Novel fusion protein specific to CD137 and GPC3

Abstract

The present disclosure provides a fusion protein specific to both CD137 and GPC3, said fusion protein can be used to co-stimulate lymphocyte activation in a GPC3-target-dependent manner. Such fusion protein can be used for many pharmaceutical applications, for example, as an anticancer agent and / or immunomodulator for the treatment or prevention of human diseases such as various tumors. The present disclosure also relates to the fusion protein described herein and a method for preparing a composition comprising such fusion protein. The present disclosure also relates to a chromatic acid molecule encoding such fusion protein and a method for producing such fusion protein and nucleic acid molecules. Furthermore, the present application discloses the therapeutic and / or diagnostic use of such fusion protein and a composition comprising one or more such fusion proteins.

Description

Technology Field

[0001] The present disclosure provides, among other things, a novel approach to simultaneously bind CD137 and GPC3 through one or more fusion proteins having binding specificity for CD137 and binding specificity for GPC3. Background Technology

[0002] Glypican-3 (GPC3, also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, and SGBS1) is an oncofetal antigen belonging to the glypican family of glycosyl-phosphatidylinositol-fixed heparin sulfate proteoglycans. GPC3 is expressed in various tissues during development and plays an important role in morphogenesis and growth, for example, through fibroblast growth factor (FGF), noncanonical Wnt, or insulin-like growth factor signaling pathways (Cheng et al., Carcinogenesis , 2008, Song et al., J Biol Chem , 2005, Song et al., J Biol Chem , 1997). However, GPC3 expression is mostly downregulated or silenced in normal adult tissues. Additionally, GPC3 can positively and negatively regulate cell growth depending on the cell type. Loss-of-function mutations in GPC3 are the cause of the rare X-chromosome-linked growth disorder, Simpson-Golabi-Behmel or Simpson dysmorphia syndrome (SGBS), and patients with SGBS have an increased risk of embryonic tumors, including Wilms' tumor (Pilia et al., Nat Genet , 1996).

[0003] 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), the major form of liver cancer accounting for 90% of all liver cancers (Capurro et al., Gastroenterology , 2003, Nakatsura et al., Biochem Biophys Res Commun , 2003, Sung et al., Cancer Sci , 2003, Zhu et al., Gut (, 2001), causes at least 500,000 deaths annually (Jelic et al., Ann OncolExtensive research is being conducted on GPC3 as a diagnostic biomarker and therapeutic target for HCC and other cancer types. Although several antibodies targeting GPC3 have been generated, including humanized mouse antibodies YP7 and GC33 and human antibodies HN3 and MDX-1414, they have not demonstrated the ability to inhibit HCC cell proliferation or induce apoptosis (Feng and Ho, FEBS Lett , 2014). Exceptionally, GC33 is currently being evaluated in clinical trials for HCC. While the mechanism of GC33 function includes antibody-dependent cytotoxicity (ADCC), 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, GC33 is in vivo ( in vivo Although it appeared to inhibit tumor growth, it did not provide satisfactory clinical efficacy. Meanwhile, knock-down and siRNA test results suggest that GPC3 is not a lethal gene to HCC cells, so it is uncertain whether the anti-GPC3 antibody can efficiently induce tumor regression.

[0004] Cluster of differentiation 137, or CD137 (also known as 4-1BB or TNFRS9), is a member of the helper-stimulating immune receptor and tumor necrosis factor receptor (TNFR) superfamily. It is primarily expressed in activated CD4+ and CD8+ T cells, activated B cells, and natural killer (NK) cells, but also in quiescent mononuclear cells and dendritic cells (Li and Liu, Clin Pharmacol , 2013), or endothelial cells (Snell et al., Immunol Rev It can also be found in ( , 2011). CD137 plays an important role in regulating immune responses, making it a target for cancer immunotherapy. The CD137 ligand (CD137L) is the only known natural ligand of CD137, is constitutively expressed in several types of antigen-presenting cells such as activated B cells, monocytes, and splenic dendritic cells, and can be induced on T lymphocytes.

[0005] CD137L is a trimer protein that exists in membrane-bound and soluble variants. However, the ability of soluble CD137L to activate CD137, for example in CD137-expressing lymphocytes, is limited, and high concentrations are required to elicit an effect (Wyzgol et al., J Immunol , 2009). The natural mechanism of CD137 activation is through the binding of CD137-positive cells and CD137L-positive cells. Subsequently, CD137 activation is induced by clustering via CD137L in the opposite cells, leading to signaling via TRAF1, 2, and 3 (Yao et al., Nat Rev Drug Discov , 2013, Snell et al., Immunol RevIt is thought to lead to additional accompanying downstream effects in CD137-positive T cells (2011). For T cells activated by the recognition of their respective cognate targets, the effects induced by co-stimulation of CD137 are enhanced activation, enhanced survival and proliferation, production of pro-inflammatory cytokines, and improved apoptosis.

[0006] The benefits of CD137 co-stimulation for cancer cell elimination have been demonstrated in various in vivo models. Forced expression of CD137L in tumors induces, for example, tumor rejection (Melero et al., Eur J Immunol , 1998). Likewise, forced expression of anti-CD137 scFv in tumors is the tumor's CD4 + Induces T-cell and NK-cell-dependent clearance (Yang et al., Cancer Res , 2007, Zhang et al., Mol Cancer Ther , 2006, Ye et al., Nat Med , 2002). Systemically administered anti-CD137 antibodies have also been shown to induce a delay in tumor growth (Martinet et al., Gene Ther , 2002).

[0007] CD137 is an excellent marker for tumor-responsive T cells that occur naturally in human tumors (Ye et al., Clin Cancer Res (, 2014), anti-CD137 antibodies are CD8 for application in selected T-cell therapies + It has been revealed that it can be used to improve the expansion and activity of melanoma tumor-infiltrating lymphocytes (Chacon et al., PLoS One , 2013).

[0008] Preclinical demonstration of the potential therapeutic benefits of CD137 co-stimulation has facilitated the development of therapeutic antibodies targeting CD137, including BMS-663513 (described in U.S. Patent No. 7,288,638) and PF-05082566 (Fisher et al., Cancer Immunol Immunother , 2012).

[0009] The present disclosure provides, among other things, a novel approach to simultaneously bind CD137 and GPC3 through one or more fusion proteins having binding specificity for CD137 and binding specificity for GPC3. The problem to be solved

[0010] The present disclosure provides, among other things, a novel approach to simultaneously bind CD137 and GPC3 through one or more fusion proteins having binding specificity for CD137 and binding specificity for GPC3. means of solving the problem

[0011] definition

[0012] The following list defines terms, phrases, and abbreviations used throughout this specification. All terms listed and defined herein are intended to include all grammatical forms.

[0013] As used herein, unless otherwise specified, “D137” means human CD137 (huCD137). Human CD137 means the full-length protein, a fragment thereof, or a variant thereof as defined by UniProt Q07011. CD137 is also known as 4-1BB, tumor necrosis factor receptor superfamily member 9 (TNFRSF9), and ILA (induced by lymphocyte activation). In some specific embodiments, CD137 of non-human species, e.g., cyanomolgus CD137 and mouse CD137, is used.

[0014] As used herein, unless otherwise specified, “Glypican-3” or “GPC3” means human GPC3 (huGPC3). Human GPC3 means the full-length protein, a fragment thereof, or a variant thereof as defined by UniProt P51654. Human GPC3 GPC3 It is encoded by a gene. GPC3 is also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS, or SGBS1. In some specific embodiments, GPC3 from non-human species, such as cyanomolus GPC3 and mouse GPC3, is used.

[0015] As used herein, “binding affinity” refers to the ability of a biomolecule of the disclosure (e.g., polypeptide or protein) (e.g., lipocalin mutain, antibody, fusion protein, or any other peptide or protein) to bind to a selected target and form a complex. Binding affinity is measured by various methods known to those skilled in the art, including, but not limited to, fluorescence titration, enzyme-linked immunosorbent assay (ELISA)-based assays including direct and competitive ELISA, calorimetry such as isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). These methods are well established in the art, and some examples of such methods are further described herein. Accordingly, binding affinity is the dissociation constant (K) measured using these methods. D ), half maximum effective concentration (EC 50 ), or half maximum inhibitory concentration (IC10). 50 It is reported as a value of ). Lower K D , EC 50 , or IC 50 The value reflects better (higher) binding ability (affinity). Therefore, the binding affinity of two biomolecules for a selected target can be measured and compared. When comparing the binding affinity of two biomolecules for a selected target, the terms "nearly identical," "substantially identical," or "substantially similar" refer to K where one biomolecule is identical or similar to another molecule within the experimental variability of binding affinity measurements. D , EC 50 , or IC 50 It means having a binding affinity reported as a value. Experimental variability in binding affinity measurements depends on the specific method used and is known to those skilled in the art.

[0016] As used herein, the term “substantially” may also refer to a qualitative state representing the whole or nearly whole range or degree of a feature or characteristic of interest. A person skilled in the art of biology will understand that biological and chemical phenomena, even if such cases exist, rarely complete, reach completion, achieve, or avoid absolute results. Accordingly, the term “substantially” is used herein to express the potential lack of completeness inherent in many biological and chemical phenomena.

[0017] As used herein, the terms “detect,” “detect,” “detectable,” or “detecting” are understood to refer to quantitative and qualitative levels, as well as combinations thereof. Accordingly, this includes quantitative, semi-quantitative, and qualitative measurements performed on the biomolecules of the present disclosure.

[0018] As used herein, "detectable affinity" generally refers to K D , EC 50 , or IC 50 The binding ability between the biomolecule and its target, reported as a value, is up to approximately 10 -5 It means that it is less than or equal to M. K D , EC 50 , or IC 50 10 reported as a value -5 Binding affinity exceeding M is generally not measurable by conventional methods such as ELISA and SPR, so it is relatively insignificant.

[0019] It is noted that the complex formation between the biomolecule of the present disclosure and its target is influenced by many different factors, such as the concentration of each target, the presence of competitors, pH and the ionic force of the buffer system used, the experimental method used to measure binding affinity (e.g., fluorescence titration, competitive ELISA (also referred to as competitive ELISA), and surface plasmon resonance), and even the mathematical algorithm used to evaluate the experimental data. Therefore, K D , EC 50 , or IC 50 It is evident to those skilled in the art that the binding affinity reported as a value may vary within a specific experimental range depending on the method and experimental setup. This is because the measured K D , EC 50 , or IC 50 This means that there may be a slight deviation in the values ​​or, for example, a margin of error depending on whether such values ​​are measured by ELISA (direct or competitive ELISA), SPR, or other methods.

[0020]

[0001] As used herein, “specific,” “specific binding,” “to bind specifically,” or “binding specificity” refers to the ability of a biomolecule to distinguish between a desired target (e.g., CD137 and GPC3) and one or more reference targets (e.g., cell receptors for neutrophil gelatinase-associated lipocalin). Such specificity is understood to be a relative rather than absolute characteristic and can be measured, for example, by SPR, Western blot, ELISA, fluorescence activated cell sorting (FACS), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), immunohistochemistry (IHC), and peptide scan.

[0021] As used herein in relation to the fusion protein of the present disclosure that binds to CD137 and GPC3, the terms “specific,” “specific binding,” “to bind specifically,” or “binding specificity” mean that, as described herein, the fusion protein binds to, reacts with, or relates to CD137 and GPC3, but does not bind to any other protein. The term “other protein” includes any protein other than CD137 or GPC3, or a protein closely related to or homologous to CD137 or GPC3. However, CD137 or GPC3 and fragments and / or variants of CD137 or GPC3 from non-human species are not excluded from the term “other protein.” The term “essentially not binding” means that the fusion protein of the present disclosure binds to other proteins with a lower binding affinity than CD137 and / or GPC3, i.e., exhibits cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably 9, 8, 7, 6, or 5%. Whether the fusion protein specifically binds as defined herein can be easily tested, among other things, by comparing the reaction of the fusion protein of the present disclosure with CD137 and / or GPC3 and the reaction of said fusion protein with other protein(s).

[0022] As used herein, the term “lipocalin” refers to a monomeric protein of approximately 18-20 kDa weight having a supersecondary structural region of a cylindrical β-pleated sheet comprising a plurality of β-strands (preferably eight β-strands designated as A to H) that are paired at one end by a plurality of (preferably four) loops to contain a ligand-binding pocket and define an entrance to the ligand-binding pocket. Preferably, the loop containing the ligand-binding pocket used in the present invention is a loop connecting the open ends of β-strands A and B, C and D, E and F, and G and H, and is designated as loops AB, CD, EF, and GH. It is well established that the diversity of the loops in other robust lipocalin scaffolds generates various different binding modes among members of the lipocalin family capable of accommodating targets of different sizes, shapes, and chemical properties (e.g., reviewed in Skerra, Biochim Biophys Acta, 2000; Flower et al., Biochim Biophys Acta, 2000; Flower, Biochem J, 1996). The lipocalin family of proteins is understood to have evolved naturally to possess highly conserved whole-folding patterns while sharing very low levels of whole-sequence conservation (often having less than 20% sequence identity) to bind to a wide range of ligands. Correspondences between positions in various lipocals are also well known to those skilled in the art (e.g., see U.S. Patent No. 7,250,297).Proteins falling under the definition of "lipocalin" as used herein include tear lipocalin (Tlc, Lcn1), lipocalin-2 (Lcn2) or neutrophil gelatinase-associated lipocalin (NGAL), apolipoprotein D (ApoD), apolipoprotein M, α1-acid glycoprotein 1, α1-acid glycoprotein 2, α1-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. It includes, but is not limited to, human lipocalin containing synthase.

[0023] As used herein, unless otherwise specified, “tear lipocalin” refers to human tear lipocalin (hTlc) and additionally refers to mature human tear lipocalin. The term “mature” used to characterize a protein essentially means a protein released from a signal peptide. “Mature hTlc” in this disclosure refers to the mature form of human tear lipocalin released from a signal peptide. Mature hTlc is described by sequence residues 19-176, deposited in the SWISS-PROT Data Bank under accession number P31025, and its amino acids are indicated in SEQ ID NO: 1.

[0024] As used herein, “lipocalin-” or “neutrophil gelatinase-associated lipocalin” refers to human lipocalin-2 (hLcn2) or human neutrophil gelatinase-associated lipocalin (hNGAL) and additionally refers to mature human lipocalin-2 or mature human neutrophil gelatinase-associated lipocalin. The term “mature” used to characterize a protein essentially means a protein released from a signal peptide. “Mature hNGAL” in this disclosure refers to a mature form of human neutrophil gelatinase-associated lipocalin released from a signal peptide. Mature hNGAL is described by sequence residues 21-198, deposited in the SWISS-PROT Data Bank under accession number P80188, and its amino acids are indicated in SEQ ID NO: 2.

[0025] As used herein, "natural sequence" refers to a protein or polypeptide having a sequence occurring in nature or a wild-type sequence, regardless of the method of its production. Such natural sequence proteins or polypeptides may be isolated from nature or produced by other means, such as by recombinant or synthetic methods.

[0026] "Natural sequence lipocalin" refers to lipocalin having the same amino acid sequence as the corresponding polypeptide derived from nature. Thus, natural sequence lipocalin may have the amino acid sequence of naturally occurring (wild-type) lipocalin from any organism, particularly mammals. When the term "natural sequence" is used in the context of lipocalin, it includes naturally occurring variant forms of lipocalin, such as naturally occurring truncated or secreted forms, alternatively spliced ​​forms, and naturally occurring allelic variants of lipocalin. The terms "natural sequence lipocalin" and "wild-type lipocalin" are used interchangeably herein.

[0027] As used herein, “mutane,” “mutated” entity (protein or nucleic acid), or “mutant” refers to the 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 mutain as described herein. The present disclosure explicitly comprises a lipocalin mutain having a ligand-binding pocket comprising eight β-strands paired by four loops at one end as described herein, and a supersecondary structural region of a cylindrical β-sheet structure defining the entrance of the ligand-binding pocket, wherein, compared to the natural sequence lipocalin, at least one amino acid of each of at least three of the four loops is mutated. The lipocalin mutain of the present invention preferably has the function of binding to CD137 as described herein.

[0028] As used herein, the term “fragment” refers to a protein or polypeptide derived from the full length of mature hT1c or hNGAL or lipocalin mutain with the N-terminus and / or C-terminus truncated, i.e., lacking at least one N-terminus and / or C-terminus amino acid, in relation to the lipocalin mutain of the present disclosure. Such a fragment may comprise at least 10, e.g. 20 or 30, consecutive amino acids of the initial sequence of the mature hT1c or hNGAL or lipocalin mutain from which it is derived and may be detected in an immunoassay of mature hT1c or hNGAL. Such a fragment may be deficient in up to 2, 3, 4, 5, 10, 15, 20, 25, or 30 N-terminus and / or C-terminus amino acids (including all numbers in between). As an illustrative example, such a fragment may be deficient in the 1, 2, 3, or 4 N-terminal (His-His-Leu-Leu) and / or 1 or 2 C-terminal amino acids (Ser-Asp) of mature hT1c. The fragment is preferably understood to be a functional fragment of the mature hT1c or hNGAL or lipocalin mutain from which it is derived, which means preferably possessing binding specificity with CD137 of the mature hT1c / hNGAL or lipocalin mutain from which it is derived. As an illustrative example, such a functional fragment may contain at least amino acids at positions 5-153, 5-150, 9-148, 12-140, 20-135, or 26-133 corresponding to the linear polypeptide sequence of mature hT1c. As another explanatory example, such a functional fragment may contain at least 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.

[0029] The “fragment” related to the corresponding target CD137 or GPC3 of the fusion protein of the present disclosure refers to a protein domain of CD137 or GPC3 or CD137 or GPC3 with the N-terminus and / or C-terminus cleaved. A fragment of CD137 or a fragment of GPC3 as described herein possesses the ability of full-length CD137 or GPC3 to be recognized and / or bound by the fusion protein of the present disclosure. As an illustrative example, the fragment may be an extracellular domain of CD137 or GPC3. As an illustrative example, such an extracellular domain may comprise the amino acid sequences of the extracellular subdomains 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), each or bound.

[0030] As used herein, the term “variant” relates to a derivative of a protein or polypeptide comprising a mutation resulting from, for example, substitution, deletion, insertion, and / or chemical modification of an amino acid sequence or nucleotide sequence. In one embodiment, such mutation and / or chemical modification does not reduce the functionality of the protein or peptide. Such substitution may be conservative, that is, an amino acid residue is substituted with a chemically similar amino acid residue. Examples of conservative substitutions are substitutions among members of the following groups: 1) alanine, serine, threonine, and valine; 2) aspartic acid, glutamic acid, glutamine, asparagine, and histidine; 3) arginine, lysine, glutamine, asparagine, and histidine; 4) isoleucine, leucine, methionine, valine, alanine, phenylalanine, threonine, and proline; and 5) isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Such variants comprise proteins or polypeptides in which one or more amino acids are substituted by their respective D-isomers or by amino acids, e.g., ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline, in addition to the 20 naturally occurring amino acids. Such variants also comprise proteins or polypeptides in which one or more amino acid residues are added or deleted at the N- and / or C-terminus, for example. Generally, the 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 natural sequence protein or polypeptide. The variant preferably retains the biological activity of the protein or polypeptide from which it is derived, for example, binding to the same target.

[0031] With respect to the protein ligand CD137 or GPC3 of the fusion protein of the present disclosure, the term “variant” as used herein relates to CD137 or GPC3 or each fragment thereof having one or more, e.g. 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 natural sequence of CD137 or GPC3 (wild-type CD137 or GPC3), e.g., CD137 deposited in UniProt Q07011 or GPC3 deposited in UniProt P51654 as described herein. Each CD137 variant or GPC3 variant preferably has at least 50%, 60%, 70%, 80%, 85%, 90%, or 95% amino acid identity with wild-type CD137 or GPC3. The CD137 variant or GPC3 variant as described herein has the ability to bind to the fusion protein specific to CD137 and GPC3 disclosed in the present invention.

[0032] With respect to lipocalin mutain as used herein, the term “variant” relates to the lipocalin mutain of this disclosure or a fragment thereof, the sequence having mutations and / or chemical modifications including substitutions, deletions, and insertions. Variants of lipocalin mutain as described herein retain the biological activity of the lipocalin mutain from which they are derived, e.g., binding to CD137. Generally, lipocalin mutain variants have at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, and 98% amino acid sequence identity with the lipocalin mutain from which they are derived.

[0033] As used herein, the term “mutagenesis” refers to the introduction of a mutation into a polynucleotide or amino acid sequence. The mutation is preferably introduced under experimental conditions so that a naturally occurring amino acid at a given position in a protein or polypeptide sequence may be altered, for example, by substitution by at least one amino acid. The term “mutagenesis” also includes (additional) alteration of the length of a sequence segment by deletion or insertion of one or more amino acids. Thus, for example, the replacement of one amino acid in a selected sequence with a three-amino acid stretch, thereby adding two amino acid residues compared to the length of each segment of the natural protein or polypeptide amino acid sequence, is within the scope of this disclosure. Such insertions or deletions may be introduced independently of each other in any sequence segment to which mutagenesis may be applied in this disclosure. In one exemplary embodiment of the present disclosure, an insertion may be introduced into an amino acid sequence segment corresponding to loop AB of natural lipocalin (see International Patent Publication No. WO2005 / 019256, the whole of which is incorporated herein by reference).

[0034] As used herein, the term “random mutagenesis” means that a predetermined mutation (change in amino acid) is not present at a specific sequence position, but at least two amino acids may be incorporated at a predetermined sequence with a certain probability during mutagenesis.

[0035] As used herein, the terms “sequence identity” or “identity” refer to characteristics of sequences that measure their similarity or relationship. As used in this disclosure, the terms “sequence identity” or “identity” refer to the ratio of paired identical residues to the number of longer residues of the two sequences after aligning the sequence of the protein or polypeptide of this disclosure with the corresponding sequence (homologous). 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.

[0036] As used herein, the term “sequence homology” or “homology” has its general meaning, and homologous amino acids include not only the same amino acid but also amino acids considered to be conservative substitutions at equivalent positions in the linear amino acid sequence of the protein or polypeptide of the present disclosure (e.g., any fusion protein or lipocalin mutain of the present disclosure).

[0037] Those skilled in the art may use available computer programs, for example, BLAST for measuring sequence homology or sequence identity using standard parameters (Altschul et al., Nucleic Acids Res , 1997), BLAST2 (Altschul et al., J Mol Biol, 1990) and Smith-Waterman (Smith and Waterman, J Mol Biol We know of the ratio of sequence homology or sequence identity, for example, the program BLASTP, version 2.2.5 (November 16, 2002; (Altschul et al., Nucleic Acids ResIt can be measured in this specification using (, 1997). In this embodiment, the ratio of homology is preferably based on the alignment of whole protein or polypeptide sequences comprising a propeptide sequence using a wild-type protein scaffold as a reference in pairwise comparison (Matrix: BLOSUM 62; Gap Cost: 11.1; Cutoff Value: 10 - 3 It is calculated as the ratio of the number of "positive" (homologous amino acids) that appear when the BLASTP program output result is divided by the total number of amino acids selected by the alignment program.

[0038] Specifically, to determine whether amino acid residues in the amino acid sequence of lipocalin mutain differ from wild-type lipocalin corresponding to specific positions in the amino acid sequence of wild-type lipocalin, a person skilled in the art may use means and methods well known in the art, e.g., manual alignment or alignment using computer programs such as BLAST 2.0 (Basic Local Alignment Search Tool) or ClustalW, or any other suitable program suitable for generating sequence alignment. Accordingly, the wild-type sequence of lipocalin may be provided as the "target sequence" or "reference sequence," and the amino acid sequence of lipocalin mutain that differs from the wild-type lipocalin described herein may be provided as the "query sequence." The terms "wild-type sequence," "reference sequence," and "target sequence" are used interchangeably herein. A preferred wild-type sequence of lipocalin is hTLc as shown in SEQ ID NO: 1 or hNGAL as shown in SEQ ID NO: 2.

[0039] A "gap" is a space resulting from the addition or deletion of amino acids during alignment. Therefore, two copies of exactly the same sequence have 100% identity, but sequences that are less conserved and have deletions, additions, or substitutions may have a lower degree of sequence identity.

[0040] As used herein, the term “position” means either the position of an amino acid within the amino acid sequence described herein or the position of a nucleotide within the nucleic acid sequence described herein. In the context of the amino acid sequence of one or more lipocalin mutains, as used herein, the term “corresponding” or “corresponding” should be understood as not being measured solely by the number of preceding nucleotides or amino acids. Accordingly, the absolute position of a given amino acid according to the present disclosure may differ from the corresponding position due to a deletion or addition of an amino acid elsewhere in the (mutant or wild-type) lipocalin. Similarly, the absolute position of a given nucleotide according to the present disclosure may differ from the corresponding position due to a deletion or addition of a nucleotide elsewhere in the 5’-untranslated region (UTR) of the mutain or wild-type lipocalin containing the promoter and / or any other regulatory sequence or gene (including exons and introns).

[0041] A “corresponding position” according to the present disclosure may be a sequence position that aligns to a corresponding sequence position in a pair or multiple sequence alignment according to the present disclosure. Preferably, in the case of a “corresponding position” according to the present disclosure, the absolute position of a nucleotide or amino acid is different from that of an adjacent nucleotide or amino acid, but it should be understood that said adjacent nucleotide or amino acid that may be exchanged, deleted, or added may be included in the same one or more “corresponding positions.”

