Affibody that specifically binds to CD137 and its applications

Abstract

The present invention relates to an affibody that specifically binds to the extracellular domain of CD137, a protein complex comprising the affibody and an antibody or antigen-binding fragment thereof that specifically binds to a protein on the surface of cancer cells, and a pharmaceutical composition for preventing or treating cancer that contains the same as an active ingredient. By utilizing the affibody of the present invention that specifically binds to CD137, it is possible to develop a protein complex that specifically binds to T cells and also specifically binds to antigens on the surface of various cancer cells, which can be useful for preventing or treating blood cancers and solid cancers.

Description

Detailed description of the invention

[0001] [Technical Field] The present invention relates to an affibody that specifically binds to CD137 and its applications. More specifically, it relates to an affibody that specifically binds to CD137, a protein complex comprising an affibody that specifically binds to CD137 and an antibody that specifically binds to EGFR, HER2, or CD19, and applications for the prevention or treatment of cancer using these.

[0002] [Background technology] In recent years, the following immunosuppressants have attracted attention as promising cancer treatments: immune checkpoint inhibitors such as ipilimumab targeting CTLA-4, pembrolizumab or nivolumab targeting PD-1, and atezolizumab targeting PD-L1; immunotherapy agents such as chimeric antigen receptor-T (CAR-T) cells; therapeutic antibodies such as antibodies that bind to antigens on cancer cells, and antibody-drug conjugates (ADCs) that combine antibodies and drugs; blinatumomab, which bispecifically targets CD3 antigen on T cells and CD19 antigen on B-cell lymphoma, and targets CD3 antigen on T cells and epithelial cell adhesion molecules on cancer cells. T-cell engaging bispecific antibodies, such as catumaxomab, that bispecifically target a specific molecule; and anti-cancer vaccines that administer cancer cell-specific antigens or antigens to enhance the body's immune response.

[0003] Among them, T cell-involved bispecific antibodies such as blinatumomab and catumaxomab are the most advanced next-generation immunotherapy approaches for cancer, but blinatumomab is only used for blood cancers and has safety issues, and catumaxomab is only applicable to the treatment of malignant ascites and has limitations for use in various cancer treatments (Lakins MA, et al. Clin Cancer Res. 2020 Aug 1;26(15):4154-4167.).

[0004] Therefore, if a T cell-involved bispecific antibody applicable to solid tumors in addition to blood cancers can be developed, it is expected to be usefully used for the treatment of various cancers.

[0005] Throughout this specification, numerous papers and patent documents are referenced and their citations are shown. The disclosed contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the level in the technical field to which the present invention pertains and the content of the present invention.

[0006] [Prior Art Documents] [Patent Documents] [Patent Document 1] Korean Patent Publication No. 10-2017-0012754 (Publication Date: February 3, 2017) [Patent Document 2] Korean Patent Publication No. 10-2017-0117330 (Publication Date: October 23, 2017)

[0007] [Summary of the Invention] [Problems to be Solved by the Invention] The inventors have made intensive research efforts to develop T cell-involved bispecific antibodies applicable to both blood cancers and solid cancers. As a result, an affibody that specifically binds to CD137, a co-stimulatory molecule whose expression increases on the surface of activated T cells, was developed. Using this, a protein complex was developed that binds not only to CD137 of activated T cells but also specifically to antigens on the surface of cancer cells such as EGFR, HER2, or CD19, leading to the completion of a T cell-involved bispecific antibody applicable to both blood cancers and solid cancers.

[0008] Therefore, an object of the present invention is to provide an affibody that specifically binds to the extracellular domain of CD137.

[0009] Another object of the present invention is to provide a protein complex comprising the above-described affibody and an antibody or an antigen-binding fragment thereof that specifically binds to a protein on the cancer cell surface.

[0010] Yet another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the above-described affibody or protein complex and a pharmaceutically acceptable carrier.

[0011] [Means for Solving the Problems] According to one aspect of the present invention, the present invention provides an affibody that specifically binds to the extracellular domain of CD137 and comprises an amino acid sequence represented by any one of SEQ ID NO: 1 to SEQ ID NO: 8.

[0012] The inventors of this invention have diligently researched and developed a T-cell-involved bispecific antibody applicable to both hematological cancers and solid tumors. As a result, they developed an affibody that specifically binds to CD137, a co-stimulatory molecule whose expression increases on the surface of activated T cells. Using this affibody, they developed a protein complex that not only binds to CD137 on activated T cells but also specifically binds to antigens on the surface of cancer cells such as EGFR, HER2, or CD19. Therefore, the bispecific antibody containing the affibody and antibodies against antigens on the surface of cancer cells, i.e., the T-cell-involved bispecific antibody, can be usefully used for the prevention or treatment of hematological cancers and solid tumors.

[0013] In this specification, the term "CD137" refers to a co-stimulatory immune checkpoint molecule, which is one of the tumor necrosis factor receptor (TNFR) family molecules, also known as TNFRSF9 (tumor necrosis factor receptor superfamily number 9), 4-1BB, and ILA (induced by lymphocyte activation).

[0014] CD137(4-1BB) is an activated CD8 + T cells, CD4 + Expressed on T cells, regulatory T cells (Treg), NKT cells (natural killer T cells), NK cells (natural killer cells), dentritic cells (DC), neutrophils, eosinophils, mast cells, and endothelial cells.

[0015] CD137, upon binding to its ligand 4-1BBL, induces T cell activation, survival, and effector function. 4-1BBL, the ligand for CD137, is a member of the TNF family and is expressed in antigen-presenting cells (APCs) such as mature DCs, activated B cells, and macrophages.

[0016] In this specification, the term "affibody" refers to the Z-domain of protein A of Staphylococcus aureus, which is the region that has affinity for IgG (immunoglobulin G), and is also called "Z body" or "Zb".

[0017] Affibodies are small proteins consisting of 58 amino acid residues. The 13 amino acids that form the binding site with IgG in such an affibo molecule can bind to various target antigens depending on their amino acid sequence. Because random sequences are possible, libraries can be constructed. Similar to antibodies, affibo molecules capable of binding to various target antigens can be selected from a library using screening methods such as phage display and yeast two-hybrid (Y2H) assays.

[0018] Furthermore, because affibodies have a very small molecular weight of 6 kDa, they diffuse systemically upon administration to the human body and are rapidly removed by renal filtration, compared to IgG antibodies, which generally have a molecular weight of 150 kDa. Affibodies have also been developed in the form of biantibodies conjugated with general IgG (Yu F et al., 2014, MAbs). Moreover, affibodies are resistant to heat and alkaline conditions and can be mass-produced using bacteria, resulting in lower production costs compared to antibodies.

[0019] An invention relating to a polypeptide scaffold based on a first-generation Z variant (a variant of the Z domain) was disclosed in PCT Publication WO95 / 19374, and an invention relating to a polypeptide scaffold based on a second-generation Z variant was disclosed in PCT Publication WO2009 / 080811.

[0020] As mentioned above, affibodies are small protein domains that can specifically bind to different target proteins and are designed by randomizing 13 surface residues of a 58-residue immunoglobulin Fc-binding Z domain derived from Staphylococcus aureus protein A.

[0021] The 13 surface residues targeted by the above randomization are Q9, Q11, N11, F13, Y14, L17, and H18 located on the first helix, and E24, E25, R27, N28, Q32, and K35 located on the surface of the second helix. The affinity of the wild-type Z domain for human Fc(IgG) is known to be approximately 10 nM to 60 nM, and protein A is used as an affinity ligand for immunoglobulin capture (see Nord, K., Gunneriusson, E., Ringdahl, J. et al. Nat Biotechnol 15, 772-777, 1997).

[0022] In this specification, the term "AffiMab" means a bispecific antibody composed of affibody molecules fused to the heavy or light chain of an antibody. Because affiMab is modular, it can be easily applied to different targets (see Volk, AL., Mebrahtu, A., Ko, BK. et al. Drugs RD 21, 157-168, 2021). The protein complexes, fusion proteins, and biantibodies according to the present invention may be in the form of affiMab.

[0023] In one embodiment of the present invention, the CD137 is derived from humans or cynomolgus monkeys.

[0024] In one embodiment of the present invention, the binding site of the affibody to the extracellular domain of CD137 is located between amino acid residues 64 to 95 of the amino acid sequence of SEQ ID NO: 27. That is, the affibody of the present invention specifically binds to the area between amino acid residues 64 to 95 of the extracellular domain of CD137 in human (Homo sapiens) or cynomolgus monkey (Macaca fascicularis).

[0025] In other embodiments of the present invention, the affibody may be in the form of an affibody homodimer that specifically binds to the extracellular domain of CD137. The homodimer means a single protein complex formed by the interaction of identical proteins. The affibody homodimer may be in the form of a multimer in which affibody monomers are linked together. The affibody homodimer may be in the form of a multimer in which affibody monomers are linked together by amino acid linkers. The affibody homodimer may be embodied in the form of a fusion protein or a conjugate. The affibody homodimer may be linked directly, for example by known organic chemical methods, or indirectly, for example by amino acid linkers.

[0026] In another embodiment of the present invention, the affibody that specifically binds to the extracellular domain of CD137 may be in the form of a complex linked to gold nanoparticles. In this case, it may be used in nanoparticle-based immunoassays that exhibit high sensitivity and specificity to antigen-presenting cells (APCs) expressing CD137, such as activated T cells, in ELISA, immunoPCR, etc.

[0027] In another embodiment of the present invention, the affibody that specifically binds to the extracellular domain of CD137 may be in the form of a complex linked to a fluorescent protein. When the affibody of the present invention is in the form of a complex linked to a fluorescent protein, specific targeting and imaging of antigen-presenting cells expressing CD137 is possible.

[0028] According to another aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an affibody according to one embodiment.

[0029] In one embodiment of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding an affibody that specifically binds to the extracellular domain of CD137.

[0030] In one embodiment of the present invention, the nucleic acid molecule includes a nucleotide sequence encoding an affibody that specifically binds to the extracellular domain of CD137, which includes the amino acid sequence shown in SEQ ID NO: 27.

[0031] In one embodiment of the present invention, the nucleic acid molecule includes a nucleotide sequence encoding an affibody that specifically binds between amino acid residues 64 to 95 of the amino acid sequence shown in Sequence ID No. 27.

[0032] In this specification, the term "nucleic acid molecule" comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and the nucleotide, which is the basic building block of a nucleic acid molecule, includes not only natural nucleotides but also analogues in which the sugar or base site has been altered (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

[0033] It will be obvious to those skilled in the art that the nucleotide sequence encoding the affibody of the present invention may be any nucleotide sequence encoding the amino acid sequence constituting the affibody, and is not limited to any specific nucleotide sequence.

[0034] This is because even if a mutation occurs in the nucleotide sequence, expressing the mutated nucleotide sequence as a protein may not result in any change in the protein sequence. This is called codon degeneracy. Therefore, the nucleotide sequence includes a nucleotide sequence containing codons that are functionally equivalent or codons that code for the same amino acid (for example, due to codon degeneracy, there are six codons for arginine or serine), or codons that code for biologically equivalent amino acids.

