3 target-dependent manner evaluated using a CD137 bioassay is shown. NFκB-luc2 / CD137 Jurkat cells were incubated with GPC3-expressing tumor cell lines (highly to moderately expressing: HepG2>Hep3B>MKN-45, GPC3 negative: NCI-N87, corresponding to Figure 6 A, 6B, 6C and 6D) were co-cultured. After 4 hours, luciferase assay reagent was added and the luminescence signal was measured. A four-parameter logistic curve analysis was performed to calculate the EC 50 Value (see Table 7). The fusion protein is sufficient in GPC3 ( Figure 6 A and 6B), but not in the absence or deficiency of GPC3 ( Figure 6 C and 6D). In contrast, in the absence of GPC3-expressing cells and in the absence of any tumor cells, the reference anti-CD137 mAbs (SEQ ID NOs: 26 and 27) displayed similar activation regardless of GPC3 expression levels.

[0067] Figure 7: Demonstrated the ability of representative fusion proteins to costimulate T cell activation in the presence of GPC3. GPC3 antibodies (SEQ ID NOs: 81 and 82), GPC3-specific lipocalin mutants (Fc fusion) (SEQ ID NO: 97), previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83), and isotype controls (SEQ ID NOs: 24 and 25) were tested in parallel. Figure 7 A) or mock transfection (human GPC3 negative, Figure 7 B) SK-Hep1 cells or cells expressing GPC3 ( Figure 7 C) Hep-G2 tumor cell line was seeded into anti-human anti-CD3 coated plates. Pan T cells and different concentrations of test molecules were added and incubated for 3 days. The level of secreted IL-2 in the supernatant was determined by electrochemiluminescence-based assay as described in Example 9. Fusion proteins SEQ ID NOs: 87 and 82 induced a strong dose-dependent increase in IL-2 secretion only in the presence of GPC3, which was stronger than the previously known CD137 / GPC3 bispecific fusion protein SEQ ID NO: 83.

[0068] Figure 8 : The ability of representative fusion proteins to costimulate T cell activation in a GPC3 target-dependent manner is shown. Figure 8 A, 8B and 8C; GPC3 negative: NCI-N87, Figure 8 D) different tumor cell lines were inoculated into plates coated with anti-human CD3. Pan T cells and different concentrations of fusion proteins and single building blocks were added and incubated for 3 days. As described in Example 10, the level of secreted IL-2 was determined by electrochemiluminescence-based assays. The fusion protein can increase IL-2 secretion in a GPC3-dependent manner. In contrast, the IL-2 secretion induced by the reference CD137 antibody (SEQ ID NOs: 26 and 27) tested in parallel was not GPC3-dependent.

[0069] Fig. 9:The ability of representative fusion proteins demonstrated in impedance-based T cell killing assays to activate the CD137 co-stimulatory signaling pathway and induce T cell-mediated cytolysis of GPC3-expressing tumor cells is shown. HepG2 cells expressing GPC3 or GPC3-negative NCI-N87 cells were seeded and adhered to electronic microtiter plates, and then anti-CD3 antibodies, test molecules, and non-adherent CD8+ T cells were added. As described in Example 11, impedance was measured every 15 minutes for 3 days as a unitless "cell index (CI)" parameter. Compared to the previously known CD137 / GPC3 bispecific fusion protein SEQ ID NO: 83, fusion proteins SEQ ID NOs: 87 and 82 were able to activate the CD137 pathway at a higher level in a GPC3-dependent manner and induce dose-dependent T cell-mediated lysis of GPC3-expressing tumor cells.

[0070] Fig.10 : Shows tumor volume over time after treatment with 0.5, 5 and 20 mg / kg of an exemplary fusion protein, equimolar concentrations (0.39 and 3.9 mg / kg) of a GPC3 antibody contained in the fusion protein, equimolar concentrations (3.9 mg / kg) of a reference CD137 antibody or a vehicle control (PBS) in a humanized mouse tumor model, as described in Example 12. NOG mice were implanted subcutaneously with HepG2 tumors and allowed to grow to 80-100 mm 3 The mice were then randomly assigned to treatment (control) groups and received 5×10 6 Fresh human PBMCs were injected intraperitoneally with the test molecules at the indicated doses on days 1, 8 and 15. Tumor growth was recorded every 3 to 4 days. Data are shown as medians with interquartile error bars. The dotted lines indicate the number of animals reduced in each group compared to the previous days. At higher dose levels (5 and 20 mg / kg), the fusion protein completely inhibited tumor growth throughout the study, while at lower concentrations (0.5 or 0.39 mg / kg, respectively), it was dose-dependent but had less effect on tumor growth.

[0071] Fig.11: Provided are the results of pharmacokinetic analysis of exemplary fusion proteins and GPC3 antibodies contained in the fusion proteins in mice compared to two previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83 and SEQ ID NO: 84), as described in Example 13. Male CD-1 mice (2 mice per time point) were intravenously injected with fusion proteins at a dose of 2 mg / kg or 10 mg / kg. Drug levels were detected using a sandwich ELISA that detects intact molecules by targeting GPC3 and anti-human Fc or anti-NGAL. Compared to previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83 and SEQ ID NO: 84), the fusion proteins of the present invention (SEQ ID NOs: 87 and 82) showed strongly enhanced pharmacokinetic behavior and extended half-life.

[0072] IV. Detailed Description of the Invention

[0073] As described herein, the present invention encompasses such recognition that bivalent CD137 binders (such as antibodies) themselves may not be sufficient to aggregate CD137 on T cells or NK cells and lead to effective activation. In addition, the latest work around TNFR family members has shown the mechanism of anti-TNFR antibodies, by which the antibodies interact with Fc-γ receptors through their Fc regions (thereby engaging activated immune cells expressing Fc-γ receptors) and promote subsequent anti-tumor activity (Bulliard et al., Immunol Cell Biol, 2014; Bulliard et al., J Exp Med, 2013). Therefore, this suggests that anti-CD137 antibodies may trigger CD137 aggregation, depending on the abundance of Fc-γ receptor-positive cells that are not selectively localized to tumors but distributed throughout the body. Therefore, the efficacy and target specificity of anti-CD137 monotherapy may be worrying. In fact, some anti-CD137 therapies in clinical studies (such as urelumab and utomilumab) show disappointing efficacy results at low doses and / or show toxicity at high or effective doses (Bulliard et al., Immunol Cell Biol, 2014; Bulliard et al., J Exp Med, 2013). Therefore, the demand for CD137 targeted therapies that are both effective and safe has not been met. The ideal CD137 targeting agent should cause CD137 aggregation and achieve CD137 aggregation on tumor-infiltrating lymphocytes in a tumor-localized manner. As described herein, in order to obtain such a CD137 targeting agent, a bispecific agent can be designed to target CD137 at one end and differentially expressed tumor targets at the other end.

[0074] The present invention encompasses the recognition that monospecific GPC3-targeting agents may not be sufficiently effective to cure tumors such as HCC, and that the anti-tumor activity of anti-GPC3 agents that are able to recruit immune cells to the tumor site and / or directly inhibit cancer cell proliferation and / or survival may be relevant. Thus, there is an unmet need for GPC3-targeted therapies that have additive anti-tumor effects and the ability to provide stronger anti-tumor activity than monospecific GPC3-targeting agents alone.

[0075] Among other things, the present invention provides a novel method for simultaneously engaging CD137 and GPC3 through a fusion protein with CD137 binding specificity and GPC3 binding specificity. In order to engage CD137 and GPC3, a fusion protein of an anti-GPC3 antibody-CD137 specific lipocalin mutant protein is described in, for example, WO2016 / 184882. However, there is still a need for such a fusion protein that induces GPC3-dependent T cell killing / immune response to inhibit the growth of tumors expressing GPC3 with high efficiency and selectivity. Different aspects of the present invention provide fusion proteins that promote CD137 aggregation by bridging CD137-positive T cells with GPC3 expressed in the tumor microenvironment, thereby combining CD137-induced T cell activation and expansion with anti-GPC3-mediated cytotoxic killing of tumor cells. The present invention also provides such a fusion protein that provides the potential for combination therapy in one molecule and simultaneously allows for the local induction of antigen-specific T cells in the tumor microenvironment, potentially reducing peripheral toxicity. In these aspects, the fusion proteins of the invention can provide enhanced IL-2 production, immune response, T cell-mediated cytolysis, and / or anti-tumor effects compared to monomeric CD137 or GPC3 therapy.

[0076] Additionally, since cynomolgus monkeys have been extensively used in pharmacokinetic or drug safety studies for the development of new therapeutics, including new biologics, and such studies may further be an essential prerequisite for regulatory approval, it is advantageous to have a fusion protein containing a GPC3 binding portion that cross-reacts with both human and cynomolgus GPC3. Such fusion proteins containing a GPC3 binding portion with such cross-reactivity characteristics have not been previously described.

[0077] In addition, as provided herein, since bispecific or multispecific molecules may differ from their building blocks in some properties (including targeting affinity and specificity, stability, pharmacokinetics in blood, and mechanism of action) (Spiess et al., Mol Immunol, 2015; Sedykh et al., Drug Des Devel Ther, 2018), careful selection of building blocks and optimization of manufacturing and other practices are provided in some embodiments to produce high-quality clinical candidates. For example, bispecific or multispecific molecules may partially or completely lose target binding affinity and specificity of building blocks, resulting in undesirable off-target binding. Off-target binding can further affect the pharmacokinetics, tissue distribution, efficacy and toxicity of such bispecific or multispecific molecules for therapeutic use. In particular, poor pharmacokinetic profiles can lead to difficulty in achieving the dose distribution required for therapeutic efficacy and patient noncompliance (Alavijeh and Palmer, IDrugs, 2004), and good pharmacokinetics allow for reduced doses, development of subcutaneous formulations and reduced COG, and extended dosing intervals to, for example, once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks (Ryman and Meibohm, CPT Pharmacometrics Syst Pharmacol, 2017). Pharmacokinetic properties are also affected by isoelectric point (pI). An increase in pI can lead to increased blood clearance and increased tissue retention, a shorter half-life, while a lower pI can reduce tissue uptake and lead to a longer half-life. Although observations on the correlation between protein clearance and pI may be contradictory, therapeutic antibodies generally have pI values ​​slightly above physiological pH 7.4 because most cell surfaces are negatively charged. In this regard, various aspects of the present invention provide bispecific fusion proteins for CD137 and GPC3 with high affinity and specificity, enhanced stability, suitable pI, and good pharmacokinetic properties.

[0078] Such fusion proteins with these features relevant to the uses provided by the present invention have not been described previously. In contrast to the fusion proteins provided herein, previously known fusion proteins targeting CD137 and GPC3 suffer from one or more of the following: poor PK, unacceptable levels of off-target binding, unacceptable levels of nonspecific (e.g., GPC3-independent) activation of the immune system, and / or reduced or degraded ability to mediate T cell activation, proliferation and / or infiltration.

[0079] A. Exemplary fusion proteins of the invention specific for CD137 and GPC3.

[0080] In some embodiments, a fusion protein is provided containing at least two subunits in any order: (1) a first subunit comprising a full-length immunoglobulin or an antigen binding domain thereof specific for GPC3, and (2) a second subunit comprising a lipocalin mutein specific for CD137.

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

[0082] In some embodiments, a fusion protein is provided that comprises at least two subunits, wherein a first subunit comprises a full-length immunoglobulin or an antigen binding domain thereof and is specific for GPC3, and wherein a second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is C-terminally linked to the N-terminus of each HC of the first subunit, optionally via a linker.

[0083] In some embodiments, a fusion protein is provided that comprises at least two subunits, wherein a first subunit comprises a full-length immunoglobulin, or an antigen binding domain thereof, and is specific for GPC3, and wherein a second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is N-terminally linked to the C-terminus of each light chain (LC) of the first subunit, optionally via a linker.

[0084] In some embodiments, a fusion protein is provided comprising at least two subunits, wherein a first subunit comprises a full-length immunoglobulin or an antigen binding domain thereof and is specific for GPC3, and wherein a second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is C-terminally linked to the N-terminus of each LC of the first subunit, optionally via a linker.

[0085] In some embodiments, a fusion protein is provided that comprises at least two subunits, wherein a first subunit comprises a full-length immunoglobulin or an antigen binding domain thereof and is specific for GPC3, and wherein a second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is N-terminally linked to the C-terminus of each heavy chain constant region (CH) of the first subunit, optionally via a linker.

[0086] In some embodiments, a fusion protein is provided that comprises at least two subunits, wherein a first subunit comprises a full-length immunoglobulin, or an antigen binding domain thereof, and is specific for GPC3, and wherein a second subunit comprises a lipocalin mutein and is specific for CD137, wherein the second subunit is N-terminally linked to the C-terminus of each light chain constant region (CL) of the first subunit, optionally via a linker.

[0087] In some embodiments, the fusion protein provided further comprises at least one additional subunit, e.g., a third subunit. For example, the fusion protein may contain a third subunit specific for CD137. In some embodiments, the third subunit may be or comprise a lipocalin mutant protein specific for CD137. For example, two lipocalin mutant proteins may be fused to a first immunoglobulin subunit, one at the C-terminus of the immunoglobulin and one at the N-terminus of the immunoglobulin. In some embodiments, the lipocalin mutant protein may be fused to a heavy chain or light chain of an immunoglobulin.

[0088] In some embodiments, provided fusion proteins may include one or more additional subunits (eg, a fourth, fifth, or sixth subunit).

[0089] In some embodiments, at least one subunit may be fused to another subunit at its N-terminus and / or its C-terminus.

[0090] In some embodiments, at least one subunit can be connected to another subunit via a linker. In some other embodiments, the linker is a peptide linker, for example, an unstructured glycine-serine (GS) linker, a glycosylated GS linker, or a proline-alanine-serine polymer (PAS) linker. In some embodiments, the GS linker is as shown in SEQ ID NO: 13 (Gly 4 Ser) 3 Linker ((G 4 S) 3 ). Other exemplary linkers are shown in SEQ ID NOs: 14-23. In some embodiments, the peptide linker may have 1 to 50 amino acids, such as 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 amino acids. For example, when the first subunit comprises a full-length immunoglobulin, the second subunit may be connected via a peptide linker between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of the immunoglobulin. In some other embodiments, the third subunit may be connected via a peptide linker between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the immunoglobulin.

[0091] In some embodiments, the Figure 1 One subunit is connected to another subunit as shown. In general, one subunit can be fused to another subunit at its N-terminus and / or its C-terminus. For example, in some embodiments, the lipocalin mutant protein subunit can be fused to an immunoglobulin subunit at its N-terminus and / or its C-terminus, preferably via a peptide bond. By way of further example, the lipocalin mutant protein subunit can be connected at its N-terminus to the C-terminus of the heavy chain domain (HC) of the immunoglobulin subunit ( Figure 1 A), the lipocalin mutein subunit can be linked at its C-terminus to the N-terminus of the HC of the immunoglobulin subunit ( Figure 1 C), the lipocalin mutein subunit can be linked at its N-terminus to the C-terminus of the light chain (LC) of the immunoglobulin subunit ( Figure 1 B), and / or the lipocalin mutein subunit may be linked at its C-terminus to the N-terminus of the LC of the immunoglobulin subunit ( Figure 1 D).

[0092] In some embodiments, the lipocalin mutein subunit may be fused to an immunoglobulin fragment at its N-terminus and / or its C-terminus. For example, in some embodiments, the lipocalin mutein subunit may be linked to the C-terminus of the heavy chain constant region (CH) of the immunoglobulin subunit, preferably via a peptide linker, at its N-terminus, or the lipocalin mutein subunit may be linked to the C-terminus of the light chain constant region (CL) of the immunoglobulin subunit, preferably via a peptide linker, at its N-terminus.

[0093] In some embodiments, when one subunit comprises a full-length immunoglobulin, a second subunit may be linked between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of the immunoglobulin.

[0094] In some embodiments, the third subunit can be linked between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the immunoglobulin.

[0095] In some embodiments, for the fusion protein of the present invention, at least one subunit may be or include a full-length immunoglobulin. When the fusion protein simultaneously binds to CD137 and GPC3, the Fc function of the full-length immunoglobulin Fc region on Fc receptor-positive cells can be retained.

[0096] In some embodiments, at least one subunit of the fusion protein provided may be or include a full-length immunoglobulin, and the Fc function of the full-length immunoglobulin Fc region to Fc receptor-positive cells can be reduced or completely inhibited by protein engineering, while the fusion protein simultaneously engages CD137 and GPC3. In some embodiments, the above situation can be achieved by, for example, converting the IgG1 skeleton to IgG4, because it is known that IgG4 shows reduced Fc-γ receptor interactions compared to IgG1. In some embodiments, in order to further reduce the residual binding to the Fc-γ receptor, mutations such as F234A and L235A can be introduced into the IgG4 skeleton. In some embodiments, the S228P mutation can also be introduced into the IgG4 skeleton to minimize the exchange of IgG4 half antibodies (Silva et al., J Biol Chem, 2015). In some embodiments, F234A and L235A mutations may be introduced to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010) and / or M428L and N434S mutations or M252Y, S254T and T256E mutations may be introduced to extend serum half-life (Dall'Acqua et al., J Biol Chem, 2006; Zalevsky et al., Nat Biotechnol, 2010). In some embodiments, an additional N297A mutation may be present in the immunoglobulin heavy chain of the fusion protein in order to remove the native glycosylation motif.

[0097] In some embodiments, the Fc portion of an immunoglobulin included in the fusion protein of the present invention can help maintain the serum level of the fusion protein. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the fusion protein can become internalized and circulate back into the bloodstream, thereby increasing its half-life in vivo.

[0098] In one aspect, the fusion protein of the present invention binds to CD137 with high affinity. In another aspect, the provided fusion protein binds to GPC3 with high affinity. In some preferred embodiments, the provided fusion protein simultaneously binds to CD137 and GPC3. In some embodiments, simultaneous binding to CD137 and GPC3 allows the provided fusion protein to exhibit a durable anti-tumor or anti-infective response.

[0099] In some embodiments, the fusion protein of the invention may have an isoelectric point (pI) of about 6.5 or higher, such as about 6.8 or higher, about 7.1 or higher, about 7.4 or higher, about 7.5 or higher, about 7.7 or higher, about 8.0 or higher, about 8.5 or higher, or about 9.0 or even higher. In some embodiments, the fusion protein of the invention has a higher pI than a previously known fusion protein that binds CD137 and GPC3 comprising the amino acid sequence of SEQ ID NO: 83.

[0100] In some embodiments, the fusion proteins of the invention may be capable of activating the K of at most about 2 nM or even lower, such as about 1.5 nM or lower, about 1 nM or lower, about 0.8 nM or lower, or about 0.7 nM or lower. D In some embodiments, the fusion proteins of the invention may be able to bind to GPC3 at a K value of 1:1. D K with equivalent or lower value D The K of a provided fusion protein can be measured, for example, in a surface plasmon resonance (SPR) assay, such as an SPR assay essentially as described in Example 2. D value.

[0101] In some embodiments, the fusion proteins of the invention may be capable of activating an EC of at most about 1 nM or even lower, such as about 0.5 nM or lower, about 0.3 nM or lower, about 0.2 nM or lower, about 0.15 nM or lower, or about 0.1 nM or lower. 50 In some embodiments, the fusion proteins of the invention may be capable of binding to GPC3 in an EC sequence with an immunoglobulin specific for GPC3 (such as an antibody having heavy and light chains provided by SEQ ID NOs: 81 and 82) contained in the particular fusion protein. 50 Equivalent or lower EC 50 The EC values ​​of the provided fusion proteins can be measured, for example, in an enzyme-linked immunosorbent assay (ELISA) assay, such as an ELISA assay essentially as described in Example 3. 50 value.

[0102] In some embodiments, the fusion proteins of the invention may be capable of activating an EC of at most about 5 nM or even lower, such as about 3 nM or lower, about 2 nM or lower, about 1 nM or lower, about 0.5 nM or lower, about 0.2 nM or lower, or about 0.1 nM or lower. 50The EC values ​​of the provided fusion proteins can be measured, for example, in an ELISA assay, such as an ELISA assay essentially as described in Example 3. 50 value.

[0103] In some embodiments, the fusion proteins of the invention may cross-react with macaque GPC3. In some embodiments, the fusion proteins provided may be capable of cross-reacting with macaque GPC3 at a K of up to about 2 nM or even lower, such as about 1.5 nM or lower, about 1 nM or lower, about 0.8 nM or lower, or about 0.7 nM or lower. D In some embodiments, the fusion proteins of the invention may be able to bind to GPC3 at a Kp of an immunoglobulin specific for GPC3 (such as an antibody having a heavy chain and a light chain provided by SEQ ID NOs: 81 and 82) contained in such fusion proteins. D K with equivalent or lower value D The K values ​​of the provided fusion proteins can be measured, for example, in an SPR assay, such as an SPR assay essentially as described in Example 2. D In some embodiments, the fusion protein provided may be capable of being expressed at an EC of at most about 1 nM or even lower, such as about 0.5 nM or lower, such as about 0.2 nM or lower, about 0.1 nM or lower. 50 The EC values ​​for the provided fusion proteins can be measured, for example, in an ELISA assay, such as an ELISA assay essentially as described in Example 3. 50 value.

[0104] In some embodiments, the fusion proteins of the invention may be capable of binding to CD137 and GPC3 simultaneously. In some embodiments, the fusion proteins provided may be capable of binding to CD137 and GPC3 at an EC of at most about 10 nM or even lower, such as 5 nM or lower, 3 nM or lower, 2 nM or lower, 1 nM or lower, or 0.5 nM or lower. 50 In some other embodiments, the fusion protein provided may be capable of binding to CD137 and GPC3 at an EC of at most about 10 nM or even lower, such as 8 nM or lower, 5 nM or lower, 3 nM or lower, or 2 nM or lower. 50 The simultaneous binding may be determined, for example, in an ELISA assay, such as an ELISA assay essentially as described in Example 4.

[0105] In some embodiments, the fusion proteins of the invention may be capable of activating an EC of at most about 50 nM or even lower, such as about 30 nM or even lower, about 25 nM or even lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, or about 1 nM or even lower. 50 The EC of the provided fusion protein can be measured, for example, in a flow cytometric analysis essentially as described in Example 6. 50 The cells expressing CD137 can be, for example, CHO cells transfected with human CD137.

[0106] In some embodiments, the fusion proteins of the invention may be capable of activating an EC of at most about 50 nM or even lower, such as about 30 nM or lower, about 25 nM or even lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, or about 1 nM or even lower. 50 The EC of the provided fusion proteins can be measured, for example, in a flow cytometric assay essentially as described in Example 6. 50 The cells expressing GPC3 may be, for example, SK-Hep1 cells transfected with human GPC3.