[0042] Furthermore, in the case of corresponding positions within lipocalin mutain based on the reference sequence according to the present disclosure, it should be understood that when evaluated by a person skilled in the art in light of the highly conserved overall folding pattern in lipocalin, the positions of nucleotides or amino acids of lipocalin mutain, preferably, may structurally correspond to other positions of reference lipocalin (wild-type lipocalin) or other lipocalin mutains, even if they differ in the absolute number of positions.

[0043] As used interchangeably herein, the terms “conjugate,” “conjugation,” “to fuse,” “fusion,” or “linked” refer to combining two or more subunits by means including, but not limited to, genetic fusion, chemical conjugation, coupling through a linker or crosslinker, and non-covalent bonding through any form of covalent or non-covalent bonding.

[0044] As used herein, the terms “fusion polypeptide” or “fusion protein” refer to a polypeptide or protein comprising two or more subunits. In one embodiment, a fusion protein as described herein comprises two or more subunits, at least one of these subunits capable of specifically binding to CD137, and an additional subunit capable of specifically binding to GPC3. Within the fusion protein, these subunits may be joined by covalent or non-covalent bonds. Preferably, the fusion protein is a translational fusion between two or more subunits. A translational fusion may be generated by genetically manipulating the coding sequence for one subunit within a reading frame together with the coding sequence of an additional subunit. The two subunits may be arranged by a nucleotide sequence encoding a linker. However, the subunits of the fusion protein of the present disclosure may also be joined through chemical conjugation. The subunits forming the fusion protein are typically joined such that the C-terminus of one subunit is joined to the N-terminus of another subunit, or the C-terminus of one subunit is joined to the C-terminus of another subunit, or the N-terminus of one subunit is joined to the N-terminus of another subunit, or the N-terminus of one subunit is joined to the C-terminus of another subunit. The subunits of the fusion protein may be joined in any order and may include one or more of any constituent subunits. Where one or more subunits are part of a protein (complex) composed of one or more polypeptide chains, the term “fusion protein” also refers to the protein comprising the fused sequence of the protein (complex) and all other polypeptide sequence(s). As an explanatory example, where full-length immunoglobulin is fused to lipocalin mutain by the heavy or light chain of the immunoglobulin, the term “fusion protein” refers to a single polypeptide chain containing lipocalin mutain and the heavy or light chain of the immunoglobulin.The term "fusion protein" may also refer to whole immunoglobulin (both light and heavy chains) and lipocalin mutain fused to one or both of its heavy and / or light chains.

[0045] As used herein, the term “subunit” of a fusion protein refers to a single protein or distinct polypeptide chain that can define a unique function of forming a stable folding structure itself and providing a binding motif to a target. In one embodiment, a preferred subunit of the present disclosure is lipocalin mutain. In some other embodiments, a preferred subunit of the present disclosure is a full-length immunoglobulin or its antigen-binding domain.

[0046] A “linker” that may be included by the fusion protein of the present disclosure joins together two or more subunits of the fusion protein as described herein. The joining may be covalent or non-covalent. A preferred covalent joining is through a peptide bond, such as a peptide bond between amino acids. A preferred linker is a peptide linker. Accordingly, in a preferred embodiment, the linker comprises one or more amino acids, e.g., 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 and include a glycine-serine (GS) linker, a glycosylated GS linker, and a proline-alanine-serine polymer (PAS) linker. In some preferred embodiments, the GS linker is (G4S)3 as described in SEQ ID NO: 13 and is used to bind the subunits of the fusion protein together. Other preferred linkers include chemical linkers.

[0047] As used herein, the term "albumin" includes all mammalian albumin, such as human serum albumin, bovine serum albumin, or rat serum albumin.

[0048] As used herein, the term “organic molecule” or “organic small molecule” means an organic molecule comprising at least two, preferably seven or fewer, carbon atoms or 12 rotatable carbon bonds, having a molecular weight in the range of 100 to 2,000 daltons, preferably 100 to 1,000 daltons, and optionally comprising one or two metal atoms.

[0049] "Sample" is defined as a biological sample obtained from any subject. Biological samples include, but are not limited to, blood, serum, urine, feces, semen, or tissues including tumor tissue.

[0050] “Subject” is a vertebrate, preferably a mammal, more preferably a human. The term “mammal” is used herein to refer to any animal classified as a mammal, including but not limited to humans, livestock and farm animals, and zoo, sports or pet animals, such as sheep, dogs, horses, cats, cattle, rats, pigs, and cyanomols, for the purpose of citing some descriptive examples. Preferably, as used herein, “mammal” is a human.

[0051] The "effective dose" is an amount sufficient to produce a beneficial or desirable result. The effective dose may be administered as one or more individual doses or doses.

[0052] As used herein, "antibody" comprises a whole antibody or any antigen-binding fragment (i.e., "antigen-binding portion") or a single chain thereof. A whole antibody refers to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) connected to each other by disulfide bonds. Each heavy chain has a heavy chain variable domain (V H or HCVR) and heavy chain invariant region (C H It consists of ). The heavy chain invariant region consists of 3 domains, C H1, C H2 and C H3 It consists of. Each light chain is a light chain variable domain (V L or LCVR) and light chain invariant region (C L It consists of ). The light chain invariant region is one domain, C L It consists of. V H and V L The region can be subdivided into a much more conserved region, also known as the Framework Region (FR), and an overvariant region, also known as the Complementarity Decision Region (CDR). Each V H and V L It consists of three CDRs and four FRs arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens (e.g., GPC3). The constant region of the antibody can selectively mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0053] As used herein, the “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., GPC3). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included within the term, “antigen-binding fragment” of an antibody, are (i) V H , V L , C L and C H1 (ii) a Fab fragment composed of domains; (ii) an F(ab')2 fragment comprising two Fab fragments joined by a disulfide bridge at the hinge region; (iii) V H , V L , C L and C H1 Domain and C H1 and C H2Fab' fragment consisting of regions between domains; (iv) V H and C H1 Fd fragment composed of a domain; (v) V of a single arm of the antibody H and V L A single-strand Fv fragment consisting of a domain, (vi) V H dAb fragments composed of domains (Ward et al., Nature (1989); and (vii) an isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs that can optionally be joined by a synthetic linker; (viii) V linked in the same polypeptide chain using a short linker. H and V L "diabody" including (e.g., patent document EP 404,097; WO 93 / 11161; and Holliger et al., Proc Natl Acad Sci USA (ix) In some examples, two or more V H Regions that are sharedly combined, V H or V L It includes a "domain antibody fragment" containing only...

[0054] Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g., humanized, chimeric, or multispecific). Antibodies may also be entirely human.

[0055] As used herein, "framework" or "FR" refers to variable domain residues other than the supervariable domain (CDR) residue.

[0056] "Fragment crystallizable region" or "Fc region" refers to the C-terminal region of the immunoglobulin heavy chain, including the natural-sequence Fc region and the variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain vary, the human IgG heavy-chain Fc region is typically defined from the amino acid residue from Pro230 to its carboxy-terminus at the Cys226 position numbered according to the EU index of Kabat (Johnson and Wu, Nucleic Acids Res , 2000). The lysine at the C-terminus of the Fc region (residue 447 according to the EU index of Kabat) may be removed, for example, during the production or purification of the antibody, or by manipulating the heavy chain encoding nucleic acid of the antibody through recombination. Thus, the composition of the intact antibody may include a population of antibodies with all K447 residues removed, a population of antibodies with K447 residues not removed, and a population of antibodies having a mixture of antibodies with or without K447 residues. Suitable natural-sequence Fc regions for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0057] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody.

[0058] As used herein, "isolated antibody" refers to an antibody that is substantially free from its natural environment. For example, the isolated antibody is substantially free from cellular material and other proteins from the cell or tissue source from which it originated. "Isolated antibody" also refers to an antibody that is substantially free from other antibodies having different antigen specificities. In this case, the isolated antibody that specifically binds to GPC3 is substantially free from antibodies that specifically bind to antigens other than GPC3. However, the isolated antibody that specifically binds to GPC3 may be cross-reactive to other antigens, such as GPC3 molecules from other species.

[0059] As used herein, "monoclonal antibody" refers to a preparation of antibody molecules of a single molecular composition. The monoclonal antibody composition exhibits single-binding specificity and affinity for a specific epitope.

[0060] As used herein, “humanized antibody” refers to an antibody composed of a CDR of an antibody derived from a non-human mammal and a FR region and a constant region derived from a human antibody or a human antibody. In some embodiments, the humanized antibody includes a variable domain having a variable region amino acid sequence that is closer to humans than to other species analyzed overall when evaluated using the Immunogenetics Information System (IMGT) DomainGapAlign tool, as described by Ehrenmann et al. (2010). In some embodiments, the humanized antibody will be useful as an active ingredient in therapeutics due to its reduced antigenicity. As used herein, the terms “therapeutic agent” or “therapeutic agent” refer to a therapeutically useful agent. A therapeutic agent may be any agent for the prevention, improvement, or treatment of a disease, physiological condition, or sign, or any agent for the evaluation or diagnosis thereof.

[0061] As used herein, “human antibody” comprises an antibody having a variable region in which both the framework and the CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody comprises a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibody of the present invention may comprise amino acid residues not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo). However, the term “humanized antibody” as used herein is not intended to include an antibody in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, is transplanted into a human framework sequence. Brief explanation of the drawing

[0062] Fig. 1 isThis application provides an overview of the design of a representative fusion protein described in this application that is bispecific to targets CD137 and GPC3. Representative fusion proteins are antibodies specific to GPC3 (e.g., provided by SEQ ID NO: 81, or containing the heavy chain variable domain of SEQ ID NO: 78, or containing the CDR sequences of GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), and TRFYSYTY (HCDR3; SEQ ID NO: 74), containing the light chain provided by SEQ ID NO: 82, or containing the light chain variable domain of SEQ ID NO: 79, or containing the CDR sequences 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 mutains specific to CD137 (e.g., the lipocalin mutain of SEQ ID NO: 40 or for SEQ ID NO: 49). It was manufactured based on a lipocalin mutain designated as CD137Ac1 with 97% sequence homology. One or more lipocalin mutains do 1A-1I Genetically fused to the C- and / or N-terminus of the heavy or light chain of a GPC3-specific antibody as described in, the fusion protein, e.g., 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 (97% of the sequence for 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 A homologous fusion protein) was generated. In addition, the generated fusion protein is divalent to CD137 (e.g., also 1A-1DAs described in), or for CD137, 4 (e.g., Fig. 1E-1H As described in), or can have a much higher bonding affinity for CD137 (e.g., Fig. 1I As described in ). Additional monospecific fusion proteins were generated by fusing one or more GPC-3 specific lipocalin mutains or CD137 specific lipocalin mutains to the C-terminus of the Fc region of the provided antibody as described herein via a peptide linker (e.g., Figure 1J-1K As described in ). The generated single-specific fusion protein is provided, for example, in SEQ ID NO: 97 and SEQ ID NO: 98. Fig. 2 Is Examples 3 As described in [document], the binding of representative fusion proteins to human CPC3 (Fig. 2A), cyanomorphic GPC3 (Fig. 2B), and human CD137 (Fig. 2C) is shown in the measured ELISA experimental results. GPC3 or C-terminal His-tagged CD137 was coated on microtiter plates, and the test substances were titrated starting at a maximum concentration of 100 nM. Under the study, the bound agents were detected via anti-human IgG Fc-horsradish peroxidase (HRP) or anti-NGAL-HRP, respectively. The data are EC 50 It was applied to a 1:1 combined model with the value and maximum signal as free parameters and a slope fixed at 1. The generated EC 50 The value Table 4 It is provided in. Fig. 3 silver Examples 4 As described in [link], this is shown by the results of an exemplary ELISA experiment in which the ability of a representative fusion protein to simultaneously bind to two targets, GPC3 and CD137, was measured. Recombinant huGPC3 ( Fig. 3A ) or huCD137-His (do 3B) was coated onto a microtiter plate, and then the fusion protein was titrated. Then, each constant concentration of biotinylated huCD137-His (Fig. 3A ) or biotinylated huGPC3 (also 3B ) was added and detected via ExtrAvidin-Peroxidase. Data are EC 50 It was applied to a 1:1 coupled model with the value and maximum signal as free parameters and a slope fixed by unity. The generated EC 50 The value Table 3 It is provided in. Fig. 4 Is Examples 6 As described in, human-CD137 expressing CHO cells ( Fig. 4A ) and human GPC3-expression SK-Hep1 ( Fig. 4B This shows the results of the evaluation of target binding of the fusion protein by flow cytometry using ) cells. No binding was observed when mock-transfected cells were used. The geometric mean of the fluorescence intensity is EC 50 It was used to calculate the value, EC 50 The value Table 6 It is provided in. Fig. 5 Is Shows the binding of the fusion protein to GPC3-positive tumor cells evaluated using flow cytometry. Tumor cell lines with different GPC3 expression levels (high to intermediate expression: HepG2 ( Fig. 5A ) > Hep3B ( Fig. 5B ) > MKN-45 ( Fig. 5C )) and GPC3-negative cell line NCI-N87 ( Fig. 5D ) was incubated with different fusion proteins or a control as in Example 7, and the corresponding binding affinity (EC 50 )silver Table 7 It is summarized in. Fig. 6 silver It demonstrates the potential of a representative fusion protein to co-stimulate T-cell activation in a GPC3-target-dependent manner, as evaluated using a CD137 bioassay. NFkB-luc2 / CD137 Jurkat cells were treated with GPC3-expressing tumor cell lines (high to intermediate expression: HepG2 > Hep3B > MKN-45, GPC3 negative: NCI-N87, respectively) in the presence of various concentrations of the fusion protein or a control. do 6A, 6B, 6C, and 6D It was co-cultured with ). After 4 hours, the luciferase assay reagent was added and the luminescence signal was measured. Four-parameter logistic curve analysis was performed to determine the EC 50 The value was calculated ( Table 7 (Reference). The fusion protein only co-stimulates T-cell activation in the presence of GPC3, but ( Figures 6A and 6B ) This is not the case when GPC3 is absent or insufficient ( Figs. 6C and 6D In contrast, reference anti-CD137 mAbs (sequence numbers: 26 and 27) exhibit similar activation regardless of GPC3 expression levels in the absence of GPC3-expressing cells and in the absence of any tumor cells. Fig. 7 silver This demonstrates the ability of representative fusion proteins to co-stimulate T-cell activation in the presence of GPC3. GPC3 antibodies included in the fusion protein (SEQNs: 81 and 82), GPC3-specific lipocalin mutain (Fc fusion) (SEQN: 97), a previously known CD137 / GPC3 bispecific fusion protein (SEQN: 83), and isotype controls (SEQNs: 24 and 25) were tested simultaneously. SK-Hep1 cells transfected with human GPC3 ( Fig. 7A ) or Mott-transfected SK-Hep1 cells (human GPC3 negative, Fig. 7B ) or GPC3 ( Fig. 7C)-expressing Hep-G2 tumor cell lines were inoculated onto plates coated with anti-human anti-CD3. Pan T cells as well as test molecules at various concentrations were added and incubated for 3 days. Examples 9 As described in [document], the levels of IL-2 secreted in the supernatant were measured by electrochemiluminescence-based analysis. Fusion proteins SEQ NO: 87 and 82 induce a strong dose-dependent increase in IL-2 secretion only in the presence of GPC3, more strongly than the previously known CD137 / GPC3 bispecific fusion protein SEQ NO: 83. Fig. 8 This demonstrates the ability of a representative fusion protein to co-stimulate T-cell activation in a GPC3-target-dependent manner. Various tumor cell lines expressing different levels of GPC3 (high to intermediate expression: HepG2 > Hep3B > MKN-45, respectively) do 8A, 8B, and 8C ; GPC3 Voice: NCI-N87, Fig. 8D Pan T cells, various concentrations of fusion protein, and single building blocks were inoculated onto plates coated with anti-human CD3. The cells were incubated for 3 days. Examples 10 As described in [document], secreted IL-2 levels were determined by electrochemiluminescence-based analysis. The fusion protein can increase IL-2 secretion in a GPC3-dependent manner. In contrast, IL-2 secretion induced by the reference CD137 antibodies (SEQNs: 26 and 27) tested concurrently is not GPC3-dependent. Fig. 9This demonstrates the ability of representative fusion proteins to activate the CD137 co-stimulatory signaling pathway and induce T-cell-mediated cytolysis of GPC3-expressing tumor cells, as demonstrated in an impedance-based T-cell death assay. After GPC3-expressing HepG2 cells or GPC3-negative NCI-N87 cells were inoculated and attached to an electronic microtiter plate, an anti-CD3 antibody, a test molecule, and non-attached CD8+ T cells were added. As described in Example 11, impedance was measured as a unitless "Cell Index (CI)" parameter every 15 minutes for 3 days. Fusion proteins SEQ NOs: 87 and 82 can activate the CD137 pathway in a GPC3-dependent manner and can induce dose-dependent T-cell-mediated lysis of GPC3-expressing tumor cells at a higher level than the previously known CD137 / GPC3 bispecific fusion protein SEQ NO: 83. Fig. 10 silver Examples 12 As described in [reference], this shows tumor volume over time in humanized mouse tumor models after treatment with 0.5, 5, and 20 mg / kg of the exemplary fusion protein, GPC3 antibody at the same molar concentration (0.39 and 3.9 mg / kg) contained in the fusion protein, reference CD137 antibody at the same molar concentration (3.9 mg / kg), or vehicle control (PBS). NOG mice have a tumor volume of 80–100 mm 3 HepG2 tumors allowed to grow to a size were subcutaneously implanted. Mice were then randomized into a treatment group (or control group) and 5x10 of the test molecule were administered at the indicated doses on days 1, 8, and 15. 6Fresh human PBMCs were injected intravenously and intraperitoneally. Tumor growth was recorded every 3 to 4 days. Data are displayed as medians with error bars in the interquartile range. The dashed line(s) indicate a decrease in the number of animals per group compared to the previous date. During the study period, the fusion protein completely inhibited tumor growth at higher dose levels (5 and 20 mg / kg), whereas it had a dose-dependent but negligible effect on tumor growth at lower concentrations (0.5 and 0.39 mg / kg, respectively). Fig. 11 silver Examples 13 As described in [the document], we provide pharmacokinetic analysis results of the GPC3 antibody contained in the fusion protein and the exemplary fusion protein in mice compared to the previously known CD137 / GPC3 bispecific fusion protein (SEQN: 83 and SEQN: 84). The fusion protein was intravenously injected into male CD-1 mice (2 mice per time point) at a dose of 2 mg / kg or 10 mg / kg. Drug levels were detected using a sandwich ELISA detecting the whole molecule via the target GPC3 and anti-human Fc or anti-NGAL. The fusion proteins of the present disclosure (SEQN: 87 and 82) exhibit strongly enhanced pharmacokinetic behavior and an extended half-life compared to the previously known CD137 / GPC3 bispecific fusion protein (SEQN: 83 and SEQN: 84). Specific details for implementing the invention

[0063] As described herein, the present disclosure includes the recognition that a divalent CD137 binder, such as an antibody, may not be sufficient on its own to cluster CD137 in T cells or NK cells and induce efficient activation. Furthermore, recent studies on members of the TNFR family demonstrate the mechanism of anti-TNFR antibodies in which antibodies interact with Fc-gamma receptors through their Fc regions to activate Fc-gamma-expressing immune cells and promote subsequent anti-tumor activity (Bulliard et al., Immunol Cell Biol , 2014, Bulliard et al., J Exp Med (e.g., 2013). Therefore, this suggests that anti-CD137 antibodies may induce CD137 clustering depending on the abundance of Fc-gamma receptor-positive cells distributed throughout the body, rather than selectively being tumor-localized. Consequently, the efficacy and target-specificity of anti-CD137 monotherapy may be of concern. In fact, under clinical studies, some anti-CD137 therapies, such as urelumab and utomilumab, have shown disappointing efficacy results at low doses and / or exhibit toxicity at high or effective doses (Bulliard et al., Immunol Cell Biol , 2014, Bulliard et al., J Exp Med (2013). Therefore, there is an unmet need for effective and safe CD137-targeted therapy regimens. An ideal CD137-targeting agent should lead to the clustering of CD137 in a tumor-localized manner on tumor-infiltrating lymphocytes. As described herein, to obtain such a CD137-targeting agent, a bispecific agent can be designed to target CD137 at one end and differentially target a tumor target at the other end.

[0064] The present disclosure includes the recognition that monospecific GPC3-targeted agents may not be sufficiently potent for the treatment of tumors such as HCC, and that the anti-tumor activity of anti-GPC3 agents capable of mobilizing immune cells to the tumor site and / or directly inhibiting cancer cell proliferation and / or survival may be relevant. Accordingly, there is an unmet need for therapeutic regimens that have an additional anti-tumor effect by targeting GPC3 and having the ability to provide enhanced anti-tumor activity compared to monospecific GPC3-targeted agents alone.

[0065] The present disclosure provides, among other things, a novel approach for simultaneously binding to CD137 and GPC3 through a fusion protein having binding specificity for CD137 and binding specificity for GPC3. For binding to CD137 and GPC3, for example, an anti-GPC3 antibody-CD137 specific lipocalin mutain fusion protein is described, for example, in WO2016 / 184882. However, it is still desirable for the fusion protein to induce a GPC3-dependent T cell apoptosis / immune response to inhibit GPC3-expressing tumor growth with high efficiency and selectivity. Various aspects of the present disclosure provide a fusion protein that promotes CD137 clustering by linking CD137-positive T cells to GPC3 expressed in the tumor microenvironment, in order to combine CD137-induced T-cell activation and expansion with anti-GPC3-mediated cytotoxic apoptosis of tumor cells. The present disclosure also provides a fusion protein that offers the potential for combination therapy in a single molecule and simultaneously allows for the local induction of antigen-specific T cells in the tumor microenvironment, thereby potentially reducing peripheral toxicity. In this regard, the fusion protein of the present disclosure may provide enhanced IL-2 production, immune response, T-cell-mediated cytolysis, and / or anti-tumor effects compared to monomeric CD137 or GPC3 therapy.

[0066] In addition, cyanomolgus monkeys have been widely used for pharmacokinetic or drug-safety studies in the development of new therapies, including novel biologics, and since such studies may be a prerequisite for regulatory approval, it is desirable to have a fusion protein having a GPC3 binding moiety that is cross-reactive with both human and cyanomolgus GPC3. Such a fusion protein having a GPC3 binding moiety with these cross-reactive features has not been previously described.

[0067] In addition, as provided herein, because bispecific or multispecific molecules may differ from their building blocks in specific characteristics including target affinity and specificity, stability, pharmacokinetics in blood, and mechanism of action (Spiess et al., Mol Immunol , 2015, Sedykh et al., Drug Des Devel Ther (Alavijeh and Palmer, 2018), in one embodiment, careful selection of building blocks and optimization of manufacturing and other practices are provided to yield high-quality clinical candidates. For example, bispecific or multispecific molecules may partially or completely lose the target binding affinity and specificity of the building blocks, resulting in unwanted off-target binding. Off-target binding may further affect the pharmacokinetics, tissue distribution, efficacy, and toxicity of these bispecific or multispecific molecules for therapeutic use. In particular, poor pharmacokinetic profiles may cause difficulties in achieving the dose profile required for therapeutic efficacy and patient non-adherence (Alavijeh and Palmer, IDrugs(Ryman and Meibohm, 2004), favorable pharmacokinetics can lower the dosage, develop subcutaneous formulations, reduce the COG, and extend the dosing interval, for example, once a week, every two weeks, every three weeks, or every four weeks (Ryman and Meibohm, CPT Pharmacometrics Syst Pharmacol , 2017). Pharmacokinetic characteristics are also influenced by the isoelectric point (pl). An increase in pl leads to increased blood clearance and a shorter half-life, resulting in increased tissue retention, whereas a low pl may lead to decreased tissue uptake and a longer half-life. Although observations regarding the correlation between protein clearance and pl may be conflicting, therapeutic antibodies often have pl values ​​slightly higher than the physiological pH of 7.4 because most cell surfaces carry a negative charge. In this regard, various aspects of the present disclosure provide a bispecific fusion protein for CD137 and GPC3 having high affinity and specificity, enhanced safety, a suitable pl, and favorable pharmacokinetic characteristics.

[0068] Such fusion proteins having such features accompanying the uses provided by the present disclosure have not been previously described. In contrast to the fusion proteins provided herein, previously known fusion proteins targeting both CD137 and GPC3 have suffered one or more of the following: poor PK, unacceptable degree of non-target binding, unacceptable degree of non-specificity (e.g., GPC3 independent), such as reduced or impaired ability to mediate immune system activation and / or T cell activation, proliferation, and / or infiltration.

[0069] A. Main disclosure CD137 and to GPC3 An exemplary fusion protein specific to [the subject].

[0070] In one embodiment, the provided fusion protein comprises at least two subunits in any order: (1) a first subunit comprising a full-length immunoglobulin specific to GPC3 or an antigen-binding domain thereof, and (2) a second subunit comprising a lipocalin mutain specific to CD137.

[0071] In one embodiment, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, the second subunit comprises a lipocalin mutane and is specific to CD137, and the second subunit is selectively linked through a linker from the N-terminus to the C-terminus of each heavy chain (HC) of the first subunit.

[0072] In one embodiment, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, the second subunit comprises a lipocalin mutane and is specific to CD137, and the second subunit is selectively connected from the C-terminus to the N-terminus of each HC of the first subunit through a linker.

[0073] In one embodiment, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, the second subunit comprises a lipocalin mutain and is specific to CD137, and the second subunit is selectively linked from the N-terminus to the C-terminus of each light chain (LC) of the first subunit through a linker.

[0074] In one embodiment, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, the second subunit comprises a lipocalin mutain and is specific to CD137, and the second subunit is selectively connected from the C-terminus to the N-terminus of each LC of the first subunit through a linker.