[0035] Considering the mutations having the same biological activity as described above, the nucleic acid molecule of the present invention that encodes the amino acid sequence constituting the affibody is interpreted to also include sequences that exhibit substantial identity with it.

[0036] The substantial identity described above is determined by aligning the sequence of the present invention described above with any other sequence to the greatest extent possible, and analyzing the aligned sequences using algorithms commonly used in the industry, resulting in at least 60% homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically, 70% homology (e.g., 71%, 72%, 73%, 74%, 75%). This means sequences exhibiting 76%, 77%, 78%, or 79% homology, more specifically 80% or more (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), particularly specifically 90% or more homology (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and most specifically 95% or more homology (e.g., 95%, 96%, 97%, 98%, or 99%). All integers between 60% and 100% and decimals between them are included within the scope of the present invention in relation to % homology.

[0037] Alignment methods for sequence comparison are publicly known in this industry. Various methods and algorithms for alignment are disclosed in Smith and Waterman, Adv.Appl.Math.2:482 (1981); Needleman and Wunsch, J.Mol.Bio.48:443 (1970); Pearson and Lipman, Methods in Mol.Biol.24:307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc.Acids Res.16:10881-90 (1988); Huang et al., Comp.Appl.BioSci.8:155-65 (1992); and Pearson et al., Meth.Mol.Biol.24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol.Biol.215:403-10(1990)) is accessible from the NBCI (National Center for Biological Information) and other sources, and can be used in conjunction with sequence analysis programs such as blastp, blastn, blastx, tblastn, and tblastx over the internet. BLAST can be accessed through the BLAST page on the NCBI website. Methods for comparing sequence homology using this program can be found on the BLAST help page on the NCBI website.

[0038] According to a specific embodiment of the present invention, the polypeptide forming the affibody for CD137 and the nucleotide sequence encoding it are included in the sequence catalog attached to this specification.

[0039] In one embodiment of the present invention, the nucleotide sequence includes one of the nucleotide sequences from SEQ ID NOs: 9 to SEQ ID NOs: 16.

[0040] According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule described above.

[0041] Specifically, the present invention provides a recombinant vector comprising a nucleic acid molecule, which comprises a nucleotide sequence encoding an affibody that specifically binds to CD137.

[0042] In one embodiment of the present invention, the recombinant vector is a vector for expressing an affibody that specifically binds to CD137, which contains the nucleic acid molecule described above.

[0043] In one embodiment of the present invention, the recombinant vector is a vector for expressing an affibody that specifically binds to the extracellular domain of CD137, which contains the nucleic acid molecule described above.

[0044] In one embodiment of the present invention, the recombinant vector is a vector for expressing an affibody that specifically binds to the extracellular domain of CD137, which includes the above-mentioned nucleic acid molecule and the amino acid sequence shown in SEQ ID NO: 27.

[0045] In one embodiment of the present invention, the recombinant vector is a vector for the expression of an affibody that specifically binds between amino acid residues 64 to 95 of the amino acid sequence shown in SEQ ID NO: 27, which includes the nucleic acid molecule described above.

[0046] In this specification, the term "vector" refers to a means for expressing a target gene in a host cell, and includes, but is not limited to, phagemide vectors; plasmid vectors; cosmid vectors; bacteriophage vectors; adenovirus vectors; retrovirus vectors; and viral vectors such as adeno-associated virus vectors.

[0047] In one embodiment of the present invention, the nucleic acid molecule encoding the affibody in the vector of the present invention is operatively linked to the promoter.

[0048] In this specification, the term “operatively linked” means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, thereby the regulatory sequence regulating the transcription and / or translation of the other nucleic acid sequence.

[0049] The recombinant vector system of the present invention may be constructed by various methods known in the art, specific methods of which are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is inserted herein by reference.

[0050] The vectors of the present invention may typically be constructed as vectors for cloning or vectors for expression. Furthermore, the vectors of the present invention may be constructed using prokaryotic or eukaryotic cells as hosts.

[0051] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, promoters derived from the genome of mammalian cells (e.g., metallothione promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Roussarcoma virus (RSV) promoter) may be used, and generally have a polyadenylated sequence as a transcription termination sequence.

[0052] The vector of the present invention may be fused with other sequences to facilitate the purification of the affibodies expressed therefrom. Examples of sequences to be fused include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0053] On the other hand, the expression vector of the present invention includes, as a selective label, antibiotic resistance genes commonly used in the industry, such as resistance genes to ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0054] Selectively, the vector can further deliver genes encoding reporter molecules (e.g., luciferase and glucuronidase).

[0055] According to one embodiment of the present invention, the expression vector is a recombinant vector for host cell expression in which a nucleic acid molecule encoding an affibody that specifically binds to human or cynomolgus monkey CD137 is inserted, and which is operatively linked to the nucleotide sequence of the nucleic acid molecule and includes a promoter that causes RNA molecule formation in the host cell and a poly(A) signal sequence that acts in the host cell to bring about polyadenylation of the 3' end of the RNA molecule.

[0056] Any host cell known in the art may be used to stably and continuously roning and express the vector of the present invention. Suitable eukaryotic host cells for the vector include, but are not limited to, yeast (Saccharomyces cerevisiae), insect cells, monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK-293 cells.

[0057] According to yet another aspect of the present invention, the present invention provides isolated host cells containing the recombinant vector described above.

[0058] In one embodiment of the present invention, the isolated host cells are transformed with the recombinant vector of the present invention.

[0059] In this specification, the terms “transformed,” “transduced,” or “transfected” refer to the process by which an exogenous nucleic acid is transmitted or introduced into a host cell. A “transformed,” “transduced,” or “transfected” cell is a cell that has been transformed, transduced, or transfected with an exogenous nucleic acid, and such cell includes the cell itself and its progeny cells obtained by subculturing.

[0060] The vector of the present invention can be delivered into host cells by microinjection (Capecchi, MR, Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, F. Let al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), ribosome-mediated phenotypic infection (Wong, T. Ket al., Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell Biol., 5:1188-1190 (1985)), and gene banbarment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)) when the host cell is a eukaryotic cell.

[0061] In the present invention, a recombinant vector injected into a host cell can express the above-mentioned affibody that has been recombinantly introduced into the host cell, and in such cases, a large amount of affibody can be obtained. For example, if the expression vector contains a galactose-inducible promoter, gene expression can be induced by treating the host cell with IPTG.

[0062] The culture is usually carried out under aerobic conditions, such as by shaking culture or rotational culture using a rotary machine. The culture temperature is preferably in the range of 10°C to 40°C, and the culture time is generally 5 hours to 7 days. The pH of the culture medium is preferably maintained in the range of 3.0 to 9.0 during culture. The pH of the culture medium can be adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. During culture, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be added if necessary to maintain and express the recombinant vector. When culturing host cells transformed with a recombinant expression vector having an induceable promoter, an appropriate inducer may be added to the culture medium if necessary. For example, if the expression vector contains a lac promoter or a lactose-inducible promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) may be added, and if it contains a trp promoter, indoleacrylic acid may be added to the culture medium.

[0063] According to yet another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising an affibody according to one embodiment and a pharmaceutically acceptable carrier.

[0064] The affibody mentioned above is a cell that expresses CD137, specifically an activated CD8 + Since it specifically binds to T cells, dendritic cells, NK cells, etc., the affibody acts as an agonist on CD137, activating T cells and causing NK cell ADCC (antibody-dependent cell-mediated cytotoxicity) or T-cell-mediated cytotoxicity, making it useful for the prevention or treatment of cancer.

[0065] Since the pharmaceutical compositions of the present invention utilize the affibody that specifically binds to CD137 as described above as an active ingredient, the common elements between these two are omitted from this specification to avoid excessive complexity.

[0066] In one embodiment of the present invention, the cancer is a hematological cancer or a solid tumor.

[0067] In one specific example of the present invention, the solid tumors include brain tumors, benign astrocytomas, malignant astrocytomas, pituitary adenomas, meningiomas, cerebral lymphomas, oligovascular gliomas, ependymal cell tumors, brainstem tumors, head and neck tumors, laryngeal cancers, oropharyngeal cancers, nasal cavity cancers, paranasal sinus cancers, nasopranosal sinus cancers, nasopharyngeal cancers, salivary gland cancers, hypopharyngeal cancers, thyroid cancers, oral cancers, chest tumors, small cell lung cancers, non-small cell lung cancers, thymic cancers, mediastinal tumors, One or more cancers selected from the group consisting of esophageal cancer, breast cancer, male breast cancer, abdominal tumors, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male reproductive organ tumors, penile cancer, prostate cancer, female reproductive organ tumors, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer, and skin cancer, but not limited to these.

[0068] In one specific example of the present invention, the hematological cancers include acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, polycythemia vera, essential thrombocytosis, myelofibrosis, mononuclear leukemia, erythroleukemia, megakaryoblastic leukemia, basophilic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma, B-cell lymphoma, marginal zone B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and diffuse large B-cell lymphoma. One or more of the following are selected from the group consisting of extranodal marginal zone lymphoma, extranodal NK / T cell lymphoma, peripheral T cell lymphoma, bucket lymphoma, progenitor cell neoplasm, mantle cell lymphoma, vesicle lymphoma, immunodeficiency-associated lymphoproliferative disorder, nodular lymphocyte-predominant Hodgkin's disease, tuberous sclerosis-type classical Hodgkin's disease, lymphocyte-rich classical Hodgkin's disease, mixed-cell type classical Hodgkin's disease, lymphopenic classical Hodgkin's disease, and chemotherapy-resistant pilocytic cell leukemia, but are not necessarily limited to these.

[0069] The pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0070] The pharmaceutical composition of the present invention can be administered orally or parenterally, for example, by intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intrasternal injection, local administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0071] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity, and a skilled, ordinary physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. In this specification, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat the diseases described above.

[0072] In this specification, the term “prevention” means the treatment of a disease or disease condition that prevents or protects against the disease. In this specification, the term “treatment” means the reduction, suppression, sedation, or eradication of a disease condition.

[0073] The pharmaceutical compositions of the present invention may be manufactured in unit dose form or encapsulated in multi-dose containers by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further contain a dispersant or stabilizer.

[0074] According to one aspect of the present invention, the present invention provides a protein complex comprising: an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment; and an antibody or antigen-binding fragment thereof that specifically binds to a protein on the surface of a cancer cell.

[0075] In this specification, the term "protein complex" means a group of at least two related polypeptide chains.

[0076] In this specification, the term "protein on the surface of cancer cells" means a protein that is embedded in or spans the cell membrane layer. The protein on the surface of cancer cells may be, but is not limited to, an integral membrane protein, a transmembrane protein, a lipid-anchored membrane protein, a peripheral membrane protein, an antigen, a proteoglycan, or a mucin that is present on or expressed on the surface of cancer cells.

[0077] The protein on the surface of the cancer cell may be a protein that is overexpressed compared to normal cells. The protein on the surface of the cancer cell may be a wild-type protein that has undergone a mutation. The protein on the surface of the cancer cell may be a cancer cell-specific protein or a tumor marker. The tumor marker may be any protein that is present in or produced from cancer cells or other cells as a result of a response to cancer or a specific noncancerous benign condition.