[0107] In some embodiments, the fusion proteins of the invention may be capable of binding to GPC3 expressed on tumor cells. In some embodiments, provided fusion proteins may be capable of binding to GPC3 expressed on tumor cells at an EC of at most about 50 nM or even lower, such as about 30 nM or lower, about 25 nM or even lower, about 20 nM or lower, about 15 nM or lower, about 10 nM or lower, about 5 nM or lower, about 3 nM or lower, or about 1 nM or even lower. 50 The EC value for binding to GPC3 expressed on tumor cells can be measured in a flow cytometric analysis, for example, essentially as described in Example 7. 50 Tumor cells expressing GPC3 may be, for example, HepG2, Hep3B and MKN-45 cells.

[0108] In some embodiments, the fusion proteins of the invention do not substantially bind to targets other than CD137 or GPC3. In some embodiments, the fusion proteins provided have reduced off-target binding to targets other than CD137 or GPC3 compared to previously known fusion proteins that bind to CD137 and GPC3 comprising the amino acid sequence of SEQ ID NO: 83. The assessment of such target binding can be performed by an ELISA assay as described in Example 5.

[0109] In some embodiments, the fusion proteins of the present invention may be capable of costimulating T cell responses. In some embodiments, the fusion proteins provided result in comparable or stronger T cell activation than the GPC3 antibodies (having heavy chains and light chains provided by SEQ ID NOs: 81 and 82) contained in the fusion protein, the reference CD137 antibodies (having heavy chains and light chains provided by SEQ ID NOs: 26 and 27), or the previously known fusion proteins that bind CD137 and GPC3 containing the amino acid sequence of SEQ ID NO: 83. In some embodiments, the fusion proteins provided result in T cell activation with comparable or better efficacy than the GPC3 antibodies (having heavy chains and light chains provided by SEQ ID NOs: 81 and 82) contained in the fusion protein, the reference CD137 antibodies (having heavy chains and light chains provided by SEQ ID NOs: 26 and 27), or the previously known fusion proteins that bind CD137 and GPC3 containing the amino acid sequence of SEQ ID NO: 83. Stimulated T cell responses or T cell activation can be measured, for example, in a CD137 bioassay essentially as described in Example 8, or in a functional T cell activation assay essentially as described in Example 9, Example 10 and Example 11.

[0110] In some embodiments, the fusion proteins of the present invention may be able to induce increased IL-2 secretion. In some preferred embodiments, the fusion proteins provided may be able to induce concentration-dependent IL-2 secretion, and / or show a trend of inducing enhanced IL-2 secretion at higher concentrations. In some embodiments, the fusion proteins provided may have comparable or better efficacy than the GPC3 antibodies (having heavy chains and light chains provided by SEQ ID NOs: 81 and 82) contained in the fusion protein or the previously known fusion proteins that bind CD137 and GPC3 containing the amino acid sequence of SEQ ID NO: 83, resulting in increased IL-2 secretion. IL-2 secretion can be measured, for example, in a functional T cell activation assay as described in Example 9 and Example 10.

[0111] In some embodiments, the fusion protein of the present invention may be able to induce T cell-mediated cytotoxicity. In some embodiments, the fusion protein provided may lead to dose-dependent T cell-mediated cell lysis. In some embodiments, compared with the GPC3 antibody (having the heavy chain and light chain provided by SEQ ID NOs: 81 and 82) contained in the fusion protein provided, the fusion protein provided can induce cytotoxic T cell activation with comparable or better efficacy. In some embodiments, compared with the CD137 antibody with heavy chain and light chain provided by SEQ ID NOs: 26 and 27, the reference CD137 antibody with heavy chain and light chain provided by SEQ ID NOs: 26 and 27, or the previously known fusion protein of binding CD137 and GPC3 comprising the amino acid sequence of SEQ ID NO: 83, the fusion protein provided can induce cytotoxic T cell activation with comparable or better efficacy. T cell-mediated cytotoxicity can be measured in, for example, an impedance-based T cell killing assay as described in Example 11.

[0112] In some embodiments, the fusion proteins of the present invention may be able to costimulate T cell responses in a GPC3-dependent manner. In some embodiments, the fusion proteins provided may result in local induction of IL-2 production in T cells near GPC3-positive cells (such as GPC3-transfected cells or tumor cells expressing GPC3). When used herein, "near GPC3-positive cells" refers to T cells and GPC3-positive cells being brought close to each other via the provided fusion proteins that simultaneously bind CD137 and GPC3. In some embodiments, the fusion proteins provided may result in local T cell-mediated killing of tumor cells expressing GPC3. GPC3-dependent T cell activation of the fusion proteins provided can be determined, for example, in a CD137 bioassay substantially as described in Example 8, or in a functional T cell activation assay substantially as described in Example 9, Example 10, and Example 11.

[0113] In some embodiments, the fusion proteins provided are unable to costimulate T cell responses in the absence of GPC3. In some embodiments, the fusion proteins provided are unable to costimulate T cell responses in the absence of cells expressing GPC3. In some embodiments, the fusion proteins provided may be able to recognize the presence of GPC3 and result in better corresponding T cell activation than CD137 antibodies having heavy and light chains provided by SEQ ID NOs: 26 and 27. The GPC3-dependent effects of the fusion proteins may be determined, for example, in a CD137 bioassay substantially as described in Example 8, or in a functional T cell activation assay substantially as described in Example 9, Example 10, and Example 11.

[0114] In some embodiments, the fusion proteins provided can result in anti-tumor activity. In some embodiments, the fusion proteins provided can inhibit tumor growth of hepatocellular carcinoma HepG2 cells. In some embodiments, the fusion proteins provided can inhibit tumor growth of hepatocellular carcinoma HepG2 cells with comparable or better efficacy than the GPC3 antibody (having a heavy chain and a light chain provided by SEQ ID NOs: 81 and 82) contained in the fusion protein. Such anti-tumor activity can be determined, for example, using a HepG2 xenograft model substantially as described in Example 12.

[0115] In some embodiments, provided fusion proteins can increase the level of tumor infiltrating lymphocytes. In some embodiments, provided fusion proteins can result in increased intratumoral filtration of CD3, CD4 or CD8 T cells compared to a GPC3 antibody (having a heavy chain and a light chain provided by SEQ ID NOs: 81 and 82) contained in the fusion protein. Tumor infiltrating lymphocytes can be analyzed, for example, as described in Example 13.

[0116] In some embodiments, the fusion proteins provided have good stability and pharmacokinetic characteristics. In some embodiments, the fusion proteins provided have pharmacokinetic characteristics comparable to the GPC3 antibodies (having heavy chains and light chains provided by SEQ ID NOs: 81 and 82) contained in the fusion proteins. In some embodiments, the fusion proteins provided have antibody-like pharmacokinetics. In some embodiments, the fusion proteins provided have a terminal half-life of about 50 hours or longer, 75 hours or longer, 100 hours or longer, 125 hours or longer, about 150 hours or longer, about 175 hours or longer, about 200 hours or longer, about 250 hours or longer, or even longer. In some embodiments, the fusion proteins provided have a terminal half-life of about 50 hours or longer, 75 hours or longer, 100 hours or longer, 125 hours or longer, about 150 hours or longer, about 175 hours or longer, about 200 hours or longer, about 250 hours or longer, or even longer in mice. The pharmacokinetic characteristics of the provided fusion proteins can be analyzed as described in Example 13.

[0117] In some embodiments, the fusion protein provided comprises the amino acid sequence set forth in any one of SEQ ID NOs: 87-96.

[0118] In some embodiments, the fusion protein provided comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to the amino acid sequence shown in any one of SEQ ID NOs:87-96.

[0119] In some embodiments, the fusion protein provided comprises the amino acid sequence shown in SEQ ID NOs:87 and 82, SEQ ID NOs:88 and 82, SEQ ID NOs:81 and 89, SEQ ID NOs:81 and 90, SEQ ID NOs:91 and 82, SEQ ID NOs:92 and 82, SEQ ID NOs:81 and 93, SEQ ID NOs:81 and 94, SEQ ID NOs:95 and 82, or SEQ ID NOs:96 and 82.

[0120] In some embodiments, the fusion protein provided comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to the amino acid sequence shown in SEQ ID NOs:87 and 82, SEQ ID NOs:88 and 82, SEQ ID NOs:81 and 89, SEQ ID NOs:81 and 90, SEQ ID NOs:91 and 82, SEQ ID NOs:92 and 82, SEQ ID NOs:81 and 93, SEQ ID NOs:81 and 94, SEQ ID NOs:95 and 82, or SEQ ID NOs:96 and 82.

[0121] B. Exemplary Immunoglobulins Included in Fusion Proteins

[0122] In some embodiments, for the fusion proteins provided, the first subunit can be or comprise a full-length immunoglobulin or an antigen binding domain thereof specific for GPC3. In some embodiments, the immunoglobulin can be, for example, IgG1, IgG2, or IgG4. In some embodiments, the immunoglobulin is or comprises IgG4. In some embodiments, the immunoglobulin is a monoclonal antibody against GPC3.

[0123] Illustrative examples of GPC3-binding antibodies of the present invention may comprise an antigen-binding region that cross-blocks or binds to the same epitope as a GPC3-binding antibody comprising a heavy chain variable domain (V H ) and the light chain variable domain (V L), such known antibodies as codrituzumab (also known as GC33 or RO5137382), YP7 (including humanized YP7), HN3 and HS20. In some embodiments, the GPC3-binding antibody of the present invention may comprise an antigen binding region from an antibody selected from codrituzumab, YP7, HN3 and HS20, such as any one of the three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2 and HCDR3) and the three light chain CDRs (LCDR1, LCDR2 and LCDR3).

[0124] In some embodiments, the provided GPC3 antibodies or antigen binding domains thereof may have a heavy chain variable region (HCVR) selected from SEQ ID NOs: 78, 114, 119, 126 and 129 and / or a light chain variable region (LCVR) selected from SEQ ID NOs: 79, 115 and 127.

[0125] In some embodiments, the heavy and light chain pairs of the provided GPC3 antibodies or antigen binding domains thereof are or comprise the following HCVR and LCVR, respectively: SEQ ID NOs: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs: 114 and 115, or SEQ ID NOs: 126 and 127.

[0126] In some embodiments, the heavy and light chain pairs of the GPC3 antibodies or antigen-binding domains thereof provided are or comprise HCVR and LCVR, respectively, having sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to the amino acid sequences set forth in SEQ ID NOs:78 and 79, SEQ ID NOs:129 and 79, SEQ ID NOs:114 and 115, or SEQ ID NOs:126 and 127.

[0127] In some embodiments, provided GPC3 antibodies or antigen binding domains thereof may have a heavy chain that is any one of SEQ ID NOs: 80 and 81, and / or a light chain that is SEQ ID NO:82.

[0128] In some embodiments, the heavy and light chain pairs of the provided GPC3 antibodies are or comprise the amino acid sequences set forth in SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82.

[0129] In some embodiments, the heavy chain and light chain pair of the GPC3 antibody provided is or comprises a heavy chain and a light chain having a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to the amino acid sequence shown in SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82.

[0130] In some embodiments, provided GPC3 antibodies or antigen binding domains thereof may have a HCVR having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to an amino acid sequence selected from SEQ ID NOs: 78, 114, 119, 126 and 129, and / or a LCVR having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to an amino acid sequence selected from SEQ ID NOs: 79, 115 and 127. In other embodiments, the GPC3 antibodies or antigen binding domains thereof provided may have a heavy chain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to an amino acid sequence selected from SEQ ID NOs: 80 and 81, and / or a light chain having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or even higher sequence identity to an amino acid sequence of SEQ ID NO: 82.

[0131] In some embodiments, the heavy chain variable region of the provided GPC3 antibody or its antigen binding domain may have three CDRs, and the three CDRs have the following sequences: GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), TRFYSYTY (HCDR3; SEQ ID NO: 74). In some embodiments, the heavy chain variable region of the provided GPC3 antibody or its antigen binding domain may have three CDRs, and the three CDRs have the following sequences: GFTFNKNA (HCDR1, SEQ ID NO: 108), IRNKTNNYAT (HCDR2, SEQ ID NO: 109), VAGNSFAY (HCDR3; SEQ ID NO: 110). In some embodiments, the heavy chain variable region of the provided GPC3 antibody or its antigen binding domain may have three CDRs, and the three CDRs have the following sequences: YFDFDSYE (HCDR1, SEQ ID NO: 116), IYHSGST (HCDR2, SEQ ID NO: 117), ARVNMDRFDY (HCDR3; SEQ ID NO: 118). In some embodiments, the heavy chain variable region of the provided GPC3 antibody or its antigen binding domain may have three CDRs, and the three CDRs have the following sequences: GFTFSSYA (HCDR1, SEQ ID NO: 120), IQKQGLPT (HCDR2, SEQ ID NO: 121), AKNRAKFDY (HCDR3; SEQ ID NO: 122).

[0132] In some embodiments, the light chain variable region of the provided GPC3 antibody or its antigen binding domain may have three CDRs, and the three CDRs have the following sequences: QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO: 77). In some embodiments, the light chain variable region of the provided GPC3 antibody or its antigen binding domain may have three CDRs, and the three CDRs have the following sequences: QSLLYSSNQKNY (LCDR1, SEQ ID NO: 111), WAS (LCDR2), QQYYNYPLT (LCDR3; SEQ ID NO: 113). In some embodiments, the light chain variable region of the provided GPC3 antibody or its antigen binding domain may have three CDRs, and the three CDRs have the following sequences: QSISSY (LCDR1, SEQ ID NO: 123), NAS (LCDR2), QQNRGFPLT (LCDR3; SEQ ID NO: 125).

[0133] In some embodiments, a GPC3 antibody or antigen-binding domain thereof is provided that comprises a heavy chain variable region having three CDRs with the following sequences: GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), TRFYSYTY (HCDR3; SEQ ID NO: 74), and a light chain variable region having three CDRs with the following sequences: QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO: 77). In some embodiments, the provided GPC3 antibody or its antigen-binding domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has three CDRs with the following sequences: GFTFNKNA (HCDR1, SEQ ID NO: 108), IRNKTNNYAT (HCDR2, SEQ ID NO: 109), VAGNSFAY (HCDR3; SEQ ID NO: 110), and the light chain variable region has three CDRs with the following sequences: QSLLYSSNQKNY (LCDR1, SEQ ID NO: 111), WAS (LCDR2), QQYYNYPLT (LCDR3; SEQ ID NO: 113). In some embodiments, a GPC3 antibody or antigen-binding domain thereof is provided that comprises a heavy chain variable region having three CDRs with the following sequences: GFTFSSYA (HCDR1, SEQ ID NO: 120), IQKQGLPT (HCDR2, SEQ ID NO: 121), AKNRAKFDY (HCDR3; SEQ ID NO: 122), and a light chain variable region having three CDRs with the following sequences: QSISSY (LCDR1, SEQ ID NO: 123), NAS (LCDR2), QQNRGFPLT (LCDR3; SEQ ID NO: 125).

[0134] Unless otherwise indicated, all CDR sequences disclosed herein are defined according to the IMGT method described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999). CDR1 consists of positions 27 to 38, CDR2 consists of positions 56 to 65, CDR3 of germline V-genes consists of positions 105 to 116, and CDR3 of rearranged VJ-genes or VDJ-genes consists of positions 105 to 117 (positions before J-PHE or J-TRP 118), with gaps at the top of the loop for rearranged CDR3-IMGTs with less than 13 amino acids, or additional positions 112.1, 111.1, 112.2, 111.2, etc. for rearranged CDR3-IMGTs with more than 13 amino acids. The positions given in this paragraph are according to the IMGT numbering as described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).

[0135] The antibody specifically binding to GPC3 included in the fusion protein of the present invention may include an Fc portion that allows the in vivo half-life of the bispecific binding molecule of the present invention to be extended. In some embodiments, this Fc portion is preferably from a human source, more preferably a human Fc portion of an IgG1 or IgG4 antibody, and even more preferably an engineered human Fc portion of an IgG1 or IgG4 with an activated or silent effector function. In some embodiments, the silent effector function may be superior to the activated effector function. In some embodiments, this Fc portion is engineered to silence the effector function, which has mutations at positions 234 and / or 235 numbered according to the EU index of Kabat (Johnson and Wu, Nucleic Acids Res, 2000). In some embodiments, mutations may be introduced at positions F234 and L235 of the anti-GPC3 antibodies provided to silence the effector function. In other embodiments, mutations may be introduced at positions D265 and P329 of the anti-GPC3 antibodies provided to silence the effector function. Both sets of these potential mutations are numbered according to the EU index of Kabat (Shields et al., J Biol Chem, 2001).

[0136] Various techniques for producing antibodies and fragments thereof are well known in the art and are described, for example, in Altshuler et al. (2010). Thus, for example, polyclonal antibodies can be obtained from the blood of an animal following immunization with an antigen mixed with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line cultures. Examples of such techniques are described, for example, in Harlow and Lane (1999), (1988), and include those originally described by and Milstein, 1975 described hybridoma technology, trioma technology, human B cell hybridoma technology (see, e.g., Li et al., Proc Natl Acad Sci USA, 2006; Kozbor and Roder, Immunol Today, 1983) and EBV-hybridoma technology to produce human monoclonal antibodies (Cole et al., Cancer Res, 1984). In addition, recombinant antibodies can be obtained from monoclonal antibodies or can be prepared from scratch by various display methods (such as phage, ribosome, mRNA or cell display). In some embodiments, suitable systems for expressing recombinant (humanized) antibodies or fragments thereof can be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., U.S. Pat. No. 6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). In addition, the described technology for producing single-chain antibodies (see, in particular, U.S. Pat. No. 4,946,778) can be suitable for producing single-chain antibodies specific to the target of the present invention. Surface plasmon resonance employed in the BIAcore system can be used to increase the efficiency of phage antibodies.

[0137] C. Exemplary Lipocalin Muteins of the Invention

[0138] Lipocalins are proteinaceous binding molecules that have evolved naturally to bind ligands. Lipocalins are found in many organisms, including vertebrates, insects, plants, and bacteria. Members of the lipocalin family (Pervaiz and Brew, FASEB J, 1987) are usually small secreted proteins and have a single polypeptide chain. They are characterized by a range of different molecular recognition properties: they bind a variety of mainly hydrophobic small molecules (e.g., retinoids, fatty acids, cholesterol, prostaglandins, biliverdin, pheromones, sweeteners (tastants) and odorants (odorants)); and they bind to specific cell surface receptors and they form macromolecular complexes. Although they were mainly classified as transport proteins in the past, it is now clear that lipocalins perform a variety of physiological functions. These functions include roles in retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins are also involved in the regulation of immune responses and in mediating the homeostasis of the cellular intracellular environment (e.g., reviewed in Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0139] The level of global sequence conservation between lipocalins is very low, typically with less than 20% sequence identity. In sharp contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded antiparallel β-sheet that closes on itself to form a continuous hydrogen-bonded β-barrel. The β-barrel forms a central cavity. One end of the barrel is sterically closed by an N-terminal peptide segment passing through its base and three peptide loops connecting the β-strands. The other end of the β-barrel is open to the solvent and contains a target binding site formed by four flexible peptide loops (AB, CD, EF and GH). It is this diversity of the loops in the supposedly rigid lipocalin backbone that produces a variety of different binding modes, each of which can accommodate targets of different sizes, shapes and chemical characteristics (e.g., Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996).

[0140] The lipocalin mutein according to the present invention can be a mutein of any lipocalin. Examples of suitable lipocalins (sometimes also referred to as "reference lipocalin", "wild-type lipocalin", "reference protein backbone" or simply "backbone") for which the mutein can be used include, but are not limited to, tear lipocalin (lipocalin-1, Tlc or von Ebner's gland protein), retinol binding protein, neutrophil lipocalin-type prostaglandin D synthase, β-lactoglobulin, bile binding protein (BBP), apolipoprotein D (APOD), neutrophil gelatinase-associated lipocalin (NGAL), α2-microglobulin-associated protein (A2m), 24p3 / uterine calcitonin (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 a related embodiment, the lipocalin mutein is derived from the group of lipocalins consisting of human tear lipocalin (hTIc), human neutrophil gelatinase-associated lipocalin (hNGAL), human apolipoprotein D (hAPOD), and bile-binding protein of Pieris brassicae.

[0141] The amino acid sequence of a lipocalin mutein according to the invention may have a high sequence identity with the reference (or wild-type) lipocalin from which it is derived (e.g. hTlc or hNGAL) compared to the sequence identity with another lipocalin (see also above). In this general case, the amino acid sequence of a lipocalin mutein according to the invention 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) due to additions or deletions of amino acids when aligned. The corresponding sequence of a lipocalin mutein according to the invention that is substantially similar to the sequence 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%, at least 90% identity, including at least 95% identity, with the sequence of the corresponding lipocalin. In this respect, the lipocalin mutein of the invention may of course comprise substitutions as described herein which render the lipocalin mutein capable of binding CD137.

[0142] Typically, the lipocalin mutant protein contains one or more mutated amino acid residues in the four loops (see above) that contain the ligand binding pocket and define the open end of the ligand binding pocket entrance relative to the amino acid sequence of the wild-type or reference lipocalin (e.g., hTlc or hNGAL). As described above, these regions are crucial in determining the binding specificity of the lipocalin mutant protein to the desired target. In some embodiments, the lipocalin mutant protein of the present invention may also contain mutated amino acid residues in regions outside the four loops. In some embodiments, the lipocalin mutant protein of the present invention may contain one or more mutated amino acid residues in one or more of the three peptide loops (named BC, DE and FG) connecting the β-chain at the closed end of the lipocalin. In some embodiments, the mutant protein derived from tear lipocalin, NGAL lipocalin or their homologs may have 1, 2, 3, 4 or more mutated amino acid residues at any sequence position in the three peptide loops BC, DE and FG arranged at the N-terminal region and / or the end of the β-barrel structure, which is located opposite the natural lipocalin binding pocket. In some embodiments, compared to the wild-type sequence of tear lipocalin, the mutein derived from tear lipocalin, NGAL lipocalin, or homologs thereof has no mutated amino acid residues in the peptide loop DE arranged at the end of the β-barrel structure.

[0143] In some embodiments, the lipocalin mutant according to the invention may comprise one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more) mutated amino acid residues compared to the corresponding reference (wild-type) lipocalin, provided that such lipocalin mutant should be able to bind to CD 137. In some embodiments, the lipocalin mutant of the invention comprises at least two (including 2, 3, 4, 5 or even more) mutated amino acid residues, wherein the native amino acid residue of the corresponding reference (wild-type) lipocalin is replaced by an arginine residue.