[0075] In one embodiment, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, the second subunit comprises a lipocalin mutain and is specific to CD137, and the second subunit is selectively connected through a linker from the N-terminus to the C-terminus of each heavy chain constant region (CH) of the first subunit.

[0076] In one embodiment, the provided fusion protein comprises at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, the second subunit comprises a lipocalin mutain and is specific to CD137, and the second subunit is selectively connected through a linker from the N-terminus to the C-terminus of each light chain constant region (CL) of the first subunit.

[0077] In one embodiment, the provided fusion protein may also contain at least one additional subunit, for example, a third subunit. For example, the fusion protein may contain a third subunit specific to CD137. In one embodiment, the third subunit may be a lipocalin mutain specific to CD137 or may comprise a lipocalin mutain specific to CD137. For example, two lipocalin mutains may be fused to the first immunoglobulin subunit, one at the C-terminus and one at the N-terminus of the immunoglobulin. In one embodiment, the lipocalin mutain may be fused to the heavy chain or light chain of the immunoglobulin.

[0078] In one embodiment, the provided fusion protein may include one or more additional subunits (e.g., a fourth, fifth, or sixth subunit).

[0079] In one embodiment, at least one subunit may be fused to another subunit at its N-terminus and / or its C-terminus.

[0080] In one embodiment, at least one subunit may be connected to another subunit via a linker. In another embodiment, the linker is a peptide linker, e.g., an unstructured glycine-serine (GS) linker, a glycosylated GS linker, or a proline-alanine-serine polymer (PAS) linker. In one embodiment, the GS linker is a (Gly4Ser)3 linker ((G4S)3) as shown in SEQ ID NO: 13. Other exemplary linkers are shown in SEQ ID NOs: 14-23. In one embodiment, the peptide linker may have 1 to 50 amino acids, e.g., 1, 2, 3, 4, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids. For example, if the first subunit 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 another embodiment, 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.

[0081] In one embodiment, one subunit is essentially do 1 It may be connected to other subunits as described in [the document]. Generally, one subunit may be fused to another subunit at its N-terminus and / or its C-terminus. For example, in one embodiment, the lipocalin mutain subunit may be fused to the immunoglobulin subunit at its N-terminus and / or its C-terminus, preferably via a peptide bond. In additional embodiments, the lipocalin mutain subunit may be connected at its N-terminus to the C-terminus of the heavy chain domain (HC) of the immunoglobulin subunit ( Fig. 1A), the lipocalin mutain subunit can be connected at its C-terminus to the N-terminus of the HC of the immunoglobulin subunit ( Fig. 1C ), the lipocalin mutain subunit can be connected at its N-terminus to the C-terminus of the light chain (LC) of the immunoglobulin subunit ( Fig. 1B ), and / or lipocalin mutain subunits can be connected at their C-terminus to the N-terminus of the LC of the immunoglobulin subunit ( Fig. 1D ).

[0082] In one embodiment, the lipocalin mutain subunit may be fused to an immunoglobulin fragment at its N-terminus and / or its C-terminus. For example, in one embodiment, the lipocalin mutain subunit may be connected at its N-terminus to the C-terminus of the heavy chain constant region (CH) of the immunoglobulin subunit, preferably via a peptide linker, or the lipocalin mutain subunit may be connected at its N-terminus to the C-terminus of the light chain constant region (CL) of the immunoglobulin subunit, preferably via a peptide linker.

[0083] In one embodiment, when one subunit comprises a full-length immunoglobulin, a second subunit may be connected between the N-terminus of the second subunit and the C-terminus of the heavy chain constant region (CH) of the immunoglobulin.

[0084] In one embodiment, the third subunit may be connected between the N-terminus of the third subunit and the C-terminus of the light chain constant region (CL) of the immunoglobulin.

[0085] In one embodiment, with respect to the fusion protein of the present disclosure, at least one subunit may be a full-length immunoglobulin or may comprise a full-length immunoglobulin, while the Fc function of the Fc region of the full-length immunoglobulin for Fc receptor-positive cells is preserved, and the fusion protein binds simultaneously with CD137 and GPC3.

[0086] In one embodiment, at least one subunit of the provided fusion protein is a full-length immunoglobulin or may contain a full-length immunoglobulin, and the Fc function of the Fc region of the full-length immunoglobulin for Fc receptor-positive cells is reduced or completely suppressed by protein manipulation, while the fusion protein binds simultaneously with CD137 and GPC3. In one embodiment, this can be achieved, for example, by switching from an IgG1 backbone to IgG4, as IgG4 is known to exhibit reduced Fc-gamma receptor interactions compared to IgG1. In one embodiment, to further reduce residual binding with the Fc-gamma receptor, mutations such as F234A and L235A may be introduced into the IgG4 backbone. In one embodiment, to minimize the exchange of IgG4 half-antibodies, the S228P mutation may also be introduced into the IgG4 backbone (Silva et al., J Biol Chem , 2015). In one embodiment, F234A and L235A mutations may be introduced for reduced ADCC and ADCP (Glaesner et al., Diabetes Metab Res Rev M428L and N434S mutations or M252Y, S254T, and T256E mutations may be introduced for an extended serum half-life (Dall'Acqua et al., 2010). J Biol Chem , 2006, Zalevsky et al., Nat Biotechnol (2010). In one embodiment, an additional N297A mutation may be present in the immunoglobulin heavy chain of the fusion protein to remove the natural glycosylation motif.

[0087] In one embodiment, the Fc portion of the immunoglobulin included in the fusion protein of the present disclosure may contribute to maintaining serum levels of the fusion protein. For example, when the Fc portion binds to Fc receptors on endothelial cells and phagocytes, the fusion protein may be internalized and recirculated into the bloodstream while enhancing its half-life in vivo.

[0088] In one embodiment, the fusion protein of the present disclosure binds to CD137 with high affinity. In another embodiment, the provided fusion protein binds to GPC3 with high affinity. In some preferred embodiments, the provided fusion protein binds to CD137 and GPC3 simultaneously. In one embodiment, simultaneous binding to CD137 and GPC3 causes the provided fusion protein to exhibit a long-term anti-tumor or anti-infection response.

[0089] In one embodiment, the fusion protein of the present disclosure may have an isoelectric point (pl) of about 6.5 or higher, e.g., 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 even about 9.0 or higher. In one embodiment, the fusion protein of the present disclosure may have a higher pl than a previously known fusion protein that binds to both CD137 and GPC3 comprising the amino acid sequence of SEQ ID NO: 83.

[0090] In one embodiment, the fusion protein of the present disclosure is even at a maximum of about 2 nM or less, e.g., about 1.5 nM or less, about 1 nM or less, about 0.8 nM or less, or about 0.7 nM or less K D It can bind to GPC3 with a value. In one embodiment, the fusion protein of the present disclosure is a K of an immunoglobulin specific to GPC3 included in such a fusion protein, such as an antibody having a heavy chain and a light chain provided by SEQ ID NOs. 81 and 82. D K that is comparable to or lower than the value DIt can bind to GPC3 with the value of the provided fusion protein's K D The value is, for example, Examples 2 It can be measured in surface-plasmon-resonance (SPR) analysis, such as SPR analysis as essentially described in [the text].

[0091] In one embodiment, the fusion protein of the present disclosure has an EC of even up to about 1 nM, e.g., about 0.5 nM, about 0.3 nM, about 0.2 nM, about 0.15 nM, or about 0.1 nM. 50 It can bind to GPC3 with a value. In one embodiment, the fusion protein of the present disclosure is an EC of an immunoglobulin specific to GPC3 contained in a specific fusion protein, such as an antibody having a heavy chain and a light chain provided by SEQ ID NOs. 81 and 82. 50 Equivalent to or lower than the value EC 50 It can bind to GPC3 with the value. EC of the provided fusion protein 50 The value is, for example, Examples It can be measured in an enzyme-linked immunosorbent assay (ELISA), such as the ELISA assay essentially described in 3.

[0092] In one embodiment, the fusion protein of the present disclosure has an EC of even up to about 5 nM, e.g., about 3 nM or less, about 2 nM or less, about 1 nM or less, about 0.5 nM or less, about 0.2 nM or less, or about 0.1 nM or less. 50 It can bind to CD137 with the value. EC of the provided fusion protein 50 The value is, for example, Examples 3 It can be measured in ELISA assays, such as the ELISA assay described in the form.

[0093] In one embodiment, the fusion protein of the present disclosure may cross-react with cyanomolgus GPC3. In one embodiment, the provided fusion protein may have even up to about 2 nM, e.g., about 1.5 nM, about 1 nM, about 0.8 nM, or about 0.7 nM of K D It can bind to cyanomolecular GPC3 with a value. In one embodiment, the fusion protein of the present disclosure is a K of an immunoglobulin specific to GPC3 included in such fusion protein, such as an antibody having a heavy chain and a light chain provided by SEQ ID NOs. 81 and 82. D K that is comparable to or lower than the value D It can bind to cyanomolgus GPC3 with the value. The K of the provided fusion protein D The value Examples 2 It can be measured in SPR assays, such as SPR assays as essentially described in [the document]. In one embodiment, the provided fusion protein has an EC of even up to about 1 nM or less, e.g., about 0.5 nM or less, e.g., about 0.2 nM or less, about 0.1 nM or less. 50 It can bind to cyanomolgus GPC3 with the value. EC of the provided fusion protein 50 The value is, for example, Examples 3 It can be measured in ELISA assays, such as the ELISA assay essentially described in [the document].

[0094] In one embodiment, the fusion protein of the present disclosure may bind simultaneously with CD137 and GPC3. In one embodiment, the provided fusion protein has an EC of even up to about 10 nM, e.g., 5 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, or 0.5 nM or less. 50It can bind simultaneously with CD137 and GPC3 by value. In some other embodiments, the provided fusion protein has an EC of even up to about 10 nM or less, e.g., 8 nM or less, 5 nM or less, 3 nM or less, or 2 nM or less. 50 It can be combined simultaneously with CD137 and GPC3 as a value. Simultaneous combination is, for example, Examples 4 It can be determined in an ELISA assay, such as the ELISA assay described essentially in [the document].

[0095] In one embodiment, the fusion protein of the present disclosure has an EC of even up to about 50 nM, e.g., even about 30 nM or less, even about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 5 nM or less, about 3 nM or less, or even about 1 nM or less. 50 It can bind to CD137 expressed on the cell at the value. EC of the provided fusion protein 50 The value is, for example, Examples 6 It can be measured by flow cytometry analysis as essentially described in ). Cells expressing CD137 may be, for example, CHO cells transfected with human CD137.

[0096] In one embodiment, the fusion protein of the present disclosure is even up to about 50 nM, e.g., about 30 nM or less, even about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 5 nM or less, about 3 nM or less, or even about 1 nM or less EC 50 It can bind to GPC3 expressed on the cell at the value. EC of the provided fusion protein 50 The value is, for example, Examples 6It can be measured by flow cytometry analysis as essentially described in ). Cells expressing GPC3 may be, for example, SK-Hep1 cells transfected with human GPC3.

[0097] In one embodiment, the fusion protein of the present disclosure may bind to GPC3 expressed on tumor cells. In one embodiment, the provided fusion protein may have an EC of even up to about 50 nM, e.g., about 30 nM or less, even about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 5 nM or less, about 3 nM or less, or even about 1 or less. 50 It can bind to GPC3 expressed on tumor cells at a value. EC of the fusion protein binding to tumor cells expressing GPC3 50 The value is, for example, Examples 7 It can be measured by flow cytometry analysis as essentially described in ). Tumor cells expressing GPC3 may be, for example, HepG2, Hep3B, and MKN-45 cells.

[0098] In one embodiment, the fusion protein of the present disclosure substantially does not bind to a target other than CD137 or GPC3. In one embodiment, the provided fusion protein has reduced off-target binding to a target other than CD137 or GPC3 compared to a previously known fusion protein that binds to both CD137 and GPC3 comprising the amino acid sequence of SEQ ID NO: 83. This evaluation of target binding is Examples 5 It can be performed by ELISA analysis as described in.

[0099] In one embodiment, the fusion protein of the present disclosure can co-stimulate a T-cell response. In one embodiment, the provided fusion protein induces T-cell activation more strongly than or comparable to, or more strongly than, a previously known fusion protein that binds to both CD137 and GPC3 containing the amino acid sequence of SEQ NO: 83, a reference CD137 antibody having a heavy chain and a light chain provided by SEQ NO: 26 and 27, or a GPC3 antibody contained in a fusion protein having a heavy chain and a light chain provided by SEQ NO: 81 and 82. In one embodiment, the provided fusion protein induces T-cell activation with efficiency comparable to or better than that of a previously known fusion protein binding to both CD137 and GPC3 comprising the amino acid sequence of SEQ NO: 83, a reference CD137 antibody having a heavy chain and a light chain provided by SEQ NOs: 26 and 27, or a GPC3 antibody contained in a fusion protein having a heavy chain and a light chain provided by SEQ NOs: 81 and 82. The stimulated T-cell response or T-cell activation is, for example, Examples 8 It can be measured in CD137 biological assays as essentially described in, or Examples 9 , Examples 10 , and Examples 11 It can be measured in functional T-cell activation assays as essentially described in [the document].

[0100] In one embodiment, the fusion protein of the present disclosure may induce increased IL-2 secretion. In one embodiment, the provided fusion protein may induce concentration-dependent IL-2 secretion and / or exhibit a tendency to induce enhanced IL-2 secretion at higher concentrations. In one embodiment, the provided fusion protein may induce increased IL-2 secretion with efficiency comparable to or better than that of a previously known fusion protein binding to both CD137 and GPC3 comprising the amino acid sequence of SEQ NO: 83, or a GPC3 antibody contained in a fusion protein having heavy and light chains provided by SEQ NOs: 81 and 82. IL-2 secretion is, for example, Examples 9 and Examples 10 It can be measured in functional T-cell activation assays as essentially described in [the document].

[0101] In one embodiment, the fusion protein of the present disclosure may induce T cell-mediated cytotoxicity. In one embodiment, the provided fusion protein may induce dose-dependent T cell-mediated cytolysis. In one embodiment, the provided fusion protein may induce cytotoxic T-cell activation with efficiency comparable to or better than that of the GPC3 antibody contained in the provided fusion protein having heavy and light chains provided by Sequence Nos. 81 and 82. In one embodiment, the provided fusion protein may induce cytotoxic T-cell activation with efficiency comparable to or better than that of a previously known fusion protein binding to both CD137 and GPC3 containing the amino acid sequence of Sequence No. 83, a reference CD137 antibody having heavy and light chains provided by Sequence Nos. 26 and 27, or a CD137 antibody having heavy and light chains provided by Sequence Nos. 26 and 27. T cell-mediated cytotoxicity is, for example, Examples 11It can be measured in an impedance-based T cell death assay as essentially described in [the document].

[0102] In one embodiment, the fusion protein of the present disclosure can co-stimulate a T-cell response in a GPC3-dependent manner. In one embodiment, the provided fusion protein can induce local induction of IL-2 production by T-cells in the vicinity of GPC3-positive cells, such as GPC3-transfected cells or GPC3-expressing tumor cells. "In the vicinity of GPC3-positive cells" as used herein refers to T-cells and GPC3-positive cells that come into close proximity to each other through the provided fusion protein that binds to CD137 and GPC3 simultaneously. In one embodiment, the provided fusion protein can induce localized T-cell-mediated apoptosis in GPC3-expressing tumor cells. GPC3-dependent T-cell activation by the provided protein is, for example, Examples 8 In the CD137 biological assay essentially described in, or Examples 9 , Examples 10 and Example 11 It can be measured in the functional T-cell activation assay essentially described in ).

[0103] In one embodiment, the provided fusion protein cannot co-stimulate a T-cell response in the absence of GPC3. In one embodiment, the provided fusion protein cannot co-stimulate a T-cell response in the absence of GPC3-expressing cells. In one embodiment, the provided fusion protein can identify the presence of GPC3 and can induce a corresponding T-cell activation more than the CD137 antibody having heavy and light chains provided by SEQ ID NOs. 26 and 27. The GPC3-dependent mode of the fusion protein is, for example, Examples 8 In the CD137 biological assay essentially described in, or Examples 9 , Examples10 , and Examples 11 It can be measured in the functional T-cell activation assay essentially described in ).

[0104] In one embodiment, the provided fusion protein may induce anti-tumor activity. In one embodiment, the provided fusion protein may inhibit tumor growth of hepatocellular carcinoma HepG2 cells. In one embodiment, the provided fusion protein may inhibit tumor growth of hepatocellular carcinoma HepG2 cells with efficiency comparable to or better than that of the GPC3 antibody contained in the fusion protein having heavy and light chains provided by SEQ ID NOs. 81 and 82. Such anti-tumor activity is, for example, Examples 12 It can be measured using the HepG2 xenograft model essentially described in ).

[0105] In one embodiment, the provided fusion protein can increase the level of tumor-infiltrating lymphocytes. In one embodiment, the provided fusion protein can increase the intratumoral infiltration of CD3, CD4, or CD8 T cells compared to the GPC3 antibody contained in the fusion protein having heavy and light chains provided by SEQ ID NOs. 81 and 82. Tumor-infiltrating lymphocytes are, for example, Examples 13 It can be analyzed as described in.

[0106] In one embodiment, the provided fusion protein has favorable stability and pharmacokinetic profiles. In one embodiment, the provided fusion protein has a pharmacokinetic profile comparable to that of the GPC3 antibody contained in the fusion protein having heavy and light chains provided by SEQ ID NOs. 81 and 82. In one embodiment, the provided fusion protein has antibody-like pharmacokinetics. In one embodiment, the provided fusion protein has a terminal half-life of about 50 hours or more, 75 hours or more, 100 hours or more, 125 hours or more, about 150 hours or more, about 175 hours or more, about 200 hours or more, about 250 hours or more, or even more. In one embodiment, the provided fusion protein has a terminal half-life in mice of about 50 hours or more, 75 hours or more, 100 hours or more, 125 hours or more, more than 150 hours or more, about 175 hours or more, about 200 hours or more, about 250 hours or more, or even more. The pharmacokinetic profile of the provided fusion protein is Examples 13 It can be analyzed as described in.

[0107] In one embodiment, the provided fusion protein comprises the amino acid sequence presented in any one of SEQ ID NOs: 87-96.

[0108] In one embodiment, the provided fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity with respect to the amino acid sequence presented in any one of SEQ ID NOs: 87-96.

[0109] In one embodiment, the provided fusion protein comprises amino acids presented 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.

[0110] In one embodiment, the provided fusion protein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or even higher sequence identity with respect to the amino acid sequences presented 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, or 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.

[0111] B. An exemplary immunoglobulin included in a fusion protein.

[0112] In one embodiment, with respect to the provided fusion protein, the first subunit may be a full-length immunoglobulin specific to GPC3 or its antigen-binding domain, or may include it. In one embodiment, the immunoglobulin may be, for example, IgG1, IgG2, or IgG4. In one embodiment, the immunoglobulin is IgG4 or includes it. In one embodiment, the immunoglobulin is a monoclonal antibody against GPC3.

[0113] Descriptive examples of GPC3-binding antibodies in this disclosure are heavy chain variable domains (V) of known antibodies such as codrituzumab (also known as GC33 or RO5137382), YP7 (including humanized YP7), HN3, and HS20. H ) and light chain variable domain (V LA GPC3-binding antibody containing a ) region may include an antigen-binding region that cross-blocks or binds to the same epitope. In one embodiment, the GPC3-binding antibody of the present disclosure may include an antigen-binding region such as any one of three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3) from an antibody selected from the group consisting of codrituzumab, YP7, HN3, and HS20.

[0114] In one embodiment, the provided GPC3 antibody or its antigen-binding domain may have a heavy chain variable region (HCVR) selected from the group consisting of SEQ ID NOs: 78, 114, 119, 126, and 129, and / or a light chain variable region (LCVR) selected from the group consisting of SEQ ID NOs: 79, 115, and 127.

[0115] In one embodiment, the heavy chain and light chain pairs of the provided GPC3 antibody or its antigen-binding domain are each HCVR and LCVR as follows or comprise: 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.

[0116] In one embodiment, the heavy chain and light chain pairs of the provided GPC3 antibody or its antigen-binding domain are, respectively, HCVR and LCVR 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: 78 and 79, SEQ ID NOs: 129 and 79, SEQ ID NOs: 114 and 115, or SEQ ID NOs: 126 and 127.

[0117] In one embodiment, the provided GPC3 antibody or its antigen-binding domain may have a heavy chain of any one of SEQ ID NO: 80 and 81, and / or a light chain of SEQ ID NO: 82.

[0118] In one embodiment, the heavy chain and light chain pairs of the provided GPC3 antibody are amino acid sequences as shown in SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82, or include the same.

[0119] In one embodiment, the heavy chain and light chain pair of the provided GPC3 antibody are or comprise a heavy chain and light chain having an amino acid sequence such as the amino acid sequence as shown in SEQ ID NOs: 80 and 82 or SEQ ID NOs: 81 and 82, and 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 a higher sequence identity.

[0120] In one embodiment, the provided GPC3 antibody or its antigen-binding domain may have an 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 78, 114, 119, 126, and 129, and / or an 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 with an amino acid sequence selected from the group consisting of SEQ ID NOs: 79, 115, and 127. In another embodiment, the provided GPC3 antibody or its antigen-binding domain 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 with an amino acid sequence selected from the group consisting of SEQ ID NO: 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 with an amino acid sequence of SEQ ID NO: 82.

[0121] In one embodiment, the provided GPC3 antibody or the heavy chain variable region of its antigen-binding domain may have three CDRs having the following sequences: GYTFTDYE (HCDR1, SEQ NO: 72), LDPKTGDT (HCDR2, SEQ NO: 73), TRFYSYTY (HCDR3; SEQ NO: 74). In one embodiment, the provided GPC3 antibody or the heavy chain variable region of its antigen-binding domain may have three CDRs having the following sequences: GFTFNKNA (HCDR1, SEQ NO: 108), IRNKTNNYAT (HCDR2, SEQ NO: 109), VAGNSFAY (HCDR3; SEQ NO: 110). In one embodiment, the provided GPC3 antibody or the heavy chain variable region of its antigen-binding domain may have three CDRs having the following sequences: YFDFDSYE (HCDR1, SEQ NO: 116), IYHSGST (HCDR2, SEQ NO: 117), ARVNMDRFDY (HCDR3; SEQ NO: 118). In one embodiment, the provided GPC3 antibody or the heavy chain variable region of its antigen-binding domain may have three CDRs having the following sequences: GFTFSSYA (HCDR1, SEQ NO: 120), IQKQGLPT (HCDR2, SEQ NO: 121), AKNRAKFDY (HCDR3; SEQ NO: 122).

[0122] The light chain variable region of the GPC3 antibody or its antigen-binding domain provided in one embodiment may have three CDRs having the following sequences: QSLVHSNRNTY (LCDR1, SEQ NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ NO: 77). The light chain variable region of the GPC3 antibody or its antigen-binding domain provided in one embodiment may have three CDRs having the following sequences: QSLLYSSNQKNY (LCDR1, SEQ NO: 111), WAS (LCDR2), QQYYNYPLT (LCDR3; SEQ NO: 113). The light chain variable region of the GPC3 antibody or its antigen-binding domain provided in one embodiment may have three CDRs having the following sequences: QSISSY (LCDR1, SEQ NO: 123), NAS (LCDR2), QQNRGFPLT (LCDR3; SEQ NO: 125).

[0123] In one embodiment, the provided GPC3 antibody or its antigen-binding domain comprises a heavy chain variable region having three CDRs having the following sequences: GYTFTDYE (HCDR1, SEQ NO: 72), LDPKTGDT (HCDR2, SEQ NO: 73), TRFYSYTY (HCDR3; SEQ NO: 74), and a light chain variable region having three CDRs having the following sequences: QSLVHSNRNTY (LCDR1, SEQ NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ NO: 77). In one embodiment, the provided GPC3 antibody or its antigen-binding domain comprises a heavy chain variable region having three CDRs having the following sequences: GFTFNKNA (HCDR1, SEQ NO: 108), IRNKTNNYAT (HCDR2, SEQ NO: 109), VAGNSFAY (HCDR3; SEQ NO: 110), and a light chain variable region having three CDRs having the following sequences: QSLLYSSNQKNY (LCDR1, SEQ NO: 111), WAS (LCDR2), QQYYNYPLT (LCDR3; SEQ NO: 113). In one embodiment, the provided GPC3 antibody or its antigen-binding domain comprises a heavy chain variable region having three CDRs having the following sequences: GFTFSSYA (HCDR1, SEQ NO: 120), IQKQGLPT (HCDR2, SEQ NO: 121), AKNRAKFDY (HCDR3; SEQ NO: 122), and a light chain variable region having three CDRs having the following sequences: QSISSY (LCDR1, SEQ NO: 123), NAS (LCDR2), QQNRGFPLT (LCDR3; SEQ NO: 125).

[0124] Unless otherwise specified, all CDR sequences described herein are defined according to the IMGT method as 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 for germ cell V-gene consists of positions 105 to 116, and CDR3 for rearranged VJ-gene or VDJ-gene consists of positions 105 to 117 (formerly J-PHE or J-TRP position 118) having a gap at the top of the loop for rearranged CDR3-IMGT having fewer than 13 amino acids, or additional positions 112.1, 111.1, 112.2, 111.2 etc. for rearranged CDR3-IMGT having 13 or more amino acids. The positions provided in this paragraph follow the IMGT numbering described in Lefranc, M.-P., The Immunologist, 7, 132-136 (1999).