[0078] In one embodiment of the present invention, the protein complex is in a polymeric form in which monomers of the affibody according to the embodiment and monomers of antibodies or antigen-binding fragments thereof that specifically bind to proteins on the surface of cancer cells are linked together.

[0079] In one embodiment of the present invention, the protein complex may be in a multimer form in which the affibody and each antibody or antigen-binding fragment thereof are covalently linked to one another.

[0080] In one embodiment of the present invention, the protein complex may be in a polymeric form in which the affibody and each antibody or antigen-binding fragment thereof are linked to each other by amino acid linkers.

[0081] In one specific example of the present invention, the protein complex may be embodied in the form of a fused protein or a conjugate.

[0082] Therefore, the affibody and the antibody or its antigen-binding fragment may be linked by chemical conjugation (known as an organic chemical method) or by other means (e.g., expressing the complex as a fusion protein, directly or indirectly by a linker (e.g., an amino acid linker)).

[0083] According to a specific embodiment of the present invention, each monomer constituting the protein complex is linked by at least one linker. In this case, the linker may consist of an amino acid sequence represented by the general formula (GnSm)p or (SmGn)p:

[0084] Here, n, m, and p are independent of each other.

[0085] n is an integer between 1 and 7;

[0086] m is an integer between 0 and 7;

[0087] The sum of n and m is an integer less than or equal to 8; and

[0088] p is an integer between 1 and 7.

[0089] In another specific embodiment of the present invention, the linker is n=1 to 5 and m=0 to 5. In a more specific embodiment, n=4 and m=1. In yet another specific embodiment, the linker is (GGGGS)3. In yet another embodiment, the linker is GGGGS. In yet another specific embodiment, the linker is VDGS. In yet another specific embodiment, the linker is ASGS.

[0090] In one embodiment of the present invention, the protein complex may be a multi-antibody having two or more targets.

[0091] In one embodiment of the present invention, the protein complex may be a monoclonal antibody, a multiclonal antibody, scFv, Fab, F(ab), F(ab)2, scFv-Fc, a minibody, a diabody, a triabody, a tetrabody, a bibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a chemobody, an optobody, or an antigen-binding fragment thereof.

[0092] In one specific embodiment of the present invention, the protein complex is a bispecific antibody. In another specific embodiment of the present invention, the protein complex is a tetravalent antibody.

[0093] In a specific embodiment of the present invention, the protein complex is a trispecific antibody.

[0094] In a specific embodiment of the present invention, the protein complex is a T-cell engaging bispecific antibody.

[0095] In another specific embodiment of the present invention, the protein complex is a monoclonal bispecific antibody.

[0096] In yet another specific embodiment of the present invention, the protein complex is a multiple clonal bispecific antibody.

[0097] In one specific example of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to the protein on the surface of the cancer cell may be a monoclonal antibody, a multiclonal antibody, scFv, Fab, F(ab), F(ab)2, scFv-Fc, a minibody, a diabody, a triabody, a tetrabody, a bispecific antibody, a triplicate specific antibody, a multispecific antibody, a bivalent antibody, a tetravalent antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof.

[0098] In one embodiment of the present invention, the affibody fuses to the heavy chain or light chain of the antibody or its antigen-binding fragment.

[0099] In one specific example of the present invention, the affibody is fused to the C-terminus of the heavy or light chain of the antibody or its antigen-binding fragment.

[0100] In another specific example of the present invention, the affibody is fused to the N-terminus of the heavy or light chain of the antibody or its antigen-binding fragment.

[0101] In yet another embodiment of the present invention, the affibody is fused to the C-terminus and N-terminus of the heavy chain or light chain of the antibody or its antigen-binding fragment.

[0102] In one embodiment of the present invention, the protein on the surface of the cancer cell is EGFR (epidermal growth factor receptor), HER2 (human epidermal growth factor receptor 2), or CD19 (cluster of differentiation 19).

[0103] In one specific example of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to EGFR includes the following:

[0104] (a) cetuximab; or

[0105] (b) Heavy chain variable region including CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 19; and light chain variable region including CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 22.

[0106] The aforementioned cetuximab is a commercially available antibody that specifically binds to human EGFR, and it will be understood by those skilled in the art that any antibody or antigen-binding fragment known in the art that specifically binds to human EGFR may be used without restriction.

[0107] In one specific example of the present invention, the protein complex comprises an affibody that specifically binds to the extracellular domain of CD137 according to one example, and cetuximab.

[0108] More specifically, a protein complex according to one specific example of the present invention comprises an affibody and cetuximab that specifically bind to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8.

[0109] In another specific embodiment of the present invention, the protein complex is an affibody that specifically binds to the extracellular domain of CD137; and

[0110] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 19; and a light chain variable region including CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 22.

[0111] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0112] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region including CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 19; and a light chain variable region including CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 22.

[0113] In one specific example of the present invention, (b) includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23; and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24.

[0114] In one specific example of the present invention, the protein complex is an affibody that specifically binds to the extracellular domain of CD137 according to one example; and

[0115] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24.

[0116] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0117] The antibody or its antigen-binding fragment comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 24.

[0118] In one specific example of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to HER2 includes the following:

[0119] (c) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28; and light chain variable region containing the amino acid sequence of SEQ ID NO: 29;

[0120] (d) Trastuzumab; or

[0121] (e) Heavy chain variable region including CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 32; and light chain variable region including CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 35.

[0122] The aforementioned trastuzumab is a commercially available antibody that specifically binds to human HER2, and it will be understood by those skilled in the art that any antibody or antigen-binding fragment known in the art that specifically binds to human HER2, or any such antibody, may be used without restriction.

[0123] In one specific example of the present invention, the protein complex is an affibody that specifically binds to the extracellular domain of CD137; and

[0124] (c) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 29.

[0125] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0126] (c) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 29.

[0127] In one specific example of the present invention, the protein complex is an affibody that specifically binds to the extracellular domain of CD137; and

[0128] (d) Contains trastuzumab.

[0129] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0130] (d) Contains trastuzumab.

[0131] In one specific example of the present invention, the protein complex is an affibody that specifically binds to the extracellular domain of CD137; and

[0132] (e) A heavy chain variable region comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 32; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 35.

[0133] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0134] (e) A heavy chain variable region comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 32; and an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 35.

[0135] In one specific example of the present invention, (e) includes a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 36; and a light chain variable region containing the amino acid sequence of SEQ ID NO: 37.

[0136] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0137] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 36, and a light chain variable region containing the amino acid sequence of SEQ ID NO: 37.

[0138] In one specific example of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to CD19 includes the following:

[0139] (f) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38; and light chain variable region containing the amino acid sequence of SEQ ID NO: 39; or

[0140] (g)FMC63

[0141] FMC63 is a commercially available antibody that specifically binds to human CD19, and it will be understood by those skilled in the art that any antibody or antigen-binding fragment thereof that specifically binds to human CD19 and is known in the art may be used without restriction.

[0142] In one specific example of the present invention, the protein complex includes an affibody that specifically binds to the extracellular domain of CD137; and

[0143] (f) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39.

[0144] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0145] (f) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39.

[0146] In one specific example of the present invention, the protein complex includes an affibody that specifically binds to the extracellular domain of CD137; and

[0147] (g) Includes FMC63

[0148] More specifically, a protein complex according to one specific example of the present invention includes an affibody that specifically binds to the extracellular domain of CD137, which contains an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0149] (g) Includes FMC63

[0150] According to another aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a protein complex according to one embodiment.

[0151] In one embodiment of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex that includes an affibody that specifically binds to the extracellular domain of CD137 and an antibody or antigen-binding fragment thereof that specifically binds to a protein on the surface of a cancer cell.

[0152] In one specific example of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex that includes an affibody that specifically binds to the extracellular domain of CD137 and an antibody or antigen-binding fragment thereof that specifically binds to EGFR.

[0153] In another specific example of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex that includes an affibody that specifically binds to the extracellular domain of CD137 and an antibody or antigen-binding fragment thereof that specifically binds to HER2.

[0154] In yet another embodiment of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex that includes an affibody that specifically binds to the extracellular domain of CD137 and an antibody or antigen-binding fragment thereof that specifically binds to CD19.

[0155] It will be obvious to those skilled in the art that the nucleotide sequence encoding the protein complex of the present invention may be any nucleotide sequence encoding the amino acid sequence constituting the protein complex, and is not limited to any specific nucleotide sequence.

[0156] According to another aspect of the present invention, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the protein complex described above.

[0157] According to one embodiment of the present invention, in the vector of the present invention, an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment and a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to a protein on the surface of cancer cells are operatively linked to the promoter.

[0158] According to one specific example of the present invention, in the vector of the present invention, an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment, and a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds to EGFR, HER2, or CD19, are operatively bound to the promoter.

[0159] According to one specific example of the present invention, in the vector of the present invention, the nucleic acid molecule encoding the heavy chain variable region and the nucleic acid molecule encoding the light chain variable region of the antibody are operatively bound to the promoter.

[0160] According to another specific example of the present invention, in the vector of the present invention, the affibody and the nucleic acid molecule encoding cetuximab that specifically bind to the extracellular domain of CD137 according to one embodiment are operatively bound to the promoter.

[0161] According to yet another specific example of the present invention, in the vector of the present invention, an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment; and

[0162] Nucleic acid molecules encoding the heavy chain variable region, which includes CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 19; and the light chain variable region, which includes CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 22, are operatively bound to the promoter.

[0163] According to yet another specific example of the present invention, in the vector of the present invention, an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment; and

[0164] The nucleic acid molecules encoding the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 24 are operatively bound to the promoter.

[0165] According to one specific example of the present invention, the vector of the present invention comprises an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment; and

[0166] A single-clonal antibody that specifically binds to HER2, or a nucleic acid molecule encoding its antigen-binding fragment, is operatively bound to the promoter.

[0167] According to yet another specific example of the present invention, in the vector of the present invention, an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment; and

[0168] (c) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28; and light chain variable region containing the amino acid sequence of SEQ ID NO: 29;

[0169] (d) Trastuzumab; or

[0170] (e) Nucleic acid molecules encoding the heavy chain variable region, including CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 32; and the light chain variable region, including CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 35, are operatively bound to the promoter.

[0171] According to yet another specific example of the present invention, in the vector of the present invention, an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment; and

[0172] The nucleic acid molecules encoding the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 36 and the light chain variable region containing the amino acid sequence of SEQ ID NO: 37 are operatively bound to the promoter.

[0173] According to another specific example of the present invention, in the vector of the present invention, an affibody specifically binds to the extracellular domain of CD137 according to one embodiment; and

[0174] A single clone antibody that specifically binds to CD19, or a nucleic acid molecule encoding its antigen-binding fragment, is operatively bound to the promoter.

[0175] According to yet another specific example of the present invention, in the vector of the present invention, an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment; and

[0176] (f) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38; and light chain variable region containing the amino acid sequence of SEQ ID NO: 39; or

[0177] (g) The nucleic acid molecule encoding FMC63 is operatively bound to the promoter.