[0144] Any type and number of mutations (including substitutions, deletions and insertions) are contemplated, so long as the provided lipocalin mutein retains its ability to bind CD137 and / or has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more sequence identity with the amino acid sequence of a reference (wild-type) lipocalin (e.g., mature hTlc or mature hNGAL).

[0145] In some embodiments, the substitution is a conservative substitution. In some embodiments, the substitution is a non-conservative substitution or one or more of the exemplary substitutions listed below.

[0146] In particular, in order to determine whether an amino acid residue of the amino acid sequence of a lipocalin mutant protein differs from the reference (wild-type) lipocalin protein at a position corresponding to the reference (wild-type) lipocalin amino acid sequence, the skilled person may use means and methods well known in the art, such as manually or by using a computer program such as BLAST 2.0 (which stands for Basic Local Alignment Search Tool) or ClustalW or any other suitable program for generating sequence alignments. Thus, the amino acid sequence of the reference (wild-type) lipocalin protein may serve as the "subject sequence" or "reference sequence", while the amino acid sequence of the lipocalin mutant protein serves as the "query sequence" (see also above).

[0147] Conservative substitutions are generally the following substitutions, which are listed according to the amino acid to be mutated, and each amino acid to be mutated is followed by one or more conservative substitutions: Ala→Ser, Thr or Val; Arg→Lys, Gln, Asn or His; Asn→Gln, Glu, Asp or His; Asp→Glu, Gln, Asn or His; Gln→Asn, Asp, Glu or His; Glu→Asp, Asn, Gln or His; His→Arg, Lys, Asn, Gln, Asp or Glu; Ile→Thr, Leu, Met, Phe, Val, Trp, Tyr, Ala or Pro; Leu→Thr, Ile, Val, Met, A La, Phe, Pro, Tyr or Trp; Lys → Arg, His, Gln or Asn; Met → Thr, Leu, Tyr, Ile, Phe, Val, Ala, Pro or Trp; Phe → Thr, Met, Leu, Tyr, Ile, Pro, Trp, Val or Ala; Ser → Thr, Ala or Val; Thr → Ser, Ala, Val, Ile, Met, Val, Phe, Pro or Leu; Trp → Tyr, Phe, Met, Ile or Leu; Tyr → Trp, Phe, Ile, Leu or Met; Val → Thr, Ile, Leu, Met, Phe, Ala, Ser or Pro. Other substitutions are also permissible and can be determined empirically or based on other known conservative or non-conservative substitutions. As another orientation, each of the following groups includes amino acids that can be generally used to define conservative substitutions of each other:

[0148] (a) Alanine (Ala), serine (Ser), threonine (Thr), valine (Val)

[0149] (b) Aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), asparagine (Asn), histidine (His)

[0150] (c) Arginine (Arg), Lysine (Lys), Glutamine (Gln), Asparagine (Asn), Histidine (His)

[0151] (d) Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val), Alanine (Ala), Phenylalanine (Phe), Threonine (Thr), Proline (Pro)

[0152] (e) Isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp).

[0153] If such conservative substitutions result in a change in biological activity, more substantial changes (such as those described below or as further described below with respect to amino acid classes) can be introduced and the products screened for the desired characteristics. Examples of such more substantial changes are: Ala→Leu or Phe; Arg→Glu; Asn→Ile, Val or Trp; Asp→Met; Cys→Pro; Gln→Phe; Glu→Arg; His→Gly; Ile→Lys, Glu or Gln; Leu→Lys or Ser; Lys→Tyr; Met→Glu; Phe→Glu, Gln or Asp; Trp→Cys; Tyr→Glu or Asp; Val→Lys, Arg, His.

[0154] 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, e.g., sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chain.

[0155] Based on general side chain properties, naturally occurring residues are divided into the following groups: (1) hydrophobic: methionine, alanine, valine, leucine, isoleucine; (2) neutral hydrophilic: cysteine, serine, threonine, asparagine, glutamine; (3) acidic: aspartic acid, glutamic acid; (4) basic: histidine, lysine, arginine; (5) residues affecting chain orientation: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine. In some embodiments, substitutions may require exchanging a member of one of these classes for another.

[0156] Any cysteine ​​residues not involved in maintaining the correct conformation of the respective lipocalin may also be replaced with serine, generally to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bonds may be added to lipocalins to increase their stability.

[0157] D. Exemplary CD137-specific lipocalin muteins of the invention

[0158] As described above, lipocalin is a polypeptide defined by its supersecondary structure, i.e., a cylindrical β-pleated sheet supersecondary structure region comprising eight β-strands connected in pairs by four loops at one end to thereby define a binding pocket. The present invention is not limited to the lipocalin mutants specifically disclosed herein. In this aspect, the present invention relates to lipocalin mutants having a cylindrical β-pleated sheet supersecondary structure region comprising eight β-strands connected in pairs by four loops at one end to thereby define a binding pocket, wherein at least one amino acid in each of at least three of the four loops has been mutated, and wherein the lipocalin effectively binds to CD137 with a detectable affinity.

[0159] In some embodiments, the lipocalin mutants disclosed herein may be or include mutants of mature human tear lipocalin (hTlc). The mutants of mature hTlc are designated herein as "hTlc mutants". In some other embodiments, the lipocalin mutants disclosed herein are mutants of mature human neutrophil gelatinase-associated lipocalin (hNGAL). The mutants of mature hNGAL are designated herein as "hNGAL mutants".

[0160] In one aspect, the present invention includes any number of lipocalin mutants derived from a reference (wild-type) lipocalin (preferably derived from mature hTlc or mature hNGAL) that bind to CD137 with detectable affinity. In a related aspect, the present invention includes various lipocalin mutants that can activate CD137 downstream signaling pathways by binding to CD137. In this sense, CD137 can be regarded as a non-natural target of a reference (wild-type) lipocalin (preferably hTlc or hNGAL), wherein "non-natural target" refers to a substance that does not bind to a reference (wild-type) lipocalin under physiological conditions. By engineering a reference (wild-type) lipocalin with one or more mutations at certain sequence positions, the inventors of the present invention have demonstrated that high affinity and high specificity for a non-natural target, i.e., CD137, is possible. In some embodiments, with the goal of generating lipocalin mutants capable of binding to CD137, random mutagenesis can be performed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more nucleotide triplets at certain sequence positions on the encoding wild-type lipocalin by replacing these positions with a subset of nucleotide triplets.

[0161] In some embodiments, the lipocalin mutant protein of the present invention may have a mutated (including substitution, deletion or insertion) amino acid residue at one or more sequence positions corresponding to the linear polypeptide sequence of the reference lipocalin (preferably hTlc or hNGAL). In some embodiments, the number of mutated amino acid residues of the lipocalin mutant protein of the present invention 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) compared to the amino acid sequence of the reference lipocalin (preferably hTlc or hNGAL), wherein preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11, more preferably 9, 10 or 11. However, preferably, the lipocalin mutant protein of the present invention is still able to bind CD137.

[0162] In some embodiments, the lipocalin muteins of the invention may lack 1, 2, 3, 4 or more amino acids at their N-terminus and / or lack 1, 2 or more amino acids at their C-terminus compared to the corresponding reference (wild-type) lipocalin; e.g., SEQ ID NOs: 32-38. In some embodiments, the invention encompasses hTlc muteins as defined above, wherein the first 4, 1, 2 or 3 N-terminal amino acid residues (His-His-Leu-Leu; positions 1-4) of mature hTlc and / or the last 1 or 2 C-terminal amino acid residues (Ser-Asp; positions 157-158) of the linear polypeptide sequence of mature hTlc are deleted (e.g., SEQ ID NOs: 32-38). In some embodiments, the invention encompasses hNGAL muteins as defined above, wherein the amino acid residues (Lys-Asp-Pro; positions 46-48) of the linear polypeptide sequence of mature hNGAL are deleted (SEQ ID NO: 43). Furthermore, the lipocalin mutein of the invention may comprise the wild-type (native) amino acid sequence of a reference (wild-type) lipocalin (preferably hTlc or hNGAL) except for the mutated amino acid sequence position.

[0163] In some embodiments, the introduction of one or more mutated amino acid residues into the lipocalin mutant protein of the present invention does not substantially impede or interfere with the binding activity and folding of the mutant protein to the specified target. Such mutations (including substitutions, deletions and insertions) can be accomplished at the DNA level using established standard methods (Sambrook and Russell, 2001, Molecular cloning: a laboratory manual). In some embodiments, mutated amino acid residues are introduced at one or more sequence positions corresponding to the linear polypeptide sequence of a reference (wild-type) lipocalin (preferably hTlc or hNGAL) by random mutagenesis of replacing one or more nucleotide triplets encoding the corresponding sequence position of the reference lipocalin with a subset of nucleotide triplets.

[0164] In some embodiments, the provided lipocalin mutants that bind to CD137 with detectable affinity may comprise at least one amino acid substitution that replaces a native cysteine ​​residue with another amino acid (e.g., a serine residue). In some embodiments, the lipocalin mutants that bind to CD137 with detectable affinity may comprise one or more non-natural cysteine ​​residues that replace one or more amino acids of a reference (wild-type) lipocalin (preferably hTlc or hNGAL). In some embodiments, the lipocalin mutants according to the present invention comprise at least two amino acid substitutions that replace natural amino acids with cysteine ​​residues, thereby forming one or more cysteine ​​bridges. In some embodiments, the cysteine ​​bridges may connect at least two loop regions. According to (Biochim Biophys Acta, 2000), Flower (1996) and Breustedt et al., (2005) the definitions of these regions are used herein.

[0165] Generally speaking, the lipocalin muteins of the invention may have at least about 70%, including at least about 80%, such as at least about 85% amino acid sequence identity to the amino acid sequence of mature hTlc (SEQ ID NO: 1) or mature hNGAL (SEQ ID NO: 2).

[0166] In some aspects, the invention provides hTlc mutant proteins that bind CD137. In this aspect, the invention provides one or more hTlc mutant proteins that can bind CD137 with a K of about 300 nM, 200 nM, 150 nM, 100 nM or less. D In some embodiments, the hTlc mutant proteins provided can bind to CD137 with an EC of about 250 nM, 150 nM, 100 nM, 50 nM, 20 nM or even lower. 50 In some other embodiments, the hTlc mutant protein that binds CD137 can cross-react with macaque CD137 (cyCD137).

[0167] In some embodiments, the hTlc mutant protein of the present invention can interfere with the binding of CD137L to CD137.

[0168] In some embodiments, the hTlc mutant proteins provided may comprise a mutated amino acid residue at one or more of positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0169] In some embodiments, provided hTlc muteins may comprise mutated amino acid residues at one or more of positions 26-34, 55-58, 60-61, 65, 104-106, and 108 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0170] In some embodiments, the provided hTlc mutant proteins may further comprise a mutated amino acid residue at one or more of positions 101, 111, 114, and 153 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0171] In some embodiments, the hTlc mutant proteins provided may comprise mutated amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or even more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In some preferred embodiments, the hTlc mutant proteins provided are capable of binding to CD137, particularly human CD137.

[0172] In some embodiments, the hTlc mutant proteins provided may comprise mutated amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even more positions corresponding to positions 26-34, 55-58, 60-61, 65, 104-106 and 108 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In some preferred embodiments, the hTlc mutant proteins provided are capable of binding to CD137, particularly human CD137.

[0173] In some embodiments, the lipocalin mutein according to the invention may comprise at least one amino acid substitution of a native cysteine ​​residue with, for example, a serine residue. In some embodiments, the hTlc mutein according to the invention comprises an amino acid substitution of a native cysteine ​​residue with another amino acid, such as a serine residue, at a position corresponding to position 61 and / or 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1). In this context, it should be noted that it has been found that (in the corresponding native Removal of the structural disulfide bond of wild-type hTlc formed by cysteine ​​residues 61 and 153 (see Breustedt et al., J Biol Chem, 2005) can provide hTlc mutant proteins that are not only stably folded, but also can bind to a given non-natural target with high affinity. In some embodiments, the elimination of structural disulfide bonds can provide further advantages, namely allowing non-natural disulfide bonds to be generated or intentionally introduced into the mutant proteins of the present invention, thereby increasing the stability of the mutant proteins. However, hTlc mutant proteins that bind to CD137 and have a disulfide bridge formed between Cys 61 and Cys 153 are also part of the present invention.

[0174] In some specific embodiments, the hTlc mutant protein of the present invention may comprise one or more of the following amino acid substitutions at positions 61 and / or 153 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): Cys61→Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp and / or Cys153→Ser or Ala.

[0175] In some embodiments, two or all three cysteine ​​codons at positions 61, 101 and 153 corresponding to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1) are replaced by codons for another amino acid. In addition, in some embodiments, the hTlc mutant protein according to the present invention comprises an amino acid substitution in which the native cysteine ​​residue is replaced by a serine residue or a histidine residue at the position corresponding to position 101 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1).

[0176] In some embodiments, the mutant protein according to the present invention comprises an amino acid replacement in which the natural amino acid at the position corresponding to position 28 or 105 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1) is replaced by a cysteine ​​residue. In addition, in some embodiments, the mutant protein according to the present invention comprises an amino acid replacement in which the natural arginine residue at the position corresponding to position 111 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1) is replaced by a proline residue. In addition, in some embodiments, the mutant protein according to the present invention comprises an amino acid replacement in which the natural lysine residue at the position corresponding to position 114 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1) is replaced by a tryptophan residue or glutamic acid.

[0177] In some embodiments, the hTlc mutant proteins that bind to CD137 provided may comprise one or more of the following mutated amino acid residues at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): Ala 5→Val or Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Gly 46→Asp; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys65→Arg or Asn; Thr 71→Ala; Val 85→Asp; Lys 94→Arg or Glu; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile; and Cys 153→Ser. In some embodiments, the hTlc mutant proteins of the present invention comprise two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more or even all mutated amino acid residues at these sequence positions of mature hTlc (SEQ ID NO: 1).

[0178] In some embodiments, compared to the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), provided CD137-binding hTlc mutant proteins may comprise one of the following groups of mutated amino acid residues:

[0179] (a) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser;

[0180] (b) Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Arg; Val 85→Asp; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys 153→Ser;

[0181] (c) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Asn; Lys 94→Arg; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys153→Ser;

[0182] (d) Ala 5→Val; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Arg; Lys 94→Glu; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys 153→Ser;

[0183] (e) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Leu56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys 153→Ser;

[0184] (f) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile; and Cys 153→Ser; and

[0185] (g)Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Gly 46→Asp; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys153→Ser.

[0186] In some embodiments, the remaining region of the hTlc mutant protein of the present invention, i.e., the region that is different from the positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1), may comprise the wild-type (native) amino acid sequence of the linear polypeptide sequence of mature hTlc outside the mutated amino acid sequence position.

[0187] In some embodiments, the hTlc mutant protein of the present invention has at least 70% sequence identity or at least 70% sequence homology with the sequence of mature hTlc (SEQ ID NO: 1). As an illustrative example, the mutant protein of SEQ ID NO: 32 has nearly 84% amino acid sequence identity or sequence homology with the amino acid sequence of mature hTlc.

[0188] In some embodiments, the hTlc mutant protein of the present invention comprises an amino acid sequence as shown in any one of SEQ ID NOs: 32-38 or a fragment or variant thereof.

[0189] In some embodiments, the hTlc mutant protein of the present invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or higher sequence identity with an amino acid sequence selected from SEQ ID NOs: 32-38.

[0190] The present invention also includes structural homologs of hTlc mutant proteins having an amino acid sequence selected from SEQ ID NOs: 32-38, wherein the structural homologs have an amino acid sequence homology or sequence identity with the hTlc mutant proteins that is greater than about 60%, preferably greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92% and most preferably greater than 95%.

[0191] In some aspects, the invention provides hNGAL mutant proteins that bind CD137. In this aspect, the invention provides one or more hNGAL mutant proteins that can bind CD137 with a K of about 800nM, 700nM, 200nM, 140nM, 100nM or lower, preferably about 70nM, 50nM, 30nM, 10nM, 5nM, 2nM or even lower. D In some embodiments, the hNGAL mutant proteins provided can bind to CD137 with an EC of about 1000 nM, 500 nM, 100 nM, 80 nM, 50 nM, 25 nM, 18 nM, 15 nM, 10 nM, 5 nM or less. 50 Values ​​for binding to CD137.

[0192] In some embodiments, the hNGAL mutant proteins provided that bind CD137 can cross-react with macaque CD137. In some embodiments, the hNGAL mutant proteins provided can cross-react with macaque CD137 by about 50nM, 20nM, 10nM, 5nM, 2nM or even lower K D In some embodiments, the hNGAL mutant proteins provided can bind to macaque CD137 with an EC of about 100 nM, 80 nM, 50 nM, 30 nM or even lower. 50 Values ​​for binding to macaque CD137.

[0193] In some embodiments, the hNGAL mutant proteins of the present invention may interfere with or compete for the binding of CD137L to CD137. In some other embodiments, the hNGAL mutant proteins of the present invention may be able to bind to CD137 and / or be able to bind to the CD137 / CD137L complex in the presence of CD137L.

[0194] In some embodiments, provided hNGAL mutant proteins can comprise a mutated amino acid residue at one or more of positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0195] In some embodiments, the hNGAL mutant proteins provided may comprise mutated amino acid residues at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or even more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the hNGAL mutant proteins provided are capable of binding to CD137, particularly human CD137.

[0196] In some embodiments, the hNGAL mutant proteins provided may comprise a mutated amino acid residue at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 87, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In some preferred embodiments, the hNGAL mutant proteins provided are capable of binding to CD137, particularly human CD137.

[0197] In some embodiments, provided hNGAL mutant proteins can comprise a mutated amino acid residue at one or more of positions 36, 87, and 96 corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2) and at one or more of positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 94, 100, 103, 106, 125, 127, 132, and 134 corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0198] In some other embodiments, the hNGAL mutant proteins provided may comprise a mutated amino acid residue at one or more of positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2).

[0199] In other embodiments, the hNGAL mutant proteins provided can comprise a mutated amino acid residue at one or more of positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO:2) and at one or more of positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101, and 122 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO:2).

[0200] In some embodiments, the lipocalin muteins according to the invention may comprise at least one amino acid substitution of a native cysteine ​​residue with, for example, a serine residue. In some embodiments, the hNGAL muteins according to the invention may comprise an amino acid substitution of a native cysteine ​​residue with another amino acid, such as a serine residue, at a position corresponding to position 76 and / or 175 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2). In this context, it should be noted that it has been found that (in the corresponding native Removal of the structural disulfide bond of wild-type hNGAL formed by cysteine ​​residues 76 and 175 (see Breustedt et al., J Biol Chem, 2005) can provide hNGAL mutant proteins that are not only stably folded, but also bind to a given non-native target with high affinity. In some embodiments, the elimination of structural disulfide bonds can provide further advantages, namely allowing non-native disulfide bonds to be generated or intentionally introduced into the mutant proteins of the present invention, thereby increasing the stability of the mutant proteins. However, hNGAL mutant proteins that bind to CD137 and have a disulfide bridge formed between Cys 76 and Cys 175 are also part of the present invention.

[0201] In some embodiments, provided CD137-binding hNGAL mutant proteins may comprise one or more of the following mutated amino acid residues at one or more of positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg or Lys; Gln 49→Val, Ile, His, Ser, or Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Met, Ala, or Gly; Leu 70→Ala, Lys, Ser or Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Met, Arg, Thr or Asn; Trp 79→Ala or Asp; Arg 81→Met, Trp or Ser; Phe 83→Leu; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu and Lys 134→Tyr. In some embodiments, the hNGAL mutant proteins of the present invention comprise two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or even more, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or all mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2).

[0202] In some embodiments, provided CD137-binding hNGAL mutant proteins may comprise one or more of the following mutated amino acid residues at one or more of positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence corresponding to mature hNGAL (SEQ ID NO: 2): Gln 20→Arg; Asn 25→Tyr or Asp; Gln 28→His; Val 33→Ile; Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Val or Asp; Gln 49→His; Tyr52→Ser or Gly; Lys 59→Asn; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys73→Asp; Asp 77→Gln or His; Tyr 78→His; Trp 79→Ile; Ile 80→Asn; Arg 81→Trp or Gln; Thr 82→Pro; Cys 87→Ser; Phe 92→Leu or Ser; Asn 96→Phe; Lys 98→Arg; Tyr 100→Asp; Pro 101→Leu; Leu 103→His or Pro; Phe 122→Tyr; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly. In some embodiments, the hNGAL mutant proteins of the invention comprise two or more, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 mutated amino acid residues at these sequence positions of mature hNGAL (SEQ ID NO: 2).

[0203] In some embodiments, the hNGAL mutant proteins that bind to CD137 provided may comprise one or more of the following mutated amino acid residues at positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, corresponding to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Ser or Gly; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln or His; Trp 79→Ile; Arg 81→Trp or Gln; Asn 96→Phe; Tyr 100→Asp; Leu 103→His or Pro; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly. In some embodiments, the hNGAL mutant proteins that bind to CD137 provided may further comprise one or more of the following mutated amino acid residues at one or more of positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101, and 122, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, of the linear polypeptide sequence corresponding to mature hNGAL (SEQ ID NO: 2): Gln 20→Arg; Asn 25→Tyr or Asp; Val 33→Ile; Glu 44→Val or Asp; Lys 59→Asn; Phe 71→Leu; Tyr 78→His; Ile 80→Asn; Thr 82→Pro; Phe 92→Leu or Ser; Lys 98→Arg; Pro 101→Leu; and Phe 122→Tyr.

[0204] In some embodiments, provided CD137-binding hNGAL muteins may comprise one of the following groups of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

[0205] (a)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Asn;Tyr 52→Met;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0206] (b)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→Ile;Tyr 52→Met;Asn 65→Asp;Ser 68→Met;Leu 70→Lys;Arg 72→Asp;Lys 73→Asp;Asp 77→Met;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0207] (c)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0208] (d)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0209] (e)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Ser;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Ser;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Met;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0210] (f)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Val;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Arg;Trp 79→Asp;Arg 81→Ser;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0211] (g)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→His;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0212] (h)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Phe 83→Leu;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;or

[0213] (i) Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ser; Tyr 52→Met; Asn 65→Asp; Ser 68→Ala; Leu 70→Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Asn; Trp 79→Ala; Arg 81→Ser; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser127→Phe; Tyr 132→Glu; and Lys 134→Tyr.

[0214] In some other embodiments, in the remaining regions, i.e., the regions that differ from positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), the hNGAL mutant proteins of the present invention may comprise the wild-type (native) amino acid sequence of mature hNGAL except for the mutated amino acid sequence positions.