[0125] Antibodies specifically binding to GPC3 included in the fusion protein of the present disclosure may include an Fc portion that enables the in vivo half-life of the bispecific binding molecule of the present disclosure. In one embodiment, such an Fc portion is preferably of human origin, more preferably a human Fc portion of an IgG1 or IgG4 antibody, and much more preferably a engineered human Fc portion of IgG1 or IgG4 that activates or silences effector function. In one embodiment, in some embodiments, silencing effector function may be preferred over activating effector function. In some embodiments, such an Fc portion is engineered with mutation(s) at positions 234 and / or 235, which are numbered according to the Kabat EU index, to silence effector function (Johnson and Wu, Nucleic Acids Res, 2000). In one embodiment, mutations may be introduced at positions F234 and L235 of the provided anti-GPC3 antibody to silence effector function. In another embodiment, mutations are introduced at the D265 and P329 positions of the provided anti-GPC3 antibody to silence effector function. The numbering for these two sets of potential mutations follows Kabat's EU index (Shields et al., J Biol Chem, 2001).

[0126] 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 by immunizing animal blood with an antigen in a mixture with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous culture of cell lines. Examples of such techniques include, for example, the hybridoma technique described in Harlow and Lane (1999) (1988), the trioma technique described originally by Kohler and Milstein, 1975, the human B cell hybridoma technique (see, for example, Li et al., Proc Natl Acad Sci USA, 2006; Kozbor and Roder, Immunol Today, 1983), and the EBV-hybridomoma technique for producing human monoclonal antibodies (Cole et al., Cancer Res, 1984). Furthermore, recombinant antibodies may be obtained from monoclonal antibodies or produced in de novo using various display methods such as phage, ribosome, mRNA, or cell display. In some embodiments, a suitable system for expressing a recombinant (humanized) antibody or a fragment thereof may be selected from, for example, bacteria, yeast, insect, mammalian cell lines, or transgenic animals or plants (e.g., see U.S. Patent No. 6,080,560; Holliger and Hudson, Nat Biotechnol, 2005). Additionally, the techniques described for producing single-strand antibodies (particularly see U.S. Patent No. 4,946,778) may be applied to produce single-strand antibodies specific to the targets of this invention. The efficiency of phage antibodies was increased by utilizing surface plasmon resonance used in the BIAcore system.

[0127] C. Main disclosure Exemplary Lipocalin Mutane .

[0128] Lipocalins are naturally evolved protein-based binding molecules that bind to ligands. Lipocalins are found in many organisms, including vertebrates, insects, plants, and bacteria. Members of the lipocalin protein family (Pervaiz and Brew, FASEB J Lipocalins (1987) are typically small secretory proteins with a single polypeptide chain. They are characterized by distinct molecular recognition properties: binding to various, primarily hydrophobic small molecules (e.g., retinoids, fatty acids, cholesterol, prostaglandins, biliverdins, pheromones, tastants, and deodorants), specific binding to cell surface receptors, and the formation of macromolecular complexes. While they were previously classified primarily as transport proteins, it is now evident that lipocalins perform various physiological functions. These include retinol transport, olfaction, pheromone signaling, and prostaglandin synthesis. Lipocalins are also involved in the regulation of immune responses and the mediation of cellular homeostasis (e.g., Flower et al., Biochim Biophys Acta , 2000, Flower, Biochem J (Reviewed in , 1996).

[0129] Lipocalins share a very low level of total sequence conservation, typically with sequence identity of less than 20%. In contrast, their total folding patterns are highly conserved. The central portion of the lipocalin structure consists of self-closed, eight-stranded antiparallel β-sheets that form a continuously hydrogen-bonded pi-barrel. This pi-barrel forms a central cavity. One end of the barrel is sterically blocked by three peptide loops connecting an N-terminal peptide segment running across the bottom and the β-strands. The other end of the pi-barrel opens to the solvent and contains a target-binding site formed by four flexible peptide loops (AB, CD, EF, and GH). This is a variation of the loops found in other rigid lipocalin scaffolds, each giving rise to different binding modes capable of accommodating targets of different sizes, shapes, and chemical properties (e.g., Skerra, Biochim Biophys Acta , 2000, Flower et al., Biochim Biophys Acta , 2000, Flower, Biochem J (Reviewed in , 1996).

[0130] The lipocalin mutain according to the present disclosure may be any lipocalin mutain. Examples of suitable lipocalins for which mutain may be used (sometimes also referred to as "reference lipocalin," "wild-type lipocalin," "reference protein scaffold," or simply "scaffold") include lipocalin (lipocalin-1, Tlc, or von Ebner gland protein), retinol-binding protein, neutrophil lipocalin-type prostaglandin D-synthase, β-lactoglobulin, bilin-binding protein (BBP), apolipoprotein D (APOD), neutrophil gelatinase-associated lipocalin (NGAL), α2-microglobulin-related protein (A2m), and 24p3 / eutecalin (24p3 / uterocalin, 24p3), von Ebner's gland protein 1 (VEGP 1) and Major allergen Can f 1 (ALL-1) are included, but not limited thereto. In related embodiments, lipocalin mutains include human tear lipocalin (hTlc), human neutrophil gelatinase-associated lipocalin (hNGAL), human apolipocalin D (hAPOD) and Pieris brassicae ( Pieris brassicae It is derived from the lipocalin group, which consists of bilin-binding proteins.

[0131] The amino acid sequence of the lipocalin mutain according to the present disclosure may have high sequence identity when comparing sequence identity with other lipocals (see also above), for example, with reference (or wild-type) lipocals derived from hTlc or hNGAL. In this general context, the amino acid sequence of the lipocalin mutain according to the present disclosure is at least substantially similar to the amino acid sequence of the corresponding reference (wild-type) lipocalin, provided that there may be gaps in alignment resulting from the addition or deletion of amino acids (as defined herein). Each sequence of the lipocalin mutain of the present disclosure that is substantially similar to the sequence of the corresponding reference (wild-type) lipocalin has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 87%, and at least 90% identity with the sequence of the corresponding lipocalin, including at least 95% identity in some embodiments. In this regard, the lipocalin mutain of the present disclosure may include a substitution as described in this specification that provides a lipocalin mutain capable of binding to CD137 as well as a lipocalin mutain.

[0132] Typically, lipocalin mutaine contains a ligand-binding pocket in comparison to the amino acid sequences of wild-type or reference lipocalin, e.g., hTlc and hNGAL, and contains one or more mutated amino acid residues within four loops at the open end defined as the entrance of the ligand-binding pocket (see above). As described above, these regions are essential for determining the binding specificity of lipocalin mutaine to a desired target. In some embodiments, the lipocalin mutaine of the present disclosure may also contain mutated amino acid residues in addition to the four loops. In some embodiments, the lipocalin mutaine of the present disclosure may contain one or more mutated amino acid residues in one or more of three peptide loops (named BC, DE, and FG) connecting the β-strand at the closed end of the lipocalin. In some embodiments, the tear lipocalin, the mutain derived from NGAL lipocalin, or its homolog 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 end of the pie-barrel structure opposite to the N-terminal region and / or the natural lipocalin binding pocket. In some embodiments, the tear lipocalin, the mutain derived from NGAL lipocalin, or its homolog may not have mutated amino acid residues in the peptide loop DE arranged at the end of the pie-barrel structure compared to the wild-type sequence of tear lipocalin.

[0133] In one embodiment, the lipocalin mutain according to the present disclosure may comprise one or more mutated amino acid residues, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, compared with the amino acid sequence of a corresponding reference (wild-type) lipocalin, provided that such lipocalin must be able to bind to CD137. In some embodiments, the lipocalin mutain of the present disclosure comprises at least two mutated amino acid residues, including 2, 3, 4, 5 or more, where a natural amino acid residue of the corresponding reference (wild-type) lipocalin is substituted with an arginine residue.

[0134] Any type and number of mutations, including substitutions, deletions, and insertions, are expected as long as the provided lipocalin mutain has the ability to bind to CD137 and / or has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or higher sequence identity with the amino acid sequence of reference (wild-type) lipocalin, e.g., mature hTlc or mature hNGAL.

[0135] 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 example substitutions below.

[0136] Specifically, to ensure that the amino acid residues of the amino acid sequence of lipocalin mutain differ from those of reference (wild-type) lipocalin, a person skilled in the art may use means and methods well known in the art, such as manual alignment or alignment using a computer program such as BLAST2.0, which represents the Basic Local Alignment Search Tool or ClustalW, or any other suitable program suitable for generating sequence alignment. Thus, the amino acid sequence of the reference (wild-type) lipocalin may be provided as the "target sequence" or "reference sequence," while the amino acid sequence of lipocalin mutain may be provided as the "query sequence" (see also above).

[0137] Conservative substitutions are generally the following substitutions listed according to the amino acid to be mutated, each followed by one or more substitutions that can be considered conservative: Ala → Ser, Thr, or Val; Arg → Lys, Gln, Asn, or His; Asn → Gln, Glu, Asp, or His; Asp → Glu, Gln, Asn, or His; Gln → Asn, Asp, Glu, or His; Glu → Asp, Asn, Gln, or His; His → Arg, Lys, Asn, Gln, Asp, or Glu; Ile → Thr, Leu, Met, Phe, Val, Trp, Tyr, Ala, or Pro; Leu → Thr, Ile, Val, Met, Ala, Phe, Pro, Tyr, or Trp; Lys → Arg, His, Gln, or Asn; Met → Thr, Leu, Tyr, Ile, Phe, Val, Ala, Pro, or Trp; Phe → Thr, Met, Leu, Tyr, Ile, Pro, Trp, Val, or Ala; Ser → Thr, Ala, or Val; Thr → Ser, Ala, Val, Ile, Met, Val, Phe, Pro, or Leu; Trp → Tyr, Phe, Met, Ile, or Leu; Tyr → Trp, Phe, Ile, Leu, or Met; Val → Thr, Ile, Leu, Met, Phe, Ala, Ser, or Pro. Other substitutions are also permitted and may be determined empirically or based on other known conservative or non-conservative substitutions. As an additional direction, the following groups each include amino acids that can typically be used to define mutually conservative substitutions:

[0138] (a) Alanine (Ala), Serine (Ser), Threonine (Thr), Valine (Val)

[0139] (b) Aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), asparagine (Asn), histidine (His)

[0140] (c) Arginine (Arg), Lysine (Lys), Glutamine (Gln), Asparagine (Asn), Histidine (His)

[0141] (d) Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val), Alanine (Ala), Phenylalanine (Phe), Threonine (Thr), Proline (Pro)

[0142] (e) Isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp)

[0143] If such conservative substitutions result in a change in biological activity, more substantial changes such as those described below, or additionally described below in reference to amino acid classes, may be introduced to screen the product for desired properties. 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.

[0144] In one embodiment, substantial modifications to the physical and biological characteristics of lipocalin (mutane) are achieved by selecting significantly different substitutions in (a) the structure of the polypeptide backbone in the substitution region as, for example, a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining large side chains.

[0145] Naturally occurring residues are grouped based on common side chain characteristics: (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 direction: glycine, proline; and (6) aromatic: tryptophan, tyrosine, phenylalanine. In one embodiment, substitution may involve exchanging a member of one of these classes for another.

[0146] Any cysteine ​​residue not involved in maintaining the proper structure of each lipocalin is also generally substituted with serine to improve the molecule's oxidative stability and prevent abnormal cross-linking. Conversely, cysteine ​​bond(s) can be added to lipocalin to improve its stability.

[0147] D. Main disclosure Exemplary CD137-specific Lipocalin Mutane .

[0148] As noted above, lipocalin is a polypeptide defined by its supersecondary structure, also known as a cylindrical β-sheet structure supersecondary structure region, comprising eight β-strands paired by four loops at one end and defined as binding pockets. The present disclosure is not limited to the lipocalin mutain disclosed in detail herein. In this regard, the present disclosure relates to a lipocalin mutain having a cylindrical β-sheet structure supersecondary structure region comprising eight β-strands paired by four loops at one end and defined as binding pockets, provided that at least one amino acid of each of at least three of the four loops is mutated, and said lipocalin is effective at binding to CD137 with detectable affinity.

[0149] In one embodiment, the lipocalin mutain disclosed herein is or may comprise a mutain of mature human tear lipocalin (hTlc). The mutain of mature hTlc is referred to herein as “hTlc mutain.” In some other embodiments, the lipocalin mutain disclosed herein is a mutain of mature human neutrophil gelatinase-associated lipocalin (hNGAL). The mutain of mature hNGAL is referred to herein as “hNGAL mutain.”

[0150] In one embodiment, the present disclosure comprises any number of lipocalin mutains derived from reference (wild-type) lipocalin, preferably derived from mature hTlc or mature hNGAL, which bind to CD137 with detectable affinity. In a related embodiment, the present disclosure comprises various lipocalin mutains capable of binding to CD137 and activating the downstream signaling pathway of CD137. In this sense, CD137 may be considered a non-natural target of reference (wild-type) lipocalin, preferably hTlc or hNGAL, where "non-natural target" refers to a substance that does not bind to reference (wild-type) lipocalin under physiological conditions. By manipulating reference (wild-type) lipocalin having one or more mutations at specific sequence positions, the inventors have demonstrated that high affinity and high specificity for the non-natural target, CD137, are possible. In some embodiments, random mutagenesis may be performed by substitution of a subset of nucleotide triplets at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotide triplet(s) encoding specific sequence positions on wild-type lipocalin for the purpose of producing a lipocalin mutain capable of binding to CD137.

[0151] In one embodiment, the lipocalin mutain of the present disclosure may have amino acid residue(s) mutated, including substitution, deletion, and insertion at one or more positions corresponding to the linear polypeptide sequence of reference lipocalin, preferably hTlc or hNGAL. In some embodiments, the number of amino acid residues of the lipocalin mutain of the present disclosure that are mutated compared to the amino acid sequence of reference lipocalin, preferably hTlc or hNGAL, is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, e.g., 25, 30, 35, 40, 45 or 50, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, and more preferably 9, 10 or 11. However, it is preferable that the lipocalin mutain of the present disclosure can still bind to CD137.

[0152] In one embodiment, the lipocalin mutain of the present disclosure may be deficient in 1, 2, 3, 4 or more amino acids at its N-terminus and / or 1, 2 or more amino acids at its C-terminus compared to each reference (wild-type) lipocalin; e.g., SEQ ID NO: 32-38. In one embodiment, the present disclosure comprises an hTlc mutain as defined above, wherein the first 1, 2, 3, or N-terminal amino acid residues of the mature hTlc sequence (His-His-Leu-Leu; positions 1-4) and / or the last 1 or 2 C-terminal amino acid residues of the linear polypeptide sequence of the mature hTlc (Ser-Asp; positions 157-158) are deleted (e.g., SEQ ID NO: 32-38). In one embodiment, the present disclosure comprises an hNGAL mutain as defined above, wherein amino acid residues (Lys-Asp-Pro, positions 46-48) of the linear polypeptide sequence of mature hNGAL are deleted (SEQN: 43). Additionally, the lipocalin mutain of the present disclosure may comprise a reference (wild-type) lipocalin, preferably a wild-type (natural) amino acid sequence of hTlc or hNGAL, in addition to the mutated amino acid sequence positions.

[0153] In one embodiment, one or more mutated amino acid residues incorporated into the lipocalin mutain of the present disclosure do not substantially impede or interfere with binding activity with a designated target and the folding of the mutain. Such mutations, including substitutions, deletions, and insertions, can be achieved at the DNA level using established standard methods (Sambrook and Russell, 2001, Molecular cloning: a laboratory manual). In one embodiment, one or more mutated amino acid residues are introduced through random mutagenesis by replacing a nucleotide triplet(s) encoding a reference (wild-type) lipocalin, preferably a corresponding sequence position of the reference lipocalin, with a subset of nucleotide triplets, to one or more sequence positions corresponding to the linear polypeptide sequence of hTlc or hNGAL.

[0154] In one embodiment, the provided lipocalin mutain that binds to CD137 with detectable affinity may comprise at least one amino acid substitution of a natural cysteine ​​residue to another amino acid, e.g., a serine residue. In one embodiment, the lipocalin mutain that binds to CD137 with detectable affinity may comprise one or more non-natural cysteine ​​residues that substitute one or more amino acids of reference (wild-type) lipocalin, preferably hTlc or hNGAL. In one embodiment, the lipocalin mutain according to the present disclosure comprises forming one or more cysteine ​​bridges through at least two amino acid substitutions to cysteine ​​residues of natural amino acids. In one embodiment, the cysteine ​​bridge may connect at least two loop regions. The definition of these regions is ( Biochim Biophys Acta It is used in this invention according to Flower (1996) and Breustedt et al. (2005), , 2000).

[0155] Generally, the lipocalin mutain of the present disclosure may have at least about 70% amino acid sequence identity with the amino acid sequence of mature hTlc (SEQ No. 1) or mature hNGAL (SEQ No. 2), including at least about 80%, e.g., at least about 85%.

[0156] In some embodiments, the present disclosure provides a CD137-bound hTlc mutain. In this regard, the present disclosure provides K of about 300 nM, 200 nM, 150 nM, and 100 nM or less. D Provides one or more hTlc mutains capable of binding to CD137 with an affinity measured by [formula]. In one embodiment, the provided hTlc mutains have an EC of about 250 nM, 150 nM, 100 nM, 50 nM, 20 nM or less. 50 It can bind to CD137 as a value. In some other embodiments, CD137-binding hTlc mutains can cross-react with cyanomolecular CD137 (cyCD137).

[0157] In one embodiment, the hTlc mutain of the present disclosure may interfere with the binding of CD137L to CD137.

[0158] In one embodiment, the provided hTlc mutain may comprise one or more mutated amino acid residues at positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature hTlc (SEVENTEEN NO: 1).

[0159] In one embodiment, the provided hTlc mutain may comprise one or more mutated amino acid residues at positions corresponding to positions 26-34, 55-58, 60-61, 65, 104-106, and 108 of the linear polypeptide sequence of mature hTlc (SEVENTEEN NO: 1).

[0160] In one embodiment, the provided hTlc mutain may further comprise one or more mutated amino acid residues at positions corresponding to positions 101, 111, 114, and 153 of the linear polypeptide sequence of mature hTlc (SEVENTEEN NO: 1).

[0161] In one embodiment, the provided hTlc mutain is at positions 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 higher 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 (SEVENTEEN NO: 1). It may contain mutated amino acid residues. In some preferred embodiments, the provided hTlc mutain may bind to CD137, particularly human CD137.

[0162] In one embodiment, the provided hTlc mutain may comprise mutated amino acid residues at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, corresponding to positions 26-34, 55-58, 60-61, 65, 104-106, and 108 of the linear polypeptide sequence of mature hTlc (SEVENTEEN NO: 1). In some preferred embodiments, the provided hTlc mutain may bind to CD137, particularly human CD137.

[0163] In one embodiment, the lipocalin mutain according to the present disclosure may comprise at least one amino acid substitution to a natural cysteine ​​residue, such as a serine residue. In one embodiment, the hTlc mutain according to the present disclosure may comprise an amino acid substitution to a natural cysteine ​​residue, such as a serine residue, at a position corresponding to the 61 and / or 153 positions of the linear polypeptide sequence of mature hTlc (SEVENTEEN NO: 1). In this context, the removal of the structural disulfide bond of wild-type hTlc formed by cysteine ​​residues 61 and 153 (at the level of each naive nucleic acid library) (Breustedt et al., J Biol Chem Note that (see , 2005) it has been found that hTlc mutains can be provided that not only fold stably but also bind to non-natural targets with high affinity. In one embodiment, the removal of structural disulfide bonds may also provide the additional advantage of increasing the stability of the mutains by creating or considering the introduction of non-natural disulfide bonds into the mutains of the present disclosure. However, hTlc mutains having a disulfide bridge formed between Cys 61 and Cys 153 and bound to CD137 are also part of the present disclosure.

[0164] In some specific embodiments, the hTlc mutain of the present disclosure may comprise one or more amino acid substitutions Cys 61→Ala, Phe, Lys, Arg, Thr, Asn, Gly, Gln, Asp, Asn, Leu, Tyr, Met, Ser, Pro or Trp and / or Cys 153→Ser or Ala at positions corresponding to the 61 and / or 153 positions of the linear polypeptide sequence of mature hTlc (SEVENTEEN NO: 1).

[0165] In one embodiment, two or three of the cysteine ​​codons are replaced with codons of other amino acids at positions corresponding to positions 61, 101, and 153 of the linear polypeptide sequence of mature hTlc (SEQ No. 1). Additionally, in one embodiment, the hTlc mutain according to the present disclosure comprises an amino acid substitution of a serine residue or a histidine residue of a natural cysteine ​​residue at a position corresponding to position 101 of the linear polypeptide sequence of mature hTlc (SEQ No. 1).

[0166] In one embodiment, the mutain according to the present disclosure comprises an amino acid substitution to a cysteine ​​residue of a natural amino acid at a position corresponding to the 28 or 105 position of the linear polypeptide sequence of mature hTlc (SEQ No. 1). Additionally, in one embodiment, the mutain according to the present disclosure comprises an amino acid substitution to a proline residue of a natural arginine residue at a position corresponding to the 111 position of the linear polypeptide sequence of mature hTlc (SEQ No. 1). Additionally, in one embodiment, the mutain according to the present disclosure comprises an amino acid substitution to a tryptophan residue or glutamic acid of a natural lysine residue at a position corresponding to the 114 position of the linear polypeptide sequence of mature hTlc (SEQ No. 1).

[0167] In one embodiment, the provided CD137-binding hTlc mutain 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 (SEVENTEEN 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; Lys 65 → Arg or Asn; Thr 71 → Ala; Val 85 → Asp; Lys 94 → Arg or Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile; and Cys 153 → Ser. In one embodiment, the hTlc mutain of the present disclosure comprises two or more, e.g., 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 (SEVENTEEN: 1) positions of mature hTlc.

[0168] In one embodiment, the provided CD137-binding hTlc mutain may comprise one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hTlc (SEQ No. 1):

[0169] (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;

[0170] (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;

[0171] (c) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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; 및 Cys 153 → Ser;

[0172] (d) Ala 5 → Val; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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; 및 Cys 153 → Ser;

[0173] (e) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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 → Island; 및 Cys 153 → Ser;

[0174] (f) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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 → Island; 및 Cys 153 → Ser; 및

[0175] (g) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Gly 46 → Asp; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile; and Cys 153 → Ser.

[0176] In one embodiment, the remaining region, i.e., the region 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 mutain of the present disclosure (SEQ No. 1), may include the wild-type (natural) amino acid sequence of the linear polypeptide sequence of mature hTlc in addition to the mutated amino acid sequence positions.

[0177] In one embodiment, the hTlc mutain of the present disclosure has at least 70% sequence identity or at least 70% sequence homology with the sequence of mature hTlc (SEQ: 1). As an illustrative example, the mutain of SEQ: 32 has approximately 84% amino acid sequence identity or sequence homology with the amino acid sequence of mature hTlc.

[0178] In one embodiment, the hTlc mutain of the present disclosure comprises an amino acid sequence or a fragment or variant thereof described in any one of SEQ ID NOs: 32-38.

[0179] In one embodiment, the hTlc mutain of the present disclosure 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 the group consisting of SEQ ID NOs: 32-38.

[0180] The present disclosure also comprises structural homologs of hTlc mutain having amino acid sequences selected from the group consisting of SEQ ID NOs: 32-38, wherein the structural homologs have at least about 60%, preferably at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, and most preferably at least 95% amino acid sequence homology or sequence identity compared to said hTlc mutain.

[0181] In some embodiments, the present disclosure provides a CD137-bound hNGAL mutain. In this regard, the present disclosure provides K of about 800 nM, 700 nM, 200 nM, 140 nM, 100 nM or less, preferably about 70 nM, 50 nM, 30 nM, 10 nM, 5 nM, 2 nM or less. D Provides one or more hNGAL mutaines capable of binding to CD137 with an affinity measured by [formula]. In one embodiment, the provided hNGAL mutaine has an EC of about 1000 nM, 500 nM, 100 nM, 80 nM, 50 nM, 25 nM, 18 nM, 15 nM, 10 nM, and 5 nM or less. 50 It can be combined with CD137 as a value.

[0182] In one embodiment, the provided CD137-binding hNGAL mutain may cross-react with cyanomolgus CD137. In one embodiment, the provided hNGAL mutain has a K of about 50 nM, 20 nM, 10 nM, 5 nM, 2 nM or less. DIt can bind to cyanomolgus CD137 with an affinity measured by [formula]. In one embodiment, the provided hNGAL mutain has an EC of about 100 nM, 80 nM, 50 nM, and 30 nM or less. 50 It can be combined with cyanomorphic CD137 as a value.

[0183] In one embodiment, the hNGAL mutain of the present disclosure may interfere with or compete with the binding of CD137L to CD137. In some other embodiments, the hNGAL mutain of the present disclosure may bind to CD137 and / or to the CD137 / CD137L complex in the presence of CD137L.

[0184] In one embodiment, the provided hNGAL mutain may comprise one or more mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEVENTEEN NO: 2).

[0185] In one embodiment, the provided hNGAL mutain may comprise mutated amino acid residues at positions 1, 2, 3, 4, 5, 6, 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 (SEVENTEEN NO: 2). In some preferred embodiments, the provided hNGAL mutain can combine with CD137, particularly human CD137.

[0186] In one embodiment, the provided hNGAL mutaine may comprise one or more mutated amino acid residues at 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 (SEVENTEEN NO: 2). In some preferred embodiments, the provided hNGAL mutaine may bind to CD137, particularly human CD137.