[0178] In this specification, the term “operatively linked” means a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, signal sequence, or array of transcription factor binding sites) and another nucleic acid sequence, thereby regulating the transcription and / or reading of the other nucleic acid sequence.

[0179] The recombinant vector system of the present invention may be constructed by various methods known in the art, specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0180] The vectors of the present invention may typically be constructed as vectors for cloning or vectors for expression. Furthermore, the vectors of the present invention may be constructed using prokaryotic or eukaryotic cells as hosts.

[0181] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, promoters derived from the genome of mammalian cells (e.g., metallothione promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Roussarcoma virus (RSV) promoter) may be used, and generally have a polyadenylated sequence as a transcription termination sequence.

[0182] The vector of the present invention may be fused with other sequences to facilitate the purification of the antibody expressed therefrom. Examples of sequences that can be fused include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0183] Furthermore, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified using a protein A column or the like without the need for further sequencing for purification.

[0184] On the other hand, the expression vector of the present invention includes, as a selective label, antibiotic resistance genes commonly used in the industry, such as resistance genes to ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0185] Selectively, the vector can further deliver a gene encoding a reporter molecule (e.g., luciferase and glucuronidase).

[0186] According to one embodiment of the present invention, the expression vector is a recombinant vector for host cell expression in which a nucleic acid molecule encoding an antibody against EGFR, HER2, or CD19 or an antigen-binding fragment thereof is inserted, and which is operatively linked to the nucleotide sequence of the nucleic acid molecule, and which includes a promoter that causes the formation of an RNA molecule in the host cell and a poly(A) signal sequence that acts in the host cell to cause polyadenylation of the 3' end of the RNA molecule.

[0187] According to one aspect of the present invention, the present invention provides isolated host cells containing the recombinant vector described above.

[0188] In one embodiment of the present invention, the isolated host cells were transformed with the recombinant vector of the present invention.

[0189] Any host cell known in the art may be used to stably and continuously clone and express the vector of the present invention. Suitable eukaryotic host cells for the vector include, but are not limited to, yeast (Saccharomyce cerevisiae), insect cells, monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT78 cells, and HEK-293 cells.

[0190] In one embodiment of the present invention, the host cells are cynomolgus monkey-derived fibroblasts (CYNOM-K1, Sigma-Aldrich) and rhesus monkey-derived kidney cells (ATCC, LLC-MK2 Original CCL-7). TM , or ATCC,FRhK-4,CRL-1688 TM ), or rhesus monkey-derived lymphoblasts (ATCC, LCL8664, CRL-1805 TM ) is acceptable.

[0191] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising a protein complex according to one embodiment and a pharmaceutically acceptable carrier.

[0192] Since the pharmaceutical compositions of the present invention utilize the aforementioned protein complex as an active ingredient, details common to both are omitted from this specification to avoid excessive complexity.

[0193] In one embodiment of the present invention, the cancer is a hematological cancer or a solid tumor.

[0194] In one specific example of the present invention, the solid tumors include brain tumors, benign astrocytomas, malignant astrocytomas, pituitary adenomas, meningiomas, cerebral lymphomas, oligovascular gliomas, ependymal cell tumors, brainstem tumors, head and neck tumors, laryngeal cancers, oropharyngeal cancers, nasal cavity cancers, paranasal sinus cancers, nasopranosal sinus cancers, nasopharyngeal cancers, salivary gland cancers, hypopharyngeal cancers, thyroid cancers, oral cancers, chest tumors, small cell lung cancers, non-small cell lung cancers, thymic cancers, mediastinal tumors, One or more cancers selected from the group consisting of esophageal cancer, breast cancer, male breast cancer, abdominal tumors, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male reproductive organ tumors, penile cancer, prostate cancer, female reproductive organ tumors, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, female urethral cancer, and skin cancer, but not limited to these.

[0195] In one specific example of the present invention, the hematological cancers include acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, polycythemia vera, essential thrombocytosis, myelofibrosis, mononuclear leukemia, erythroleukemia, megakaryoblastic leukemia, basophilic leukemia, eosinophilic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma, B-cell lymphoma, marginal zone B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, and diffuse large B-cell lymphoma. One or more of the following are selected from the group consisting of extranodal marginal zone lymphoma, extranodal NK / T cell lymphoma, peripheral T cell lymphoma, bucket lymphoma, progenitor cell neoplasm, mantle cell lymphoma, vesicle lymphoma, immunodeficiency-associated lymphoproliferative disorder, nodular lymphocyte-predominant Hodgkin's disease, tuberous sclerosis-type classical Hodgkin's disease, lymphocyte-rich classical Hodgkin's disease, mixed-cell type classical Hodgkin's disease, lymphopenic classical Hodgkin's disease, and chemotherapy-resistant pilocytic cell leukemia, but are not necessarily limited to these.

[0196] In one embodiment of the present invention, the protein complex induces the activation of T cells.

[0197] Specifically, the protein complex of the present invention specifically binds to proteins on the surface of cancer cells and simultaneously specifically binds to CD137 on T cells. Therefore, the protein complex acts as a T cell agonist to induce T cell activation, thereby reducing the number of cancer cells or suppressing their increase, making it useful for the prevention or treatment of cancer. In other words, the protein complex of the present invention induces T cell activation in a protein-dependent manner on the surface of cancer cells.

[0198] More specifically, the protein complex of the present invention specifically binds to EGFR on the surface of cancer cells and simultaneously specifically binds to CD137 on T cells. Therefore, the protein complex acts as a T cell agonist to induce T cell activation, thereby reducing the number of cancer cells expressing EGFR or suppressing the increase in the number of EGFR-expressing cancer cells, and can be usefully used for the prevention or treatment of cancer. In other words, the protein complex of the present invention induces T cell activation in an EGFR-dependent manner.

[0199] The aforementioned cancer may be EGFR-positive.

[0200] The aforementioned cancer is characterized by overexpression of EGFR.

[0201] The aforementioned cancers include, but are not limited to, non-small cell lung cancer (NSCLC), lung cancer, colorectal cancer, brain tumor, astrocytoma, esophageal cancer, cervical cancer, or synovial sarcoma.

[0202] The aforementioned cancer may be triple-negative breast cancer (TNBC). TNBC is known to overexpress EGFR. TNBC refers to breast cancer in which the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal receptor 2 (Her2neu) is less than 1%.

[0203] More specifically, the protein complex of the present invention specifically binds to HER2 on the surface of cancer cells and simultaneously specifically binds to CD137 on T cells. Therefore, the protein complex acts as a T cell agonist to induce T cell activation, thereby reducing the number of cancer cells expressing HER2 or suppressing the increase in the number of HER2-expressing cancer cells, and can be usefully used for the prevention or treatment of cancer. In other words, the biantibody of the present invention induces T cell activation in a HER2-dependent manner.

[0204] The aforementioned cancer may be HER2-positive.

[0205] The aforementioned cancer is characterized by overexpression of HER2.

[0206] The cancer may be invasive breast cancer or metastatic breast cancer. The cancer may be, but is not limited to, breast cancer, bladder cancer, pancreatic cancer, ovarian cancer, and gastric cancer.

[0207] More specifically, the protein complex of the present invention specifically binds to CD19 on the surface of cancer cells and simultaneously specifically binds to CD137 on T cells. Therefore, the protein complex acts as a T cell agonist to induce T cell activation, thereby reducing the number of cancer cells expressing CD19 or suppressing the increase in the number of cancer cells expressing CD19, and can be usefully used for the prevention or treatment of cancer. In other words, the protein complex of the present invention induces T cell activation in a CD19-dependent manner.

[0208] The aforementioned cancer may be CD19 positive.

[0209] The aforementioned cancer is characterized by overexpression of CD19.

[0210] The cancer may be a lymphoma. The lymphoma may be a B-cell lymphoma. The cancer may be, but is not limited to, marginal lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloid leukemia, or chemotherapy-resistant pilocytic cell leukemia.

[0211] In one embodiment of the present invention, activation of the T cells increases interferon-gamma (IFN-γ).

[0212] In another embodiment of the present invention, the pharmaceutical composition of the present invention increases interferon-gamma in a cancer cell surface protein-dependent manner.

[0213] In one specific example of the present invention, the pharmaceutical composition of the present invention increases interferon-gamma in an EGFR protein-dependent manner. That is, when the composition comes into contact with cancer cells that are EGFR-positive or overexpress EGFR, it activates T cells and increases the concentration of interferon-gamma.

[0214] In another specific example of the present invention, the pharmaceutical composition of the present invention increases interferon-gamma in a HER2 protein-dependent manner. That is, when the composition comes into contact with cancer cells that are HER2-positive or overexpress HER2, it activates T cells and increases the concentration of interferon-gamma.

[0215] In yet another specific example of the present invention, the pharmaceutical composition of the present invention increases interferon-gamma in a CD19 protein-dependent manner. That is, when the composition comes into contact with cancer cells that are CD19-positive or overexpress CD19, it activates T cells and increases the concentration of interferon-gamma.

[0216] In another embodiment of the present invention, the pharmaceutical composition of the present invention induces T cell activation in a cancer cell surface protein-dependent manner.

[0217] In one specific example of the present invention, the pharmaceutical composition of the present invention induces T cell activation in an EGFR protein-dependent manner. That is, the composition induces T cell activation when it comes into contact with cancer cells that are EGFR-positive or that overexpress EGFR.

[0218] In another specific example of the present invention, the pharmaceutical composition of the present invention induces T cell activation in a HER2 protein-dependent manner. That is, the composition induces T cell activation when it comes into contact with cancer cells that are HER2-positive or that overexpress HER2.

[0219] In yet another specific example of the present invention, the pharmaceutical composition of the present invention induces T cell activation in a CD19 protein-dependent manner. That is, the composition induces T cell activation when it comes into contact with cancer cells that are CD19-positive or overexpress CD19.

[0220] The pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to these components, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0221] The pharmaceutical composition of the present invention can be administered orally or parenterally, for example, by intravenous injection, subcutaneous injection, intramuscular injection, peritoneal injection, intrasternal injection, local administration, intranasal administration, intrapulmonary administration, and intrarectal administration.

[0222] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity, and a skilled, ordinary physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. In this specification, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat the diseases described above.

[0223] In this specification, the term "prevention" means the treatment of a disease or disease condition that prevents or protects it. In this specification, the term "treatment" means the reduction, suppression, sedation, or eradication of a disease condition.

[0224] The pharmaceutical compositions of the present invention may be manufactured in unit dose form or encapsulated in multi-dose containers by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further contain a dispersant or stabilizer.

[0225] [Effects of the invention] The features and advantages of the present invention can be summarized as follows:

[0226] This invention provides an affibody that specifically binds to the extracellular domain of CD137.

[0227] The present invention provides an affibody that specifically binds to the extracellular domain of CD137; and a protein complex comprising an antibody or an antigen-binding fragment thereof that specifically binds to a protein on the surface of cancer cells.

[0228] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the above-mentioned affibody or protein complex and a pharmaceutically acceptable carrier.