[0215] In some other embodiments, the provided CD137-binding hNGAL mutant proteins may comprise one of the following groups of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

[0216] (a) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly;

[0217] (b) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Trp 79→Ile; Arg 81→Trp; Phe 92→Leu; Asn 96→Phe; Lys 98→Arg; Tyr 100→Asp; Pro 101→Leu; Leu 103→His; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly;

[0218] (c) Asn 25 → Tyr; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Gln; Phe 92 → Ser; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly;

[0219] (d) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Tyr 78 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly;

[0220] (e) Asn 25 → Asp; Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Gln 49 → His; Tyr 52 → Gly; Ser 68 → Asp; Leu 70 → Met; Arg 72 → Leu; Lys 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Lys 125 → Ser; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly;

[0221] (f)Val 33→Ile;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0222] (g)Gln 20→Arg;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Glu 44→Val;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Phe 122→Tyr;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0223] (h)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Ser;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Ile 80→Asn;Arg 81→Trp;Thr 82→Pro;Asn 96→Phe;Tyr 100→Asp;Pro 101→Leu;Leu 103→Pro;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0224] (i) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Gln 49→His; Tyr 52→Gly; Lys 59→Asn; Ser 68→Asp; Leu 70→Met; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln; Trp 79→Ile; Arg 81→Trp; Phe 92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly; and

[0225] (j) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Asp; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys 73→Asp; Asp 77→His; Trp 79→Ile; Arg 81→Trp; Phe 92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly.

[0226] In some embodiments, the hNGAL mutant proteins that bind to CD137 provided may comprise the following groups of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ile; Tyr 52→Met; Asn 65→Asp; Ser 68→Met; Leu 70→Lys; Arg 72→Asp; Lys 73→Asp; Asp 77→Met; Trp 79→Asp; Arg 81→Trp; Cys87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser127→Phe; Tyr 132→Glu; and Lys 134→Tyr and / or the provided mutant protein may have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity with the amino acid sequence of SEQ ID NO:40.

[0227] In some embodiments, the remaining regions of the hNGAL mutant proteins of the invention, i.e., the regions that differ from positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2), may comprise the wild-type (native) amino acid sequence of mature hNGAL outside of the mutated amino acid sequence positions.

[0228] In some embodiments, the hNGAL mutant protein of the present invention has at least 70% sequence identity or at least 70% sequence homology with the sequence of mature hNGAL (SEQ ID NO: 2). As an illustrative example, the mutant protein of SEQ ID NO: 40 has nearly 87% amino acid sequence identity or sequence homology with the amino acid sequence of mature hNGAL.

[0229] In some embodiments, the hNGAL mutant protein of the present invention comprises an amino acid sequence as shown in any one of SEQ ID NOs: 39-57 or a fragment or variant thereof.

[0230] In some embodiments, the hNGAL mutant protein of the invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity to an amino acid sequence selected from SEQ ID NOs: 39-57.

[0231] The present invention also includes a structural homolog of a hNGAL mutant protein having an amino acid sequence selected from SEQ ID NOs: 39-57, wherein the structural homolog has an amino acid sequence homology or sequence identity of greater than about 60%, preferably greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 92% and most preferably greater than 95% to the hNGAL mutant protein.

[0232] In some embodiments, the invention provides a method for preparing a novel molecule having a K of about 5 nM or less. D A lipocalin mutein that binds to CD137 with a measured affinity, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0233] In some embodiments, the invention provides a method for preparing a novel molecule having a K of about 5 nM or less. DA lipocalin mutein that binds to CD137 with measured affinity, wherein the lipocalin mutein has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 49.

[0234] In some embodiments, the lipocalin mutant protein of the present invention may contain a heterologous amino acid sequence at its N- or C-terminus, preferably at the C-terminus, such as a Strep II tag (SEQ ID NO: 12) or a cleavage site sequence for certain restriction enzymes, without affecting the biological activity of the lipocalin mutant protein (binding to its target, such as CD137).

[0235] In some embodiments, other modifications of the lipocalin mutant protein can be introduced to modulate certain characteristics of the mutant protein or introduce new characteristics into the mutant protein, such as improving folding stability, serum stability, protein resistance or water solubility, or reducing aggregation tendency. In some embodiments, the modification can result in two or more (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10) characteristics of the provided mutant protein being modulated.

[0236] For example, one or more amino acid sequence positions of the lipocalin mutein can also be mutated to introduce new reactive groups, for example, for conjugation with other compounds such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides or proteins, or for forming non-naturally occurring disulfide linkages. In some cases, the conjugated compounds (e.g., PEG and HES) can increase the serum half-life of the corresponding lipocalin mutein.

[0237] In some embodiments, the reactive groups of the lipocalin mutant protein may be naturally present in its amino acid sequence, such as cysteine ​​residues naturally present in the amino acid sequence. In some other embodiments, such reactive groups may be introduced by mutagenesis. In the case of introducing such reactive groups by mutagenesis, one possibility is that the amino acid at the appropriate position is mutated to a cysteine ​​residue. Exemplary possibilities of such mutations of introducing cysteine ​​residues into the amino acid sequence of the hTlc mutant protein include substitutions Thr 40→Cys, Glu 73→Cys, Arg 90→Cys, Asp 95→Cys, and Glu 131→Cys of the wild-type sequence of hTlc (SEQ ID NO: 1). Exemplary possibilities of such mutations of introducing cysteine ​​residues into the amino acid sequence of the hNGAL mutant protein include introducing cysteine ​​residues at one or more sequence positions corresponding to sequence positions 14, 21, 60, 84, 88, 116, 141, 145, 143, 146, or 158 of the wild-type sequence of hNGAL (SEQ ID NO: 2). The resulting thiol moiety can be used to PEGylate or HESylate the mutein, for example, in order to increase the serum half-life of the corresponding lipocalin mutein.

[0238] In some embodiments, in order to provide suitable amino acid side chains as new reactive groups for conjugating one of the above compounds to the lipocalin mutant protein, artificial amino acids can be introduced into the amino acid sequence of the lipocalin mutant protein. Generally speaking, these artificial amino acids are designed to be more reactive, thereby facilitating conjugation with the desired compound. Such an artificial amino acid that can be introduced by mutagenesis (e.g., using artificial tRNA) is p-acetyl-phenylalanine.

[0239] In some embodiments, the lipocalin mutein of the present invention is fused to a protein, protein domain or peptide, such as an antibody, a signal sequence and / or an affinity tag, at its N-terminus or its C-terminus. In some other embodiments, the lipocalin mutein of the present invention is conjugated to a partner, which is a protein, protein domain or peptide, such as an antibody, a signal sequence and / or an affinity tag, at its N-terminus or its C-terminus.

[0240] Examples of suitable fusion partners are affinity tags such as Strep-tag or Strep-tag II (Schmidt et al., J Mol Biol, 1996), c-myc-tag, FLAG-tag, His-tag or HA-tag or proteins such as glutathione-S-transferase, which allow easy detection and / or purification of recombinant proteins. Proteins with chromogenic or fluorescent properties, such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), are also suitable fusion partners for the lipocalin muteins of the invention. In general, the lipocalin muteins of the invention can be labeled with any suitable chemical substance or enzyme that directly or indirectly produces a detectable compound or signal in a chemical, physical, optical or enzymatic reaction. For example, fluorescent or radioactive labels can be conjugated to the lipocalin muteins to produce fluorescence or X-rays as a detectable signal. Alkaline phosphatase, horseradish peroxidase and β-galactosidase are examples of enzyme labels (which are also optical labels) that catalyze the formation of chromogenic reaction products. In general, all labels commonly used for antibodies (except those used exclusively with carbohydrate moieties in the Fc portion of immunoglobulins) can also be used for conjugation to the lipocalin muteins of the invention.

[0241] In some embodiments, the lipocalin mutant protein of the present invention can be fused or conjugated with a moiety that extends the serum half-life of the mutant protein (see also International Patent Publication No. WO 2006 / 056464 in this regard, which describes such a fusion or conjugation strategy with reference to a mutant protein of human neutrophil gelatinase-associated lipocalin (hNGAL) with CTLA-4 binding affinity). The moiety that extends the serum half-life can be a PEG molecule, a HES molecule, a fatty acid molecule such as palmitic acid (Vajo and Duckworth, Pharmacol Rev, 2000), an Fc portion of an immunoglobulin, a C portion of an immunoglobulin, or a C portion of an immunoglobulin. H 3 domains, immunoglobulin C H 4 domains, albumin binding peptides, albumin binding proteins or transferrin, to name a few.

[0242] In some embodiments, 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, U.S. Patent No. 6,177,074 or U.S. Patent No. 6,403,564 for interferon, or as described for other proteins such as PEG-modified asparaginase, PEG-adenosine deaminase (PEG-ADA) or PEG-superoxide dismutase (Fuertges and Abuchowski, Journal of Controlled Release, 1990). The molecular weight of such polymers (such as polyethylene glycol) can be from about 300 to about 70,000 daltons, including, for example, polyethylene glycol with a molecular weight of about 10,000, about 20,000, about 30,000 or about 40,000 daltons. In addition, carbohydrate oligomers and polymers such as HES may be conjugated to the muteins of the present invention for the purpose of increasing serum half-life as described, for example, in US Pat. Nos. 6,500,930 or 6,620,413.

[0243] In some embodiments, if the Fc portion of an immunoglobulin is used for the purpose of extending the serum half-life of the lipocalin mutein of the present invention, SynFusion TM The application of this Fc-fusion technology allows the production of longer-acting biopharmaceuticals and can, for example, consist of two copies of the mutant protein linked to the Fc region of an antibody to improve pharmacokinetics, solubility and production efficiency.

[0244] Examples of albumin binding peptides that can be used to extend the serum half-life of lipocalin muteins are, for example, peptides having a Cys-Xaa residue as described in U.S. Patent Publication No. 20030069395 or Dennis et al. (2002). 1 -Xaa 2 -Xaa 3 -Xaa 4 -Cys consensus sequences, where Xaa 1 Asp, Asn, Ser, Thr or Trp; Xaa 2 Asn, Gln, His, Ile, Leu or Lys; Xaa 3 is Ala, Asp, Phe, Trp or Tyr; and Xaa 4is Asp, Gly, Leu, Phe, Ser or Thr. The albumin binding peptide fused or conjugated to the lipocalin mutein to extend the serum half-life can be a bacterial albumin binding protein, an antibody, an antibody fragment including a domain antibody (see, e.g., U.S. Pat. No. 6,696,245), or a lipocalin mutein with albumin binding activity. Examples of bacterial albumin binding proteins include Streptococcal protein G (Konig and Skerra, J Immunol Methods, 1998).

[0245] In some embodiments, if the albumin binding protein is an antibody fragment, it may be a domain antibody. Domain antibodies (dAbs) are engineered to allow precise control of biophysical properties and in vivo half-life, thereby producing an optimal safety and efficacy product profile. Domain antibodies are commercially available, for example, from Domantis Ltd. (Cambridge, UK and MA, USA).

[0246] In some embodiments, albumin itself (Osborn et al., J Pharmacol Exp Ther, 2002) or a biologically active fragment of albumin can be used as a partner of the lipocalin mutein of the present invention to 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 its fragments can be recombinantly produced as described in U.S. Patent No. 5,728,553 or European Patent Publication Nos. EP0330451 and EP0361991. Accordingly, recombinant human albumin (e.g., from Novozymes Delta Ltd., Nottingham, UK) can be used. ) conjugated or fused to the lipocalin mutein of the invention.

[0247] In some embodiments, if transferrin is used as a partner to extend the serum half-life of the lipocalin mutein of the present invention, the mutein 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 the transferrin fusion protein will similarly have an extended half-life. The transferrin carrier also provides high bioavailability, biodistribution, and circulatory 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 TM) is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

[0248] Another option for extending the half-life of the lipocalin muteins of the invention is to fuse long, non-structural, flexible glycine-rich sequences (e.g., polyglycine having about 20 to 80 consecutive glycine residues) to the N- or C-terminus of the mutein. This approach is disclosed in International Patent Publication No. WO2007 / 038619, also known as "rPEG" (recombinant PEG).

[0249] E. Exemplary Uses and Applications of Fusion Proteins Specific for CD137 and GPC3

[0250] In some embodiments, the fusion protein of the present invention can produce a synergistic effect by dual-targeting CD137 and GPC3. In some embodiments, the fusion protein of the present invention can produce a local anti-tumor effect by dual-targeting CD137 and GPC3. Therefore, the fusion protein of the present invention has many possible applications in medicine.

[0251] In some embodiments, the present invention encompasses the use of one or more fusion proteins disclosed herein, or one or more compositions comprising such fusion proteins, for simultaneously binding CD137 and GPC3.

[0252] The present invention also relates to the use of one or more of the fusion proteins for forming a complex with CD137 and / or GPC3.

[0253] Therefore, in one aspect of the present invention, the fusion proteins provided can be used to detect CD137 and GPC3. Such uses may include the following steps: contacting one or more of the fusion proteins with a sample suspected of containing CD137 and / or GPC3 under suitable conditions, thereby allowing a complex to form between the fusion protein and CD137 and / or GPC3, and detecting the complex by a suitable signal. The detectable signal may be caused by a label as described above, or by a change in a physical property caused by the binding (i.e., complex formation) itself. An example is surface plasmon resonance, the value of which changes during the binding of a binding partner, one of which is fixed on a surface, such as gold foil.

[0254] The fusion protein of the present invention can also be used to separate CD137 and / or GPC3. Such use may include the following steps: contacting one or more of the fusion proteins with a sample suspected of containing CD137 and / or GPC3 under suitable conditions, thereby allowing a complex to form between the fusion protein and CD137 and / or GPC3, and separating the complex from the sample.

[0255] In some embodiments, the present invention provides diagnostic and / or analytical kits comprising one or more fusion proteins according to the present invention.

[0256] In addition to its use in diagnostics, in yet another aspect, the present invention contemplates a pharmaceutical composition comprising one or more fusion proteins of the present invention and a pharmaceutically acceptable excipient.

[0257] In addition, in some embodiments, the present invention provides fusion proteins that simultaneously bind CD137 and / or GPC3, used as anti-tumor agents and / or anti-infective agents and immunomodulators. In some embodiments, it is envisioned that the fusion proteins of the present invention are used in methods for preventing, improving or treating human diseases such as various cancers (including GPC3-positive cancers, such as hepatocellular carcinoma (HCC), melanoma, Merkel cell tumor, Wilm's tumor and hepatoblastoma). Therefore, a method for preventing, improving or treating human diseases such as various cancers (including GPC3-positive cancers, such as hepatocellular carcinoma (HCC), melanoma, Merkel cell tumor) in a subject in need is also provided, the method comprising administering to the subject a therapeutically effective amount of one or more fusion proteins of the present invention.

[0258] Other examples of cancers that can be treated using the fusion proteins of the invention include: liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, skin or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer (carcinoma), endometrial cancer (carcinoma), cervical cancer (carcinoma), vaginal cancer (carcinoma), vulvar cancer (carcinoma), non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer (carcinoma) , central nervous system (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal cord axis tumors, brain stem gliomas, pituitary tumors, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, environmentally induced cancers including those induced by asbestos, hematological malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides fungoides), anaplastic large cell lymphoma, T-cell lymphoma, precursor T-lymphoblastic lymphoma, and any combination of the foregoing cancers. In some embodiments, the present invention is also used to treat metastatic cancers.

[0259] In some embodiments, the fusion proteins of the present invention can simultaneously target tumor cells (such as HCC, melanoma, Merkel cell tumor, Wilm's tumor, and hepatoblastoma) in which GPC3 is expressed, and activate lymphocytes of the host immune system near such tumor cells. In some embodiments, the fusion proteins of the present invention can increase the activity of targeted anti-tumor T cells, enhance anti-tumor immunity, induce T cell-mediated cell lysis, and / or have a direct inhibitory effect on tumor growth, thereby producing anti-tumor results. In some embodiments, the fusion proteins of the present invention can activate immune responses in the tumor microenvironment. In some embodiments, the fusion proteins of the present invention can reduce the side effects of effector lymphocytes on healthy cells, i.e., off-target toxicity, for example, by locally inhibiting oncogene activity and inducing cell-mediated cytotoxicity through NK cells and / or T cells.

[0260] In some embodiments, the present invention encompasses the use of fusion proteins of the present invention or compositions comprising provided fusion proteins for inducing local lymphocyte responses near GPC3-positive tumor cells (such as HCC, melanoma, Merkel cell tumor, Wilm's tumor, and hepatoblastoma). Thus, in some embodiments, the present invention provides methods for inducing local lymphocyte responses near GPC3-positive tumor cells (such as HCC, melanoma, Merkel cell tumor, Wilm's tumor, and hepatoblastoma), the methods comprising applying one or more fusion proteins of the present invention or one or more compositions comprising such fusion proteins. "Local" means that after simultaneously binding to T cells and engaging GPC3-positive tumor cells via CD137, T cells produce cytokines, particularly IL-2 and / or IFNγ, near GPC3-positive cells. Such cytokines reflect the activation of T cells, which are then able to kill GPC3-positive cells directly or indirectly by activating other killer cells (such as T cells or NK cells).

[0261] In some embodiments, the present invention encompasses the use of fusion proteins of the present invention or compositions comprising such fusion proteins for costimulating T cells and / or activating CD137 downstream signaling pathways. Preferably, when tumor cells expressing GPC3 are engaged therein, the fusion proteins provided costimulate T cells and / or activate CD137 downstream signaling pathways. Therefore, the present invention provides, preferably when tumor cells expressing GPC3 therein (such as HCC, melanoma, Merkel cell tumor, Wilm's tumor and hepatoblastoma), a method of inducing T lymphocyte proliferation and / or activating CD137 downstream signaling pathways, the method comprising applying one or more fusion proteins of the present invention and / or one or more compositions comprising such fusion proteins.

[0262] In some embodiments, the present invention encompasses the use of fusion proteins of the present invention or compositions comprising such fusion proteins for inducing CD137 aggregation and activation on T cells and directing such T cells to tumor cells in which GPC3 is expressed (such as HCC, melanoma, Merkel cell tumor, Wilm's tumor, and hepatoblastoma).

[0263] For those skilled in the art, additional objects, advantages and features of the present invention will become apparent upon review of the following examples and the accompanying drawings (which are not intended to be limiting). Therefore, it should be understood that although the present invention is specifically disclosed by exemplary embodiments and optional features, those skilled in the art may adopt modifications and variations of the disclosures disclosed herein, and such modifications and variations are considered to be within the scope of the present invention.

[0264] F. Generation of Exemplary Fusion Proteins Specific for CD137 and GPC3

[0265] In some embodiments, the invention provides nucleic acid molecules (DNA and RNA) comprising the nucleotide sequence of the fusion protein provided by coding. In some embodiments, the present invention encompasses host cells containing the nucleic acid molecules provided. Because the degeneracy of genetic code allows some codons to be replaced by other codons of specified identical amino acids, therefore the present invention is not limited to the specific nucleic acid molecules of encoding fusion protein as described herein, but encompasses all nucleic acid molecules of the nucleotide sequence comprising the encoding function fusion protein. In this respect, the invention further relates to the nucleotide sequence of the fusion protein provided by coding.

[0266] A nucleic acid molecule, such as DNA, is said to be "capable of expressing a nucleic acid molecule" or "capable of allowing expression of a nucleotide sequence" if it contains sequence elements containing information about transcriptional and / or translational regulation, and such sequences are "operably linked" to the nucleotide sequence encoding the protein. An operable link is a link in which the regulatory sequence elements and the sequence to be expressed are linked in a manner that enables gene expression. The exact nature of the regulatory regions necessary for gene expression may vary from species to species, but generally these regions include a promoter, which contains the promoter itself (i.e., a DNA element that directs transcriptional initiation) and a DNA element that will send a translation initiation signal when transcribed into RNA in prokaryotes. Such a promoter region generally includes 5' non-coding sequences involved in transcription and translation initiation, such as -35 / -10 boxes and Shine-Dalgarno elements in prokaryotes or TATA boxes, CAAT sequences and 5'-capping elements in eukaryotes. These regions may also include enhancer or repressor elements and translation signals and leader sequences for targeting native proteins to specific compartments of the host cell.

[0267] Furthermore, the 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences are not satisfactorily functional in a particular host cell, they may be replaced by signals that are functional in that cell.

[0268] Therefore, nucleic acid molecules of the present invention can be "operably linked" to one or more regulatory sequences (such as promoter sequences) to allow the expression of the nucleic acid molecules. In some embodiments, nucleic acid molecules of the present invention include promoter sequences and transcription termination sequences. Suitable prokaryotic promoters are, for example, tet promoters, lacUV5 promoters or T7 promoters. Examples of promoters that can be used for expression in eukaryotic cells are SV40 promoters or CMV promoters.

[0269] In some embodiments, a nucleic acid molecule encoding a lipocalin mutein disclosed herein may be "operably linked" to another nucleic acid molecule encoding an immunoglobulin of the invention to allow for expression of a fusion protein disclosed herein.

[0270] In some embodiments, the provided methods can include subjecting at least one nucleic acid molecule encoding mature hTlc to mutagenesis at a nucleotide triplet encoding one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of hTlc (SEQ ID NO: 1) to obtain a lipocalin mutant protein contained in a provided fusion protein. In some embodiments, the provided methods can include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at a nucleotide triplet encoding one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 2) to obtain a lipocalin mutein contained in a provided fusion protein. In some embodiments, the provided methods can include subjecting at least one nucleic acid molecule encoding mature hNGAL to mutagenesis at a nucleotide triplet encoding one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of hNGAL (SEQ ID NO: 2) to obtain a lipocalin mutein contained in a provided fusion protein.

[0271] In addition, for the hTlc mutant protein or hNGAL mutant protein of the present invention contained in the fusion protein, in some embodiments, the naturally occurring disulfide bond between Cys 61 and Cys 153 or Cys 76 and Cys 175 can be removed, respectively. Therefore, such mutant proteins can be produced in a cell compartment with a reduced redox environment (redox milieu), such as in the cytoplasm of Gram-negative bacteria.

[0272] Further, for the hTlc mutant protein or hNGAL mutant protein provided by the present invention contained in the fusion protein, the present invention also includes nucleic acid molecules encoding such mutant proteins, which in some embodiments may contain one or more additional mutations outside the specified sequence position of the experimental mutagenesis. Such mutations are usually tolerable or even proven to be advantageous, for example, if they help to improve the folding efficiency, serum stability, thermal stability or ligand binding affinity of the lipocalin mutant protein and / or fusion protein.

[0273] In some embodiments, the provided nucleic acid molecules may also be part of a vector or any other kind of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or artificial chromosome.