[0187] In one embodiment, the provided hNGAL mutain may comprise one or more mutated amino acid residues corresponding to positions 36, 87, and 96 of the linear polypeptide sequence of mature hNGAL (SEQ No. 2) and one or more positions corresponding to positions 28, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 94, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ No. 2).

[0188] In another embodiment, the provided hNGAL mutain may comprise one or more mutated amino acid residues at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEVENTEEN NO: 2).

[0189] In another embodiment, the provided hNGAL mutain may comprise one or more mutated amino acid residues corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ No. 2) and one or more positions corresponding to 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 No. 2).

[0190] In one embodiment, the lipocalin mutain according to the present disclosure may comprise at least one amino acid substitution of a natural cysteine ​​residue by, for example, a serine residue. In one embodiment, the hNGAL mutain according to the present disclosure may comprise an amino acid substitution of a natural cysteine ​​residue by another amino acid, such as a serine residue, at a position corresponding to positions 76 and / or 175 of the linear polypeptide sequence of mature hNGAL (SEQ No. 2). In this context, the removal of structural disulfide bonds of wild-type hNGAL formed by cysteine ​​residues 76 and 175 (at the level of each naive nucleic acid library) (Breustedt et al., J Biol Chem It is noted that hNGAL mutaine (see , 2005) can provide a structure that not only folds stably but also binds to a given non-natural target with high affinity. In one embodiment, the removal of structural disulfide bonds may provide the additional advantage of increasing the stability of the mutaine by creating or deterring the introduction of non-natural disulfide bonds into the mutaine of the present disclosure. However, hNGAL mutaine having a disulfide bridge formed between Cys 76 and Cys 175 and bound to CD137 is also part of the present disclosure.

[0191] In one embodiment, the provided CD137-binding hNGAL mutain may comprise one or more of the following mutated amino acid residues at one or more positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEVENTEEN 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 one embodiment, the hNGAL mutain of the present disclosure comprises two or more, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more, e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or all mutated amino acid residues at these sequence (SEVENTEEN: 2) positions of mature hNGAL.

[0192] In one embodiment, the provided CD137-binding hNGAL mutain may comprise one or more of the following mutated amino acid residues at one or more positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEVENTEEN 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. In one embodiment, the hNGAL mutain of the present disclosure comprises two or more mutated amino acid residues at these sequence (SEVENTEEN: 2) positions of mature hNGAL, e.g., 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.

[0193] In one embodiment, the provided CD137-binding hNGAL mutain may comprise one or more of the following mutated amino acid residues corresponding to positions 36, 40, 41, 49, 52, 68, 70, 72, 73, 77, 79, 81, 96, 100, 103, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEVENTEEN NO: 2), e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19: 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 one embodiment, the provided CD137-binding hNGAL mutain may further comprise one or more of the following mutated amino acid residues corresponding to positions 20, 25, 33, 44, 59, 71, 78, 80, 82, 87, 92, 98, 101, and 122 of the linear polypeptide sequence of mature hNGAL (SEVENTEEN NO: 2), e.g., positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14: 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.

[0194] In one embodiment, the provided CD137-binding hNGAL mutain may comprise one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ No. 2):

[0195] (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;

[0196] (b) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; District 41 → Arg; Gn 49 → Island; Tyr 52 → Meth; 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 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0197] (c) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; District 41 → Arg; Gn 49 → Asn; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Wing; Leu 70 → Wing; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0198] (d) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gn 49 → Asn; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Wing; Leu 70 → Wing; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0199] (e) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gn 49 → Ser; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Ser; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → No; Arg 81 → Meth; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0200] (f) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gln 49 → Val; Tyr 52 → Meth; 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 → The; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0201] (g) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; District 41 → Arg; Gln 49 → His; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → No; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0202] (h) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gn 49 → Asn; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → No; Arg 81 → Ser; Phy 83 → Leu; Cys 87 → Ser; Leu 94 → The; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr; 또는

[0203] (i) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; District 41 → Arg; Gn 49 → Ser; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Wing; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Asn; Trp 79 → No; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr.

[0204] In some other embodiments, in the remaining region, i.e., in a region different from positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence of mature hNGAL (SEQ No. 2), the hNGAL mutain of the present disclosure may include the wild-type (natural) amino acid sequence of mature hNGAL in addition to the mutated amino acid sequence positions.

[0205] In some other embodiments, the provided CD137-binding hNGAL mutain may comprise one of the following sets of amino acid residues mutated compared to the linear polypeptide sequence of mature hNGAL (SEQ No. 2):

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

[0207] (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;

[0208] (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;

[0209] (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;

[0210] (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;

[0211] (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;

[0212] (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;

[0213] (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;

[0214] (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

[0215] (j) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Asp; Gln 49 → His; Taurus 52 → Sees; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Light 73 → Asp; Asp 77 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Light 125 → Looks; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly.

[0216] In one embodiment, the provided CD137-binding hNGAL mutain may comprise the following set of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ No. 2) and Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ile; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met; Leu 70 → Lys; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr and / or the provided mutain may have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0217] In one embodiment, in the remaining region, i.e., in a region different from positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ No. 2), the hNGAL mutain of the present disclosure may include the wild-type (natural) amino acid sequence of mature hNGAL in addition to the mutated amino acid sequence positions.

[0218] In one embodiment, the hNGAL mutain of the present disclosure has at least 70% sequence identity or at least 70% sequence homology with the sequence of mature hNGAL (SEQ: 2). In an exemplary embodiment, the mutain of SEQ: 40 has approximately 87% amino acid sequence identity or sequence homology with the amino acid sequence of mature hNGAL.

[0219] In one embodiment, the hNGAL mutain of the present disclosure comprises an amino acid sequence or a fragment thereof or a variant thereof described in any one of SEQ ID NOs: 39-57.

[0220] In one embodiment, the hNGAL mutain of the present disclosure 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 the group consisting of SEQ ID NOs: 39-57.

[0221] The present disclosure also comprises a structural homolog of hNGAL mutain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-57, wherein the structural homolog has about 60%, preferably 65% ​​or more, 70% or more, 755% or more, 80% or more, 85% or more, 90% or more, 92% or more, and most preferably 95% or more amino acid sequence homology or sequence identity compared to said hNGAL mutain.

[0222] In one embodiment, the present disclosure has a K of about 5 nM or less. D Provides a lipocalin mutain that binds to CD137 with an affinity measured by [amount], wherein the lipocalin mutain has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0223] In one embodiment, the present disclosure has a K of about 5 nM or less. DProvides a lipocalin mutain that binds to CD137 with an affinity measured by [amount], wherein the lipocalin mutain has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or higher sequence identity with the amino acid sequence of SEQ ID NO: 49.

[0224] In one embodiment, the lipocalin mutain of the present disclosure may include a heterogeneous amino acid sequence, such as a Strep II tag (SEVENTEEN NO: 12) or a cleavage site sequence for a specific restriction enzyme, at its N- or C-terminus, preferably the C-terminus, without affecting the biological activity of the lipocalin mutain (binding with its target, e.g., CD137).

[0225] In one embodiment, additional modifications of lipocalin mutain may be introduced to modify specific characteristics of the mutain, for example, to improve folding stability, serum stability, protein tolerance, or water solubility, or to reduce the tendency to aggregation, or to introduce new characteristics to the mutain. In one embodiment, the modification(s) may result in two or more characteristics (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the provided mutain being modified.

[0226] For example, it is possible to mutate one or more amino acid sequence positions of lipocalin mutain to introduce a new reactive group, for example, through conjugation with other compounds such as polyethylene glycol (PEG), hydroxyethyl starch (HES), biotin, peptides, or proteins, or to form non-naturally occurring disulfide bonds. The conjugated compounds, for example, PEG and HES, may increase the serum half-life of the corresponding lipocalin mutain in some cases.

[0227] In some embodiments, the reactive group of lipocalin mutaine may occur naturally in its amino acid sequence, such as a cysteine ​​residue that occurs naturally in the amino acid sequence. In some other embodiments, such a reactive group may be introduced through mutagenesis. When the reactive group is introduced through mutagenesis, one possibility is a mutation of the amino acid at a suitable position by a cysteine ​​residue. Exemplary possibilities of such a mutation introducing a cysteine ​​residue into the amino acid sequence of hTlc mutaine include the Thr 40→Cys, Glu 73→Cys, Arg 90→Cys, Asp 95→Cys, and Glu 131→Cys substitutions in the wild-type sequence of hTlc (SEQ No. 1). Exemplary possibilities of such mutations introducing cysteine ​​residues into the amino acid sequence of hNGAL mutain include the introduction of 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 No. 2). The resulting thiol moiety may be used, for example, to PEGylate or HESylate the mutain to increase the serum half-life of each lipocalin mutain.

[0228] In one embodiment, an artificial amino acid may be introduced into the amino acid sequence of lipocalin mutain to provide an amino acid side chain suitable as a new reactor for conjugating one of the compounds to lipocalin mutain. Generally, such artificial amino acids are designed to be more reactive so as to facilitate conjugation with the desired compound. Such artificial amino acids may be introduced, for example, by mutagenesis using para-acetylphenylalanine, an artificial tRNA.

[0229] In one embodiment, the lipocalin mutain of the present disclosure is fused at its N-terminus or its C-terminus to a protein, protein domain, or peptide, e.g., an antibody, a signal sequence, and / or an affinity tag. In some other embodiments, the lipocalin mutain of the present disclosure is conjugated at its N-terminus or its C-terminus to a partner, which is a protein, protein domain, or peptide; e.g., an antibody, a signal sequence, and / or an affinity tag.

[0230] Strep-tag or Strep-tag II enabling easy detection and / or purification of recombinant proteins (Schmidt et al., J Mol Biol (e.g., 1996), affinity tags such as c-myc-tag, FLAG-tag, His-tag, or HA-tag, or proteins such as glutathione-S-transferase are examples of suitable fusion partners. Proteins having chromogenic or fluorescent properties, such as green fluorescent protein (GFP) or yellow fluorescent protein (YFP), are also suitable fusion partners for the lipocalin mutain of the present disclosure. In general, it is possible to generate a compound or signal detectable by chemical, physical, optical, or enzymatic reactions, directly or indirectly, by labeling the lipocalin mutain of the present disclosure with any suitable chemical or enzyme. For example, fluorescent or radioactive labels may be conjugated to the lipocalin mutain to generate fluorescence or X-rays as a detectable signal. Alkaline phosphatase, horseradish peroxidase, and β-galactosidase are examples of enzymatic labels (and simultaneously optical labels) that catalyze the formation of chromogenic reaction products. Generally, all labels commonly used on antibodies (excluding those in which a sugar moiety is exclusively used in the Fc portion of the immunoglobulin) may also be used for conjugation with the lipocalin mutain of the present disclosure.

[0231] In one embodiment, the lipocalin mutain of the present disclosure may be fused or conjugated with a moiety that extends the serum half-life of the mutain (in this regard, also refer to International Patent Publication No. WO2006 / 056464, in which such a strategy is described by reference to a mutain of human neutrophil gelatinase-associated lipocalin (hNGAL) having binding affinity for CTLA-4). Moietys that extend the serum half-life include, to name just a few, PEG molecules, HES molecules, fatty acid molecules, e.g., palmitic acid (Vajo and Duckworth, Pharmacol Rev , 2000), Fc portion of immunoglobulin, C of immunoglobulin H 3 domains, immunoglobulin C H It may be a 4-domain, albumin-binding peptide, albumin-binding protein, or transferrin.

[0232] In one embodiment, when PEG is used as a conjugation partner, the PEG molecule may be substituted, unsubstituted, linear, or branched. Additionally, it may be an activated polyethylene derivative. Examples of suitable compounds are those described in International Patent Publication No. WO1999 / 64016, U.S. Patent No. 6,177,074, or U.S. Patent No. 6,403,564 in relation to interferon, or those 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) is a PEG molecule. The molecular weight of such polymers, such as polyethylene glycol, may be in the range of about 300 to about 70,000 daltons, including polyethylene glycol having a molecular weight of about 10,000, about 20,000, about 30,000, or about 40,000 daltons. Furthermore, polymers such as carbohydrate oligomers and HES, as described, for example, in U.S. Patent No. 6,500,930 or No. 6,620,413, may be conjugated to the mutain of the present disclosure for the purpose of extending serum half-life.

[0233] In one embodiment, where the Fc portion of the immunoglobulin is used for the purpose of extending the serum half-life of the lipocalin mutain of the present disclosure, SynFusion commercially available from Syntonix Pharmaceuticals, Inc. (MA, USA) TM The technology can be used. The use of this Fc-fusion technology can create longer-lasting biopharmaceuticals, and, for example, can improve pharmacokinetics, availability, and production efficiency by consisting of two copies of a mutain linked to the Fc region of an antibody.

[0234] Examples of albumin-binding peptides that can be used to extend the serum half-life of lipocalin mutain are those having the common sequence Cys-Xaa1-Xaa2-Xaa3-Xaa4-Cys as described in, for example, U.S. Patent Publication No. 20030069395 or Dennis et al. (2002), where Xaa1 is Asp, Asn, Ser, Thr, or Trp; Xaa2 is Asn, Gln, His, Ile, Leu, or Lys; Xaa3 is Ala, Asp, Phe, Trp, or Tyr; and Xaa4 is Asp, Gly, Leu, Phe, Ser, or Thr. An albumin-binding protein fused or conjugated to lipocalin mutain to prolong serum half-life may be a bacterial albumin-binding protein, an antibody, an antibody fragment including a domain antibody (see, e.g., U.S. Patent No. 6,696,245), or a lipocalin mutain having binding activity for albumin. Examples of bacterial albumin-binding proteins include streptococcal protein G (Konig and Skerra, J Immunol Methods , 1998).

[0235] In one embodiment, if the albumin-binding protein is an antibody fragment, it may be a domain antibody. Domain antibodies (dAbs) are engineered to allow for precise control over biophysical properties and in vivo half-life to produce an optimal safety and efficacy product profile. Domain antibodies are commercially available, for example, from Domantis Ltd. (Cambridge, UK, and MA, USA).

[0236] In one embodiment, albumin itself (Osborn et al., J Pharmacol Exp Ther(e.g., 2002), or a biologically active fragment of albumin may be used as a partner to the lipocalin mutain of the present disclosure to extend the serum half-life. The term “albumin” includes all mammalian albumins, such as human serum albumin, bovine serum albumin, or rat albumin. Albumin or a fragment thereof may be produced recombinantly as described in U.S. Patent No. 5,728,553 or European Patent Publications EP0330451 and EP0361991. Accordingly, recombinant human albumin (e.g., Recombumin® of Novozymes Delta Ltd., Nottingham, UK) may be conjugated or fused to the lipocalin mutain of the present disclosure.

[0237] In one embodiment, when transferrin is used as a partner to extend the serum half-life of the lipocalin mutain of the present disclosure, the mutain may be genetically fused to the N or C terminus, or both, of non-glycosylated transferrin. The non-glycosylated transferrin has a half-life of 14–17 days, and the transferrin fusion protein will have a similarly extended half-life. The transferrin carrier also provides high bioavailability, in vivo distribution, and circulatory stability. This technology is commercially available from BioRexis (BioRexis Pharmaceutical Corporation, PA, USA). Recombinant human transferrin (DeltaFerrin™) for use as a protein stabilization / half-life extension partner is also commercially available from Novozymes Delta Ltd. (Nottingham, UK).

[0238] Another alternative for extending the half-life of the lipocalin mutain of the present disclosure is to fuse a long, unstructured, flexible glycine-rich sequence (e.g., a polyglycine having about 20 to 80 consecutive glycine residues) to the N- or C-terminus of the mutain. For example, this approach disclosed in International Patent Publication No. WO2007 / 038619 is referred to as "rPEG" (recombinant PEG).

[0239] E. CD137 and to GPC3 Exemplary Uses and Applications of Specific Fusion Proteins

[0240] In one embodiment, the fusion protein of the present disclosure can produce a synergistic effect through dual targeting of CD137 and GPC3. In one embodiment, the fusion protein of the present disclosure can produce a localized anti-tumor effect through dual targeting of CD137 and GPC3. Thus, numerous possible applications for the fusion protein of the present disclosure exist in medicine.

[0241] In one embodiment, the present disclosure includes the use of one or more fusion proteins disclosed herein or one or more compositions comprising such fusion proteins for the simultaneous binding of CD137 and GPC3.

[0242] The present disclosure also relates to the use of one or more fusion proteins as described for the formation of a complex with CD137 and / or GPC3.

[0243] Accordingly, in one embodiment of the present disclosure, the provided fusion protein may be used for the detection of CD137 and GPC3. Such use may include the steps of contacting a sample suspected of containing CD137 and / or GPC3 with one or more of the fusion proteins under suitable conditions to form a complex 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 change in physical characteristics resulting from labeling or binding, i.e., the formation of the complex itself, as described above. One example is surface plasmon resonance, the value of which changes during the binding of a binding partner immobilized on a surface such as gold foil.

[0244] The fusion protein of the present disclosure may also be used for the isolation of CD137 and / or GPC3. Such use may include the steps of contacting a sample presumed to contain CD137 and / or GPC3 with one or more of the fusion proteins under suitable conditions to form a complex between the fusion protein and CD137 and / or GPC3, and isolating the complex from the sample.

[0245] In one embodiment, the present disclosure provides a diagnostic and / or analysis kit comprising one or more fusion proteins according to the present disclosure.

[0246] In addition to their use in diagnosis, in another aspect, the present disclosure considers a pharmaceutical composition comprising one or more fusion proteins of the present disclosure and a pharmaceutically acceptable excipient.

[0247] Additionally, in one embodiment, the present disclosure provides a fusion protein that binds to CD137 and / or GPC3 simultaneously for use as an anti-tumor and / or anti-infective agent and as an immunomodulator. In one embodiment, the fusion protein of the present disclosure is expected to be used in a method for preventing, improving, or treating various cancers, such as human diseases, including GPC3-positive cancers such as hepatocellular carcinoma (HCC), melanoma, Merkel cell carcinoma, Wilms tumor, and hepatoblastoma. Accordingly, a method is also provided for preventing, improving, or treating human diseases such as various cancers, including GPC3-positive cancers such as hepatocellular carcinoma (HCC), melanoma, Merkel cell carcinoma, Wilms tumor, and hepatoblastoma, in subjects requiring such treatment, comprising administering a therapeutically effective amount of one or more fusion proteins of the present disclosure to a subject.

[0248] Other examples of cancers that can be treated using the fusion protein of the present disclosure include liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, malignant melanoma of the skin or eye, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, gastric cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tube, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin lymphoma, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, orphan solid tumor, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis carcinoma, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, vertebral axis tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, sarcoma of the sarcoma of the brainstem, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, blood cancers including, e.g., multiple myeloma, B-cell lymphoma, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, immunoblastic large cell lymphoma, precursor B-lymphocytic lymphoma, mantle cell lymphoma, acute lymphocytic lymphoma, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma and precursor T-lymphocytic lymphoma, and any combination of said cancers. In some embodiments, the present invention may also be applied to the treatment of metastatic cancer.

[0249] In one embodiment, the fusion protein of the present disclosure may target tumor cells expressing GPC3, such as HCC, melanoma, Merkel cell carcinoma, Wilms tumor, and hepatoblastoma, and may simultaneously activate lymphocytes of the host immune system surrounding such tumor cells. In one embodiment, the fusion protein of the present disclosure may increase targeted anti-tumor T cell activity, enhance anti-tumor immunity, induce T-cell-mediated cytolysis, and / or have a direct inhibitory effect on tumor growth, thereby producing anti-tumor results. In one embodiment, the fusion protein of the present disclosure may activate an immune response in the tumor microenvironment. In one embodiment, the fusion protein of the present disclosure may reduce non-target toxicity by locally inhibiting effector lymphocytes on healthy cells, i.e., oncogene activity, and inducing cell-mediated cytotoxicity by NK cells and / or T-cells.

[0250] In one embodiment, the present disclosure comprises the use of the fusion protein of the present disclosure, or a composition comprising the provided fusion protein, to induce a localized lymphocyte response around GPC3-positive tumor cells, such as HCC, melanoma, Merkel cell carcinoma, Wilms tumor, and hepatoblastoma. Accordingly, in one embodiment, the present disclosure provides a method for inducing a localized lymphocyte response around GPC3-positive tumor cells, such as HCC, melanoma, Merkel cell carcinoma, Wilms tumor, and hepatoblastoma, comprising applying one or more fusion proteins of the present disclosure or one or more compositions comprising such fusion proteins. "Localized" means that when T-cells bind via CD137 and simultaneously bind to GPC3-positive tumor cells, the T-cells produce cytokines, particularly IL-2 and / or IFN gamma, around the GPC3-positive cells. Such cytokines can cause T-cell activation and then attract other killer cells, such as T-cells or NK cells, to directly or indirectly kill GPC3-positive cells.

[0251] In one embodiment, the present disclosure comprises the use of the fusion protein of the present disclosure or a composition comprising such fusion protein to co-stimulate T-cells and / or activate the downstream signaling pathway of CD137. Preferably, the provided fusion protein co-stimulates T-cells and / or activates the downstream signaling pathway of CD137 when bound to tumor cells expressing GPC3. Accordingly, the present disclosure provides a method for inducing T lymphocyte proliferation and / or activating the downstream signaling pathway of CD137, preferably when bound to tumor cells expressing GPC3, such as HCC, melanoma, Merkel cell carcinoma, Wilms tumor, and hepatoblastoma, by applying one or more fusion proteins of the present disclosure and / or one or more compositions comprising such fusion proteins.

[0252] In one embodiment, the present disclosure includes the use of a fusion protein of the present disclosure or a composition comprising such a fusion protein to induce CD137 clustering and activation of T-cells and to deliver such T-cells to tumor cells expressing GPC3, such as HCC, melanoma, Merkel cell carcinoma, Wilms tumor, and hepatoblastoma.

[0253] Further purposes, disadvantages, and features of this disclosure will be apparent to those skilled in the art at the time of experimentation with the following embodiments and the accompanying drawings, and are not intended to be limiting. Accordingly, although this disclosure is disclosed in detail by exemplary embodiments and optional features, variations and modifications of this disclosure as embodied herein may be influenced by those skilled in the art, and such variations and modifications should be understood as being within the scope of this disclosure.

[0254] F. CD137 and to GPC3 Generation of an exemplary fusion protein specific to the provided.

[0255] In one embodiment, the present disclosure provides a nucleic acid molecule (DNA and RNA) comprising a nucleotide sequence encoding the provided fusion protein. In one embodiment, the present disclosure comprises a host cell comprising the provided nucleic acid molecule. Since the degeneracy of the genetic code allows for the substitution of a specific codon with another codon that specifies the same amino acid of that specific codon, the present disclosure is not limited to a specific nucleic acid molecule encoding a fusion protein as described herein, but rather comprises any nucleic acid molecule comprising a nucleotide sequence encoding a functional fusion protein. In this regard, the present disclosure also relates to a nucleotide sequence encoding the provided fusion protein.

[0256] Nucleic acid molecules, such as DNA, are referred to as "capable of expressing a nucleic acid molecule" or "capable of enabling the expression of a nucleotide sequence" if they contain sequence elements containing information regarding the regulation of transcription and / or translation, and such sequences are "operably linked" to nucleotide sequences encoding proteins. An operable link is a connection in which a regulatory sequence element and an expressed sequence are linked in a manner that enables gene expression. While the precise nature of regulatory regions essential for gene expression may vary between species, generally, these regions include the promoter itself in prokaryotes—that is, a promoter containing both DNA elements that guide the initiation of transcription and DNA elements that signal the initiation of translation when transcribed into RNA. Such promoter regions usually include 5' non-coding sequences involved in the initiation of transcription and translation, such as the -35 / -10 box and Shine-Dalgarno element in prokaryotes, and the TATA box, CAAT sequence, and 5'-capping elements in eukaryotes. These regions may also include enhancer or repressor elements, and translated signal and leader sequences for targeting natural proteins in specific compartments of the host cell.

[0257] Furthermore, 3' non-coding sequences may contain regulatory elements related to transcription termination, polyadenylation, etc. However, if these termination sequences are not satisfactory in a particular host cell, they may be replaced by functional signals in that cell.

[0258] Accordingly, the nucleic acid molecule of the present disclosure may be “operably linked” to one or more regulatory sequences, such as a promoter sequence, to enable the expression of the nucleic acid molecule. In one embodiment, the nucleic acid molecule of the present disclosure comprises a promoter sequence and a transcription termination sequence. Suitable eukaryotic promoters are, for example, the tet promoter, the lacUV5 promoter, or the T7 promoter. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter.

[0259] In one embodiment, a nucleic acid molecule encoding the lipocalin mutain disclosed in this application may be “operably linked” to another nucleic acid molecule encoding the immunoglobulin of this disclosure to enable the expression of the fusion protein disclosed herein.

[0260] In one embodiment, the provided method may comprise applying mutagenicity to at least one nucleic acid molecule encoding mature hTlc at one or more nucleotide triplets encoding 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 of hTlc (SEVENTEEN NO: 1) to obtain a lipocalin mutain included in the provided fusion protein. In one embodiment, the provided method may comprise applying mutagenicity to at least one nucleic acid molecule encoding mature hNGAL at one or more nucleotide triplets encoding 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 (SEVENTEEN NO: 2) to obtain a lipocalin mutain included in the provided fusion protein. In one embodiment, the provided method may comprise applying mutagenicity to at least one nucleic acid molecule encoding mature hNGAL at one or more nucleotide triplets encoding 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 (SEVENTEEN NO: 2) to obtain a lipocalin mutain contained in the provided fusion protein.