[0229] The present invention provides an affibody that specifically binds to CD137, enabling the development of a protein complex that specifically binds to T cells and simultaneously to antigens on the surface of various cancer cells, which can be usefully used for the prevention or treatment of hematological cancers and solid tumors.

[0230] [Brief explanation of the drawing] Figure 1 shows the results of identifying affibodies that bind to the CD137 protein in the form of a periplasmic extract.

[0231] Figure 2 shows the results of identifying CD137-binding affibodies expressed on CD137 using activated CEMT cells, which are CD137-positive cells, based on the morphology of the antibody-affibodies that bind to CD137.

[0232] Figure 3 shows the results of testing whether a biantibody composed of CD137 affibodies was produced based on two types of EGFR-binding antibodies (CET or 15E3) and whether the biantibody bound to the CD137 protein and the EGFR protein.

[0233] Figure 3A shows confirmation that a biantibody composed of a CD137 affibody based on cetuximab (CET) antibody binds to the EGFR protein.

[0234] Figure 3B shows confirmation that a biantibody composed of a cetuximab (CET) antibody-based CD137 affibody binds to the CD137 protein.

[0235] Figure 3C shows confirmation that a biantibody composed of a 15E3 antibody-based CD137 affibody binds to the EGFR protein.

[0236] Figure 3D shows confirmation that a biantibody composed of a 15E3 antibody-based CD137 affibody binds to the CD137 protein.

[0237] Figure 4A shows that a biantibody composed of a cetuximab (CET) antibody-based CD137 affibody binds to activated CEMT cells expressing CD137.

[0238] Figure 4B shows that a biantibody composed of a 15E3 antibody-based CD137 affibody binds to activated CEMT cells expressing CD137.

[0239] Figure 5A shows that a biantibody composed of a cetuximab (CET) antibody-based CD137 affibody binds to HT29 cells that express EGFR.

[0240] Figure 5B shows that a biantibody composed of a 15E3 antibody-based CD137 affibody binds to HT29 cells expressing EGFR.

[0241] Figure 6 shows the results of EGFR-dependent T cell activation, confirmed by IFN-gamma secretion analysis, for the selection of CD137 affibodies.

[0242] Figure 7 shows which extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to.

[0243] Figure 7A is a schematic diagram of hCD137-ECD, hCD137-ECD-ΔD1, hCD137-ECD-ΔD12, hCD137-ECD-ΔD123p, hCD137-ECD-ΔD123, hCD137-ECD-ΔD3, and hCD137-ECD-ΔD5, which are composed of a signal peptide and a combination of different ECD domains 1 to 5. The amino acid sequences for human CD137 ECD composed of domains 1 to 5 are shown in SEQ ID NO: 27.

[0244] Figure 7B shows confirmation that urelumab binds to domain 1 of the CD137 ECD.

[0245] Figure 7C shows that utomilumab binds to domain 3 of the CD137 ECD.

[0246] Figure 7D shows that cetuximab (Negative Ab) does not bind to any domain of the CD137 ECD.

[0247] Figure 7E shows confirmation that the affibody ZAAD01 binds to domain 3 of CD137 ECD.

[0248] Figure 7F shows confirmation that the affibody ZAAD05 binds to domain 3 of CD137 ECD.

[0249] Figure 8 shows the cross-reactivity observed between two selected CD137 affibodies and other proteins belonging to the TNFR superfamily: CD27, CD30, CD134, CD270, CD357, and TNF-α.

[0250] Figure 8A shows that the affibody ZAAD01 specifically binds only to CD137.

[0251] Figure 8B shows confirmation that the affibody ZAA0D5 specifically binds only to CD137.

[0252] Figure 9 shows the interspecies cross-reactivity between two selected CD137 affibodies and the CD137 protein in humans, mice, and cynomolgus monkeys.

[0253] Figure 9A shows the confirmation that the affibody ZAAD01 binds to the CD137 protein in humans and cynomolgus monkeys.

[0254] Figure 9B shows the confirmation that the affibody ZAAD05 binds to the CD137 protein in humans and cynomolgus monkeys.

[0255] Figure 10 shows the results of BioLayer Interferometry (BLI) confirming that four types of biantibodies in antibody-affibody form simultaneously bind to both the CD137 antigen and the EGFR antigen targets. Figure 10A shows the BLI results for CET-ZAAD01 biantibody, Figure 10B for CET-ZAAD05 biantibody, Figure 10C for 15E3-ZAAD01 biantibody, and Figure 10D for 15E3-ZAAD05 biantibody.

[0256] Figure 11 shows the cell-specific expression levels of EGFR-expressing cells, confirmed by flow cytometry, and demonstrates a correlation between the degree of T cell activation by the biantibody and the EGFR expression level.

[0257] Figure 11A shows the results of EGFR expression in EGFR-positive cells (A431, HT29, and SW403) and EGFR-negative cells (MOLT4).

[0258] Figure 11B shows the EGFR-dependent T cell activation by the 15E3-ZAAD01 biantibody, as confirmed by IFN-gamma secretion analysis based on EGFR expression levels.

[0259] Figure 12 shows the confirmation that biantibodies produced using a CD137 affibody and either a HER2 antibody or a CD19 antibody bind to CD137 and HER2 or CD19, respectively.

[0260] Figure 12A shows the confirmation that trastuzumab-ZAAD01 biantibody (anti-HER2 Ab1_ZAAD01) and hz1E11.10-ZAAD01 biantibody (anti-HER2 Ab2_ZAAD01) bind to CD137.

[0261] Figure 12B shows the confirmation that trastuzumab-ZAAD01 biantibody and hz1E11.10-ZAAD01 biantibody bind to HER2.

[0262] Figure 12C shows confirmation that the FMC63-ZAAD01 biantibody (anti-CD19 Ab_ZAAD01) binds to CD137.

[0263] Figure 12D shows confirmation that the FMC63-ZAAD01 biantibody binds to CD19.

[0264] Figure 13 shows the results of IFN-gamma secretion analysis confirming that biantibodies produced using a CD137 affibody and a HER2 antibody or CD19 antibody activate T cells in a HER2 or CD19-dependent manner.

[0265] Figure 13A shows that trastuzumab-ZAAD01 biantibody and hz1E11.10-ZAAD01 biantibody activated T cells in a HER2-dependent manner compared to utomirumab, as confirmed by IFN-gamma secretion analysis.

[0266] Figure 13B shows that IFN-gamma secretion analysis confirmed that the FMC63-ZAAD01 biantibody activated T cells in an EGFR-dependent manner compared to utomirumab.

[0267] Figure 14A shows that DLD1-luc cells are killed by the activation of immune cells by the biantibody (CET_ZAAD01 and CET_ZAAD05).

[0268] Figure 14B shows that the activation of immune cells by the biantibodies (CET_ZAAD01 and CET_ZAAD05) increases the secretion of IFN-gamma.

[0269] Figure 15 shows the extended survival time due to the anticancer effect of the biantibody (CET_ZAAD01 and CET_ZAAD05) in a humanized CD137 model.

[0270] Figure 16A shows the tumor size in the vehicle-treated group in the humanized CD137 model.

[0271] Figure 16B is a diagram showing the tumor sizes of the cetuximab-treated group in the humanized CD137 model.

[0272] Figure 16C is a diagram showing the tumor sizes of the bispecific antibody (CET_ZAAD01)-treated group in the humanized CD137 model.

[0273] Figure 16D is a diagram showing the tumor sizes of the bispecific antibody (CET_ZAAD05)-treated group in the humanized CD137 model.

[0274] [Mode for Carrying Out the Invention] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are merely for explaining the present invention more specifically, and it will be apparent to those having ordinary knowledge in the art that the scope of the present invention is not limited to these examples by the gist of the present invention.

[0275] Examples Throughout this specification, “%” used to indicate the concentration of a specific substance is (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise specified.

[0276] Example 1. Development of Affibody Against CD137

[0277] 1-1: Screening of 10 Affibody Clones Specifically Binding to Human CD137

[0278] Using CD137 protein, clones specifically binding to CD137 were selected from an affibody (Zb) library by panning. The selected clones were produced using animal cells as bispecific antibodies in the form of AffiMab (a bispecific antibody composed of a single-clone antibody-based affibody), and clones binding to CD137-expressing cells were selected.

[0279] Panning-Affinity Maturation

[0280] The affibody library was rescued in phage form using the VSCM13 helper phage and used for panning. The initial number of library phages bound to the antigen was 10. 13 The sample size was greater than 1, and four panning cycles were performed. As the number of panning cycles increased, the amount of antigen (10 μg, 5 μg, 2 μg, and 1 μg) was progressively reduced, while the number of washes (3, 5, 7, and 10) was progressively increased. This strategy was used to selectively select phages with high affinity. Binder phages obtained from each panning cycle were used to infect E. coli ER2537 (New England Biolabs® Inc.), and the resulting colonies were examined for antigen binding using the ELISA method.

[0281] Periplasm extraction - Affibody separation

[0282] Colonies obtained by infecting with binder phages were inoculated into SB medium (MOPS 10g / L, Bact yeast extract 20g / L, and tryptone 30g / L), and then OD 600 After culturing until the optical density at 600 nm reached 0.8, 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside, LPS Solution Co., Ltd.) was added and the culture was shaken at 30°C to induce overexpression of the affibody (Zb). Periplasmic extraction (PE) of the affibody was performed using BBS buffer (200 mM boric acid, 150 mM NaCl, and 1 mM EDTA). The binder was then screened using the ELISA method.

[0283] ELISA method - Binder screening

[0284] For ELISA, the periplasmic extract of the affibody was treated on a plate coated with hCD137-ECD-Fc (human CD137-extracellular domain-fragment crystallizable region) protein at a concentration of 2 μg / mL. After treatment with the secondary antibody (anti-HA-HRP, Roche, 12013819001, human influenza hemagglutinin (HA) protein), a color reaction was carried out with TMB (3,3’,5,5’-tetramethylbenzidine, BioFX TM , TMBC-1000-01), and the OD 450 value was measured using an ELISA reader (Victor X3, PerkinElmer, Inc.), and the results are shown in Figure 1. For the control, plates coated with mouse CD137 ECD Fc protein (mouse CD137) or no protein at all (Negative protein) were also used. Negative PE is the extract of Escherichia coli ER2537 without the affibody plasmid.

[0285] ELISA Results - Selection of 10 Affibody Clones

[0286] As shown in Figure 1, for negative PE, almost no OD 450 value was measured, and for mouse CD137 protein, almost no OD 450 value was measured either. None of the 10 affibodies bound to these, but it was confirmed that all 10 affibodies bound to human CD137 protein due to the high OD 450 value.

[0287] From the results of the above ELISA, 10 affibodies, ZAAD01 to ZAAD10, that bind to human CD137 protein were selected. As a result of confirming the sequences of the affibodies, 10 unique clones (unique clone, specific cell lines that have been cloned) that bind to human CD137 were confirmed.

[0288] 1-2: Selection of 8 types of affiliate bodies

[0289] The ten unique clones mentioned above were cloned in the Zb-Fc form and produced using animal cells. Subsequently, cell fusion was confirmed using CD137-expressing cells. Fc stands for Fragment crystallizable region.