[0274] In some embodiments, the provided nucleic acid molecules may be contained in a phagemid. As used in this context, a phagemid vector refers to a vector encoding an intergenic region of a temperate phage (such as M13 or f1), or a functional portion thereof fused to a target cDNA. For example, in some embodiments, after superinfection of a bacterial host cell via such a provided phagemid vector and an appropriate helper phage (such as M13K07, VCS-M13 or R408), a complete phage particle is produced, thereby enabling the encoded heterologous cDNA to be physically coupled to a polypeptide displayed on the surface of its corresponding phage (Lowman, Annu Rev Biophys Biomol Struct, 1997; Rodi and Makowski, Curr Opin Biotechnol, 1999).

[0275] According to different embodiments, in addition to the above-mentioned regulatory sequences and the nucleic acid sequences encoding the fusion proteins as described herein, the cloning vector can include replication and control sequences derived from species compatible with the host cell for expression, and selection markers that confer selectable phenotypes on transformed or transfected cells. A large number of suitable cloning vectors are known in the art and are commercially available.

[0276] In some embodiments, the present invention also relates to methods for producing fusion proteins of the present invention starting from nucleic acids encoding fusion proteins or any subunits thereof using genetic engineering methods. In some embodiments, the provided methods can be performed in vivo, wherein the provided fusion proteins can be produced, for example, in bacteria or eukaryotic host organisms and then isolated from the host organisms or their cultures. Fusion proteins of the present invention can also be produced in vitro, for example, using an in vitro translation system.

[0277] When fusion proteins are produced in vivo, nucleic acids encoding such proteins can be introduced into suitable bacteria or eukaryotic host organisms using recombinant DNA techniques well known in the art. In some embodiments, DNA molecules encoding fusion proteins as described herein, particularly cloning vectors containing the coding sequence of such fusion proteins, can be transformed into host cells capable of expressing the gene. Standard methods can be used for transformation. Therefore, the present invention also relates to host cells containing nucleic acid molecules disclosed herein.

[0278] In some embodiments, the transformed host cell can be cultured under conditions suitable for expression of the nucleotide sequence encoding the fusion protein of the present invention. In some embodiments, the host cell can be prokaryotic such as Escherichia coli (E.coli) or Bacillus subtilis (Bacillus subtilis), or eukaryotic such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), SF9 or High5 insect cells, immortalized mammalian cell lines (such as HeLa cells or CHO cells) or primary mammalian cells.

[0279] In some embodiments, where the lipocalin muteins of the invention (including those contained in the fusion proteins disclosed herein) comprise intramolecular disulfide bonds, it may be preferred to use an appropriate signal sequence to direct the nascent protein to a cellular compartment having a redox environment. Such an oxidative environment may be provided by the periplasm of gram-negative bacteria (such as E. coli), in the extracellular environment of gram-positive bacteria, or in the lumen of the endoplasmic reticulum of eukaryotic cells, and generally favors the formation of structural disulfide bonds.

[0280] In some embodiments, the fusion protein of the present invention can also be produced in the cytosol of a host cell (preferably E. coli). In this case, the fusion protein provided can be obtained directly in a soluble and folded state, or recovered in the form of inclusion bodies and then renatured in vitro. Another option is to use a specific host strain that has an oxidative intracellular environment, which can thus allow the formation of disulfide bonds in the cytosol (Venturi et al., J Mol Biol, 2002).

[0281] In some embodiments, the fusion proteins of the present invention as described herein may not necessarily be generated or produced in whole or in part by using genetic engineering. Rather, such proteins may also be obtained by any of a number of conventional and well-known techniques, such as common organic synthesis strategies, solid phase related synthesis techniques, commercially available automatic synthesizers, or by in vitro transcription and translation. For example, it is possible to use molecular modeling to identify promising fusion proteins or lipocalin mutant proteins contained in such fusion proteins, synthesize them in vitro, and study their binding activity to the target of interest. Methods for solid phase and / or solution phase synthesis of proteins are well known in the art (see, e.g., Bruckdorfer et al., CurrPharm Biotechnol, 2004).

[0282] In some embodiments, fusion proteins of the invention can be produced by in vitro transcription / translation using well-established methods known to those skilled in the art.

[0283] In some further embodiments, the fusion proteins described herein can also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques.

[0284] In addition, in some embodiments, the fusion protein according to the present invention can be obtained by conjugating a separate subunit (such as an immunoglobulin) with a mutant protein contained in the fusion protein. Such conjugation can be achieved, for example, by all forms of covalent or non-covalent linkage using conventional methods.

[0285] Those skilled in the art will appreciate methods for preparing fusion proteins contemplated by the present invention, but whose protein or nucleic acid sequences are not explicitly disclosed herein. As an overview, such modifications of the amino acid sequence include directed mutagenesis such as at a single amino acid position to simplify the subcloning of a protein gene or a portion thereof by introducing a cleavage site for certain restriction enzymes. In addition, these mutations can also be introduced to further improve the affinity of the fusion protein to its target (e.g., CD137 and GPC3). In addition, if desired, mutations can be introduced to adjust one or more features of the protein, such as to improve folding stability, serum stability, protein resistance or water solubility or reduce aggregation tendency.

[0286] The present invention is also characterized by the following items.

[0287] Item 1. A fusion protein capable of binding to CD137 and GPC3, wherein the fusion protein comprises at least two subunits in any order, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutant protein and is specific for CD137.

[0288] Item 2. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutant protein and is specific for CD137, wherein the second subunit is optionally N-terminally linked to the C-terminus of each heavy chain of the first subunit via a linker.

[0289] Item 3. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutant protein and is specific for CD137, wherein the second subunit is optionally connected at the C-terminus to the N-terminus of each heavy chain of the first subunit via a linker.

[0290] Item 4. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutant protein and is specific for CD137, wherein the second subunit is optionally N-terminally linked to the C-terminus of each light chain of the first subunit via a linker.

[0291] Item 5. A fusion protein, wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutant protein and is specific for CD137, wherein the second subunit is optionally connected at the C-terminus to the N-terminus of each light chain of the first subunit via a linker.

[0292] Item 6. A fusion protein according to any one of Items 1-5, wherein the fusion protein is capable of activating a K of up to about 1 nM. D value for binding to GPC3 or capable of binding to the immunoglobulin or its antigen binding domain contained in the first subunit alone D K with equivalent or lower value D Values ​​combined with GPC3.

[0293] Item 7. The fusion protein according to Item 6, wherein K is determined by surface plasmon resonance (SPR) analysis. D value.

[0294] Item 8. A fusion protein according to any one of Items 1 to 7, wherein the fusion protein is capable of activating an EC of at most about 0.5 nM. 50 The value of binding to GPC3 or being able to bind to the immunoglobulin or its antigen binding domain contained in the first subunit alone50 Equivalent or lower EC 50 Values ​​combined with GPC3.

[0295] Item 9. A fusion protein according to any one of Items 1 to 8, wherein the fusion protein is capable of activating an EC of at most about 3 nM. 50 The value of binding to CD137 or being able to bind to the CD137-specific lipocalin mutant protein contained in the second subunit alone EC 50 Equivalent or lower EC 50 Values ​​for binding to CD137.

[0296] Item 10. The fusion protein according to any one of Items 8-9, wherein the EC is determined by enzyme-linked immunosorbent assay (ELISA) analysis. 50 value.

[0297] Item 11. A fusion protein according to any one of Items 1-10, wherein the fusion protein cross-reacts with macaque GPC3.

[0298] Item 12. A fusion protein according to any one of Items 1-11, wherein when the fusion protein is measured in an ELISA assay, the fusion protein is capable of quantifying the activity of the fusion protein with an EC value of at most about 10 nM. 50 Values ​​for simultaneous binding of CD137 and GPC3.

[0299] Item 13. A fusion protein according to any one of Items 1 to 12, wherein when the fusion protein is measured in a flow cytometric analysis, the fusion protein is capable of having an EC of at most about 30 nM. 50 The values ​​were compared with those of CD137 expressed on the binding cells.

[0300] Item 14. A fusion protein according to any one of Items 1-13, wherein when the fusion protein is measured in a flow cytometric analysis, the fusion protein is capable of having an EC of at most about 30 nM. 50 The values ​​were in combination with GPC3 expressed on cells.

[0301] Item 15. A fusion protein according to any one of Items 1-14, wherein the fusion protein is capable of binding to tumor cells expressing GPC3.

[0302] Item 16. A fusion protein according to any one of Items 1-15, wherein the fusion protein is capable of stimulating T cell responses.

[0303] Item 17. A fusion protein according to any one of Items 1 to 16, wherein the fusion protein is capable of inducing increased secretion of IL-2.

[0304] Item 18. A fusion protein according to any one of Items 1-17, wherein compared with SEQ ID NO:83, the fusion protein is capable of inducing increased secretion of IL-2 to a higher level; and / or compared with SEQ ID NO:83, the fusion protein is capable of inducing increased secretion of IL-2 with better efficiency.

[0305] Item 19. A fusion protein according to any one of Items 1 to 18, wherein the fusion protein is capable of inducing lymphocyte-mediated cytotoxicity.

[0306] Item 20. A fusion protein according to any one of Items 1-19, wherein compared with SEQ ID NO:83, the fusion protein is capable of inducing enhanced T cell-mediated killing of tumor cells expressing GPC3; and / or compared with SEQ ID NO:83, the fusion protein is capable of inducing cytotoxic T cell activation with better efficiency.

[0307] Item 21. A fusion protein according to any one of Items 1-20, wherein the fusion protein is capable of co-stimulating T cell responses in a GPC3-dependent manner.

[0308] Item 22. A fusion protein according to any one of Items 1-21, wherein the fusion protein is capable of co-stimulating T cell responses in the tumor microenvironment.

[0309] Item 23. A fusion protein according to any one of Items 1-22, wherein the fusion protein does not co-stimulate T cell response in the absence of GPC3.

[0310] Item 24. A fusion protein according to any one of Items 1-23, wherein the fusion protein has antibody-like pharmacokinetic characteristics.

[0311] Item 25. A fusion protein according to any one of Items 1-24, wherein the fusion protein has a half-life in mice of at least 50 hours, at least 75 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 175 hours, at least 200 hours, at least 250 hours or even longer, and / or wherein the fusion protein has a half-life in mice that is longer than the half-life of SEQID NO:83.

[0312] Item 26. A fusion protein according to any one of items 1-25, wherein the fusion protein has an isoelectric point of at least 6.5, at least 6.8, at least 7.1, at least 7.4, at least 7.5, at least 7.7 or even higher, and / or wherein the fusion protein has an isoelectric point higher than the isoelectric point of SEQ ID NO:83.

[0313] Item 27. A fusion protein according to any one of items 1-26, wherein the lipocalin mutant protein comprises one or more mutated amino acid residues at positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence corresponding to mature human tear lipocalin (SEQ ID NO: 1).

[0314] Item 28. A fusion protein according to any one of items 1 to 27, wherein the amino acid sequence of the lipocalin mutant protein comprises one or more of the following mutated amino acid residues at one or more positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150 and 153 of the linear polypeptide sequence of mature hTlc (SEQ ID NO: 1): Ala 5→Val or Thr; Arg26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Gly 46→Asp; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Arg or Asn; Thr 71→Ala; Val 85→Asp; Lys 94→Arg or Glu; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile and Cys 153→Ser.

[0315] Item 29. A fusion protein according to any one of items 1 to 28, wherein the amino acid sequence of the lipocalin mutant protein comprises one of the following groups of mutated amino acid residues compared to the linear polypeptide sequence of mature human tear lipocalin (SEQ ID NO: 1):

[0316] (a) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; and Cys 153→Ser;

[0317] (b) Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Arg; Val 85→Asp; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys 153→Ser;

[0318] (c) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Asn; Lys 94→Arg; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys153→Ser;

[0319] (d) Ala 5→Val; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Lys 65→Arg; Lys 94→Glu; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Lys 121→Glu; Ala 133→Thr; and Cys 153→Ser;

[0320] (e) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Thr 42→Ser; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys 153→Ser;

[0321] (f) Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Lys 52→Glu; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ala 133→Thr; Arg 148→Ser; Ser 150→Ile; and Cys 153→Ser; and

[0322] (g)Ala 5→Thr; Arg 26→Glu; Glu 27→Gly; Phe 28→Cys; Pro 29→Arg; Glu 30→Pro; Met 31→Trp; Leu 33→Ile; Glu 34→Phe; Gly 46→Asp; Leu 56→Ala; Ser 58→Asp; Arg 60→Pro; Cys 61→Ala; Thr 71→Ala; Cys 101→Ser; Glu 104→Val; Leu 105→Cys; His 106→Asp; Lys 108→Ser; Arg 111→Pro; Lys 114→Trp; Ser 150→Ile; and Cys153→Ser.

[0323] Item 30. A fusion protein according to any one of Items 1-29, wherein the amino acid sequence of the lipocalin mutant protein comprises an amino acid sequence selected from SEQ ID NOs: 32-38 or a fragment or variant thereof.

[0324] Item 31. A fusion protein according to any one of Items 1-30, wherein the amino acid sequence of the lipocalin mutant protein has at least 85% sequence identity with an amino acid sequence selected from SEQ ID NOs: 32-38.

[0325] Item 32. A fusion protein according to any one of items 1-26, wherein the lipocalin mutant protein comprises one or more mutated amino acid residues at positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence (SEQ ID NO: 2) corresponding to mature human neutrophil gelatinase-associated lipocalin (hNGAL).

[0326] Item 33. A fusion protein according to any one of items 1-26 and 32, wherein the amino acid sequence of the lipocalin mutant protein comprises one or more of the following mutated amino acid residues at positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature human neutrophil gelatinase-associated lipocalin (hNGAL) (SEQ ID NO: 2): Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg or Lys; Gln 49→Val, Ile, His, Ser or Asn; Tyr 52→Met; Asn 65→Asp; Ser 68→Met, Ala or Gly; Leu 70→Ala, Lys, Ser or Thr; Arg 72→Asp; Lys 73→Asp; Asp 77→Met, Arg, Thr or Asn; Trp 79→Ala or Asp; Arg81→Met, Trp or Ser; Phe 83→Leu; Cys 87→Ser; Leu 94→Phe; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu and Lys134→Tyr.

[0327] Item 34. A fusion protein according to any one of items 1-26 and 33, wherein the lipocalin mutant protein comprises one or more mutated amino acid residues at positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 of the linear polypeptide sequence (SEQID NO: 2) corresponding to mature human neutrophil gelatinase-associated lipocalin (hNGAL).

[0328] Item 35. A fusion protein according to any one of items 1-26 and 34, wherein the amino acid sequence of the lipocalin mutant protein comprises one or more of the following mutated amino acid residues at positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln 20→Arg; Asn 25→Tyr or Asp; Gln 28→His; Val 33→Ile; Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Val or Asp; Gln 49→His; Tyr 52→Ser or Gly; Lys 59→Asn; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys 73→Asp; Asp 77→Gln or His; Tyr 78→His; Trp 79→Ile; Ile 80→Asn; Arg 81→Trp or Gln; Thr 82→Pro; Cys 87→Ser; Phe 92→Leu or Ser; Asn 96→Phe; Lys 98→Arg; Tyr 100→Asp; Pro 101→Leu; Leu 103→His or Pro; Phe 122→Tyr; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly.

[0329] Item 36. A fusion protein according to any one of items 1-26 and 32-35, wherein the amino acid sequence of the lipocalin mutant protein comprises one of the following groups of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2):

[0330] (a)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Asn;Tyr 52→Met;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0331] (b)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→Ile;Tyr 52→Met;Asn 65→Asp;Ser 68→Met;Leu 70→Lys;Arg 72→Asp;Lys 73→Asp;Asp 77→Met;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0332] (c)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0333] (d)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Ala;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Asp;Arg 81→Trp;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0334] (e)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Ser;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Ser;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Met;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0335] (f)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Val;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Arg;Trp 79→Asp;Arg 81→Ser;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0336] (g)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→His;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0337] (h)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Lys;Gln 49→Asn;Tyr 52→Met;Asn 65→Asp;Ser 68→Gly;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Thr;Trp 79→Ala;Arg 81→Ser;Phe 83→Leu;Cys 87→Ser;Leu 94→Phe;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0338] (i)Gln 28→His;Leu 36→Gln;Ala 40→Ile;Ile 41→Arg;Gln 49→Ser;Tyr 52→Met;Asn 65→Asp;Ser 68→Ala;Leu 70→Thr;Arg 72→Asp;Lys 73→Asp;Asp 77→Asn;Trp 79→Ala;Arg 81→Ser;Cys 87→Ser;Asn 96→Lys;Tyr 100→Phe;Leu 103→His;Tyr 106→Ser;Lys 125→Phe;Ser 127→Phe;Tyr 132→Glu;and Lys 134→Tyr;

[0339] (j)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Ser;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0340] (k)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Ser;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Lys 98→Arg;Tyr 100→Asp;Pro 101→Leu;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0341] (l)Asn 25→Tyr;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Phe 71→Leu;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Gln;Phe 92→Ser;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0342] (m)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Tyr 78→His;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0343] (n)Asn 25→Asp;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0344] (o)Val 33→Ile;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0345] (p)Gln 20→Arg;Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Glu 44→Val;Gln 49→His;Tyr 52→Gly;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Phe 122→Tyr;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0346] (q)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Ser;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Ile 80→Asn;Arg 81→Trp;Thr 82→Pro;Asn 96→Phe;Tyr 100→Asp;Pro 101→Leu;Leu 103→Pro;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;

[0347] (r)Leu 36→Met;Ala 40→Asn;Ile 41→Leu;Gln 49→His;Tyr 52→Gly;Lys 59→Asn;Ser 68→Asp;Leu 70→Met;Arg 72→Leu;Lys 73→Asp;Asp 77→Gln;Trp 79→Ile;Arg 81→Trp;Phe 92→Leu;Asn 96→Phe;Tyr 100→Asp;Leu 103→His;Lys 125→Ser;Ser 127→Ile;Tyr 132→Trp;and Lys 134→Gly;and

[0348] (s) Leu 36→Met; Ala 40→Asn; Ile 41→Leu; Glu 44→Asp; Gln 49→His; Tyr 52→Ser; Ser 68→Asp; Leu 70→Met; Phe 71→Leu; Arg 72→Leu; Lys 73→Asp; Asp 77→His; Trp 79→Ile; Arg 81→Trp; Phe 92→Leu; Asn 96→Phe; Tyr 100→Asp; Leu 103→His; Lys 125→Ser; Ser 127→Ile; Tyr 132→Trp; and Lys 134→Gly.

[0349] Item 37. A fusion protein according to any one of items 1-26 and 32-36, wherein the amino acid sequence of the lipocalin mutant protein comprises an amino acid sequence selected from SEQ ID NOs: 39-57 or a fragment or variant thereof.

[0350] Item 38. A fusion protein according to any one of items 1-26 and 32-36, wherein the amino acid sequence of the lipocalin mutant protein has at least 85% sequence identity with an amino acid sequence selected from SEQ ID NOs: 39-57.

[0351] Item 39. A fusion protein according to any one of items 1-26 and 32-38, wherein the amino acid sequence of the lipocalin mutant protein comprises the following mutated amino acid residue groups compared to the linear polypeptide sequence of mature hNGAL (SEQ ID NO: 2): Gln 28→His; Leu 36→Gln; Ala 40→Ile; Ile 41→Arg; Gln 49→Ile; Tyr 52→Met; Asn 65→Asp; Ser 68→Met; Leu 70→Lys; Arg 72→Asp; Lys 73→Asp; Asp77→Met; Trp 79→Asp; Arg 81→Trp; Cys 87→Ser; Asn 96→Lys; Tyr 100→Phe; Leu 103→His; Tyr 106→Ser; Lys 125→Phe; Ser 127→Phe; Tyr 132→Glu; and Lys 134→Tyr, and / or wherein the lipocalin mutein has at least 85% sequence identity to the amino acid sequence of SEQ ID NO:40.

[0352] Item 40. A fusion protein according to any one of Items 1 to 39, wherein one subunit is connected to another subunit via a linker.

[0353] Item 41. A fusion protein according to any one of Items 1-40, wherein the N-terminus of the second subunit is connected to the N- or C-terminus of each heavy chain constant region (CH) of the first subunit or to the N- or C-terminus of each light chain constant region (CL) of the first subunit via a linker.

[0354] Item 42. A fusion protein according to any one of Items 1-41, wherein the N-terminus of the third subunit is connected to the N- or C-terminus of each heavy chain constant region (CH) of the first subunit, to the N- or C-terminus of each light chain constant region (CL) of the first subunit, or to the C-terminus of each second subunit via a linker.

[0355] Item 43. A fusion protein according to any one of Items 40-42, wherein the linker is non-structural (Gly-Gly-Gly-Gly-Ser) 3 Linker (SEQ ID NO: 13).

[0356] Item 44. A fusion protein according to any one of Items 40-43, wherein the linker is a non-structural glycine-serine linker, a polyproline linker, a proline-alanine-serine polymer, or a linker selected from SEQ IDNOs: 13-23.

[0357] Item 45. A fusion protein according to any one of Items 1-44, wherein the first subunit is an antibody.

[0358] Item 46. A fusion protein according to any one of Items 1-45, wherein the heavy chain variable region of the antibody is selected from SEQ ID NOs: 78, 114, 119, 126 and 129 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 78, 114, 119, 126 and 129, and wherein the light chain variable region of the antibody is selected from SEQ ID NOs: 79, 115 and 127 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 79, 115 and 127.

[0359] Item 47. A fusion protein according to any one of Items 1-46, wherein the antibody comprises a heavy chain or a light chain, the heavy chain is any one of SEQ ID NOs: 80 or 81 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 80 or 81, and the light chain is SEQ ID NO: 82 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or more sequence identity with the amino acid sequence shown in SEQ ID NOs: 82.

[0360] Item 48. A fusion protein according to any one of Items 1-47, wherein the antibody comprises a heavy chain variable region and a light chain variable region as follows, respectively: SEQ ID NOs: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs: 114 and 115 or SEQ ID NOs: 126 and 127, or comprises a heavy chain variable region and a light chain variable region having a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or higher sequence identity with the amino acid sequence shown in SEQ ID NOs: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs: 114 and 115 or SEQ ID NOs: 126 and 127.

[0361] Item 49. A fusion protein according to any one of Items 1-48, wherein the antibody comprises heavy chains and light chains as follows: SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82, respectively, or comprises heavy chains and light chains having sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or higher sequence identity with the amino acid sequences shown in SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82.