[0261] Furthermore, regarding the hTlc mutain or hNGAL mutain of the present disclosure included in the fusion protein, in one embodiment, naturally occurring disulfide bonds between Cys 61 and Cys 153 or Cys 76 and Cys 175, respectively, may be removed. Thus, such mutains may be produced, for example, in a cell compartment having a reducing redox environment in the cytoplasm of Gram-negative bacteria.

[0262] In addition to the hTlc mutain or hNGAL mutain provided in the disclosure and included in the fusion protein, the disclosure also comprises a nucleic acid molecule encoding such a mutain, which may include one or more additional mutations in addition to the specified sequence positions of the experimental mutagenic. Such mutations may often be proven to be advantageous, such as by conferring resistance or even by contributing, for example, to improved folding efficiency, serum stability, thermal stability, or ligand binding affinity of the lipocalin mutain and / or fusion protein.

[0263] In one embodiment, the provided nucleic acid molecule may also be a vector or any other type of cloning vehicle, such as a plasmid, phagemid, phage, baculovirus, cosmid, or part of an artificial chromosome.

[0264] In one embodiment, the provided nucleic acid molecule may be incorporated into the phagemid. As used in this context, the phagemid vector represents a vector encoding the intergeneric region of the template phage, e.g., M13 or f1, or its functional portion fused to the target cDNA. For example, in one embodiment, an intact phage particle is produced after primary infection of a bacterial host cell with such provided phagemid vector and a suitable helper phage (e.g., M13K07, VCS-M13, or R408), enabling physical coupling of the heterologous cDNA encoded for its corresponding polypeptide displayed on the surface of the phage (Lowman, Annu Rev Biophys Biomol Struct , 1997, Rodi and Makowski, Curr Opin Biotechnol , 1999).

[0265] According to various embodiments, the cloning vehicle may comprise, in addition to the regulatory sequence described above and the nucleic acid sequence encoding the fusion protein as described herein, a species-derived cloning and control sequence comparable to that of the host cell used as a selection marker for expression and to confer a selectable phenotype on cells that are transformed or transfected. A large number of suitable cloning vectors are known in the art and are commercially available.

[0266] The present disclosure also relates, in one embodiment, to a method for producing a fusion protein of the present disclosure starting from a nucleic acid encoding the fusion protein or any subunit thereof using a genetic engineering method. In one embodiment, the provided method may be carried out in vivo, and the provided fusion protein may be produced, for example, in a bacterial or eukaryotic host organism and then isolated from the host organism or its culture. It is also possible to produce the fusion protein of the present disclosure in vitro using an in vivo translation system.

[0267] When producing a fusion protein in vivo, the nucleic acid encoding such a fusion protein may be introduced into a suitable bacterial or eukaryotic host organism using recombinant DNA technology well known in the art. In some embodiments, a cloning vector comprising a DNA molecule encoding a fusion protein as described herein, and in particular a coding sequence of such a fusion protein, may be transformed into a host cell capable of expressing a gene. Transformation may be performed using standard technology. Accordingly, the present disclosure relates to a host cell comprising a nucleic acid molecule as disclosed herein.

[0268] In one embodiment, the transformed host cell may be cultured under conditions suitable for the expression of a nucleotide sequence encoding the fusion protein of the present disclosure. In some embodiments, the host cell is a prokaryote, e.g., Escherichia coli ( E. coli ) or Bacillus subtilis( Bacillus subtilis ), or eukaryotes, such as Saccharoma ises cerevisiae ( Saccharomyces cerevisiae ), Piccia Pastoris( Pichia pastoris It may be ), SF9 or High5 insect cells, immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.

[0269] In one embodiment, the lipocalin mutain of the disclosure included in the fusion protein disclosed herein may preferably be directed to a cell compartment having an oxidizing redox environment using a suitable signal sequence where the lipocalin mutain of the disclosure contains intramolecular disulfide bonds. Such an oxidizing environment is the extracellular space of Gram-positive bacteria or the lumen of the endoplasmic reticulum of eukaryotic cells, in E. coli ( E. coliIt can be provided by the periplasm of Gram-negative bacteria such as ), and usually prefers the formation of structural disulfide bonds.

[0270] In one embodiment, also, a host cell, preferably E. coli ( E. coli It is possible to produce the fusion protein of the present initiation in the cytoplasm of ). In this case, the provided fusion protein can be obtained directly in a soluble folded state or recovered in the form of an inclusion body and restored in vitro. Another option is to use a specific host strain having an oxidizing intracellular environment to induce the formation of disulfide bonds in the cytoplasm (Venturi et al., J Mol Biol , 2002).

[0271] In one embodiment, the fusion protein of the disclosure as described herein may not necessarily be generated or produced in whole or in part using genetic engineering. Rather, such a protein may also be obtained by many traditional and well-known techniques, such as simple organic synthesis strategies, solid-phase supported synthesis techniques, any one of commercially available automated synthesizers, or by in vitro transcription and translation. For example, it is possible to identify a promising fusion protein or lipocalin mutain included in such a fusion protein using molecular modeling, synthesize it in vitro, and investigate its binding activity to the target(s). Methods for solid-phase and / or solution-phase synthesis of proteins are well known in the art (e.g., Bruckdorfer et al., Curr Pharm Biotechnol (See , 2004).

[0272] In one embodiment, the fusion protein of the present disclosure can be produced by in vitro transcription / translation using well-established methods known to those skilled in the art.

[0273] In some additional embodiments, fusion proteins as described herein may also be produced using traditional recombinant technology alone or in combination with traditional synthesis technology.

[0274] Furthermore, in one embodiment, the fusion protein according to the present disclosure may be obtained by conjugating individual subunits, such as immunoglobulin and mutains included in the fusion protein. Such conjugation may be achieved, for example, through any form of covalent or non-covalent bonding using traditional methods.

[0275] A skilled worker will understand a method useful for preparing a fusion protein that is considered by this disclosure but whose protein or nucleic acid sequence is not explicitly disclosed herein. As a summary, such modifications to the amino acid sequence include direct mutagenesis of a single amino acid position to simplify the sub-cloning of a protein gene or part thereof by, for example, incorporating a cleavage site for a specific restriction enzyme. Additionally, these mutations may be included to further improve the affinity of the fusion protein for its targets (e.g., CD137 and GPC3). Furthermore, if necessary, mutations may be introduced to modify one or more characteristics of the protein, such as improving folding stability, serum stability, protein tolerance or solubility, or reducing aggregation tendencies.

[0276] The present invention may be further characterized by the following items.

[0277] Item 1. A fusion protein capable of binding to both CD137 and GPC3, comprising at least two subunits in any order, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, and the second subunit comprises a lipocalin mutane and is specific to CD137.

[0278] Item 2. A fusion protein comprising at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or its antigen-binding domain and is specific to GPC3, the second subunit comprises a lipocalin mutane and is specific to CD137, and the second subunit is optionally connected at the N-terminus to the C-terminus of each heavy chain of the first subunit via a linker.

[0279] Item 3. A fusion protein comprising at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific to GPC3, and 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.

[0280] Item 4. A fusion protein comprising at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific to GPC3, and the second subunit is optionally connected at the N-terminus to the C-terminus of each light chain of the first subunit via a linker.

[0281] Item 5. A fusion protein comprising at least two subunits, wherein the first subunit comprises a full-length immunoglobulin or an antigen-binding domain thereof and is specific to GPC3, and 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.

[0282] Item 6. In any one of Items 1 to 5, the fusion protein is up to about 1 nM or K of the immunoglobulin or its antigen-binding domain contained in the first subunit alone. D K that is comparable to or lower than the value D A fusion protein capable of binding to GPC3 at a value.

[0283] Item 7. In Item 6, K D The value of the fusion protein is determined by surface-plasmon-resonance (SPR) analysis.

[0284] Item 8. In any one of Items 1 to 7, the fusion protein is at most about 0.5 nM or EC of the immunoglobulin or its antigen-binding domain contained in the first subunit alone. 50 Equivalent to or lower than the value EC 50 A fusion protein that can bind to GPC3 at a value.

[0285] Item 9. In any one of Items 1 to 8, the fusion protein is at most about 3 nM or the EC of a CD137-specific lipocalin mutain contained in the second subunit alone. 50 Equivalent to or lower than the value EC 50 A fusion protein capable of binding to CD137 at a certain value.

[0286] Item 10. With respect to either Item 8 or 9, EC 50 The value of the fusion protein is determined by enzyme-linked immunosorbent assay (ELISA).

[0287] Item 11. In any one of Items 1 to 10, the fusion protein is a fusion protein that cross-reacts with cyanomorphic GPC3.

[0288] Item 12. In any one of Items 1 to 11, the fusion protein has an EC of up to about 10 nM when the fusion protein is measured in an ELISA assay. 50 A fusion protein capable of simultaneously binding to CD137 and GPC3.

[0289] Item 13. In any one of Items 1 to 12, the fusion protein has an EC of up to about 30 nM when the fusion protein is measured by flow cytometry analysis. 50 A fusion protein capable of binding to CD137 expressed in cells.

[0290] Item 14. In any one of Items 1 to 13, the fusion protein has an EC of up to about 30 nM when the fusion protein is measured in flow cytometry analysis. 50 A fusion protein capable of binding to GPC3 expressed in cells.

[0291] Item 15. In any one of Items 1 to 14, the fusion protein is a fusion protein capable of binding to GPC3-expressing tumor cells.

[0292] Item 16. In any one of Items 1 to 15, the fusion protein is a fusion protein capable of stimulating a T-cell response.

[0293] Item 17. In any one of Items 1 to 16, the fusion protein is a fusion protein capable of inducing increased secretion of IL-2.

[0294] Item 18. In any one of Items 1 to 17, the fusion protein is capable of inducing increased IL-2 secretion at a higher level than SEQ ID NO: 83 and / or with better efficiency than SEQ ID NO: 83.

[0295] Item 19. In any one of Items 1 to 18, the fusion protein is a fusion protein capable of inducing lymphocyte-mediated cytotoxicity.

[0296] Item 20. In any one of Items 1 to 19, the fusion protein is capable of inducing enhanced death of T cell-mediated GPC3-expressing tumor cells than SEQ ID NO: 83 and / or inducing cytotoxic T cell activation with better efficiency than SEQ ID NO: 83.

[0297] Item 21. In any one of Items 1 to 20, the fusion protein is a fusion protein capable of co-stimulating a T-cell response in a GPC3-dependent manner.

[0298] Item 22. In any one of Items 1 to 21, the fusion protein is a fusion protein capable of co-stimulating a T-cell response in a tumor microenvironment.

[0299] Item 23. In any one of Items 1 to 22, the fusion protein is a fusion protein that cannot co-stimulate a T-cell response in the absence of GPC3.

[0300] Item 24. In any one of Items 1 to 23, the fusion protein is a fusion protein having an antibody-like pharmacokinetic profile.

[0301] Item 25. In any one of Items 1 to 24, the fusion protein has a half-life in mouse 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 longer than that, and / or the fusion protein has a half-life in mouse longer than SEQ ID NO: 83.

[0302] Item 26. In any one of Items 1 to 25, the fusion protein has an isopotential point of at least 6.5, at least 6.8, at least 7.1, at least 7.4, at least 7.5, at least 7.7, or higher therein, and / or the fusion protein has an isopotential point higher than SEQ ID NO: 83.

[0303] Item 27. A fusion protein comprising, in any one of Items 1 to 26, lipocalin mutain, one or more mutated amino acid residues at positions corresponding to positions 5, 26-31, 33-34, 42, 46, 52, 56, 58, 60-61, 65, 71, 85, 94, 101, 104-106, 108, 111, 114, 121, 133, 148, 150, and 153 of the linear polypeptide sequence of mature human tear lipocalin (SEQ No. 1).

[0304] Item 28. In any one of Items 1 to 27, the fusion protein comprising 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 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; Lys 65 → Arg or Asn; Thr 71 → Ala; Val 85 → Asp; Lys 94 → Arg or Glu; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Lys 121 → Glu; Ala 133 → Thr; Arg 148 → Ser; Ser 150 → Ile and Cys 153 → Ser.

[0305] Item 29. A fusion protein according to any one of Items 1 to 28, wherein the amino acid sequence of lipocalin mutain comprises one of the following sets of mutated amino acid residues compared to the linear polypeptide sequence of mature human tear lipocalin (SEQ No. 1):

[0306] (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; Cys 153 → Ser;

[0307] (b) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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; 및 Cys 153 → Ser;

[0308] (c) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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; 및 Cys 153 → Ser;

[0309] (d) Ala 5 → Val; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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; 및 Cys 153 → Ser;

[0310] (e) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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 → Island; 및 Cys 153 → Ser;

[0311] (f) Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Island; 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 → Island; 및 Cys 153 → Ser; 및

[0312] (g) Ala 5 → Thr; Arg 26 → Glu; Glu 27 → Gly; Phe 28 → Cys; Pro 29 → Arg; Glu 30 → Pro; Met 31 → Trp; Leu 33 → Ile; Glu 34 → Phe; Gly 46 → Asp; Leu 56 → Ala; Ser 58 → Asp; Arg 60 → Pro; Cys 61 → Ala; Thr 71 → Ala; Cys 101 → Ser; Glu 104 → Val; Leu 105 → Cys; His 106 → Asp; Lys 108 → Ser; Arg 111 → Pro; Lys 114 → Trp; Ser 150 → Ile; and Cys 153 → Ser.

[0313] Item 30. A fusion protein according to any one of Items 1 to 29, wherein the amino acid sequence of lipocalin mutain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32-38 or fragments or variants thereof.

[0314] Item 31. A fusion protein according to any one of Items 1 to 30, wherein the amino acid sequence of lipocalin mutain has at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 32-38.

[0315] Item 32. A fusion protein comprising, in any one of items 1 to 26, lipocalin mutain, one or more mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132 and 134 of the linear polypeptide sequence (SEQ No. 2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL).

[0316] Item 33. In any one of Items 1 to 32, the fusion protein comprising one or more of the following mutated amino acid residues at positions corresponding to positions 28, 36, 40-41, 49, 52, 65, 68, 70, 72-73, 77, 79, 81, 83, 87, 94, 96, 100, 103, 106, 125, 127, 132, and 134 of the linear polypeptide sequence of mature human neutrophil gelatinase-associated lipocalin (hNGAL) (SEQ 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.

[0317] Item 34. Any one of Items 1 to 26 and 33, wherein the lipocalin mutain comprises one or more mutated amino acid residues at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence (SEQ No. 2) of mature human neutrophil gelatinase-associated lipocalin (hNGAL).

[0318] Item 35. In any one of Items 1 to 26 and 34, the fusion protein comprising one or more of the following mutated amino acid residues at positions corresponding to positions 20, 25, 28, 33, 36, 40-41, 44, 49, 52, 59, 68, 70-73, 77-82, 87, 92, 96, 98, 100, 101, 103, 122, 125, 127, 132, and 134 of the linear polypeptide sequence of mature hNGAL (SEQ 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.

[0319] Item 36. A fusion protein according to any one of Items 1 to 26 and 32 to 35, wherein the amino acid sequence of lipocalin mutain comprises one of the following sets of amino acid residues mutated compared with the linear polypeptide sequence of mature hNGAL (SEQ No. 2):

[0320] (a) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gn 49 → Asn; Tyr 52 → Meth; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → No; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0321] (b) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; District 41 → Arg; Gn 49 → Island; Tyr 52 → Meth; 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 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0322] (c) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; District 41 → Arg; Gn 49 → Asn; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Wing; Leu 70 → Wing; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0323] (d) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gn 49 → Asn; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Wing; Leu 70 → Wing; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0324] (e) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gn 49 → Ser; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Ser; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → No; Arg 81 → Meth; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0325] (f) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gln 49 → Val; Tyr 52 → Meth; 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 → The; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0326] (g) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; District 41 → Arg; Gln 49 → His; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → No; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0327] (h) Gln 28 → His; Leu 36 → Gln; Only 40 → Gone; Island 41 → Lys; Gn 49 → Asn; Tyr 52 → Meth; Asn 65 → Asp; Ser 68 → Gly; Leu 70 → Thr; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Thr; Trp 79 → No; Arg 81 → Ser; Phy 83 → Leu; Cys 87 → Ser; Leu 94 → The; Asn 96 → Lys; Tyr 100 → No; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Ph; Tyr 132 → Glu; 및 Lys 134 → Tyr;

[0328] (i) Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Sees; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Ala; Leu 70 → Thr; Arg 72 → Asp; Light 73 → Asp; Asp 77 → Asn; Trp 79 → Ala; Arg 81 → Ser; Cys 87 → Ser; Asn 96 → Light; Tyr 100 → Phe; Leu 103 → His; Taurus 106 → Ser; Light 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Light 134 → Taurus.

[0329] (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;

[0330] (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;

[0331] (l) Asn 25 → Taurus; 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; Light 73 → Asp; Asp 77 → Gln; Trp 79 → Ile; Arg 81 → Gln; Phe 92 → Ser; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Light 125 → Looks; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly;

[0332] (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;

[0333] (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;

[0334] (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;

[0335] (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;

[0336] (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;

[0337] (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

[0338] (s) Leu 36 → Met; Ala 40 → Asn; Ile 41 → Leu; Glu 44 → Asp; Gln 49 → His; Taurus 52 → Sees; Ser 68 → Asp; Leu 70 → Met; Phe 71 → Leu; Arg 72 → Leu; Light 73 → Asp; Asp 77 → His; Trp 79 → Ile; Arg 81 → Trp; Phe 92 → Leu; Asn 96 → Phe; Tyr 100 → Asp; Leu 103 → His; Light 125 → Looks; Ser 127 → Ile; Tyr 132 → Trp; and Lys 134 → Gly.

[0339] Item 37. A fusion protein according to any one of Items 1 to 26 and 32 to 36, wherein the amino acid sequence of lipocalin mutain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39-57 or fragments or variants thereof.

[0340] Item 38. A fusion protein according to any one of Items 1 to 26 and 32 to 36, wherein the amino acid sequence of lipocalin mutain has at least 85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 39-57.

[0341] Item 39. In any one of Items 1 to 26 and 32 to 38, the amino acid sequence of lipocalin mutain comprises the following set of mutated amino acid residues compared to the linear polypeptide sequence of mature hNGAL (SEQ No. 2): Gln 28 → His; Leu 36 → Gln; Ala 40 → Ile; Ile 41 → Arg; Gln 49 → Ile; Tyr 52 → Met; Asn 65 → Asp; Ser 68 → Met; Leu 70 → Lys; Arg 72 → Asp; Lys 73 → Asp; Asp 77 → Met; Trp 79 → Asp; Arg 81 → Trp; Cys 87 → Ser; Asn 96 → Lys; Tyr 100 → Phe; Leu 103 → His; Tyr 106 → Ser; Lys 125 → Phe; Ser 127 → Phe; Tyr 132 → Glu; and Lys 134 → Tyr and / or the lipocalin mutain are fusion proteins having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 40.

[0342] Item 40. A fusion protein according to any one of Items 1 to 39, wherein one subunit is connected to another subunit through a linker.

[0343] Item 41. A fusion protein according to any one of Items 1 to 40, wherein the second subunit is connected at the N- or C-terminus of each heavy chain constant region (CH) of the first subunit through a linker, or connected at the N- or C-terminus of each light chain constant region (CL) of the first subunit.

[0344] Item 42. A fusion protein according to any one of Items 1 to 41, wherein the third subunit is connected via a linker at the N-terminus to the N- or C-terminus of each heavy chain constant region (CH) of the first subunit, the N- or C-terminus of each light chain constant region (CL) of the first subunit, or the C-terminus of each second subunit.

[0345] Item 43. A fusion protein in which, in any one of items 40 to 42, the linker is an unstructured (Gly-Gly-Gly-Gly-Ser)3 linker (SEQ ID: 13).

[0346] Item 44. A fusion protein in which, in any one of Items 40 to 43, the linker is a non-structural glycine-serine linker, a polyproline linker, a proline-alanine-serine polymer, or a linker selected from the group consisting of SEQ ID NOs 13-23.

[0347] Item 45. A fusion protein in which, in any one of Items 1 to 44, the first subunit is an antibody.

[0348] Item 46. In any one of Items 1 to 45, the heavy chain variable region of the antibody is selected from the group consisting of SEQ ID NOs: 78, 114, 119, 126, and 129, or is 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 sequences shown in SEQ ID NOs: 78, 114, 119, 126, and 129, and the light chain variable region of the antibody is selected from the group consisting of SEQ ID NOs: 79, 115, and 127, or is a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least A fusion protein having a sequence with 95%, at least 97%, at least 98%, or higher sequence identity.

[0349] Item 47. A fusion protein comprising, in any one of Items 1 to 46, an antibody comprising a heavy chain of either SEQ ID NO: 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 higher sequence identity with the amino acid sequence shown in SEQ ID NO: 80 or 81, and a light chain of SEQ ID NO: 82 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or higher sequence identity with the amino acid sequence shown in SEQ ID NO: 82.

[0350] Item 48. A fusion protein comprising, in any one of Items 1 to 47, an antibody comprising 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 sequences 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.

[0351] Item 49. A fusion protein comprising, in any one of Items 1 to 48, an antibody comprising 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 higher sequence identity with the amino acid sequences shown in SEQ ID NOs: 80 and 82, or SEQ ID NOs: 81 and 82.

[0352] Item 50. In Item 36, the fusion protein, wherein the heavy chain of the antibody comprises one of the following sets of CDR sequences:

[0353] (a) GYTFTDYE (HCDR1, sequence number: 72), LDPKTGDT (HCDR2, sequence number: 73), TRFYSYTY (HCDR3; sequence number: 74);

[0354] (b) GFTFNKNA (HCDR1, sequence number: 108), IRNKTNNYAT (HCDR2, sequence number: 109), VAGNSFAY (HCDR3, sequence number: 110);

[0355] (c) YFDFDSYE (HCDR1, sequence number: 116), IYHSGST (HCDR2, sequence number: 117), ARVNMDRFDY (HCDR3, sequence number: 108); or

[0356] (d) GFTFSSYA (HCDR1, sequence number: 120), IQKQGLPT (HCDR2, sequence number: 121), AKNRAKFDY (HCDR3, sequence number: 122).

[0357] Item 51. In any one of Items 1 to 50, the fusion protein wherein the light chain of the antibody comprises one of the following sets of CDR sequences:

[0358] (a) QSLVHSNRNTY (LCDR1, sequence number: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; sequence number: 77);

[0359] (b) QSLLYSSNQKNY (LCDR1, sequence number: 111), WAS (LCDR2), QQYYNYPLT (LCDR3, sequence number: 113); or

[0360] (c) QSISSY (LCDR1, sequence number: 123), NAS (LCDR2), QQNRGFPLT (LCDR3, sequence number: 125).

[0361] Item 52. A fusion protein according to any one of Items 1 to 51, wherein the heavy chain of the antibody comprises the following set of CDR sequences: GYTFTDYE (HCDR1, SEQ NO: 72), LDPKTGDT (HCDR2, SEQ NO: 73), TRFYSYTY (HCDR3; SEQ NO: 74), and the light chain of the antibody comprises the following set of CDR sequences: QSLVHSNRNTY (LCDR1, SEQ NO: 75), KVS (LCDR2), SQNTHVPPT (LCDR3; SEQ NO: 77).

[0362] Item 53. In any one of Items 1 to 51, the antibody comprises a fusion protein comprising the following set of CDR sequences:

[0363] (a) GFTFNKNA (HCDR1, sequence number: 108), IRNKTNNYAT (HCDR2, sequence number: 109), VAGNSFAY (HCDR3, sequence number: 110), QSLLYSSNQKNY (LCDR1, sequence number: 111), WAS (LCDR2), QQYYNYPLT (LCDR3, sequence number: 113); or

[0364] (b) GFTFSSYA (HCDR1, sequence number: 120), IQKQGLPT (HCDR2, sequence number: 121), AKNRAKFDY (HCDR3, sequence number: 122), QSISSY (LCDR1, sequence number: 123), NAS (LCDR2), QQNRGFPLT (LCDR3, sequence number: 125).

[0365] Item 54. In any one of Items 1 to 53, the antibody is a fusion protein having an IgG4 backbone.

[0366] Item 55. In Item 54, the IgG4 backbone is a fusion protein having one or more of the following mutations: S228P, N297A, F234A, L235A, M428L, N434S, M252Y, S254T, and T256E.

[0367] Item 56. In any one of Items 1 to 55, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NOs: 87-96, and said 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.

[0368] Item 57. In any one of Items 1 to 56, the fusion protein comprises the amino acids described in SEQ ID NOs: 87 and 82, the amino acids described in SEQ ID NOs: 88 and 82, the amino acids described in SEQ ID NOs: 81 and 89, the amino acids described in SEQ ID NOs: 81 and 90, the amino acids described in SEQ ID NOs: 91 and 82, the amino acids described in SEQ ID NOs: 92 and 82, the amino acids described in SEQ ID NOs: 81 and 93, the amino acids described in SEQ ID NOs: 81 and 94, the amino acids described in SEQ ID NOs: 95 and 82, or the amino acids described in SEQ ID NOs: 96 and 82.

[0369] Item 58. In any one of Items 1 to 57, 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 sequences 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, or 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.

[0370] Item 59. A nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein of any one of Items 1 to 58.

[0371] Item 60. In Item 59, the nucleic acid molecule is operably linked to a regulatory sequence that enables the expression of said nucleic acid molecule.

[0372] Item 61. In Item 59 or 60, the nucleic acid molecule is a nucleic acid molecule that may be included in a vector or a phagemid vector.

[0373] Item 62. A host cell containing a nucleic acid molecule of any one of items 59 to 61.

[0374] Item 63. A method for producing a fusion protein according to any one of items 1 to 58, wherein the fusion protein is produced starting from a nucleic acid encoding the fusion protein.