[0290] T cell activation

[0291] CEMT cells (ATCC(R), CCL-119TM, T lymphoblast, acute lymphocytic leukemia) 1×10 7 A 10 mL sample was prepared and treated with PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich®, P1585) at a concentration of 50 ng / mL and ionomycin (ionomycin, Sigma-Aldrich®, I9657) at a concentration of 1 μg / mL. The sample was then incubated for 16 hours to activate it.

[0292] PMA and ionomycin are known to activate T cells. PMA activates protein kinase C, and ionomycin is a calcium ionophore. Both compounds pass through the T cell membrane receptor complex, inducing the activation of various intracellular signaling pathways and thereby activating T cells, which in turn induces the production of various cytokines (see Ai, W., et al. (2013). Int J Environ Res Public Health. 2013;10(9):3834-3842.). Ionomycin, together with PMA, is known to stimulate the intracellular production of cytokines, interferon, perforin, IL-2, and IL-4. Furthermore, it is known that unstimulated naive T cells do not express CD137, while stimulated activated T cells temporarily express CD137 at high levels.

[0293] 5 × 10⁶ activated CEMT cells 5 After preparation in tubes, cells were collected by centrifugation at 1,200 rpm for 3 minutes, washed with 5% FBS in PBS, treated with 5 μg / mL of the 10 Zb-Fc compounds, and incubated on ice for 1 hour. The cells were washed three times by centrifugation at 1,200 rpm for 3 minutes using 200 μL of 5% FBS in PBS. Inactivated CEMT cells were also prepared as a control.

[0294] Thereafter, the cells were treated with anti-human-Fc-FITC (anti-human-Fc-FITC, Life Technologies®, A11013) at 1 μg / mL and incubated on ice for 45 minutes with light blocked. The cells were washed three times by centrifuging at 1,200 rpm for 3 minutes using 5% FBS in 200 μL of PBS, and then the fluorescence intensity was measured using a FACS (Fluorescence-activated cell sorting) instrument from Beckman Coulter, Inc., and the results are shown in Figure 2. In the FACS result graph, the more the peak moves to the right, the stronger the binding to the cells. FITC only is the case where only the buffer is present when binding Zb-Fc, and Negative Ab is the case when hIgG (human IgG) is used.

[0295] As shown in Figure 2, as a result of FACS measurement, eight affibodies, ZAAD01, ZAAD02, ZAAD03, ZAAD04, ZAAD05, ZAAD07, ZAAD09, and ZAAD010, which specifically bind to CD137-expressing cells, were selected.

[0296] Specifically, the eight affibodies showed only a peak for FITC in non-activated CEMT cells, but showed a peak shifted to the right of the peak for FITC in activated CEMT cells. From this, it was confirmed that the eight affibodies do not bind to non-activated CEMT cells but bind to activated CEMT cells.

[0297] However, affibodies ZAAD06 and ZAAD08 did not show a peak difference between inactivated or activated CEMT cells compared to the control group (negative Ab) with hIgG added, and did not specifically bind to cells expressing CD137, that is, activated CEMT cells, so ZAAD06 and ZAAD08 were not selected.

[0298] Example 2. Affibody selection for CD137

[0299] 2-1: Production of 16 types of biantibodies

[0300] Based on cetuximab (CET), which specifically binds to EGFR (Epidermal Growth Factor Receptor), and two proprietary 15E3 antibodies (see Korean Patent Publication No. 10-2017-0012754), eight CD137 affibodies selected in Examples 1-2 above were cloned in the form of biantibodies, and a total of 16 biantibodies were produced using animal cells. When the eight affibodies mentioned above were cloned in the form of a biantibody with cetuximab (CET), they were referred to as CET-ZAAD01, CET-ZAAD02, CET-ZAAD03, CET-ZAAD04, CET-ZAAD05, CET-ZAAD07, CET-ZAAD09, and CET-ZAAD010, respectively. When they were cloned in the form of a biantibody with the 15E3 antibody, they were referred to as 15E3-ZAAD01, 15E3-ZAAD02, 15E3-ZAAD03, 15E3-ZAAD04, 15E3-ZAAD05, 15E3-ZAAD07, 15E3-ZAAD09, and 15E3-ZAAD010, respectively.

[0301] As disclosed in the aforementioned Korean Patent Publication No. 10-2017-0012754, the inventors have shown that both cetuximab and the 15E3 antibody bind to the EGFR domain 3, but that the two antibodies bind to different epitopes.

[0302] 2-2: Confirmation of biantibody binding to CD137 and EGFR proteins, respectively.

[0303] Using the purified biantibody form, CD137 affibodies that bind to both EGFR and CD137 proteins were selected by protein binding, cell binding, and T cell activation tests.

[0304] The binding of each CD137 to EGFR protein was confirmed using ELISA with the aforementioned 16 types of bivalent antibodies (8 types of Cetuximab-Zb and 8 types of 15E3-Zb) produced in animal cells. For the ELISA, plates coated with hCD137-ECD-his and hEGFR-ECD-his proteins at a concentration of 2 μg / mL were treated with the 16 purified bivalent antibodies at 7 points, starting at 60 nM and diluting by 1 / 5. After treatment with a secondary antibody (Goat anti-human IgG-F(ab')2-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097), TMB (BioFX) was used. TM A color reaction is generated using TMBC-1000-01, and an ELISA reader (Victor X3, PerkinElmer, Inc.) is used for OD 450 The values ​​were measured using GraphPad Prism software. 50 The (half maximal effective concentration) value was calculated, and the results are shown in Figures 3A to 3D.

[0305] Figures 3A and 3B show the binding test results for biantibodies composed of CD137 affibodies based on cetuximab (CET) antibody (hereinafter referred to as CET-based CD137 affibodies biantibodies, Cetuximab-Zb, or CET_ZAAD01~CET_ZAAD10), and Figures 3C and 3D show the binding test results for biantibodies composed of CD137 affibodies based on 15E3 antibody (hereinafter referred to as 15E3-based CD137 affibodies biantibodies, 15E3-Zb, or 15E3_ZAAD01~15E3_ZAAD10). Figures 3A and 3C show the results confirming binding to the EGFR protein, and Figures 3B and 3D show the results confirming binding to the CD137 protein.

[0306] From the above results, it was confirmed that all 16 types of biantibodies of the present invention, the CET-based CD137 affibody biantibody, and the 15E3-based CD137 affibody biantibody bind to both EGFR and CD137 proteins.

[0307] 2-3: Confirmation of biantibody binding in CD137-expressing cells

[0308] To confirm the binding of the 16 types of biantibody forms in CD137-expressing cells, CEMT cells were subjected to a 1 × 10⁶ experiment. 7 Prepare 10 mL of cells, treat with 50 ng / mL of PMA and 1 μg / mL of ionomycin, and then incubate for 16 hours to activate them. Activated CEMT cells were then divided into 5 × 10⁶ cells. 5 After preparation in tubes, cells were collected by centrifugation at 1,200 rpm for 3 minutes, washed with 5% FBS in PBS, treated with 5 μg / mL of biantibody, and incubated on ice for 1 hour. The cells were washed three times by centrifugation at 1,200 rpm for 3 minutes using 200 μL of 5% FBS in PBS. Subsequently, the cells were treated with 1 μg / mL of anti-human-Fc-FITC (Life Technologies®, A11013) and incubated on ice for 45 minutes while blocking light. The cells were washed three times by centrifugation at 1,200 rpm for 3 minutes using 200 μL of 5% FBS in PBS, and the fluorescence intensity was measured using a Beckman Coulter FACS instrument. The results are shown in Figures 4A and 4B. Utomilumab was used as the positive control group (Positive Ab), and human IgG (hIgG) was used as the negative control group.

[0309] Figure 4A shows the FACS results for CET-based CD137 affibody biantibodies. The peaks shifted to the right, similar to the peaks in the positive control group (Positive Ab), confirming that all eight CET-based CD137 affibody biantibodies bound to activated CEMT cells expressing CD137.

[0310] Figure 4B shows the FACS results for 15E3-based CD137 affibody biantibodies. The peaks shifted to the right, similar to the peaks in the positive control group, confirming that all eight 15E3-based CD137 affibody biantibodies bound to activated CEMT cells expressing CD137.

[0311] From the results above, it was confirmed that all 16 types of biantibodies of the present invention specifically bind to cells expressing CD137.

[0312] 2-4: Confirmation of biantibody binding in EGFR-expressing cells

[0313] Similarly, to confirm the binding of the 16 types of biantibody forms in EGFR-expressing cells, HT29 cells (Korean Cell Line Bank, KCLB, 30038, human colorectal cancer cell line), known to exhibit high EGFR expression, were used in a 5 × 10⁶ experiment. 5 After preparation in tubes, cells were collected by centrifugation at 1,200 rpm for 3 minutes. After washing with 5% FBS in PBS, the cells were treated with the 16 bivalent antibodies at 5 μg / mL and incubated on ice for 1 hour. The cells were washed three times by centrifugation at 1,200 rpm for 3 minutes using 200 μL of 5% FBS in PBS. Subsequently, the cells were treated with anti-human-Fc-FITC (Life Technologies A11013) at 1 μg / mL and incubated on ice for 45 minutes while blocking light. After washing the cells three times by centrifugation at 1,200 rpm for 3 minutes using 200 μL of 5% FBS in PBS, the fluorescence intensity was measured using a Beckman Coulter FACS instrument, and the results are shown in Figures 5A and 5B. Furthermore, for the positive control group (Positive Ab), the CET-IgG form was used for CET-based evaluations, and the 15E3-IgG form was used for 15E3-based evaluations. hIgG was used as the negative control group.

[0314] Figure 5A shows the FACS results for CET-based CD137 affibody biantibodies. The peaks shifted to the right, similar to the peaks in the positive control group (CET-IgG), confirming that all eight CET-based CD137 affibody biantibodies bind to EGFR-expressing HT29 cells.

[0315] Figure 5B shows the FACS results for the 15E3-based CD137 affibody biantibody, which showed a peak shifted to the right, similar to the peak of the positive control group (15E3-IgG). This confirms that all eight types of 15E3-based CD137 affibody biantibodies bind to HT29 cells that express EGFR.

[0316] From the results above, it was confirmed that all 16 types of biantibodies of the present invention specifically bind to cells that express EGFR.

[0317] 2-5: EGFR-dependent T cell activation test - Selection of affibody ZAAD01 and ZAAD05

[0318] Affibody selection was performed using the EGFR-dependent T cell activation test on a total of 16 types of biantibodies produced.

[0319] Testing Method

[0320] One day before the test, take an anti-CD3 antibody (Invitrogen) at a concentration of 3 μg / mL. TM50 μL of hEGFR-ECD-Fc (16-0037-85) was coated onto 96-well round plates. The following day, the plates were washed three times with 100 μL of sterile PBS. 50 μL of hEGFR-ECD-Fc was coated again onto each plate, and the control group (wells without protein) were treated with PBS only. The plates were incubated at 37°C for 3 hours. 100 μL of R10 medium (RPMI1640, 10% FBS and 10 mM HEPES) was used to wash the plates three times, and 100 μL of R10 medium was used for blocking at 37°C for 1 hour. The R10 medium on the plates was completely removed, and 50 μL of 16 types of biantibodies (8 types of Cetuximab-Zb and 8 types of 15E3-Zb) were treated at concentrations of 0.1 nM or 1 nM for 30 minutes.