[0362] Item 50. The fusion protein according to Item 36, wherein the heavy chain of the antibody comprises one of the following groups of CDR sequences:

[0363] (a) GYTFTDYE (HCDR1, SEQ ID NO:72), LDPKTGDT (HCDR2, SEQ ID NO:73), TRFYSYTY (HCDR3; SEQ ID NO:74);

[0364] (b) GFTFNKNA (HCDR1, SEQ ID NO: 108), IRNKTNNYAT (HCDR2, SEQ ID NO: 109), VAGNSFAY (HCDR3, SEQ ID NO: 110);

[0365] (c) YFDFDSYE (HCDR1, SEQ ID NO:116), IYHSGST (HCDR2, SEQ ID NO:117), ARVNMDRFDY (HCDR3, SEQ ID NO:108); or

[0366] (d) GTFFSSYA (HCDR1, SEQ ID NO:120), IQKQGLPT (HCDR2, SEQ ID NO:121), AKNRAKFDY (HCDR3, SEQ ID NO:122).

[0367] Item 51. A fusion protein according to any one of Items 1 to 50, wherein the light chain of the antibody comprises one of the following groups of CDR sequences:

[0368] (a) QSLVHSNRNTY (LCDR1, SEQ ID NO:75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO:77);

[0369] (b) QSLLYSSNQKNY(LCDR1, SEQ ID NO:111), WAS(LCDR2), QQYYNYPLT(LCDR3, SEQ ID NO:113); or

[0370] (c) QSISSY (LCDR1, SEQ ID NO:123), NAS (LCDR2), QQNRGFPLT (LCDR3, SEQ ID NO:125).

[0371] Item 52. A fusion protein according to any one of Items 1-51, wherein the heavy chain of the antibody comprises the following group of CDR sequences: GYTFTDYE (HCDR1, SEQ ID NO:72), LDPKTGDT (HCDR2, SEQ ID NO:73), TRFYSYTY (HCDR3; SEQ ID NO:74); and the light chain of the antibody comprises the following group of CDR sequences: QSLVHSNRNTY (LCDR1, SEQ ID NO:75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO:77).

[0372] Item 53. A fusion protein according to any one of Items 1 to 51, wherein the antibody comprises the following set of CDR sequences:

[0373] (a) GFTFNKNA (HCDR1, SEQ ID NOs: 108), IRNKTNNYAT (HCDR2, SEQ ID NOs: 109), VAGNSFAY (HCDR3, SEQ ID NOs: 110), QSLLYSSNQKNY (LCDR1, SEQ ID NOs: 111), WAS (LCDR2), QQYYNYPLT (LCDR3, SEQ ID NOs:113); or

[0374] (b) GTFFSSYA (HCDR1, SEQ ID NOs: 120), IQKQGLPT (HCDR2, SEQ ID NOs: 121), AKNRAKFDY (HCDR3, SEQ ID NOs: 122), QSISSY (LCDR1, SEQ ID NOs: 123), NAS (LCDR2), QQNRGFPLT (LCDR3, SEQ ID NOs: 125).

[0375] Item 54. A fusion protein according to any one of Items 1-53, wherein the antibody has an IgG4 backbone.

[0376] Item 55. A fusion protein according to Item 54, wherein the IgG4 backbone has one or more of the following mutations: S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T and T256E.

[0377] Item 56. A fusion protein according to any one of items 1-55, wherein the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 87-96, or wherein the fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or higher sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 87-96.

[0378] Item 57. A fusion protein according to any one of items 1-56, wherein the fusion protein comprises amino acids shown in SEQ ID NOs: 87 and 82, amino acids shown in SEQ ID NOs: 88 and 82, amino acids shown in SEQ ID NOs: 81 and 89, amino acids shown in SEQ ID NOs: 81 and 90, amino acids shown in SEQ ID NOs: 91 and 82, amino acids shown in SEQ ID NOs: 92 and 82, amino acids shown in SEQ ID NOs: 81 and 93, amino acids shown in SEQ ID NOs: 81 and 94, amino acids shown in SEQ ID NOs: 95 and 82, or amino acids shown in SEQ ID NOs: 96 and 82.

[0379] Item 58. A fusion protein according to any one of items 1-57, wherein the fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or higher sequence identity with the amino acid sequence shown in SEQID NOs:87 and 82, SEQ ID NOs:88 and 82, SEQ ID NOs:81 and 89, SEQ ID NOs:81 and 90, SEQ ID NOs:91 and 82, SEQ ID NOs:92 and 82, SEQ ID NOs:81 and 93, SEQ ID NOs:81 and 94, SEQ ID NOs:95 and 82 or SEQ ID NOs:96 and 82.

[0380] Item 59. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of Items 1-58.

[0381] Item 60. A nucleic acid molecule according to Item 59, wherein the nucleic acid molecule is operably linked to a regulatory sequence that allows expression of the nucleic acid molecule.

[0382] Item 61. A nucleic acid molecule according to Item 59 or 60, wherein the nucleic acid molecule is contained in a vector or a phagemid vector.

[0383] Item 62. A host cell containing the nucleic acid molecule according to any one of Items 59 to 61.

[0384] Item 63. A method for producing a fusion protein according to any one of items 1-58, wherein the fusion protein is produced starting from a nucleic acid encoding the fusion protein.

[0385] Item 64. A method according to Item 63, wherein the fusion protein is produced in a bacterial or eukaryotic host organism and isolated from the host organism or a culture thereof.

[0386] Item 65. Use of the fusion protein described in any one of Items 1 to 58 or a composition comprising such a fusion protein for simultaneously activating the downstream signaling pathway of CD137 and engaging GPC3-positive tumor cells.

[0387] Item 66. A method for simultaneously activating the downstream signaling pathway of CD137 and engaging GPC3-positive tumor cells, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0388] Item 67. A method for simultaneously co-stimulating T cells and engaging GPC3-positive tumor cells, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0389] Item 68. A method for simultaneously inducing lymphocyte activity and engaging GPC3-positive tumor cells, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0390] Item 69. A method for inducing CD137 aggregation and activation on T cells and directing the T cells to GPC3-positive tumor cells, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0391] Item 70. A method for inducing a local lymphocyte response in the vicinity of GPC3-positive tumor cells, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0392] Item 71. A method for inducing increased IL-2 secretion by T cells in the vicinity of GPC3-positive tumor cells, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0393] Item 72. A method for inducing increased lymphocyte-mediated cytolysis of GPC3-positive tumor cells, the method comprising applying to a tissue containing a tumor one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins.

[0394] Item 73. A pharmaceutical composition comprising one or more fusion proteins described in any one of Items 1-58.

[0395] Item 74. A method for preventing, ameliorating or treating GPC3-positive cancer, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0396] Item 75. A method for preventing, improving or treating hepatocellular carcinoma, the method comprising applying one or more fusion proteins described in any one of Items 1-58 or one or more compositions comprising such fusion proteins to a tissue containing a tumor.

[0397] Item 76. A fusion protein according to any one of Items 1-58, for use in therapy.

[0398] Item 77. A fusion protein for use according to Item 76, wherein the use is in cancer treatment.

[0399] Item 78. Use of the fusion protein according to any one of Items 1-58 in the preparation of a drug.

[0400] Item 79. The use according to Item 78, wherein the drug is used for cancer treatment. V. Examples

[0401] Example 1: Expression and analysis of representative fusion proteins

[0402] In this example, the GPC3-specific antibody is linked to a CD137-specific lipocalin mutant of SEQ ID NO: 40 or a CD137-specific lipocalin having 97% sequence identity to SEQ ID NO: 49 and designated as CD137Ac1 via a linker (e.g., the nonstructural (GPC3) residue of SEQ ID NO: 13). 4 S) 3Linker) fused together to simultaneously engage GPC3 and CD137, generating representative antibody-lipocalin mutein fusion proteins, the GPC3-specific antibody having a heavy chain provided by SEQ ID NO: 81, or a heavy chain variable domain comprising SEQ ID NO: 78, or a CDR comprising GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73) and TRFYSYTY (HCDR3; SEQ ID NO: 74), and a light chain provided by SEQ ID NO: 82, or a heavy chain variable domain comprising SEQ ID NO: 79, or a CDR comprising QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2, SEQ ID NO: 76) and SQNTHVPPT (LCDR3; SEQ ID NO: 77). The different forms generated are as follows Figure 1For example, by fusing one or more of the lipocalin muteins of SEQ ID NO: 40 or 97% sequence identity to SEQ ID NO: 49 to one or more of the four termini of the antibody, such as SEQ ID NOs: 87 and 82, SEQ ID NOs: 88 and 82, SEQ ID NOs: 81 and 89, SEQ ID NOs: 81 and 90, and CD137Ac1-fusion 1, CD137Ac1-fusion 2, CD137Ac1-fusion 3, CD137Ac1-fusion 4, CD137Ac1-fusion 5, CD137Ac1-fusion 6, and CD137Ac1-fusion 7 (with SEQ ID NOs: 91 and 82, SEQ ID NOs: 92 and 82, SEQ ID NOs: 81 and 93, SEQ ID NOs: 81 and 94, SEQ ID NOs: 95 and 82, or SEQ ID NOs: 96 and 82) are produced. NOs: 96 and 82 have 97% sequence identity) such fusion protein, the antibody comprises a heavy chain provided by SEQ ID NO: 81, or a heavy chain variable domain comprising SEQ ID NO: 78, or a CDR comprising GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73) and TRFYSYTY (HCDR3; SEQ ID NO: 74), and a light chain provided by SEQ ID NO: 82, or a heavy chain variable domain comprising SEQ ID NO: 79, or a CDR comprising QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2, SEQ ID NO: 76) and SQNTHVPPT (LCDR3; SEQ ID NO: 77). The resulting fusion protein can be bivalent to CD137 (e.g., such as Figure 1 A-1D) or a tetravalent anti-CD137 (e.g., Figure 1 E-1H), or have an even higher affinity for CD137 (e.g., Figure 1 I).

[0403] The GPC3-specific antibodies and all antibody-lipocalin mutant protein fusion proteins described in this example have an engineered IgG4 backbone that includes the S228P mutation to minimize IgG4 half-antibody exchange in vitro and in vivo (Silva et al., J Biol Chem, 2015). Additional mutations in the IgG4 backbone may also be present in all antibodies and fusion proteins described herein, including any one or more of the mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E. F234A and L235A mutations may also be introduced to reduce ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev, 2010). M428L and N434S mutations, or M252Y, S254T and T256E mutations may also be introduced to extend serum half-life (Dall'Acqua et al., J Biol Chem, 2006; Zalevsky et al., Nat Biotechnol, 2010). All antibodies were expressed without the carboxyl terminal lysine to avoid heterogeneity.

[0404] In addition, if Figure 1 As shown in J-1K, one or more of the CD137-specific lipocalin mutant protein of SEQ ID NO: 40 or the GPC3-specific lipocalin of SEQ ID NO: 64 are transported via a linker (such as the non-structural (GPC3) of SEQ ID NO: 13). 4 S) 3 The monospecific lipocalin mutein Fc fusion protein was obtained by fusing the mutein Fc region of the antibody provided in SEQ ID NO: 28 with a linker) and the C-terminus of the Fc region of the antibody provided in SEQ ID NO: 28. The resulting construct is provided in SEQ ID NOs: 98.

[0405] The invention also encompasses asymmetric antibody-lipocalin mutein fusion formats, wherein, for example, one light chain of the antibody may be fused to the lipocalin mutein while the other light chain is not fused.

[0406] The fusion protein construct was generated by gene synthesis and cloned into a mammalian expression vector. The fusion protein construct was then transiently expressed in Expi293FTM cells (Life Technologies). The concentration of the fusion protein in the cell culture medium was measured by HPLC (Agilent Technologies) using a protein A affinity chromatography column (Applied Biosystems). The titers of the fusion proteins are summarized in Table 1.

[0407] The fusion protein was purified using protein A chromatography followed by size exclusion chromatography (SEC) in phosphate buffered saline (PBS). After SEC purification, fractions containing monomeric protein were pooled and reanalyzed using analytical SEC and percent monomer content.

[0408] After SEC purification, the monomeric protein content and isoelectric point (pi) of the exemplary fusion proteins are summarized in Table 1. The pi of the provided fusion proteins (SEQ ID NOs: 87 and 82) was increased compared to the previously known CD137 / GPC3 bispecific fusion protein SEQ ID NO: 83.

[0409] Table 1: Transient expression titers

[0410] Monomer protein content [%] Determined pI SEQ ID NOs:87 and 82 99.9 7.5 CD137Ac1-Fusion2 98.3 -- SEQ ID NOs:81 and 82 99.0 -- SEQ ID NO:83 100.0 6.2

[0411] Example 2: Determination of the binding of fusion protein to GPC3 by surface plasmon resonance (SPR)

[0412] The binding kinetics and affinity of exemplary fusion proteins to recombinant human GPC3 (huGPC3) and cynomolgus monkey GPC3 (cyGPC3) (R&D Systems) were determined by surface plasmon resonance (SPR) using a Biacore T200 instrument (GE Healthcare).

[0413] Anti-human IgG Fc antibody (GE Healthcare) was immobilized on a CM5 sensor chip using standard amine chemistry: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to activate the carboxyl groups on the chip. Subsequently, a 25 μg / mL solution of anti-human IgG Fc antibody (GE Healthcare) in 10 mM sodium acetate (pH 5.0) was applied at a flow rate of 5 μL / min until an immobilization level of 6000-10000 resonance units (RU) was reached. Remaining unreacted NHS-esters were blocked by passing a 1 M ethanolamine solution over the surface. The reference channel was treated in a similar manner. Then, the test fusion proteins (SEQ ID NOs: 87 and 82 and CD137Ac1-fusion 1) or GPC3 antibodies contained in the fusion proteins (SEQ ID NOs: 81 and 82) were captured on the chip surface at a flow rate of 10 μL / min with anti-human IgG Fc antibodies for 180 s at 0.2 μg / mL in HBS-EP+ buffer. After each capture step, the needle was washed.

[0414] For affinity determination, dilutions of huGPC3 or cyGPC3 (100 nM, 25 nM, 6.25 nM and 1.56 nM) were prepared in HBS-EP+ buffer (GE Healthcare) and applied to the prepared chip surface. Binding assays were performed using a contact time of 180 s, a dissociation time of 900 s and a flow rate of 30 μL / min. After each injection, the needle was washed with 40 mM NaOH + 20% isopropanol. All measurements were performed at 25 °C. The cytochrome P400 was injected with 3 M MgCl 2 The chip surface was regenerated for 120 s. Three priming cycles were performed for conditioning purposes before protein measurement. Data were evaluated using Biacore T200 evaluation software (V2.0). Dual references were used and a 1:1 binding model was used to fit the raw data.

[0415] The k for the exemplary fusion protein on , k off and the obtained equilibrium dissociation constant (K D ) are summarized in Table 2. The tested fusion proteins (SEQ ID NOs: 87 and 82 and CD137Ac1-Fusion 1) bound huGPC3 and cyGPC3 with subnanomolar affinity, which is comparable to the GPC3 antibody contained in the fusion protein and the previously known CD137 / GPC3 bispecific fusion protein (SEQ ID NO: 83).

[0416] Table 2: Kinetic constants and affinities of fusion proteins determined by SPR

[0417]

[0418]

[0419] Example 3: Binding of fusion protein to GPC3 or CD137 in ELISA

[0420] Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding potency of the exemplary fusion proteins to GPC3 and CD137.

[0421] Recombinant huGPC3 (R&D Systems) at a concentration of 1 μg / mL in PBS was coated on microtiter plates overnight at 4°C. After washing five times 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-0.1% T-2% BSA) for 1 hour at room temperature. After washing five times with 100 μL PBS-0.05% T (PBS supplemented with 0.05% (v / v) Tween 20), different concentrations of exemplary fusion proteins (SEQ ID NOs: 87 and 92 and CD137Ac1-fusions 1 to 7) or GPC3 antibodies contained in the fusion proteins (SEQ ID NOs: 81 and 82) were added to the wells and incubated for 1 h at room temperature, followed by another washing step. The bound molecules under investigation were detected by incubation with anti-human IgG Fc-HRP (Jackson Laboratory) diluted 1:5000 in PBS-0.1% T-2% BSA. After an additional washing step, fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and fluorescence intensity was measured using a fluorescence microplate reader.

[0422] The binding potency of the fusion proteins to macaque GPC3 and huCD137 was also measured using the same ELISA setup, where recombinant cyGPC3 (R&D Systems) or huCD137-His (recombinant human CD137 with a C-terminal polyhistidine tag, R&D Systems) was coated on microtiter plates. Test reagents were titrated similarly and bound reagents were detected via anti-NGAL-HRP.

[0423] Figure 2 Exemplary results are depicted in A-2D, along with the fitted curves generated by a 1:1 combined sigmoidal fit, where EC 50 The value and the maximum signal are free parameters, and the slope is fixed to unity. 50 The values ​​are provided in Table 3.

[0424] EC observed for the provided fusion proteins (SEQ ID NOs: 87 and 92 and CD137Ac1-fusions 1 to 7) on human GPC3 50The values ​​were very similar or comparable to those of previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83) and / or GPC3 antibodies contained in the fusion proteins (SEQ ID NOs: 81 and 82). Some of the tested fusion proteins (SEQ ID NOs: 87 and 82, CD137Ac1-fusion 3 and CD137Ac1-fusion 4) retained strong binding affinity to human CD137. In addition, some of the tested fusion proteins (SEQ ID NOs: 87 and 82) also showed cross-reactivity with macaque GPC3 at a level comparable to that of human GPC3, i.e., at a level comparable to that of human GPC3 at the EC values ​​of the corresponding human GPC3. 50 EC in the same range 50 Values ​​for binding to macaque GPC3.

[0425] Table 3: ELISA data of GPC3 or CD137 binding

[0426]

[0427] Example 4: Simultaneous binding of fusion protein to GPC3 and CD137 in ELISA

[0428] To demonstrate that the exemplary fusion protein binds simultaneously to GPC3 and CD137, a dual binding ELISA format was used.

[0429] Recombinant huCD137-His (R&D Systems) (1 μg / mL) in PBS was coated on microtiter plates overnight at 4°C. After each incubation step, the plates were washed 5 times with 100 μL PBS-0.05% T. The plates were blocked with PBS-0.1% T-2% BSA for 1 hour at room temperature and then washed again. Different concentrations of the test fusion protein were added to the wells and incubated for 1 hour at room temperature, followed by a washing step. Subsequently, biotinylated huGPC3 was added at a constant concentration of 1 μg / mL in PBS-0.1% T-2% BSA for 1 hour. After washing, a 1:5000 dilution of ExtrAvidin-HRP (Sigma-Aldrich) in PBS-0.1% T-2% BSA was added to the wells and incubated for 1 h. After additional washing steps, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescent microplate reader.

[0430] Dual binding of the fusion proteins was also tested using a reverse setup, where 1 μg / ml recombinant huGPC3 (R&D Systems) was coated on a microtiter plate and bound fusion protein was detected by adding 5 μg / mL biotinylated huCD137-His.

[0431] Figure 3 Exemplary dual binding data are shown, along with the fitted curve generated by a 1:1 binding sigmoidal binding fit, where EC 50 The value and maximum signal are free parameters, and the slope is fixed to unity. 50 The values ​​are summarized in Table 4. Fusion proteins (SEQ ID NOs: 87 and 82 and CD137Ac1-fusions 1 to 7) showed clear binding signals, indicating that these fusion proteins can simultaneously bind GPC3 and CD137. Most of these fusion proteins can simultaneously bind GPC3 and CD137 at levels comparable to the previously known CD137 / GPC3 bispecific fusion protein SEQ ID NO: 83.

[0432] Table 4: ELISA data for simultaneous target binding of GPC3 and CD37

[0433]

[0434] Example 5: Off-target binding of fusion proteins analyzed by ELISA

[0435] ELISA-based analysis was used to evaluate the off-target binding of fusion proteins to 32 different targets including GPC3, GPC5 and other TNF receptor proteins (Frese et al., MAbs, 2013). The target was coated overnight on a microtiter plate at a concentration of 5 μg / mL in PBS at 4°C. After washing with PBS-0.05% T, the plate was blocked with PBS-0.1% T-2% BSA for 1 hour at room temperature. After washing 5 times with 100 μL PBS-0.05% T, 100 nM or 10 nM of the test fusion protein was added to the well and incubated for 1 hour at room temperature, followed by another washing step. The bound antibodies in the study were detected by incubation with goat anti-human IgG Fc-HRP (Jackson Laboratory) diluted 1:5000 in PBS-0.1% T-2% BSA. After an additional washing step, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and incubated for 1 h. Fluorescence intensity was detected using a fluorescence microplate reader and standardized relative to the signal of a control antibody (SEQ ID NOs: 106 and 107). The standardized signals of each test molecule combined with 32 targets were added to produce the cumulative binding rate of the antibody at a given concentration (i.e., 100 nM or 10 nM). The cumulative binding rates of each test molecule at 100 nM and 10 nM were further added to produce the sum of the cumulative binding rates. For example, when measured at 100 nM or 10 nM, the cumulative binding rate of the control antibody (SEQ ID NOs: 106 and 107) (the fluorescence intensity generated by combining each of the 32 targets was standardized for the control antibody) was 32, and the sum of the cumulative binding rates was 64. The results are shown in Table 5. Higher cumulative binding rates are associated with stronger off-target binding.

[0436] The provided fusion proteins (SEQ ID NOs: 87 and 82) and the GPC3 antibodies and CD137-specific lipocalin muteins (Fc fusions) contained therein (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively) showed no or negligible off-target binding (the sum of cumulative binding rates was <150), whereas the previously known CD137 / GPC3 bispecific fusion protein (SEQ ID NO: 83) showed undesired off-target binding (the sum of cumulative binding rates was >250).

[0437] Table 5: Off-target binding of fusion proteins

[0438]

[0439] Example 6: Flow cytometric analysis of fusion proteins binding to cells expressing CD137 or GPC3

[0440] The target-specific binding of exemplary fusion proteins to cells expressing human CD137 and cells expressing human GPC3 was assessed by flow cytometry.

[0441] CHO cells were stably transfected with human CD137 or mock control using the Flp-In system (Life technologies) according to the manufacturer's instructions. SK-Hep1 cells were stably transfected with human GPC3 or mock control using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Transfected CHO cells were maintained in Ham's F12 medium (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 500μg / ml hygromycin B (Roth). Transfected SK-Hep1 cells were cultured in RPMI 1640+GlutaMAX medium (Gibco) supplemented with 20% fetal bovine serum (Sigma-Aldrich) and 500μg / mL G418 (Gibco). The cells were cultured according to the manufacturer's instructions (37°C, 5% CO 2 atmosphere), and culture cells in cell culture flasks.