[0375] Item 64. In Item 63, the fusion protein is produced in a bacterial or eukaryotic host organism and isolated from this host organism or a culture thereof.

[0376] Item 65. Use of a fusion protein according to any one of Items 1 to 58 or a composition comprising such fusion protein to activate the downstream signaling pathway of CD137 and simultaneously bind to GPC3-positive tumor cells.

[0377] Item 66. A method for activating the downstream signaling pathway of CD137 and simultaneously binding to GPC3-positive tumor cells, comprising the step of applying one or more fusion proteins of any one of Items 1 to 58 or one or more compositions containing such fusion proteins to a tissue including a tumor.

[0378] Item 67. A method for co-stimulating T-cells and simultaneously binding to GPC3-positive tumor cells, comprising the step of applying one or more fusion proteins of any one of items 1 to 58 or one or more compositions containing such fusion proteins to a tissue including a tumor.

[0379] Item 68. A method for inducing lymphocyte activation and simultaneously binding to GPC3-positive tumor cells, comprising the step of applying one or more fusion proteins of any one of Items 1 to 58 or one or more compositions containing such fusion proteins to a tissue including a tumor.

[0380] Item 69. A method for inducing CD137 clustering and activation of T-cells by guiding T cells to GPC3-positive tumor cells, comprising the step of applying one or more fusion proteins of any one of items 1 to 58 or one or more compositions containing such fusion proteins to a tissue including a tumor.

[0381] Item 70. A method for inducing a localized lymphocyte response around GPC3-positive tumor cells, comprising the step of applying one or more fusion proteins of any one of Items 1 to 58 or one or more compositions containing such fusion proteins to a tissue containing a tumor.

[0382] Item 71. A method for inducing increased IL-2 secretion by T-cells around GPC3-positive tumor cells, comprising the step of applying one or more fusion proteins of any one of Items 1 to 58 or one or more compositions containing such fusion proteins to a tissue containing a tumor.

[0383] Item 72. A method for inducing increased lymphocyte-mediated cell lysis in GPC3-positive tumor cells, comprising the step of applying one or more fusion proteins of any one of Items 1 to 58 or one or more compositions comprising such fusion proteins to a tissue including a tumor.

[0384] Item 73. A pharmaceutical composition comprising one or more fusion proteins of any one of Items 1 to 58.

[0385] Item 74. A method for preventing, improving, or treating GPC3-positive cancer, comprising the step of applying a fusion protein of any one of Items 1 to 58 or one or more compositions containing such fusion protein to a tissue including a tumor.

[0386] Item 75. A method for the prevention, improvement, or treatment of hepatocellular carcinoma, comprising the step of applying a fusion protein of any one of Items 1 to 58 or one or more compositions containing such fusion protein to a tissue including a tumor.

[0387] Item 76. A fusion protein of any one of Items 1 to 58 for therapeutic use.

[0388] Item 77. In Item 76, the above use is a fusion protein for the treatment of cancer.

[0389] Item 78. Use of a fusion protein of any one of Items 1 to 58 for manufacturing a medicament.

[0390] Item 79. In Item 78, the drug is intended for the treatment of cancer.

[0391] Examples

[0392] Examples 1: Expression and Analysis of Representative Fusion Proteins

[0393] In this example, the representative antibody-lipocalin mutain fusion protein comprises a GPC3-specific antibody having the heavy chain provided by SEQ ID NO: 81, or comprising the heavy chain variable domain of SEQ ID NO: 78, or comprising the CDR of GYTFTDYE (HCDR1, SEQ ID NO: 72), LDPKTGDT (HCDR2, SEQ ID NO: 73), and TRFYSYTY (HCDR3; SEQ ID NO: 74), and the light chain provided by SEQ ID NO: 82, or comprising the heavy chain variable domain of SEQ ID NO: 79, or comprising the CDR of QSLVHSNRNTY (LCDR1, SEQ ID NO: 75), KVS (LCDR2, SEQ ID NO: 76), and SQNTHVPPT (LCDR3; SEQ ID NO: 77), and the CD137-specific lipocalin mutain of SEQ ID NO: 40 or SEQ ID NO: 49 and the designed CD137Ac1 and 97% of It is generated by simultaneously combining GPC3 and CD137 through the fusion of a CD137-specific lipokillin having sequence identity via a linker, e.g., the unstructured (G4S)3 linker of SEQ ID NO: 13. The different formats generated are Fig. 1It is described in. For example, such fusion proteins, e.g., SEQ NOs: 87 and 82, SEQ NOs: 88 and 82, SEQ NOs: 81 and 89, SEQ 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 having 97% sequence identity with SEQ NOs: 91 and 82, SEQ NOs: 92 and 82, SEQ NOs: 81 and 93, SEQ NOs: 81 and 94, SEQ NOs: 95 and 82, or SEQ NOs: 96 and 82) have 97% sequence identity with SEQ NO: 40 or SEQ NO: 49 One or more of lipocalin mutains may be produced by fusing one or more of the four ends of an antibody comprising a heavy chain provided by SEQ NO: 81, or a heavy chain variable domain of SEQ NO: 78, or a CDR of GYTFTDYE (HCDR1, SEQ NO: 72), LDPKTGDT (HCDR2, SEQ NO: 73), and TRFYSYTY (HCDR3; SEQ NO: 74), and a light chain provided by SEQ NO: 82, or a heavy chain variable domain of SEQ NO: 79, or a CDR of QSLVHSNRNTY (LCDR1, SEQ NO: 75), KVS (LCDR2, SEQ NO: 76), and SQNTHVPPT (LCDR3; SEQ NO: 77). The resulting fusion protein is divalent to CD137 (e.g., Fig. 1A-1D As described in) or for CD137, 4 (e.g., do 1E-1H As described in), or a much higher bond value for CD137 (e.g., Fig. 1I It can be as described in ).

[0394] GPC3-specific antibodies and all antibody lipocalin mutain fusion proteins described in this example contain the S228P mutation and are in vitro ( in-vitro ) and in vivo( in-vivo It has an engineered IgG4 backbone that minimizes IgG4 anti-antibody exchange (Silva et al., J Biol Chem (, 2015). Additional mutations in the IgG4 backbone may also be present in all antibodies, and the fusion proteins described herein containing any one or more mutations F234A, L235A, M428L, N434S, M252Y, S254T, and T256E can 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 can be introduced for an extended serum half-life (Dall'Acqua et al., J Biol Chem , 2006, Zalevsky et al., Nat Biotechnol , 2010). All antibodies were expressed without carboxy-terminal lysine to avoid heterogeneity.

[0395] In addition, monospecific lipocalin mutain Fc fusion is Figure 1J-1K As described in [the text], one or more of the CD137-specific lipocalin mutain of [SEQN] 40 or the GPC3-specific lipocalin mutain of [SEQN] 64 were fused to the C-terminus of the Fc region of the antibody provided in [SEQN] 28 via a linker, e.g., the unstructured (G4S)3 linker of [SEQN] 13. The resulting construct is provided in [SEQN] 98.

[0396] The present invention also implements an asymmetric antibody-lipocalin mutain fusion format, for example, in which the light chain of one of the antibodies can be fused to the lipocalin mutain but the other cannot.

[0397] The constructs of the fusion proteins are generated by gene synthesis and cloned into a mammalian expression vector. They are then transiently expressed in Expi293FTM cells (Life Technologies). The concentration of the fusion proteins in the cell culture medium was measured by HPLC (Agilent Technologies) using a POROS® protein A affinity column (Applied Biosystems). The titers of the fusion proteins Table 1 It was summarized in.

[0398] The fusion protein was purified using size-exclusion chromatography (SEC) in phosphate-buffered saline (PBS) following protein A chromatography. After SEC purification, the fraction containing the monomeric protein was collected and analyzed again using analytical SEC.

[0399] The monomer protein content and isoelectric points (pIs) of the exemplary fusion protein after SEC purification Table 1 It is summarized in ). The pl of the provided fusion proteins (sequence numbers: 87 and 82) was increased compared to the previously known specific CD137 / GPC3 dual-specific fusion protein sequence number: 83.

[0400] Table 1: Transient manifestation potency

[0401]

[0402] Examples 2: Surface Plasmon Resonance ( SPR Measured by ) to GPC3 Binding of fusion proteins

[0403] The binding kinetics and affinity of exemplary fusion proteins for recombinant human GPC3 (huGPC3) and cyanomorphic GPC3 (cyGPC3) (R&D Systems) were measured by surface plasmon resonance (SPR) using a Biacore T200 instrument (GE Healthcare).

[0404] Anti-human IgG Fc antibodies (GE Healthcare) were immobilized on the CM5 sensor chip using standard amine chemistry: carboxyl groups on the chip were activated using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Subsequently, a solution of anti-human IgG Fc antibodies (GE Healthcare) at a concentration of 25 µg / mL in 10 mM sodium acetate (pH 5.0) was applied at a flow rate of 5 μl until an immobilization level of 6,000–10,000 resonance units (RU) was reached. The remaining non-reactive NHS esters were blocked by passing a 1 M ethanolamine solution across the surface. The reference channel was processed analogously. Subsequently, GPC3 antibodies contained in the test fusion proteins (SEQ Nos. 87 and 82 and CD137Ac1-Fusion 1) or fusion proteins (SEQ Nos. 81 and 82) at 0.2 μg / ml in HBS-EP+ buffer were captured on the chip surface by an anti-human IgG-Fc antibody at a flow rate of 10 μg for 180 seconds. After each capture step, the needle was washed.

[0405] For affinity measurements, 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 fabricated chip surface. Binding analysis was performed with a contact time of 180 seconds, a dissociation time of 900 seconds, and a flow rate of 30 μL. After each injection, the needle was washed with 40 mM NaOH + 20% isopropanol. All measurements were performed at 25°C. Regeneration of the chip surface was achieved by injecting 3 M MgCl2 for 120 seconds. Prior to protein measurements, three startup cycles were performed for conditioning purposes. Data were evaluated using Biacore T200 Evaluation software (v2.0). Dual referencing was used, and raw data were fitted using a 1:1 binding model.

[0406] k for an exemplary fusion protein on , k off , and the generated equilibrium dissociation constant (K D The value measured for ) Table 2 This is summarized in [document]. The tested fusion proteins (Sequence Nos. 87 and 82 and CD137Ac1-Fusion 1) bind to huGPC3 and cyGPC3 with subnanomolar affinity comparable to that of the GPC3 antibody contained in the fusion protein and the previously known specific CD137 / GPC3 bispecific fusion protein (Sequence No. 83).

[0407] Table 2: Kinetic constants and affinities of fusion proteins determined by SPR

[0408]

[0409] Examples 3. In ELISA GPC3 or fusion protein for CD137 combination

[0410] The binding propensity of exemplary fusion proteins with GPC3 and CD137 was measured using ELISA.

[0411] Recombinant huGPC3 (R&D Systems) at a concentration of 1 µg / mL in PBS was coated onto microtiter plates overnight at 4°C. After washing with 100 μL of PBS-0.05%T (PBS supplemented with 0.05% (v / v) Tween 20), the plates were blocked at room temperature for 1 hour with 2% BSA (w / v) in PBS-0.1%T (PBS-0.1%T-2% BSA). After washing five times with 100 μL PBS-0.05%T (PBS supplemented with 0.05% (v / v) Tween 20), GPC3 antibodies containing fusion proteins (SEQ Nos. 81 and 82) or exemplary fusion proteins (SEQ Nos. 87 and 92 and CD137Ac1-Fusion 1 to 7) at different concentrations were added to the wells and incubated at room temperature for 1 hour, followed by an additional washing step. The molecules bound during the study were detected by incubation with anti-human IgG Fc-HRP (Jackson Laboratory) diluted 1:5000 in PBS-0.1%T-2% BSA. After the additional washing step, fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well, and fluorescence intensity was measured using a fluorescent microplate reader.

[0412] In addition, the binding potential of the fusion proteins to cyanomolecular GPC3 and huCD137 was measured using the same ELISA setup, with recombinant cyGPC3 (R&D Systems) or huCD137-His (recombinant human CD137 tagged with polyhistidine at the C-terminus, R&D Systems) coated instead on microtiter plates. The test reagents were titrated similarly, and the binding reagents were detected via anti-NGAL-HRP.

[0413] The exemplary result is along with the fit curve generated from the 1:1 combined S-shaped fit. do 2A-2D It was described in, EC 50 The values ​​and maximum signals are free parameters, and the slope is fixed as a single value. The generated EC 50 The value Table 3 It is provided in.

[0414] EC observed for human GPC3 of the provided fusion proteins (SEQ Nos. 87 and 82 and CD137Ac1-Fusion 1 to 7) 50 The values ​​were similar to or comparable to the GPC3 antibodies contained in the fusion proteins (SEQNs: 81 and 82) and / or the previously known CD137 / GPC3 bispecific fusion protein (SEQN: 83). Some tested fusion proteins (SEQNs: 87 and 82, CD137Ac1-Fusion 3, and CD137Ac1-Fusion 4) maintained strong binding affinity with human CD137. Furthermore, specific tested fusion proteins (SEQNs: 87 and 82) also exhibited cross-reactivity to cyanomolous GPC3 at a level comparable to human GPC3, i.e., corresponding EC for human GPC3 50 EC of the same range as 50 Combines cyanomolgus with GPC3 as a value.

[0415] Table 3. ELISA data for GPC3 or CD137 binding

[0416]

[0417] Examples 4. In ELISA GPC3 and fusion protein for CD137 Simultaneous combination

[0418] To demonstrate the simultaneous binding of exemplary fusion proteins with GPC3 and CD137, a double-binding ELISA format was used.

[0419] Recombinant huCD137-His (R&D Systems) (1 µg / mL) in PBS was coated onto microtiter plates overnight at 4°C. After each incubation step, the plates were washed five times with 100 μL. The plates were blocked with PBS-0.1% T-2% BSA for 1 hour at room temperature and then washed again. Tested fusion proteins at different concentrations were added to the wells and incubated at room temperature for 1 hour, followed by the 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 hour. After an additional washing step, a fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well, and the fluorescence intensity was detected using a fluorescent microplate reader.

[0420] In addition, the double binding of the fusion protein was tested by inverting 1 μg / ml of recombinant huGPC3 (R&D Systems) coated on a microtiter plate, and the bound fusion protein was detected by the addition of 5 μg / ml of biotinylated huCD137-His.

[0421] Exemplary double joint data, along with a fit curve generated from a 1:1 S-shaped joint fit. Fig. 3 Appears in, EC 50 The value and maximum signal are free parameters, and the slope is fixed as a single value. EC 50 The value Table 4As summarized in [document]. Fusion proteins (Sequence Nos. 87 and 82 and CD137Ac1-Fusion 1 to 7) exhibit clear binding signals, which demonstrate that fusion proteins can bind to GPC3 and CD137 simultaneously. Most of such fusion proteins can bind to GPC3 and CD137 simultaneously to a level comparable to the previously known CD137 / GPC3 bispecific fusion protein Sequence No. 83.

[0422] Table 4. ELISA data on the simultaneous binding of targets for both GPC3 and CD37

[0423]

[0424] Examples 5. Non-target binding of fusion proteins analyzed by ELISA

[0425] Non-target binding of fusion proteins to 32 different targets, including GPC3, GPC5, and other TNF receptor proteins, was evaluated using an ELISA-based assay (Frese et al., MAbs(e.g., 2013). Targets at a concentration of 5 μg / mL in PBS were coated onto microtiter plates overnight at 4°C. After washing with PBS-0.05%T, the plates were blocked with PBS-0.1%T-2%BSA for 1 hour at room temperature. After washing 5 times with 100 μm, 100 nM or 10 nM of the test fusion protein was added to the wells and incubated at room temperature for 1 hour, followed by another washing step. The binding antibodies under 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 the additional washing step, fluorescent HRP substrate (QuantaBlu, Thermo) was added to each well and incubated for 1 hour. Fluorescence intensity was detected using a fluorescent microplate reader and normalized by the signal of the control antibodies (SEQNs: 106 and 107). The normalized signals of each test molecule binding to 32 targets were summed to calculate the cumulative binding ratio for the antibodies at a given concentration, namely 100 nM or 10 nM. The cumulative binding ratios at 100 nM and 10 nM for each test molecule were added together to calculate the sum of the cumulative binding ratios. For example, the control antibodies (SEQNs: 106 and 107), whose fluorescence intensity was standardized due to binding to each of the 32 targets, have a cumulative binding ratio of 32 when tested at 100 nM or 10 nM, and a sum of cumulative binding ratios of 64. The results are Table 5 It appears in. A higher cumulative binding rate is correlated with stronger non-target binding.

[0426] The provided fusion proteins of SEQ NOs. 87 and 82 show no or negligible non-target binding (sum of cumulative binding ratios < 150), as well as CD137 (Fc fusion) specific to the GPC3 antibody and lipocalin mutain contained in the fusion proteins (SEQ NOs. 81 and 82 and SEQ NO. 98, respectively), whereas a previously known specific CD137 / GPC3 bispecific fusion protein (SEQ NO. 83) shows unwanted non-target binding (sum of cumulative binding ratios > 250).

[0427] Table 5: Non-target binding of fusion proteins.

[0428]

[0429] Examples 6. CD137 or GPC3 fusion protein that binds to expressing cells Flow Cytometry analyze

[0430] Target-specific binding of exemplary fusion proteins to human CD137-expressing cells and human GPC3-expressing cells was evaluated by flow cytometry.

[0431] CHO cells were stably transfected with human CD137 or mock controls using the Flp-In system (Life Technologies) according to the manufacturer's instructions. SK-Hep1 cells were stably transfected with human GPC3 or mock controls using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transfected CHO cells were maintained in Ham's F12 medium supplemented with 10% Fetal Calf Serum (Sigma-Aldrich) and 500 µg / mL Hygromycin B (Roth). Transfected SK-Hep1 cells were cultured in RPMI 1640 + GlutaMAX medium (Gibco) supplemented with 20% Fetal Calf Serum (Sigma-Aldrich) and 500 µg / mL G418 (Gibco). Cells were cultured in cell culture flasks according to the manufacturer's instructions (37°C, 5% CO2 atmosphere).

[0432] For flow cytometry analysis, each cell line was incubated with CD137-specific lipocalin mutain (Fc fusion) contained in exemplary fusion proteins (SEQNs: 87 and 82), fusion proteins (SEQNs: 81 and 82 and SEQN: 98, respectively), or GPC3 antibodies, and detected using reference CD137 antibodies SEQNs: 26 and 27, or isotype controls (SEQNs: 24 and 25) and fluorescently labeled anti-human IgG antibodies in FACS analysis as described below:

[0433] 5 x 10 per well 4Cells were incubated for 1 hour in ice-cold PBS containing 5% fetal bovine serum (PBS-FCS). A series of dilutions of the test molecule were added to the cells and incubated on ice for 1 hour. Cells were washed twice with PBS, then incubated on ice for 30 minutes with either the goat anti-hIgG Alexa647-labeled antibody or the goat anti-hIgG Alexa488-labeled antibody. Subsequently, cells were washed and analyzed using an iQue Flow cytometer (Intellicyte Screener). The average geometric fluorescence signals were plotted and loaded into Graphpad software. The geometric mean of fluorescence intensity was calculated using bottom-fixed-to-background four-parameter logistic regression EC₀ 50 It was used to calculate the value.

[0434] The ability of the tested molecule to bind to human GPC3 and CD137 Fig. 4 Binding affinity of fusion proteins SEQ NOs: 87 and 82 for GPC3 and human CD137-expressing cells (EC 50 ) is a low nanomolar, comparable to the respective GPC3 antibodies (SEQ Nos. 81 and 82) and CD137-specific lipocalin mutain (SEQ No. 98) or antibody (SEQ Nos. 26 and 27) ( Table 6 (summarized in ). However, a previously disclosed specific CD137 / GPC3 bispecific fusion protein (SEQ No. 83) shows limited dose-dependent binding to GPC3-expressing cells because the binding curve did not reach a steady phase at a maximum concentration of 100 nM ( Fig. 4B None of the tested molecules bound to the Mock-transfected cells (data not shown).

[0435] Table 6. Binding affinity of fusion proteins for cells expressing GPC3 or CD137

[0436]

[0437] Examples 7. GPC3 - Benign tumor fusion proteins for cells Binding affinity

[0438] The binding of fusion proteins to tumor cells expressing various levels of GPC3—HepG2, Hep3B, MKN-45, and NCI-N87—was evaluated by flow cytometry.

[0439] HepG2 cell lines were cultured in Dulbecco's modified Eagle's medium (DMSO, Pan Biotech) supplemented with 10% fetal bovine serum (Sigma-Aldrich). Hep3B cell lines were cultured in Minimal Essential Medium (containing Earle's salt) (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. All tumor cell lines were cultured in cell culture flasks according to the manufacturer's instructions (37°C, 5% CO2 atmosphere).

[0440] For flow cytometry analysis, tumor cell lines with different GPC3 expression levels (high to intermediate expression: HepG2 > Hep3B > MKN-45) and the GPC3-negative cell line NCI-N87 were incubated with exemplary fusion proteins SEQ NOs: 87 and 82, a GPC3 antibody, or CD137-specific lipocalin mutain (Fc fusion) contained in the fusion proteins (SEQ NOs: 81 and 82 and SEQ NO: 98, respectively), reference CD137 antibodies SEQ NOs: 26 and 27, or isotype controls (SEQ NOs: 24 and 25), and Examples6 As described in [the document], detection was performed using a fluorescently labeled anti-human IgG antibody.

[0441] The ability of fusion proteins SEQ Nos. 87 and 82 against GPC3-positive tumor cells Fig. 5 As illustrated in and corresponding binding affinity (EC 50 )silver Table 7 It is summarized in ). The binding affinity of the fusion protein to GPC3-expressing tumor cells was in the low nanomolar range, comparable to the GPC3 antibodies contained in the fusion protein (SEQ Nos: 81 and 82). Additionally, the results suggest that the fusion protein does not bind to tumor cells that are negative for GPC3.

[0442] Table 7. Binding affinity of fusion protein to GPC3-positive tumor cells

[0443]

[0444] Examples 8. Using the CD137 bioassay GPC3 T-cell assisted stimulation

[0445] The potential of selected fusion proteins to induce activation of the CD137 signaling pathway in a GPC3-dependent manner is CD137 and luc2 While evaluated using commercially available double-stable transfected Jurkat cell lines expressing the gene (humanized version of firefly luciferase), luc2 Expression is driven by NFkB-responsive factors. In this biological assay, CD137 binding triggers CD137 intracellular signaling, inducing NFkB-mediated luminescence.

[0446]

[0001] Hepatocellular carcinoma cell lines HepG2 and Hep3B expressing high levels of GPC3, gastric cancer cell line MKN-45 expressing intermediate levels of GPC3, and GPC3-negative NCI-N87 Examples 7Cultured as described in [document]. One day before the test, each tumor cell was cultured at 6.25 x 10⁶ per well. 3 Cells were plated and allowed to attach overnight at 37°C in a humidified 5% CO2 atmosphere.

[0447] The next day, 3.75 x 10 4 NF-kB-Luc2 / CD137 Jurkat cells were added to each well, and then various concentrations of CD137-specific lipocalin mutain (Fc fusion) contained in the fusion proteins (SEQ NOs. 81 and 82 and SEQ NO. 98, respectively), GPC3 antibody, or exemplary fusion proteins (SEQ NOs. 87 and 82), reference CD137 antibodies SEQ NOs. 26 and 27, or isotype controls (SEQ NOs. 24 and 25), typically ranging from 0.00488 nM to 10 nM, were added. The plates were covered with a gas-permeable sealant and incubated at 37°C in a humidified 5% CO2 atmosphere. After 4 hours, 30 μL of reagent was added to each well, and the bioluminescence signal was quantified using a PHERAstar luminometer. Four-parameter logistic curve analysis was performed using GraphPad Prism®, Table 8 EC summarized in 50 The values ​​(shared below) were calculated. To demonstrate the GPC3 dependence of CD137 binding by the fusion protein, the same experiment was performed in parallel in the absence of tumor cells using the highest concentration of the test molecule. The analysis was performed in triplicate.

[0448] Representative experimental results are Fig. 6 It is depicted in. Fig. 6A and 6B The data shown in demonstrate that exemplary fusion protein sequence numbers: 87 and 82 induced strong CD137-mediated T-cell helper stimulation in the presence of tumor cell lines with high GPC3 expression levels. Figs. 6C-6EThis indicates that the activation of CD137 by the fusion protein is GPC3-dependent, as NF-kB-Luc2 / CD137 Jurkat inactivation was detected in the presence of tumor cells with intermediate or low GPC3 expression or in the absence of tumor cells expressing GPC3. In contrast, reference anti-CD137 mAbs (SEQNs: 26 and 27) exhibited CD137-mediated T-cell co-stimulation regardless of GPC3 expression levels and the absence of target cells.

[0449] Table 8. Evaluation of T-cell activation using CD137 biological assay

[0450]

[0451] Examples 9. Induced by fusion protein GPC3 Assessment of T-cell activation dependent on T-cells

[0452] GPC3 target-dependent T-cell co-stimulation by the fusion protein was analyzed using a T-cell activation assay. The fusion protein was administered to anti-CD3 stimulated T cells at various concentrations and co-cultured with human GPC3-transfected or Mock-transfected SK-Hep1 cells or the GPC3-positive tumor cell line HepG2. IL-2 secretion levels were measured in the supernatant.