[0321] During this time, blood samples from two donors were diluted 1 / 2 with PBS, then placed in Leucosep tubes (Greiner Bio-One Ltd., 227290) with Ficoll (GE Healthcare, 17-1440-12) floating on top, and centrifuged at 1,000g for 30 minutes. Only the layer of peripheral blood mononuclear cells (PBMCs) located on the Ficoll was taken. After repeating the washing process twice, using 2% PBMCs in PBS and centrifuging at 300g for 10 minutes, the number of PBMCs was measured. PBMCs were then analyzed using CD8. + T cell isolation kit (CD8 + CD8 according to the protocol of the T cell isolation kit (Miltenyi Biotec Inc., 130-096-495) + T cells were prepared.

[0322] CD8 + The number of T cells was measured and 1.4 × 10 6After dissolving the solutions in R10 medium to a concentration of / mL, 50 μL each solution was applied to plates treated with the 16 types of biantibodies. Three days after processing, the plates were centrifuged at 2,000 rpm for 10 minutes, and the supernatant was collected. Interferon-gamma (IFN-gamma, IFN-γ) levels were measured using this supernatant according to the protocol of the Human Interferon-Gamma ELISA Kit (IFN-gamma ELISA set, BD Biosciences, 555142), and the results are shown in Figure 6.

[0323] Test results

[0324] As shown in Figure 6, the degree of T cell activation with and without EGFR was confirmed by measuring IFN-γ using T cell activation analysis. As a result, it was confirmed that the concentration of IFN-γ significantly increased when treated with CET-based ZAAD01 affibody biantibody, CET-based ZAAD05 affibody biantibody, 15E3-based ZAAD01 affibody biantibody, and 15E3-based ZAAD05 affibody biantibody at a concentration of 1 nM. Therefore, among the eight affibodies, ZAAD01 and ZAAD05, which showed an increase in IFN-γ concentration, were selected.

[0325] Specifically, in the EGFR-dependent T cell activation test, Cetuximab-Zb or 15E3-Zb binds to hEGFR-ECD-Fc coated on the plate, and CD8 expresses CD3 on the anti-CD3 antibody coated on the plate. + T cells bind to it. The affibody of cetuximab-Zb or 15E3-Zb bound to hEGFR-ECD-Fc is bound to CD8 with an anti-CD3 antibody. + By binding to CD137 on the surface of T cells, CD8 + T cells are activated, and activated CD8 + This will allow for the measurement of IFN-γ secreted by T cells. It is known that T cells are activated by the action (agonism) of CD137.

[0326] The Affibodies ZAAD01 and ZAAD05 according to the present invention are CD8 + CD137 was activated by binding to the CD137 ECD on the surface of T cells, and CD8 + We stimulated T cells to secrete IFN-γ.

[0327] This result is consistent with prior art in which urelumab (BMS-663513) and utomilumab, monoclonal antibodies against CD137, are known agonists that bind to CD137 (4-1BB) and activate T cells (see U.S. Patent Registration No. US7288638B2, 2007 Oct 30; U.S. Patent Registration No. US7288638B2; U.S. Patent Registration No. US8821867B2).

[0328] Based on the above results, the inventors confirmed that the biantibodies Cetuximab-ZAAD01, 15E3-ZAAD01, Cetuximab-ZAAD01, and 15E3-ZAAD01 activate T cells in an EGFR-dependent manner, and selected affibodies ZAAD01 and ZAAD05 for the final selection.

[0329] Example 3. Characterization of selected affibodies

[0330] 3-1: Confirmation of Affibody Joining Characteristics using CD137 ECD Domain Mapping

[0331] The binding properties of two selected affibodies, ZAAD01 and ZAAD05, were confirmed by CD137 domain mapping. The 15E3-based ZAAD01 and ZAAD05 affibodiment biantibodies will be referred to as 15E3ZAAD01 and 15E3ZAAD05, respectively, below. The binding domains of the single-clonal antibodies against CD137, urelumab (BMS-663513) and utomilumab (PF-05082566), were also investigated.

[0332] To determine which part of the extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to, the CD137 ECD was divided into five domains, and domain mutants were cloned. A schematic diagram of the domain mutant is shown in Figure 7A.

[0333] As shown in Figure 7A, the cloned domain mutations include signal peptides and ECD domains 1 to 5 in different combinations, specifically as follows: hCD137-ECD containing all signal peptides and domains 1 to 5; hCD137-ECD-ΔD1 lacking domain 1; hCD137-ECD-ΔD12 lacking domains 1 and 2; hCD137-ECD-ΔD123p lacking all of domains 1 and 2 and part of domain 3 (deleted up to amino acid 104); hCD137-ECD-ΔD123 lacking domains 1 to 3; and hCD137-ECD-ΔD3 and hCD137-ECD-ΔD5 lacking domain 5.

[0334] Figure 7A is a schematic diagram of hCD137 ECD including the signal peptide and domains 1-5. Sequence ID 27 shows the amino acid sequences for domains 1-5, excluding the amino acid sequence for the signal peptide.

[0335] Cloned domain mutations were produced using animal cells. Plates coated with the produced domain mutations at a concentration of 2 μg / mL were treated with purified urelumab, utomilumab, 15E3, 15E3ZAAD01, and 15E3ZAAD05 at concentrations of 10 μg / mL, 2 μg / mL, and 0.4 μg / mL, respectively. After treatment with a secondary antibody (anti-hIgG-Fab-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097), TMB (BioFX) was used. TMA color reaction is performed using TMBC-1000-01, and OD is performed using an ELISA reader (Victor X3, PerkinElmer, Inc.). 450 The values ​​were measured, and the results are shown in Figures 7B to 7F.

[0336] As shown in Figure 7B, urelumab did not bind to the domain mutations hCD137ECD ΔD1, hCD137ECD ΔD12, hCD137ECD ΔD123P, and hCD137ECD ΔD123, thus confirming that urelumab binds to domain 1 of CD137 ECD.

[0337] As shown in Figure 7C, utomirumab did not bind to the domain mutations hCD137ECD ΔD123P, hCD137ECD ΔD123, and hCD137ECD ΔD3, thus confirming that utomirumab binds to domain 3 of CD137 ECD.

[0338] As shown in Figure 7D, Negative Ab (cetuximab) did not bind to the entire human CD137 ECD or any of its domain mutations, thus confirming that it does not bind to CD137ECD.

[0339] As shown in Figure 7E, 15E3ZAAD01 did not bind to the domain mutations hCD137ECD ΔD123P, hCD137ECD ΔD123, and hCD137ECD ΔD3, thus confirming that 15E3ZAAD01 binds to domain 3 of CD137 ECD.

[0340] As shown in Figure 7F, 15E3ZAAD05 did not bind to the domain mutations hCD137ECD ΔD123P, hCD137ECD ΔD123, and hCD137ECD ΔD3, thus confirming that 15E3ZAAD05 binds to domain 3 of CD137 ECD.

[0341] From the above results, the inventors confirmed that the two selected affibodies, ZAAD01 and ZAAD05, bind to domain 3 of CD137 ECD.

[0342] 3-2: Confirmation of cross-reactivity between TNFRSF of selected affibodies

[0343] The cross-reactivity of the two selected affibodies, ZAAD01 and ZAAD05, with respect to TNFRSF (TNF-alpha receptor superfamily) was confirmed using ELISA.

[0344] Plates coated with CD137 (TNFRSF9), CD27 (TNFRSF7), CD30 (TNFRSF8), CD134 (TNFRSF4), CD270 (TNFRSF14), CD357 (TNFRSF18), and TNF-alpha protein belonging to TNFRSF at a concentration of 2 μg / mL were treated with 15E3ZAAD01 and 15E3ZAAD05 at 1 μg / mL each, and then treated with a secondary antibody (anti-hIgG-Fab-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097) before being treated with TMB (BioFX). TM A color reaction was performed using TMBC-1000-01, and an ELISA reader (Victor X3 PerkinElmer, Inc.) was used to obtain the OD (Oral Color) result. 450 The values ​​were measured, and the results are shown in Figures 8A and 8B.

[0345] As shown in Figure 8A, ZAAD01 specifically bound to CD137 only, and as shown in Figure 8B, ZAAD05 also specifically bound to CD137 only.

[0346] Based on the above results, the inventors confirmed that the two selected affibodies, ZAAD01 and ZAAD05, do not cross-react with other proteins belonging to the TNFRSF (TNF-alpha receptor superfamily).

[0347] 3-3: Interspecies cross-reaction of selected aphibodies

[0348] The interspecies cross-reactivity of two selected affibodies, ZAAD01 and ZAAD05, with CD137 was confirmed using ELISA.

[0349] Plates coated with human CD137, mouse CD137, and cynomolgus monkey CD137 proteins at a concentration of 2 μg / mL were treated with 15E3ZAAD01 and 15E3ZAAD05 at a concentration of 5 μg / mL, and then treated with a secondary antibody (anti-hIgG-Fab-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097) before being treated with TMB (BioFX). TM A color reaction is performed using TMBC-1000-01, and an ELISA reader (Victor X3, PerkinElmer, Inc.) is used to obtain an OD (Oral Data Analysis). 450 The values ​​were measured, and the results are shown in Figures 9A and 9B.

[0350] As shown in Figure 9A, affibody ZAAD01 bound to human and cynomolgus monkey CD137 proteins, and as shown in Figure 9B, affibody ZAAD05 also bound to human and cynomolgus monkey CD137 proteins.

[0351] The results above confirm that both selected CD137 affibodies exhibited interspecies cross-reactivity with the CD137 protein in both humans and cynomolgus monkeys.

[0352] Example 4. Target protein binding of biantibodies and EGFR-dependent efficacy confirmation.

[0353] 4.1: Confirm whether the biantibody binds to both targets simultaneously.

[0354] We used BioLayer Interferometry (BLI) to confirm whether four biantibodies, CET-ZAAD01, CET-ZAAD05, 15E3-ZAAD01, and 15E3-ZAAD05, which consist of cetuximab (an antibody that binds to EGFR) and ZAAD01 and ZAAD05 (affibodies that bind to 15E3 and CD137), bind simultaneously to their respective target proteins.

[0355] EGFR-ECD-Fc protein was immobilized on an AR2G sensor chip (Amine Reactive Second-Generation Biosensor, SATORIUS Corporation, 18-5092) at a concentration of 5 μg / mL using an amine coupling method with EDC / NHS. The four types of biantibodies described above were conjugated to the sensor chip with the immobilized EGFR-ECD-Fc at a concentration of 10 μg / mL each for 5 minutes and stabilized for 5 minutes. CD137-ECD-his protein was added to the sensor chip with the immobilized EGFR protein and biantibodies conjugated at a concentration of 30 μg / mL, conjugated for 5 minutes and stabilized for 5 minutes. The results are shown in Figures 10A to 10D. As a control group, the results when treated with CET or 15E3 are shown by the red line, and the results when treated with the four types of biantibodies of the present invention are shown by the blue line.