[0442] For flow cytometry analysis, the corresponding cell lines were incubated with exemplary fusion proteins (SEQ ID NOs: 87 and 82), GPC3 antibodies or CD137-specific lipocalin muteins (Fc fusions) contained therein (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively), reference CD137 antibodies SEQ ID NOs: 26 and 27 or isotype controls (SEQ ID NOs: 24 and 25), and detected using fluorescently labeled anti-human IgG antibodies in FACS analysis as described below:

[0443] 5×10 4 The cells were incubated in ice-cold PBS containing 5% fetal calf serum (PBS-FCS) for 1 h. A dilution series of the test molecule was added to the cells and incubated on ice for 1 h. The cells were washed twice with PBS and then incubated on ice for 30 min with goat anti-hIgG Alexa647-labeled antibody or goat anti-hIgG Alexa488-labeled antibody. The cells were then washed and analyzed using an iQue flow cytometer (Intellicyte Screener). The average geometric fluorescence signal was plotted and fitted using Graphpad software. The geometric mean of the fluorescence intensity was used to calculate the EC using a four-parameter logistic regression with the bottom fixed to the background. 50 value.

[0444] Figure 4The ability of the test molecules to bind to human GPC3 and CD137 is depicted. The binding affinity (EC 50 ) were in the low nanomolar range, comparable to GPC3 antibodies (SEQ ID NOs: 81 and 82) and CD137-specific lipocalin muteins (SEQ ID NO: 98) or antibodies (SEQ ID NOs: 26 and 27), respectively (summarized in Table 6). However, a previously known CD137 / GPC3 bispecific fusion protein (SEQ ID NO: 83) showed limited dose-dependent binding to GPC3-expressing cells, as its binding curve did not reach a plateau at concentrations as high as 100 nM ( Figure 4 B) No test molecule binds to mock-transfected cells (data not shown).

[0445] Table 6: Binding affinity of fusion proteins to cells expressing GPC3 or CD137

[0446]

[0447]

[0448] Example 7: Binding affinity of fusion protein to GPC3-positive tumor cells

[0449] The binding of the fusion protein to tumor cells (HepG2, Hep3B, MKN-45, and NCI-N87) expressing different levels of GPC3 was evaluated by flow cytometry.

[0450] HepG2 cell line was cultured in Dulbecco's Modified Eagle's medium (DMSO, Pan Biotech) supplemented with 10% fetal bovine serum (Sigma-Aldrich). Hep3B cell line was cultured in Minimum Essential medium (with Earle's salts) (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 2 mM L-glutamine (Gibco). MKN-45 and NCI-N87 cell lines were cultured in RPMI-1640 medium + GlutaMAX (Gibco) supplemented with 20% or 10% fetal bovine serum (Sigma-Aldrich), respectively. Cells were cultured according to the manufacturer's instructions (37°C, 5% CO 2 All tumor cell lines were cultured in cell culture flasks.

[0451] For flow cytometry analysis, tumor cell lines with different GPC3 expression levels (high to moderate expression: HepG2>Hep3B>MKN-45) and the GPC3-negative cell line NCI-N87 were incubated with exemplary fusion proteins (SEQ ID NOs: 87 and 82), GPC3 antibodies or CD137-specific lipocalin muteins (Fc fusion) contained in the fusion proteins (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively), reference CD137 antibodies SEQ ID NOs: 26 and 27 or isotype controls (SEQ ID NOs: 24 and 25), and detection was performed using fluorescently labeled anti-human IgG antibodies as described in Example 6.

[0452] Figure 5 The ability of the fusion proteins (SEQ ID NOs: 87 and 82) to bind to GPC3-positive tumor cells was characterized and the corresponding binding affinities (EC 50 ) are summarized in Table 7. The binding affinity of the fusion protein to GPC3-expressing tumor cells is in the low nanomolar range, comparable to the GPC3 antibodies (SEQ ID NOs: 81 and 82) contained in the fusion protein. The results also showed that the fusion protein did not bind to GPC3-negative tumor cells.

[0453] Table 7: Binding affinity of fusion proteins to GPC3-positive tumor cells

[0454]

[0455]

[0456] Example 8: GPC3-dependent T cell co-stimulation using CD137 bioassay

[0457] The potential of the selected fusion proteins to induce activation of the CD137 signaling pathway in a GPC3-dependent manner was assessed using a commercially available dual stably transfected Jurkat cell line expressing CD137 and luc2 genes (a humanized version of firefly luciferase), wherein the NFκB response element drives luc2 expression. In this bioassay, CD137 engagement causes CD137 intracellular signaling, resulting in NFκB-mediated luminescence.

[0458] Hepatocellular carcinoma cell lines HepG2 and Hep3B expressing high levels of GPC3, gastric cancer cell line MKN-45 expressing moderate levels of GPC3, and GPC3-negative NCI-N87 were cultured as described in Example 7. The day before the assay, the corresponding tumor cells were plated at 6.25×10 3 Cells were plated and incubated at 37°C in a humidified 5% CO2 Stay overnight in the atmosphere.

[0459] The next day, 3.75x10 4 NF-kB-Luc2 / CD137 Jurkat cells were added to each well, followed by the addition of different concentrations (generally ranging from 0.00488 nM to 10 nM) of exemplary fusion proteins (SEQ ID NOs: 87 and 82), GPC3 antibodies or CD137-specific lipocalin muteins (Fc fusions) contained in the fusion proteins (SEQ ID NOs: 81 and 82 and SEQ ID NO: 98, respectively), reference CD137 antibodies SEQ ID NOs: 26 and 27 or isotype controls (SEQ ID NOs: 24 and 25). The plates were covered with a breathable sealing film and incubated at 37°C in a humidified 5% CO 2 After 4 h, 30 μL Bio-Glo TM The reagents were added to each well and the bioluminescent signal was quantified using a photometer (PHERAstar). Four-parameter logistic curve analysis was performed to calculate EC 50 Value (shared below), EC 50 The values ​​are summarized in Table 8. To demonstrate the GPC3-dependency of the fusion protein engagement with CD137, the same experiment was performed in parallel using the highest concentration of the test molecule in the absence of tumor cells. The assay was performed in triplicate.

[0460] Figure 6 Results from a representative experiment are depicted. Figure 6 The data shown in A and 6B demonstrate that the exemplary fusion proteins (SEQ ID NOs: 87 and 82) induced strong CD137-mediated T cell co-stimulation in the presence of tumor cell lines with high GPC3 expression levels. Figure 6 C-6E shows that CD137 activation by the fusion protein is GPC3-dependent, as no activation of NF-kB-Luc2 / CD137 Jurkat cells was detected in the presence of tumor cells with moderate or low GPC3 expression or in the absence of tumor cells expressing GPC3. In contrast, the reference anti-CD137 mAb (SEQ ID NOs: 26 and 27) showed CD137-mediated T cell co-stimulation regardless of the expression level of GPC3 and in the absence of target cells.

[0461] Table 8: Assessment of T cell activation using CD137 bioassay

[0462]

[0463] Example 9: Evaluation of GPC3-dependent T cell activation induced by fusion protein

[0464] T cell activation assay was used to analyze GPC3 target-dependent T cell co-stimulation by fusion proteins. Fusion proteins were applied at different concentrations to anti-CD3 stimulated T cells and co-cultured with human GPC3 transfected or mock-transfected SK-Hep1 cells or GPC3-positive tumor cell line HepG2. IL-2 secretion levels were measured in the supernatant.

[0465] According to the Biochrom protocol, PBMCs from healthy volunteer donors were isolated from the buffy coat by centrifugation through a polysucrose density gradient (Biocoll 1.077 g / mL, Biochrom). T lymphocytes were further purified from PBMCs by magnetic cell sorting using a Pan T cell purification kit (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Purified Pan T cells were resuspended in a buffer consisting of 90% FCS and 10% DMSO, immediately frozen and stored in liquid nitrogen until further use. For the assay, T cells were thawed and placed in a culture medium (RPMI-1640 culture medium + GlutaMAX, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified 5% CO 2 SK-Hep1 and HepG2 cells were cultured as described in Example 6 and Example 7, respectively.

[0466] For each experimental condition, the following procedure was performed in triplicate: Flat-bottom tissue culture plates were pre-coated with 0.25 μg / mL anti-CD3 antibody at 37°C for 2 h and then washed twice with PBS. Human GPC3-transfected or mock-transfected SK-Hep1 cells and GPC3-positive tumor cells HepG2 were treated with 30 μg / ml mitomycin C (Sigma Aldrich) for 30 min to block proliferation. The mitomycin-treated cells were washed twice with PBS and plated at 1.0×10 4 Cells were plated in culture medium and allowed to grow at 37°C in a humidified atmosphere with 5% CO 2 Cells expressing the target that had been previously grown under standard conditions were detached using Accutase (PAA Laboratories) and then resuspended in culture medium.

[0467] In the following days, the plates were washed twice with PBS and 2.5 x 10 4T cells. A dilution series (usually ranging from 0.003 nM to 10 nM) of fusion proteins (SEQ ID NOs: 87 and 82), GPC3 antibodies contained in fusion proteins (SEQ ID NOs: 81 and 82), previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83), GPC3-specific lipocalin mutant proteins (Fc fusion) (SEQ ID NOs: 97), reference CD137 antibodies (SEQ ID NOs: 26 and 27) or isotype controls (SEQ ID NOs: 24 and 25) were added to the corresponding wells. The plate was covered with a breathable sealing film and incubated at 37°C in a humidified 5% CO 2 The cells were incubated in air for 3 days. Subsequently, IL-2 levels in the supernatant were assessed using the Human IL-2 DuoSet Kit (R&D Systems) as described in the following procedure.

[0468] 384-well plates were coated with 1 μg / mL "human IL-2 capture antibody" in PBS for 2 h at room temperature. Subsequently, the wells were washed 5 times with 80 μL PBS-0.05% T. After blocking for 1 h in PBS-0.05% T containing 1% casein (w / w), the supernatant and the IL-2 standards of the concentration series diluted in the culture medium were transferred to the corresponding wells and incubated overnight at 4 ° C. The next day, a mixture of 100 ng / mL goat anti-hIL-2-Bio detection antibody (R&D Systems) and 1 μg / mL Sulfotag-labeled streptavidin (Mesoscale Discovery) in PBS-0.05% T containing 0.5% casein was added and incubated for 1 h at room temperature. After washing, 25 μL of reading buffer (Mesoscale Discovery) was added to each well, and the electrochemiluminescence (ECL) signal obtained was detected by the Mesoscale Discovery reader. Mesoscale Discovery software was used for analysis and quantification.

[0469] The ability of additional previously known CD137 / GPC3 bispecific fusion proteins, SEQ ID NOs: 99 and 27, SEQ ID NO: 83, and SEQ ID NO: 84, to co-stimulate T cell responses was also evaluated using a T cell activation assay in a similar manner. In this experiment, tissue culture plates were pre-coated with 200 μL of 0.25 μg / mL anti-CD3 antibody at 37°C for 1 h and then washed twice with PBS. 1.25×10 4 HepG2 tumor cells were plated and allowed to grow at 37°C in a humidified 5% CO 2The plates were allowed to adhere overnight in an atmosphere and treated with 10 μg / mL mitomycin C for 2 hours at 37°C. The plates were washed twice with PBS and 5×10 4 The plate was covered with a gas-permeable sealing film and incubated at 37°C in a humidified atmosphere with 5% CO 2 The cells were incubated in air for 3 days. Subsequently, the IL-2 concentration in the supernatant was assessed.

[0470] Figure 7 Exemplary data are shown. In the presence of fusion proteins (SEQ ID NOs: 87 and 82), Pan T cells and human GPC3-transfected SK-Hep1 cells ( Figure 7 B) or HepG2 tumor cells expressing GPC-3 ( Figure 7 C) resulted in strong dose-dependent IL-2 secretion, and was stronger than GPC3 antibodies (SEQ ID NOs: 81 and 82), GPC3-specific lipocalin muteins (SEQ ID NO: 129), or previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83), where no or slight increases in IL-2 secretion were observed at high dose levels. When co-cultured with mock-transfected SK-Hep1 cells (GPC3 negative), none of the tested molecules exhibited a dose-dependent increase in IL-2 secretion. The results indicate that T cell activation by the provided fusion proteins is GPC3-dependent.

[0471] Example 10: Evaluation of T cell activation in the presence of tumor cells expressing different levels of GPC3

[0472] Another T cell assay was used to evaluate the ability of the exemplary fusion protein to costimulate T cell activation in a GPC3 target-dependent manner. In the presence of tumor cell lines with different GPC3 expression levels, the fusion protein was applied to anti-CD3 stimulated T cells at different concentrations. The tumor cell lines tested included HepG2, Hep3B, MKN-45 and NCI-N87 (high to moderate expression: HepG2>Hep3B>MKN-45, GPC3 negative: NCI-N87). The IL-2 secretion level in the supernatant was measured.

[0473] PBMCs from healthy volunteer donors were isolated from buffy coats and lymphocytes were purified from the PBMCs as described in Example 9.

[0474] For the assay, T cells were thawed and placed in culture medium (RPMI-1640 medium + GlutaMAX, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco) at 37°C in a humidified atmosphere with 5% CO. 2 In the air for 16 hours.

[0475] For each experimental condition, the following procedure was performed in triplicate: Flat-bottom tissue culture plates were pre-coated with 0.25 μg / mL anti-CD3 antibody at 37°C for 2 h and washed twice with PBS. Tumor cell lines HepG2, Hep3B, MKN-45, or NIC-N87 were treated with 30 μg / ml mitomycin C (Sigma Aldrich) for 30 min to block proliferation. Tumor cells treated with mitomycin were then washed twice with PBS and plated at 8.3×10 3 Cells were plated in culture medium and allowed to grow at 37°C in a humidified atmosphere with 5% CO 2 Target cells that had been previously grown under standard conditions were detached using Accutase (PAA Laboratories) and then resuspended in culture medium.

[0476] The next day, the plates were washed twice with PBS and 1.25 x 10 4 T cells were added to the tumor cells. A dilution series (usually ranging from 0.26 nM to 10 nM) of the exemplary fusion proteins (SEQ ID NOs: 87 and 82), GPC3 antibodies or CD137-specific lipocalin mutant proteins (Fc fusion) contained in the fusion proteins (SEQ ID NOs: 81 and 82 and SEQ ID NOs: 98, respectively), reference CD137 antibodies (SEQ ID NOs: 26 and 27) or isotype controls (SEQ ID NOs: 24 and 25) were added to the corresponding wells. The plate was covered with a gas-permeable sealing film and incubated at 37°C in a humidified 5% CO 2 Incubate in atmosphere for 3 days.

[0477] After 3 days of co-culture, IL-2 levels in the supernatant were assessed as described in Example 9.

[0478] Figure 8Exemplary data are shown. In the presence of fusion protein (SEQ ID NOs: 87 and 82), co-culture of Pan T cells with HepG2 and Hep3B cells expressing high levels of GPC3 resulted in a significant increase in IL-2 secretion compared to hIgG4 isotype control. In addition, co-culture of MKN-45 (GPC3 moderate) or NIC-N87 (GPC3 negative) with fusion protein did not increase IL-2 secretion levels. The data show that the functional activity of the fusion protein measured by its ability to activate T cells or increase IL-2 secretion is GPC3 dependent. In contrast, T cell activation or IL-2 secretion induced by reference to CD137 antibodies (SEQ ID NOs: 26 and 27) is not necessarily GPC3 dependent and difficult to predict.

[0479] Example 11: Evaluation of T cell-mediated cytolysis of GPC3-expressing tumor cells induced by fusion protein

[0480] The ability of fusion protein to activate CD137 costimulatory signaling pathway and induce T cell-mediated cell lysis of tumor cells expressing GPC3 was evaluated by impedance-based T cell killing assay. For this purpose, adherent tumor cells were seeded in the holes of electronic microtiter plates (E plates). The adhesion of cells to gold microelectrodes hindered the flow of current between electrodes. Impedance was measured as a unitless "cell index" parameter. Cell index (CI) increases with the attachment of cells and subsequent proliferation over time. Non-adhesive CD8+ T cells, anti-CD3 antibodies to independent activated T cell antigens, and test molecules of different concentrations were added to HepG2 cells. Its addition itself does not cause impedance changes. If the test molecule co-stimulates cytotoxic T cells by activating the CD137 signaling pathway, the cell lysis of T cell-mediated target cells will increase, and the destruction of adhesive tumor cells by effector cells can be detected as a reduced CI, so that a real-time killing curve can be generated.

[0481] PBMCs from healthy volunteer donors were isolated from buffy coats. CD8+ T cells were isolated from these PBMCs and stored in liquid nitrogen until further use. For the assay, CD8+ T cells were thawed and placed in assay medium consisting of RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO. 2 In the atmosphere for 24h.

[0482] For each experimental condition, the following procedure was performed in triplicate. Tumor cell lines HepG2 (hepatocellular carcinoma cell line, expressing GPC3) or NCI-N87 (gastric carcinoma cell line, negative for GPC3) were plated in test medium in E plates and incubated at 37°C in a humidified atmosphere with 5% CO2 The cells were incubated in air for 24 h to allow them to adhere and proliferate.

[0483] On the second day, anti-CD3 antibodies were added to the tumor cells, followed by a dilution series of exemplary fusion proteins (SEQ ID NOs: 87 and 82), GPC3 antibodies or CD137-specific lipocalin muteins (Fc fusions) contained in fusion proteins (SEQ ID NOs: 81 and 82 and SEQ ID NOs: 98, respectively), reference CD137 antibodies (SEQ ID NOs: 26 and 27), previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83), GPC3-specific lipocalin muteins (Fc fusions) (SEQ ID NO: 97), or isotype controls (SEQ ID NOs: 24 and 25). Resting CD8+ T cells were then added to the tumor cells at a ratio of 5: 1. The plates were covered with a gas-permeable seal and incubated for 3 days, during which impedance was measured regularly.

[0484] CI values ​​were plotted as a function of time using RTCA HT software V1.0.1 (ACEA Biosciences). Standardized CI was calculated as the CI at a given time point (20h, 30h, 40h, 50h, 60h, 70h after effector cell addition) divided by the CI at the standardized time point (first measurement after effector cell addition). For each triplicate, the average standardized CI value and the corresponding SD value were used to calculate the specific killing value using the following formula: 100-(standardized average CI / standardized average CI of the test molecule / target cells and effector cells) x100. Specific killing values ​​were exported to GraphPad Prism v7 and plotted in an XY graph with corresponding SD values ​​for the time interval of interest.

[0485] Fig. 9 The results of an exemplary experiment are depicted. The data show that the fusion proteins SEQ ID NOs: 87 and 82 induce dose-dependent T cell-mediated lysis of HepG2 cells expressing GPC3 ( Fig. 9 A-9C), but no specific lysis was observed in target-negative NCI-N87 cells ( Fig. 9D), demonstrating that fusion protein-induced activation of the CD137 pathway (which leads to T cell-mediated killing) is GPC3-dependent. The provided fusion proteins SEQ ID NOs: 87 and 82 induced higher levels of GPC3-positive target cell killing by cytotoxic T cells compared to equivalent molar amounts of previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NO: 83), GPC3 antibodies (SEQ ID NOs: 81 and 82), or GPC3-specific lipocalin mutant proteins (Fc fusion) (SEQ ID NO: 97) ( Fig. 9 C). In addition, CD137-specific lipocalin mutein (Fc fusion) (SEQ ID NO: 98), reference CD137 antibodies (SEQ ID NOs: 26 and 27), and hIgG4 isotype controls (SEQ ID NOs: 24 and 25) did not induce CD8+ T cell-mediated killing ( Fig. 9 C).

[0486] Additional experiments were performed to demonstrate that the strong T cell-mediated lysis of HepG2 cells of the CD137 / GPC3 bispecific fusion protein (SEQ ID NOs: 87 and 82) depends on its bispecific design rather than just the combination of anti-GPC3 and anti-CD137. The experiment was performed essentially as described above, but all constructs were added at 10 nM and compared with a cocktail of GPC3 antibodies (SEQ ID NOs: 81 and 82) and reference CD137 antibodies (SEQ ID NOs: 26 and 27) and a cocktail of GPC3 antibodies (SEQ ID NOs: 81 and 82) and CD137-specific lipocalin mutant proteins (Fc fusion) (SEQ ID NO: 98). Although strong T cell-mediated lysis of the fusion proteins SEQ ID NOs: 87 and 82 was detected again, the specific lysis mediated by the two mixtures was only of moderate intensity, demonstrating the benefits of this bispecific format for achieving T cell-mediated lysis.

[0487] Example 12: Evaluation of functional in vivo activity in a xenograft mouse model engrafted with human PBMCs

[0488] To investigate the in vivo activity of the provided fusion proteins, immunodeficient NOG mice implanted with human HepG2 tumor cells and human PBMCs were used.

[0489] 4-6 week old NOG mice were injected subcutaneously (sc) with 5×10 6 HepG2 cells. Allow tumors to grow to 80-100 mm 3The size of the tumor was determined and this time point was defined as day 0 of the experiment. On day 0, the mice were randomly divided into treatment groups (or control groups) according to the tumor size and animal weight. 5×10 6 Fresh human PBMCs. After the ivPBMC injection on day 1, mice were treated or controlled (PBS) by intraperitoneal (ip) injection and again treated or controlled on days 8 and 15. The molecules studied included exemplary fusion proteins SEQ ID NOs: 87 and 82 (0.5, 5 or 20 mg / kg), GPC3 antibodies (SEQ ID NOs: 81 and 82) contained in the fusion proteins (0.39 or 3.9 mg / kg, equimolar concentrations with 0.5 or 5 mg / kg treatment with fusion proteins) and reference CD137 antibodies (SEQ ID NOs: 26 and 27) (3.9 mg / kg, equimolar concentrations with 5 mg / kg treatment with fusion proteins). Tumor growth was recorded every 3 to 4 days. Animals without drug exposure were excluded from data analysis.

[0490] At the end of the study (the 16th day), mice were put to death, and tumors were fixed with formalin and embedded in paraffin (FFPE). Histology and immunohistochemical analysis of tumors were carried out at BioSiteHisto. FFPE xenograft tumor tissue sections were sliced ​​and stained with H&E or the staining of T cell markers CD3, CD4 or CD8 was carried out. The 3DHistech Pannoramic MIDI instrument with 20x object lens was used to image and digitize the sheet of staining. CaseViewer 2.2 was used for the microscopy (microscoping) of digital sheets, and Image J was used for the analysis based on tumor area. Calculate the percentage of tumor infiltrating lymphocytes (TIL) accounting for tumor area minus necrotic area.