[0453] PBMCs from healthy volunteer donors were separated from buffy coats by centrifugation through a polysucrose density gradient (Biocoll, 1.077 g / mL, Biochrom) according to Biochrom's protocol. 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. The 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 rested overnight at 37°C in a humidified 5% CO2 atmosphere in culture medium (RPMI-1640 Medium + GlutaMAX, Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco). SK-Hep1 and HepG2 cells, respectively Examples 6 and Examples 7 It was cultured as described in [the document].

[0454] The following procedure was performed three times for each experimental condition: Flat-bottom tissue culture plates were pre-coated with 25 µg / mL anti-CD3 antibody at 37°C for 2 hours and then washed twice with PBS. To block proliferation, SK-Hep1 cells transfected with human GPC3 or Mock, and GPC3-positive tumor cell HepG2, were treated with 30 µg / ml mitomycin C (Sigma Aldrich) for 30 minutes. The mitomycin-treated cells were then washed twice with PBS and cultured at 1.0 x 10⁶ per well in culture medium. 4Cells were plated and allowed to adhere overnight at 37°C in a humidified 5% CO2 atmosphere. Target-expressing cells were previously grown under standard conditions, isolated using Accutase (PAA Laboratories), and resuspended in culture medium.

[0455] The next day, after washing the plates twice with PBS, 2.5 x 10 per well 4 T cells were added. A series of dilutions ranging from 0.003 nM to 10 nM of the fusion proteins (SEQ Nos. 87 and 82), the GPC3 antibody contained in the fusion proteins (SEQ Nos. 81 and 82), the previously known CD137 / GPC3 bispecific fusion protein (SEQ No. 83), the GPC3-specific lipocalin mutain (Fc fusion) (SEQ No. 97), the reference CD137 antibody (SEQ Nos. 26 and 27), or the isotype controls (SEQ Nos. 24 and 25) were added to the corresponding wells. The plates were covered with a gas-permeable sealant and incubated at 37°C in a humidified 5% CO2 atmosphere for 3 days. IL-2 levels in the supernatant were evaluated using the human IL-2 DuoSet kit (R&D Systems) as described in the following procedure.

[0456] A 384-well plate was coated with 1 µg / mL "human IL-2 capture antibody" in PBS at room temperature for 2 hours. Subsequently, the wells were washed 5 times with 80 µl of PBS-0.05%T. After blocking for 1 hour in PBS-0.05%T containing 1% casein (w / w), the IL-2 standard concentration series diluted in the assay supernatant and culture medium were transferred to each well 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) was added to PBS-0.05%T containing 0.5% casein and incubated for 1 hour at room temperature. After washing, 25 μL of reading buffer (Mesoscale Discovery) was added to each well, and the generated electrochemiluminescence (ECL) signal was detected using a Mesoscale Discovery reader. Analysis and quantification were performed using Mesoscale Discovery software.

[0457]

[0001] In addition, a T-cell activation assay was used in a similar manner to evaluate the ability of additional previously known CD137 / GPC3 bispecific fusion proteins SEQ NOs. 99 and 27, SEQ NOs. 83, and SEQ NOs. 84 to co-stimulate T-cell responses. In the experiments, tissue culture plates were pre-coated with 200 μL of 0.25 μg / mL anti-CD3 antibody at 37°C for 1 hour and washed twice with PBS. 1.25 x 10⁶ per well 4 HepG2 tumor cells were plated and allowed to adhere overnight at 37°C in a humidified 5% CO2 atmosphere, and treated with 10 µg / mL mitomycin C at 37°C for 2 hours. The plates were washed twice with PBS, and 5 x 10 4T cells and test molecules at a concentration of 1 µg / mL were added to each well. The plates were covered with a gas-permeable sealant and incubated at 37°C in a humidified 5% CO2 atmosphere for 3 days. Subsequently, the IL-2 concentration in the supernatant was evaluated.

[0458] The exemplary data is Fig. 7 It is shown in. SK-Hep1 cells transfected with human GPC3 in the presence of fusion proteins (sequence numbers: 87 and 82) Fig. 7B ) or GPC-3 expressing HepG2 tumor cells ( Fig. 7C Co-culture of ) and Pan T cells resulted in strong dose-dependent IL-2 secretion compared to isotype controls, which is much more potent than GPC3 antibodies (SEQ Nos. 81 and 82, GPC3-specific lipocalin mutane (SEQ No. 129), or the previously known CD137 / GPC3 bispecific fusion protein (SEQ No. 83), and no increased IL-2 or slightly increased IL-2 secretion was observed at high dose levels. When co-cultured with Mock-transfected SK-Hep1 cells (GPC3 negative), the test molecule did not show a dose-dependent increase in IL-2 secretion. The results demonstrate that T cell activation by the provided fusion protein is GPC3-dependent.

[0459] Examples 10. Different levels GPC3 Evaluation of T-cell activation in the presence of expressing tumor cells

[0460] Further T-cell analyses were performed to evaluate the ability of the exemplary fusion protein to co-stimulate T-cell activation in a GPC3 target-dependent manner. The fusion protein was administered at different concentrations to anti-CD3 stimulated T-cells in the presence of tumor cell lines with different GPC3 expression levels. The tumor cell lines tested included HepG2, Hep3B, MKN-45, and NCI-N87 (high to intermediate expression: HepG2 > Hep3B > MKN-45; GPC3 negative: NCI-N87). IL-2 secretion levels were measured in the supernatant.

[0461] PBMC from healthy resource donors Examples 9 As described in [document], lymphocytes were isolated from buffy coats and purified from PBMCs.

[0462] For analysis, T cells were thawed and rested for 16 hours at 37°C in a humidified 5% CO2 atmosphere in RPMI-1640 medium + GlutaMAX (Gibco), a culture medium supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin (Gibco).

[0463] The following procedure was performed in triplicate for each experimental condition: Flat-bottom tissue culture plates were pre-coated with 0.25 µg / mL anti-CD3 antibody at 37°C for 2 hours and then washed twice with PBS. To block proliferation, tumor cell lines HepG2, Hep3B, MKN-45, or NIC-N87 were treated with 30 µg / ml mitomycin C (Sigma Aldrich) for 30 minutes. The mitomycin-treated tumor cells were then washed twice with PBS and cultured at 8.3 x 10⁶ per well in culture medium. 3Cells were plated and attached overnight at 37°C in a humidified 5% CO2 atmosphere. Target cells were previously grown under standard conditions, isolated using Accutase (PAA Laboratories), and resuspended in culture medium.

[0464] The next day, after washing the plates twice with PBS, 2.5 x 10 per well 4 T cells were added to the tumor cells. A series of dilutions ranging from 0.26 nM to 10 nM of exemplary fusion proteins (SEQ Nos. 87 and 82), GPC3 antibodies or CD137-specific lipocalin mutains (Fc fusions) contained in fusion proteins (SEQ Nos. 81 and 82 and SEQ No. 98, respectively), reference CD137 antibodies (SEQ Nos. 26 and 27), or isotype controls (SEQ Nos. 24 and 25) were added to the corresponding wells. The plates were covered with a gas-permeable sealant and incubated at 37°C in a humidified 5% CO2 atmosphere for 3 days.

[0465] After 3 days of co-culture, Examples 9 As described in [document], the IL-2 level in the supernatant was evaluated.

[0466] The exemplary data is Fig. 8As shown in [document]. Co-culturing HepG2 and Hep3B cells expressing high levels of GPC3 in the presence of fusion proteins SEQ NOs: 87 and 82 with Pan T cells induces a clear increase in IL-2 secretion compared to hIgG4 isotype controls. Additionally, co-culturing with MKN-45 (GPC3 intermediate) or NIC-N87 (GPC3 negative) did not increase IL-2 secretion levels with the fusion proteins. The data indicate that the functional activity of the fusion proteins, measured by their 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 CD137 antibodies (SEQ NOs: 26 and 27) is not necessarily GPC3-dependent and is difficult to predict.

[0467] Examples 11. Induced by fusion protein GPC3 Evaluation of T-cell-mediated cell lysis in expressing tumor cells

[0468] Using an impedance-based T cell apoptosis assay, we evaluated the ability of a fusion protein to induce T cell-mediated cytolysis of GPC3, which expresses tumor cells and activates the CD137 co-stimulation signaling pathway. For this purpose, adherent tumor cells were seeded into the wells of electronic microtiter plates (E-plates). Cell adhesion to gold microelectrodes impedes current flow between the electrodes. This impedance is measured as a unitless “cell index” parameter. The cell index (CI) increases as cells adhere and proliferate over time. Non-adherent CD8+ T cells and HepG2 cells were added to the cell index with an anti-CD3 antibody that independently activates T cell antigens, along with different concentrations of the test molecule. Adding the molecule itself does not cause a change in impedance. When the test molecule co-stimulates cytotoxic T cells by activating the CD137 signaling pathway, an increase in T cell-mediated cytolysis of target cells and a decrease in destruction of adherent tumor cells by effector cells can be detected via CI and real-time apoptosis curves.

[0469] PBMCs from healthy resource donors were isolated in a buffer coat. CD8+ T cells were isolated from these PBMCs and stored in liquid nitrogen until further use. For analysis, CD8+ T cells were thawed and rested for 24 hours at 37°C in a humidified 5% CO2 atmosphere in an analysis medium consisting of RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin.

[0470] The following procedure was performed in triplicate for each experimental condition. Tumor cell lines HepG2 (hepatocellular carcinoma cell line, GPC3-expressing) or NCI-N87 (gastric carcinoma cell line, GPC3-negative) were plated on E plates of analysis medium and incubated at 3°C ​​for 24 hours in a humidified 5% CO2 atmosphere to allow cell adhesion and proliferation.

[0471] The following day, anti-CD3 antibodies were added to the tumor cells, followed by the addition of a series of dilutions of exemplary fusion proteins (SEQ Nos. 87 and 82), CD137-specific lipocalin mutains (Fc fusions) contained in GPC3 antibodies or fusion proteins (SEQ Nos. 81 and 82 and SEQ No. 98, respectively), reference CD137 antibodies (SEQ Nos. 26 and 27), previously known specific CD137 / GPC3 bispecific fusion proteins (SEQ No. 83), GPC3-specific lipocalin mutains (Fc fusions) (SEQ No. 97), or isotype controls (SEQ Nos. 24 and 25). Then, resting CD8+ T cells were added to the tumor cells at a ratio of 5:1. The plates were covered with a gas-permeable sealant, and impedance was measured periodically during incubation for 3 days.

[0472] CI values ​​were displayed as a function of time using RTCA HT Software V 1.0.1 (ACEA Biosciences). Normalized CIs were calculated by dividing the CI at a given time point (20, 30, 40, 50, 60, and 70 hours after the addition of effector cells) by the CI at the normalized time point (the first measurement after the addition of effector cells). Specific death values ​​were calculated using the following formula with each triplicate mean normalized CI value and its corresponding SD value: 100 - (Normalized mean CI of test molecules / Normalized mean CI of target cells and effector cells) x 100. Specific death values ​​were exported to GraphPad Prism v7 and displayed as the respective SD values ​​for the time intervals of interest on an XY graph.

[0473] The exemplary experimental results are Fig. 9 This was illustrated in [figure]. Data showed that fusion proteins SEQ ID NOs: 87 and 82 induced dose-dependent T cell-mediated lysis of GPC3 expressing HepG2 cells, whereas ( Figs. 9A-9C), showing that specific lysis of target-negative NCI-N87 cells was not observed ( Fig. 9D ), this demonstrates that the fusion protein-induced activation of the CD137 pathway inducing T cell-mediated apoptosis is GPC3-dependent. The provided fusion proteins SEQ NOs. 87 and 82 induced much higher levels of GPC3-positive target cell death by cytotoxic T cells compared to the equimolar values ​​of the previously known CD137 / GPC3 bispecific fusion protein (SEQ NO. 83), GPC3 antibody (SEQ NOs. 81 and 82), or GPC3-specific lipocalin mutain (Fc fusion) (SEQ NO. 97). Fig. 9C In addition, CD137-specific lipocalin mutain (Fc fusion) (SEQN: 98), reference CD137 antibody (SEQN: 26 and 27), and hlgG4 isotype control (SEQN: 24 and 25) did not induce CD8+ T cell-mediated apoptosis ( Fig. 9C ).

[0474] Additional experiments were performed to demonstrate that potent T cell-mediated lysis of HepG2 cells by CD137 / GPC3 bispecific fusion proteins (SEQ Nos. 87 and 82) depends on their bispecific design rather than the combination of anti-GPC3 and anti-CD137 alone. The experiments were basically performed as described above, but all constructs were added at 10 nM and compared with a cocktail of GPC3 antibodies (SEQ Nos. 81 and 82) and reference CD137 antibodies (SEQ Nos. 26 and 27) and a cocktail of GPC3 antibodies (SEQ Nos. 81 and 82) and CD137-specific lipocalin mutain (Fc fusion) (SEQ No. 98). Strong T-cell mediated lysis was detected for fusion proteins SEQ ID NOs: 87 and 82, but specific lysis mediated by the two cocktails was only moderate, which demonstrates the advantage of this dual-specific format for achieving T-cell mediated lysis.

[0475] Examples 12. Human with PBMCs Evaluation of functional in vivo activity in transplanted xenograft mouse models

[0476] To investigate the in vivo activity of the provided fusion protein, human HepG2 tumor cells and immunodeficient NOG mice transplanted with human PBMCs were used.

[0477] 5 x 10⁶ of Matrigel / PBS (1:1) solution in 4 to 6-week-old NOG mice 6 HepG2 cells were injected subcutaneously (sc). The tumor was 80-100 mm 3 The tumors were allowed to grow to a size of [size], and this point was defined as Experiment Day 0. On Day 0, mice were randomized into treatment (or control) groups based on tumor size and animal body weight. Mice were injected with 5 x 10 via tail vein injection. 6 Fresh human PBMCs were administered intravenously (iv). Mice received either treatment or control (PBS) via intraperitoneal (ip) injection on days 8 and 15, following iv PBMC injection on day 1. The molecules under study included exemplary fusion proteins SEQ NOs. 87 and 82 (0.5, 5, or 20 mg / kg), GPC3 antibodies contained in fusion proteins (SEQ NOs. 81 and 82) (at 0.39 or 3.9 mg / kg, and at 0.5 or 5 mg / kg, the same molar concentration for fusion protein treatment), and reference CD137 antibodies (SEQ NOs. 26 and 27) (at 3.9 mg / kg, and at 5 mg / kg, the same molar concentration for fusion protein treatment). Tumor growth was recorded every 3–4 days. Animals not exposed to the drug were excluded from the data analysis.

[0478] At the end of the study (Day 16), mice were sacrificed, and tumors were formalin-fixed and paraffin-embedded (FFPE). Histological and immunohistochemical analyses of the tumors were performed on BioSiteHisto. FFPE xenograft tumor tissues were excised and stained with H&E or T cell markers CD3, CD4, or CD8. Stained slides were imaged and digitized using a 3D Histech Panoramic MIDI instrument equipped with a 20x objective lens. CaseViewer 2.2 was used for microscopic examination of the digital slides, and Image J was used for tumor area-based analysis. The percentage of tumor-infiltrating lymphocytes (TILs) per tumor area was calculated by subtracting the necrotic area.

[0479] Fig. 10 This reflects the change in tumor volume measured on days 2, 6, 9, 13, and 16 of the study. Tumor growth inhibition was achieved by the GPC3 antibodies contained in fusion proteins SEQ NOs: 87 and 82 and fusion proteins SEQ NOs: 81 and 82. Both the fusion proteins and the GPC3 antibodies completely inhibited tumor growth during the course of the study (at concentrations of 5 or 3.9 mg / kg, respectively). The effects of the fusion proteins and the GPC3 antibodies were dose-dependent, as lower concentrations (0.5 or 0.39 mg / kg) had a negligible effect on tumor growth. Only a limited effect on tumor growth was achieved by treatment with the reference CD137 antibodies (SEQ NOs: 26 and 27).

[0480] Table 9This summarizes the data obtained from the histological and immunohistochemical analysis of tumor-infiltrating lymphocytes. Intratumoral CD3, CD4, or CD8 T cell infiltration is expressed as % TILs per (tumor area - necrotic area). Treatment with fusion proteins resulted in T cell infiltration of up to 10% of (tumor area - necrotic area) (all treatment groups). Compared to the vehicle control, there was no intratumoral T cell infiltration induced by treatment with the GPC3 antibody or reference CD137 antibody.

[0481] Table 9. Tumor Area - Percentage of Tumor-Infiltrating Lymphocytes (TILs) per Necrotic Area

[0482]

[0483] Examples 13. Pharmacokinetics of fusion proteins in mice

[0484] Pharmacokinetic analyses of GPC3 antibodies contained in representative fusion proteins (SEQNs: 87 and 82) and fusion proteins (SEQNs: 81 and 82) were performed in mice and compared with two previously known CD137 / GPC3 bispecific fusion proteins (SEQNs: 83 and 84). Male CD-1 mice approximately 5 weeks of age (two mice per time point; Charles River Laboratories) were injected with each construct via tail vein at a dose of either 2 mg / kg (SEQNs: 87 and 82, SEQNs: 81 and 82) or 10 mg / kg (SEQNs: 83, SEQNs: 84). Mouse plasma samples were collected at time points of 5 min, 24 hours, 168 hours, and 336 hours for antibody-based constructs, and at 5 min. It was obtained at 2, 4, 8, 24 hours, 2, 3, 4, 7, 9, 11, 14, and 21 hours. Sufficient whole blood was collected under isoflurane anesthesia, and at least 30-50 μL of Li-heparin plasma was obtained per animal and hour. Plasma drug levels were then analyzed by ELISA.

[0485] 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 onto microtiter plates overnight at 4°C. After each incubation step, the plates were washed five times with 80 μL of PBS-0.05%T. The plates were blocked with PBS-0.1%T-2%BSA for 1 hour at room temperature and then washed. Plasma samples were diluted to a 20% plasma concentration in PBS-0.1%T-2%BSA, added to the wells, and incubated at room temperature for 1 hour. Another washing step followed. The binding substance under study was detected after 1 hour incubation with sulfo-tag anti-human antibody (Mesoscale Discovery) or 1 μg / mL of 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) signals from all wells were read using a Mesoscale Discovery reader.

[0486] 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 onto microtiter plates overnight at 4°C. After each incubation step, the plates were washed with 80 μL of PBS-0.05%T. The plates were blocked with PBS-0.1%T-2%BSA for 1 hour at room temperature and then washed. Plasma samples were diluted to a 20% plasma concentration in PBS-0.1%T-2%BSA, added to wells, and incubated for 1 hour at room temperature. Another washing step followed. The binding substances under study were detected after 1 hour incubation with 1 μg / mL of human glypican-bio and streptavidin sulfo-tag, respectively, diluted in PBS-0.1%T-2%BSA. After the additional washing step, 25 μL of reading buffer was added to each well, and the ECL signals of all wells were read using a Mesoscale Discovery reader.

[0487] For data analysis and quantification, a calibration curve using standard protein diluents was also prepared. In an exemplary experiment, the plasma concentration of the test molecule over time was Fig. 12 It is shown in [figure]. Non-compartmental analysis was applied to the data using Phoenix WinNonlin version 8.1, and the results are Table 10 It is summarized in.

[0488] The data demonstrate that while the provided fusion proteins SEQ NOs: 87 and 82 exhibit typical antibody pharmacokinetics, the previously known CD137 / GPC3 bispecific fusion proteins SEQ NOs: 83 and 84 exhibit significantly impaired pharmacokinetic profiles.

[0489] Table 10. Pharmacokinetics in mice

[0490]

[0491] Examples 14: Evaluation of Thermal Stability of Fusion Proteins

[0492] The melting temperature (T) of a fusion protein, a general indicator of overall stability m To determine s), test molecules at a protein concentration of 1 mg / mL in PBS (Gibco) were scanned at 1°C / min using a capillary nanoDSC instrument (CSC 6300, TA Instruments) (25–100°C). m s was calculated from the displayed thermogram using integrated Nano Analyze software.

[0493] The maximum melting temperature and melting onset temperature generated for exemplary fusion proteins are listed in Table 11 below. Fusion proteins SEQ NOs: 72 and 82 and CD137Ac1-Fusion 2 have improved thermal stability compared to specific CD137 / GPC3 bispecific fusion proteins SEQ NOs: 83, SEQ NOs: 84, and SEQ NOs: 26 and 102 previously known.

[0494] Table 11 : T determined by DSCd m and melting point

[0495]

[0496] The embodiments described descriptively in this specification may be appropriately practiced in the absence of any elements or elements, or limitations or limits not specifically disclosed in this specification. Accordingly, for example, terms such as "comprising," "including," "containing," etc., should be read broadly and without limitation. Additionally, terms and expressions used in this specification are used for descriptive rather than limiting purposes, and there is no intention to use such terms and expressions to exclude any equivalents of the features shown and described or any part thereof, but various modifications are recognized as possible within the scope of the claimed invention. Accordingly, even though the embodiments are specifically disclosed by preferred embodiments and optional features, modifications and variations thereof may be made to those skilled in the art, and such modifications and variations are considered to be within the scope of this invention. Thus, all patents, patent applications, textbooks, and academic publications described in this specification are incorporated by reference in their entirety. Furthermore, if the definition or use of a term in the references incorporated herein by reference does not match or contradicts the definition of such term provided herein, the definition provided herein shall apply, and the definition of such term in the references shall not apply. Each narrower species and subgroup belonging to the general disclosure also forms part of the invention. This includes the general description of the invention with any conditional or negative limitation removing any essence from the genus, regardless of whether the truncated material is specifically mentioned herein. Additionally, where a feature is described in the sense of the Markush group, those skilled in the art will recognize that the disclosure is also described in the sense of any individual member or subgroup of the members of the Markush group. Further embodiments will become apparent from the following claims.

[0497] Equivalents: Those skilled in the art will be able to recognize or identify many equivalents to the specific embodiments of the invention described herein by using only routine experimentation. Such equivalents are intended to be included in the claims below. All publications, patents, and patent applications mentioned herein are incorporated herein by reference to the same extent that each individual publication, patent, or patent application is specifically and individually directed to be incorporated herein by reference.

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Claims

Claim 1 A fusion protein capable of binding to both CD137 (cluster of differentiation 137) and GPC3 (glypican-3), wherein the fusion protein comprises at least two subunits, the first subunit comprises a full-length immunoglobulin and is specific to GPC3, the second subunit comprises a lipocalin mutain and is specific to CD137, the second subunit is connected to the C-terminus of the first subunit at the N-terminus by a peptide linker, and the fusion protein comprises two chains of SEQ ID NOs 87 and 82. Claim 2 In claim 1, (a) the fusion protein is K at most 1 nM D K of the value or immunoglobulin alone included in the first subunit D K that is comparable to or lower than the value D (b) the fusion protein can bind to GPC3 with a value of up to 3 nM EC 50 EC of lipocalin mutain alone that is specific to CD137 included in the value or the second subunit 50 Equivalent to or lower than the value EC 50 (c) the fusion protein can bind to CD137 with a value; (d) the fusion protein cross-reacts with GPC3 of cyanomolgus; and (d) the fusion protein has an EC of up to 10 nM when the fusion protein is measured in an ELISA assay. 50 (e) the fusion protein can bind simultaneously with CD137 and GPC3; (f) the fusion protein can bind to GPC3-expressing tumor cells; (g) the fusion protein can induce increased secretion of IL-2; (h) the fusion protein can induce increased IL-2 secretion to a higher level than SEQ NO: 83 and / or with better efficiency than SEQ NO: 83; (h) the fusion protein can induce lymphocyte-mediated cytotoxicity; (i) the fusion protein can induce enhanced apoptosis of GPC3-expressing tumor cells mediated by T cells than SEQ NO: 83 and / or induce cytotoxic T cell activation with better efficiency than SEQ NO: 83; (j) the fusion protein can co-stimulate T-cell responses in a GPC3-dependent manner; or (k) the fusion protein can co-stimulate T-cell responses in the tumor microenvironment. Claim 3 The fusion protein according to claim 1 or 2, wherein the fusion protein has a half-life 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 longer than that in a mouse, and / or the fusion protein has a half-life longer than SEQ ID NO: 83 in a mouse, and / or the fusion protein has an isopotential point of at least 6.5, at least 6.8, at least 7.1, at least 7.4, at least 7.5, at least 7.7, or higher than that, and / or the fusion protein has an isopotential point higher than SEQ ID NO:

83. Claim 4 A fusion protein according to claim 1 or 2, wherein the second subunit is connected to the C-terminus of the heavy chain constant region (CH) of the first subunit at the N-terminus through a linker. Claim 5 A fusion protein according to claim 1 or 2, wherein the first subunit comprises a set of CDRs of GYTFTDYE (HCDR1, SEQ NO: 72), LDPKTGDT (HCDR2, SEQ NO: 73), and TRFYSYTY (HCDR3; SEQ NO: 74), and a set of CDRs of QSLVHSNRNTY (LCDR1, SEQ NO: 75), KVS (LCDR2, SEQ NO: 76), and SQNTHVPPT (LCDR3; SEQ NO: 77). Claim 6 In claim 1 or 2, the immunoglobulin is a fusion protein having an IgG4 backbone. Claim 7 A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of claim 1 or 2. Claim 8 In vitro (producing the fusion protein of paragraph 1 or 2) in vitro A method wherein the fusion protein is produced starting from a nucleic acid encoding the fusion protein. Claim 9 A pharmaceutical composition for the prevention, improvement, or treatment of cancer comprising one or more fusion proteins of claim 1 or 2. Claim 10 A fusion protein for the prevention, improvement, or treatment of GPC3-positive cancer in accordance with claim 1 or 2. Claim 11 In claim 9, the above-mentioned cancer is a GPC3-positive cancer, a pharmaceutical composition. Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete

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