[0491] Fig.10 The changes in tumor volume measured on days 2, 6, 9, 13, and 16 of the study are reflected. Tumor growth inhibition was achieved by the fusion proteins SEQ ID NOs: 87 and 82 and the GPC3 antibodies (SEQ ID NOs: 81 and 82) contained in the fusion proteins. During the study, both the fusion protein and the GPC3 antibody (at a concentration of 5 or 3.9 mg / kg, respectively) completely inhibited the growth of the tumor. The effects of the fusion protein and the GPC3 antibody were dose-dependent, as lower concentrations (0.5 or 0.39 mg / kg) had little effect on tumor growth. Only limited effects on tumor growth were achieved by treatment with the reference CD137 antibody (SEQ ID NOs: 26 and 27).

[0492] Table 9 summarizes the data obtained by histological and immunohistochemical analysis of tumor infiltrating lymphocytes. Intratumoral CD3, CD4, or CD8 T cell infiltration is shown as % TIL / (tumor area-necrotic area). Treatment with fusion protein resulted in T cell infiltration of up to 10% (tumor area-necrotic area) (all treatment groups). Treatment with GPC3 antibody or reference CD137 antibody did not induce intratumoral T cell infiltration compared to vehicle control.

[0493] Table 9: Percentage of tumor infiltrating lymphocytes (TIL) in the tumor area minus the necrotic area

[0494]

[0495] Example 13: Pharmacokinetics of fusion protein in mice

[0496] Pharmacokinetic analysis of representative fusion proteins (SEQ ID NOs: 87 and 82) and GPC3 antibodies contained in the fusion proteins (SEQ ID NOs: 81 and 82) was performed in mice and compared with the pharmacokinetics of two previously known CD137 / GPC3 bispecific fusion proteins (SEQ ID NOs: 83 and SEQ ID NO: 84). Male CD-1 mice (2 mice per time point; Charles River Laboratories) at approximately 5 weeks of age were injected with the corresponding constructs into the tail vein at a dose of 2 mg / kg (SEQ ID NOs: 87 and 82, SEQ ID NOs: 81 and 82) or 10 mg / kg (SEQ ID NO: 83, SEQ ID NO: 84). For antibody-based constructs, plasma samples from mice were obtained at time points 5min, 24h, 168h and 336h and at 5min, 2h, 4h, 8h, 24h, 2d, 3d, 4d, 7d, 9d, 11d, 14d and 21d. Enough whole blood (collected under isoflurane anesthesia) was collected to obtain at least 30-50 μL of Li-Heparin plasma / animal and time. ELISA was then used to analyze plasma drug levels.

[0497] For SEQ ID NOs: 87 and 82 or SEQ ID NOs: 81 and 82, the following protocol was used: Human GPC3 was dissolved in PBS (1 μg / mL) and coated on microtiter plates overnight at 4°C. After each incubation step, the plates were washed 5 times with 80 μL PBS-0.05% T. The plates were blocked with PBS-0.1% T-2% BSA for 1 hour at room temperature and then washed. Plasma samples were diluted to 20% plasma concentration in PBS-0.1% T-2% BSA, added to the wells and incubated for 1 hour at room temperature. This was followed by another washing step. The bound reagents under study were detected after incubation for 1 hour with 1 μg / mL of SULFO-TAG anti-human antibody (Mesoscale Discovery) or anti-NGAL affinity purified polyclonal antiserum diluted in PBS-0.1% T-2% BSA. After an additional washing step, 25 μL of reading buffer was added to each well and the electrochemiluminescence (ECL) signal of each well was read by a Mesoscale Discovery reader.

[0498] For SEQ ID NO:83 or SEQ ID NO:84, the following protocol was used: human CD137 was dissolved in PBS (1 μg / mL) and coated on a microtiter plate at 4°C overnight. After each incubation step, the plate was washed with 80 μL PBS-0.05% T. The plate was blocked with PBS-0.1% T-2% BSA for 1 hour at room temperature and then washed. The plasma sample was diluted to 20% plasma concentration in PBS-0.1% T-2% BSA, added to the well and incubated for 1 hour at room temperature. This was followed by another washing step. The combined reagents in the study were detected after incubation for 1 hour with 1 μg / mL of human Glypican-bio and streptavidin SULFO-tag diluted in PBS-0.1% T-2% BSA. After an additional washing step, 25 μL of reading buffer was added to each well, and the ECL signal of each well was read by a Mesoscale Discovery reader.

[0499] For data analysis and quantification, calibration curves were also prepared using standard protein dilutions. The changes in plasma concentrations of the test molecules in the exemplary experiment over time are plotted in Figure 12. The data were analyzed non-compartmentally using Phoenix WinNonlin version 8.1, and the results are summarized in Table 10.

[0500] The data showed that the provided fusion proteins SEQ ID NOs: 87 and 82 exhibited typical antibody pharmacokinetics, whereas the previously known CD137 / GPC3 bispecific fusion proteins SEQ ID NO: 83 and SEQ ID NO: 84 exhibited significantly impaired pharmacokinetic characteristics.

[0501] Table 10: Pharmacokinetics in mice

[0502]

[0503] Example 14: Thermal stability assessment of fusion proteins

[0504] To determine the melting temperature (T) of the fusion protein as a general indicator of overall stability m ), using a capillary nanoDSC instrument (CSC 6300, TA Instruments) at 1°C / min scanning (25°C-100°C) of the test molecule at a protein concentration of 1 mg / mL in PBS (Gibco). T was calculated from the thermogram shown using the integral Nano analysis software. m .

[0505] The maximum melting temperatures and melting onset temperatures of the obtained exemplary fusion proteins are listed in the following Table 11. Compared to some previously known CD137 / GPC3 bispecific fusion proteins SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NOs: 26 and 102, the fusion proteins SEQ ID NOs: 72 and 82 and CD137Ac1-Fusion 2 have improved thermal stability.

[0506] Table 11: T determined by nanoDSC m and the onset melting temperature

[0507] Onset of melting [℃] <![CDATA[T m [℃]]]> SEQ ID NOs:87 and 82 60.0 66.6 / 71.1 CD137Ac1-Fusion2 60.0 66.6 / 70.9 SEQ ID NOs:81 and 82 60.0 66.6 / 71.1 SEQ ID NO:83 57.5 67.3 / 71.7 SEQ ID NO:84 59.0 67.6 / 70.7 SEQ ID NOs: 26 and 102 46.0 56.9 / 70.8

[0508] The embodiments described illustratively herein can be appropriately implemented in the absence of any one or more elements, one or more restrictions not specifically disclosed herein. Therefore, for example, the terms "comprise", "include", "contain", etc. should be interpreted broadly and without limitation. In addition, the terms and expressions adopted herein are used as descriptive terms rather than restrictive terms, and there is no intention to exclude any equivalents or parts of the features displayed and described when using these terms and expressions, but to recognize that various modifications can be within the scope of the present invention. Therefore, it should be understood that although the embodiments of the present invention have been specifically disclosed by preferred embodiments and optional features, those skilled in the art can seek their modifications and changes, and such modifications and changes are considered to be within the scope of the present invention. All patents, patent applications, textbooks and peer-reviewed publications described herein are incorporated herein by reference in their entirety. In addition, when the definition or term used in the reference incorporated herein by reference is inconsistent or contrary to the definition of the term provided herein, the definition of the term provided herein is applicable, and the definition of the term in the reference is not applicable. Each narrower category and sub-genus grouping falls within the disclosure scope of the genus and also forms a part of the present invention. This includes the generic description of the invention, with a proviso or negative limitation removing any subject matter from that genus, regardless of whether the removed material is specifically recited herein. In addition, where features are described in terms of Markush groups, one skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of that Markush group. Further embodiments will become apparent from the following claims.

[0509] 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. The following claims are intended to encompass such equivalents. All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this specification as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.

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Sequence Listing <110> Piris Pharmaceuticals Ltd. <120> Novel fusion protein specific for CD137 and GPC3 <130> LC21310011P <150> EP 19000100.8 <151> 2019-02-26 <160> 129 <170> PatentIn version 3.5 <210> 1 <211> 158 <212> PRT <213> Human <400> 1 His His Leu Leu Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr 1 5 10 15 Trp Tyr Leu Lys Ala Met Thr Val Asp Arg Glu Phe Pro Glu Met Asn 20 25 30 Leu Glu Ser Val Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn 35 40 45 Leu Glu Ala Lys Val Thr Met Leu Ile Ser Gly Arg Cys Gln Glu Val 50 55 60 Lys Ala Val Leu Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp 65 70 75 80 Gly Gly Lys His Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His 85 90 95 Tyr Ile Phe Tyr Cys Glu Gly Glu Leu His Gly Lys Pro Val Arg Gly 100 105 110 Val Lys Leu Val Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu 115 120 125 Asp Phe Glu Lys Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile 130 135 140 Leu Ile Pro Arg Gln Ser Glu Thr Cys Ser Pro Gly Ser Asp 145 150 155 <210> 2 <211> 178 <212> PRT <213> human <400> 2 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe Gln Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro 35 40 45 Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile 65 70 75 80 Arg Thr Phe Val Pro Gly Cys Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90 95 Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln 115 120 125 Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 3 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Lipocalin mutant protein <400> 3 Gln Asp Ser Thr Ser Asp Leu Ile Pro Ala Pro Pro Leu Ser Lys Val 1 5 10 15 Pro Leu Gln Gln Asn Phe Gln Asp Asn Gln Phe His Gly Lys Trp Tyr 20 25 30 Val Val Gly Leu Ala Gly Asn Ala Ile Leu Arg Glu Asp Lys Asp Pro 35 40 45 Gln Lys Met Tyr Ala Thr Ile Tyr Glu Leu Lys Glu Asp Lys Ser Tyr 50 55 60 Asn Val Thr Ser Val Leu Phe Arg Lys Lys Lys Cys Asp Tyr Trp Ile 65 70 75 80 Arg Thr Phe Val Pro Gly Ser Gln Pro Gly Glu Phe Thr Leu Gly Asn 85 90 95 Ile Lys Ser Tyr Pro Gly Leu Thr Ser Tyr Leu Val Arg Val Val Ser 100 105 110 Thr Asn Tyr Asn Gln His Ala Met Val Phe Phe Lys Lys Val Ser Gln 115 120 125 Asn Arg Glu Tyr Phe Lys Ile Thr Leu Tyr Gly Arg Thr Lys Glu Leu 130 135 140 Thr Ser Glu Leu Lys Glu Asn Phe Ile Arg Phe Ser Lys Ser Leu Gly 145 150 155 160 Leu Pro Glu Asn His Ile Val Phe Pro Val Pro Ile Asp Gln Cys Ile 165 170 175 Asp Gly <210> 4 <211> 152 <212> PRT <213> Artificial sequence <220> <223> Lipocalin mutant protein <400> 4 Ala Ser Asp Glu Glu Ile Gln Asp Val Ser Gly Thr Trp Tyr Leu Lys 1 5 10 15 Ala Met Thr Val Asp Arg Glu Cys Pro Glu Met Asn Leu Glu Ser Val 20 25 30 Thr Pro Met Thr Leu Thr Thr Leu Glu Gly Gly Asn Leu Glu Ala Lys 35 40 45 Val Thr Met Leu Ile Ser Gly Arg Ser Gln Glu Val Lys Ala Val Leu 50 55 60 Glu Lys Thr Asp Glu Pro Gly Lys Tyr Thr Ala Asp Gly Gly Lys His 65 70 75 80 Val Ala Tyr Ile Ile Arg Ser His Val Lys Asp His Tyr Ile Phe Tyr 85 90 95 Ser Glu Gly Glu Cys His Gly Lys Pro Val Pro Gly Val Trp Leu Val 100 105 110 Gly Arg Asp Pro Lys Asn Asn Leu Glu Ala Leu Glu Asp Phe Glu Lys 115 120 125 Ala Ala Gly Ala Arg Gly Leu Ser Thr Glu Ser Ile Leu Ile Pro Arg 130 135 140 Gln Ser Glu Thr Ser Ser Pro Gly 145 150 <210> 5 <211> 4 <212> PRT <213> Human <400> 5 His His Leu Leu 1 <210> 6 <211> 535 <212> PRT <213> Person <400> 6 Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser 1 5 10 15 Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro 20 25 30 Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys 35 40 45 Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn 50 55 60 Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile 65 70 75 80 Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg 85 90 95 His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser 100 105 110 Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val 115 120 125 Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn 130 135 140 Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn 145 150 155 160 Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly 165 170 175 Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met 180 185 190 Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala 195 200 205 Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe 210 215 220 Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr 225 230 235 240 Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val 245 250 255 Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp 260 265 270 Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg 275 280 285 Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His 290 295 300 Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr 305 310 315 320 Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala 325 330 335 Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala 340 345 350 His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile 355 360 365 Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr 370 375 380 Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn 385 390 395 400 Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly 405 410 415 Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu 420 425 430 Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu 435 440 445 Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu 450 455 460 Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu 465 470 475 480 Cys Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu 485 490 495 Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro 500 505 510 Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe 515 520 525 His Asn Leu Gly Asn Val His 530 535 <210> 7 <211> 232 <212> PRT <213> Human <400> 7 Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn 1 5 10 15 Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser 20 25 30 Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val 35 40 45 Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp 50 55 60 Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu 65 70 75 80 Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp 85 90 95 Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro 100 105 110 Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr 115 120 125 Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro 130 135 140 Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His 145 150 155 160 Ser Pro Gln Ile Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu 165 170 175 Leu Phe Leu Leu Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg 180 185 190 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 195 200 205 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 210 215 220 Glu Glu Glu Gly Gly Cys Glu Leu 225 230 <210> 8 <211> 535 <212> PRT <213> Cynomolgus monkey (Macaca fascicularis) <400> 8 Gln Pro Pro Pro Pro Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser 1 5 10 15 Phe Phe Gln Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro 20 25 30 Val Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys 35 40 45 Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn 50 55 60 Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe Leu Ile 65 70 75 80 Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu Ile Val Val Arg 85 90 95 His Ala Lys Asn Tyr Thr Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser 100 105 110 Leu Thr Pro Gln Ala Phe Glu Phe Val Gly Glu Phe Phe Thr Asp Val 115 120 125 Ser Leu Tyr Ile Leu Gly Ser Asp Ile Asn Val Asp Asp Met Val Asn 130 135 140 Glu Leu Phe Asp Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn 145 150 155 160 Pro Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly 165 170 175 Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met 180 185 190 Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu Gln Ala 195 200 205 Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp His Leu Lys Phe 210 215 220 Ser Lys Asp Cys Gly Arg Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr 225 230 235 240 Cys Gln Gly Leu Met Met Val Lys Pro Cys Gly Gly Tyr Cys Asn Val 245 250 255 Val Met Gln Gly Cys Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp 260 265 270 Arg Glu Tyr Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg 275 280 285 Ile Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His 290 295 300 Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr 305 310 315 320 Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala 325 330 335 Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala 340 345 350 His Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile 355 360 365 Gln Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr 370 375 380 Ile Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn 385 390 395 400 Gly Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly 405 410 415 Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu 420 425 430 Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu 435 440 445 Leu Arg Thr Met Ser Val Pro Lys Gly Arg Val Leu Asp Lys Asn Leu 450 455 460 Asp Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu 465 470 475 480 Cys Ile Gly Gly Ser Gly Asp Gly Met Met Lys Val Lys Asn Gln Leu 485 490 495 Arg Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Val Pro 500 505 510 Gly Asn Asn Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe 515 520 525 His Asn Leu Gly Asn Val His 530 535 <210> 9 <211> 231 <212> PRT <213> Cynomolgus monkey (Macaca fascicularis) <400> 9 Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn 1 5 10 15 Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser 20 25 30 Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val 35 40 45 Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp 50 55 60 Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys Ser Met Cys Glu 65 70 75 80 Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp 85 90 95 Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro 100 105 110 Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr 115 120 125 Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro 130 135 140 Gly Ala Ser Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His 145 150 155 160 Ser Pro Gln Ile Ile Phe Phe Leu Ala Leu Thr Ser Thr Val Val Leu 165 170 175 Phe Leu Leu Phe Phe Leu Val Leu Arg Phe Ser Val Val Lys Arg Ser 180 185 190 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 195 200 205 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 210 215 220 Glu Glu Gly Gly Cys Glu Leu 225 230 <210> 10 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Cleavage site <400> 10 Ile Glu Gly Arg 1 <210> 11 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Fusion peptide <400> 11 Ser Ala Trp Ser His Pro Gln Phe Glu Lys 1 5 10 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Strep-Tag II Peptide <400> 12 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 13 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 13 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 14 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 14 Pro Ser Thr Pro Pro Thr Asn Ser Ser Ser Thr Pro Pro Thr Pro Ser 1 5 10 15 Pro Ser <210> 15 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 15 Gly Gly Ser Gly Asn Ser Ser Gly Ser Gly Gly Ser Pro Val 1 5 10 <210> 16 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 16 Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala Ala Pro Ala Pro 1 5 10 15 Ser Ala Pro Ala 20 <210> 17 <211> 66 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 17 Ala Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Pro Val Pro Ser 1 5 10 15 Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser 20 25 30 Gly Gly Ser Gly Asn Ser Ser Gly Ser Gly Gly Ser Pro Val Pro Ser 35 40 45 Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser 50 55 60 Ala Ser 65 <210> 18 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 18 Pro Ser Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr Pro Ser 1 5 10 15 Pro Ser Gly Gly Ser Gly Asn Ser Ser Gly Ser Gly Gly Ser Pro Val 20 25 30 <210> 19 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 19 Ala Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Pro Val Pro Ser 1 5 10 15 Thr Pro Pro Thr Asn Ser Ser Ser Thr Pro Pro Thr Pro Ser Pro Ser 20 25 30 Pro Val Pro Ser Thr Pro Pro Thr Asn Ser Ser Ser Thr Pro Pro Thr 35 40 45 Pro Ser Pro Ser Pro Val Pro Ser Thr Pro Pro Thr Asn Ser Ser Ser 50 55 60 Thr Pro Pro Thr Pro Ser Pro Ser Ala Ser 65 70 <210> 20 <211> 40 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 20 Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala Ala Pro Ala Pro 1 5 10 15 Ser Ala Pro Ala Ala Ser Pro Ala Ala Pro Ala Pro Ala Ser Pro Ala 20 25 30 Ala Pro Ala Pro Ser Ala Pro Ala 35 40 <210> twenty one <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker <400> twenty one Val Asp Asp Ile Glu Gly Arg Met Asp Glu 1 5 10 <210> twenty two <211> 11 <212> PRT <213> Artificial sequence <220> <223> Linker <400> twenty two Glu Asn Leu Tyr Phe Gln Gly Arg Met Asp Glu 1 5 10 <210> twenty three <211> 10 <212> PRT <213> Artificial sequence <220> <223> Linker <400> twenty three Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> twenty four <211> 327 <212> PRT <213> Artificial sequence <220> <223> Antibody heavy chain <400> twenty four Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 25 <211> 214 <212> PRT <213> artificial sequence <220> <223> Antibodies <400> 25 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 26 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Antibody Heavy Chain <400> 26 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly 100 105 110 Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 27 <211> 216 <212> PRT <213> artificial sequence <220> <223> Antibody <400> 27 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 28 <211> 228 <212> PRT <213> artificial sequence <220> <223> AntibodyFc district <400> 28 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly 225 <210> 29 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> Antibody Fc Region <400> 29 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly 225 <210> 30 <211> 228 <212> PRT <213> Artificial Sequence <220> <223> Antibody Fc Region <400> 30 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly 225 <210> 31 <211> 228 <212> PRT <213> Artificial sequence <220> <223> Antibody Fc region <400> 31 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln ...

Claims

1. A fusion protein capable of binding to cluster of differentiation (CD137) and glypican-3 (GPC3), wherein the fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin and is specific for GPC3, and wherein the second subunit comprises a lipocalin mutant protein and is specific for CD137, wherein the second subunit is linked to the C-terminus of the first subunit at the N-terminus via a peptide linker, and wherein the fusion protein comprises two chains of SEQ ID NOs: 87 and 82.

2. The fusion protein according to claim 1, (a) wherein the fusion protein is capable of binding to D value for binding to GPC3 or being able to bind to the immunoglobulin contained in the first subunit alone with K D K with equivalent or lower value D Value binding GPC3; (b) wherein the fusion protein is capable of activating an EC of at most 3 nM 50 The value of binding to CD137 or being able to bind to the CD137-specific lipocalin mutant protein contained in the second subunit alone EC 50 Equivalent or lower EC 50 Value binding CD137; (c) wherein the fusion protein cross-reacts with macaque GPC3; (d) wherein the fusion protein is capable of quantifying the expression of the fusion protein at an EC of at most 10 nM when the fusion protein is measured in an ELISA assay. 50 Values ​​for simultaneous binding of CD137 and GPC3; (e) wherein the fusion protein is capable of binding to tumor cells expressing GPC3; (f) wherein the fusion protein is capable of inducing increased secretion of IL-2; (g) wherein the fusion protein is capable of inducing increased secretion of IL-2 to a higher level compared to SEQ ID NO: 83; and / or the fusion protein is capable of inducing increased secretion of IL-2 with better efficiency compared to SEQ ID NO: 83; (h) wherein the fusion protein is capable of inducing lymphocyte-mediated cytotoxicity; (i) wherein the fusion protein is capable of inducing enhanced T cell-mediated killing of tumor cells expressing GPC3 compared to SEQ ID NO: 83; and / or the fusion protein is capable of inducing cytotoxic T cell activation with better efficiency compared to SEQ ID NO: 83; (j) wherein the fusion protein is capable of costimulating a T cell response in a GPC3-dependent manner; or (k) wherein the fusion protein is capable of co-stimulating T cell responses in the tumor microenvironment.

3. The fusion protein of claim 1 or 2, wherein the fusion protein has a half-life in mice of at least 50 hours, at least 75 hours, at least 100 hours, at least 125 hours, at least 150 hours, at least 175 hours, at least 200 hours, at least 250 hours, or even longer, and / or wherein the fusion protein has a half-life in mice that is longer than the half-life of SEQ ID NO:

83.

4. The fusion protein of claim 1, wherein the fusion protein has an isoelectric point of at least 6.5, at least 6.8, at least 7.1, at least 7.4, at least 7.5, at least 7.7 or even higher, and / or wherein the fusion protein has an isoelectric point higher than the isoelectric point of SEQ ID NO:

83. The fusion protein according to claim 1 , wherein the N-terminus of the second subunit is connected to the C-terminus of the heavy chain constant region (CH) of the first subunit via a linker.

6. The fusion protein of claim 1, wherein the first subunit comprises a group of the following CDR sequences: GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), TRFYSYTY (HCDR3; SEQ ID NO: 74), and a group of the following CDR sequences: QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ ID NO: 77). The fusion protein of claim 1 , wherein the antibody has an IgG4 backbone.

8. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 7.

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

10. A pharmaceutical composition comprising one or more fusion proteins according to any one of claims 1 to 7.

11. Use of the fusion protein according to any one of claims 1 to 7 or one or more compositions comprising the fusion protein in the preparation of a medicament for preventing, improving or treating GPC3-positive liver cancer.

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