[0356] As shown in Figure 10A, when the CD137-ECD-Fc protein was added to the sensor chip after treating the control group with CET, there was no change in the binding signal (red line). In contrast, when the CD137-ECD-Fc protein was added to the sensor chip after treating it with the CET-ZAAD01 biantibody, the binding signal increased (blue line). This means that the CET-ZAAD01 biantiantibody simultaneously binds to the EGFR protein that was immobilized on the sensor chip beforehand and to the CD137-ECD-Fc protein that was added afterward.

[0357] Similar to the results for the CET-ZAAD01 biantibody, the CET-ZAAD05 biantibody (Figure 10B), 15E3-ZAAD01 biantibody (Figure 10C), and 15E3-ZAAD05 biantibody (Figure 10D) also showed an increase in binding signal when the CD137-ECD-his protein was added to the sensor chip, confirming that these biantibodies also bind simultaneously to the EGFR protein and the CD137 protein.

[0358] 4.2: T cell activation by biantibodies based on EGFR expression levels

[0359] We investigated whether a dual antibody regime using an affibody against CD137 and an antibody against EGFR increases T cell activation depending on the level of EGFR expression in the cell line.

[0360] EGFR expression levels of cell lines

[0361] Three types of EGFR-expressing cells, A431 (ATCC, CRL-1555, epidermis, epidermoid carcinoma), HT29 (KCLB, 30038, colon, adenocarcinoma), and SW403 (KCLB, 10230, colon, adenocarcinoma), were compared with EGFR-negative cells, MOLT4 (KCLB, 21582, adult T acute lymphoblastic leukemia), in 5 × 10⁻¹⁰ samples. 5 Prepared with a tube.

[0362] Subsequently, cells were collected by centrifugation at 1,200 rpm for 3 minutes, washed with 5% FBS in PBS, and then treated with 5 μg / mL of 15E3ZAAD01 biantibody. The cells were incubated on ice for 1 hour. The cells were washed three times using 200 μL of PBS with 5% FBS by centrifugation at 1,200 rpm for 3 minutes. Then, the cells were treated with 1 μg / mL of anti-human Fc FITC and incubated on ice for 45 minutes while blocking light. After washing the cells three times using 200 μL of PBS with 5% FBS by centrifugation at 1,200 rpm for 3 minutes, the fluorescence intensity was measured using a Beckman Coulter FACS instrument to obtain the Mean Fluorescent Intensity (MFI) value, which is shown in Figure 11A. The MFI value increases as the cells bind more 15E3ZAAD01 biantiantibody, indicating that the cells have high EGFR expression levels.

[0363] As shown in Figure 11A, EGFR expression was examined in EGFR-positive cells A431, HT29, and SW403, and EGFR-negative cells MOLT4. The MFI values ​​were 101.5, 23.9, 17.1, and 0.9 for cell lines A431, H29, SW403, and MOLT4, respectively, confirming that EGFR expression was highest in this order.

[0364] EGFR-dependent T cell activation of double antibodies

[0365] As described above, T cell assays were performed using cells with varying levels of EGFR expression. The day before, anti-CD3 antibody (Invitrogen) was administered at a concentration of 3 μg / mL. TM 50 μL of (16-0037-85) was coated onto each 96-well round plate. The following day, the plates were washed three times with 100 μL of sterile PBS. The plates were then washed three times with 100 μL of R10 medium, treated with 100 μL of R10 medium, and blocked at 37°C for 1 hour. Four cell lines, A431, H29, SW403, and MOLT4, were placed on each plate in a 2.4 × 10⁶ layer. 550 μL was added in increments to achieve a concentration of / mL. The 15E3ZAAD01 biantibody was treated with 50 μL at a concentration of 5 μg / mL for 30 minutes.

[0366] Meanwhile, blood samples from two donors were diluted 1 / 2 with PBS, placed in Leucosep tubes (Greiner Bio-One Ltd., 227290) with Ficoll (GE healthcare, 17-1440-12) floating in them, and centrifuged at 1,000g for 30 minutes.

[0367] Only the PBMC layer located on the Ficol was taken. After repeating the washing process twice, which involved centrifuging 300g of PBMC in PBS for 10 minutes, the number of PBMCs was measured. PBMCs were then converted to CD8 + T cell isolation kit (CD8 + CD8 according to the protocol of the T cell isolation kit (Miltenyi Biotec Inc., 130-096-495) + T cells were prepared.

[0368] CD8 + The number of T cells was measured and 1.4 × 10 6 After dissolving the 15E3ZAAD01 biantibody in R10 medium to the desired concentration ( / mL), 50 μL of the solution was added to a plate treated with the biantibody. Three days after treatment, the plate was centrifuged at 2,000 rpm for 10 minutes, and the supernatant was collected. The IFN-γ concentration was then measured using this supernatant according to the protocol of the IFN-γ measurement kit (BD Biosciences, 555142). The activation of T cells by the biantibody of the present invention was confirmed by the amount of IFN-γ secreted by T cells, depending on the EGFR expression level, and the results are shown in Figure 11B.

[0369] As shown in Figure 11B, T cell activation analysis revealed that the concentration of IFN-γ increased as EGFR expression increased, confirming that increased EGFR expression leads to increased T cell activity.

[0370] Specifically, the order of highest IFN-γ secretion levels coincided with the order of highest EGFR expression levels mentioned above. Specifically, cell lines A431, H29, SW403, and MOLT4 showed IFN-γ concentration fold values ​​of 5.9, 2.2, 1.9, and 1.0, respectively, confirming that IFN-γ concentrations were highest in this order.

[0371] Based on the above results, the inventors confirmed EGFR-dependent T cell activation by the 15E3-ZAAD01 biantibody depending on the level of EGFR expression, which indicates that the degree of T cell activation by the biantibody depending on the level of EGFR expression is correlated with the amount of EGFR expression.

[0372] When using the EGFR antibody-based CD137 affibody biantibody according to the present invention, T cells expressing CD137 can be located nearby, bind to, and activate cancer cells and tissues that exhibit excessive EGFR expression. Therefore, it can be usefully used for the prevention or treatment of various types of cancer that overexpress EGFR.

[0373] Example 5. Potential for developing various antigen-targeted biantibodies based on CD137 affibody - HER2 and CD19

[0374] Based on the above results, the inventors attempted to confirm, as described below, whether T cells can be activated not only with EGFR as a cancer-specific target protein, but also with antibody-based CD137 affibody biantibodies against other cancer or tumor-specific target proteins.

[0375] 5.1: Confirmation of binding to target protein, HER2, or CD19

[0376] Selected CD137 affibodies and ZAAD01-based antibodies that bind to HER2 and CD19, other proteins besides EGFR, were cloned in the form of biantibodies. Biantibodies composed of HER2 antibody and CD137 affibodies were denoted as anti-HER2 Ab1-ZAAD01 and anti-HER2 Ab2-ZAAD01, respectively, while biantibodies composed of CD19 antibody and CD137 affibo were denoted as anti-CD19 Ab-ZAAD01.

[0377] Specifically, in anti-HER2 Ab1-ZAAD01, anti-HER2 Ab1 is trastuzumab, which specifically binds to human HER2, and in anti-HER2 Ab2-ZAAD01, anti-HER2 Ab2 is a proprietary hz1E11.10 antibody (see Korean Published Patent No. 10-2017-0117330) that specifically binds to human HER2.

[0378] The anti-HER2 Ab may be an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 29.

[0379] Specifically, in anti-CD19 Ab-ZAAD01, anti-CD19 Ab is FMC63, which specifically binds to human CD19.

[0380] The anti-CD19 Ab may be an antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 39.

[0381] The aforementioned biantibodies were produced using animal cells. Protein binding and T-cell activation tests were performed on the purified biantibodies to investigate whether the CD137 affibody of the present invention can be developed in a modular form as a biantibody targeting various other antigens and is substantially usable.

[0382] For the ELISA, plates coated with CD137 protein and hHer2-ECD-Fc protein and hCD19-ECD-Fc protein at a concentration of 2 μg / mL to match the target proteins of each antibody were treated with the purified biantibodies described above at 7 points, starting at 60 nM and diluting by 1 / 5. After treatment with a secondary antibody (anti-hIgG-Fab-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097), TMB (BioFX) was used. TM A color reaction is performed using TMBC-1000-01, and OD is performed using an ELISA reader (Victor X3, PerkinElmer, Inc.). 450 The values ​​were measured. The results are shown in Figures 12A to 12D.

[0383] As shown in Figures 12A to 12D, the biantibodies produced using the CD137 affibody and either the HER2 antibody or the CD19 antibody bound to CD137 and HER2 or CD19, respectively.

[0384] Specifically, compared to the negative control group (hIgG), both the anti-HER2 Ab1-ZAAD01 and anti-HER2 Ab2-ZAAD01 biantibodies, composed of an anti-HER2 antibody and a ZAAD01 affibody, showed a concentration-dependent increase in binding to the target proteins CD137 (Figure 12A) and HER2 (Figure 12B), respectively.

[0385] Furthermore, compared to the negative control group, the binding of the anti-CD19 Ab-ZAAD01 biantibody, composed of anti-CD19 antibody and ZAAD01 affibody, to its target proteins CD137 (Figure 12C) and CD19 (Figure 12D) increased in a concentration-dependent manner.

[0386] From the above results, it was confirmed that the biantibodies anti-HER2 Ab1-ZAAD01 and anti-HER2 Ab2-ZAAD01 of the present invention bind to both CD137 and HER2 proteins, and that the biantibodies anti-CD19 Ab-ZAAD01 of the present invention bind to both CD137 and CD19 proteins.

[0387] 5.2: Target protein-dependent T cell activation test

[0388] As described above, the efficacy of the biantibody was confirmed by performing T cell activation tests dependent on other extended proteins, namely HER2 or CD19, using the produced biantibody that was confirmed to bind to all of the target proteins.

[0389] Testing Method

[0390] One day prior, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) at a concentration of 3 μg / mL was coated onto each 96-well round plate. The following day, the plates were washed three times with 100 μL of sterile PBS. 50 μL of HER2 or CD19 at a concentration of 5 μg / mL was again coated onto each plate. As a control group, wells without protein (not treated with HER2 or CD19) were treated with PBS only, and then incubated at 37°C for 3 hours. The plates were washed three times with 100 μL of R10 medium, and then blocked at 37°C for 1 hour after being treated with 100 μL of R10 medium. The R10 medium on the plates was completely removed, and the plates were treated with the aforementioned biantibody at a concentration of 5 μg / mL with 50 μL for 30 minutes.

[0391] Meanwhile, blood samples from two donors were diluted 1 / 2 with PBS, then placed in Leucosep tubes (Greiner Bio-One Ltd., 227290) with Ficoll (GE healthcare, 17-1440-12) floating on top, and centrifuged at 1,000 g for 30 minutes. Only the PBMC layer located on the Ficoll was taken. After repeating the washing process twice, using 2% PBMC in PBS and centrifuging at 300 g for 10 minutes, the number of PBMCs was measured.

[0392] PBMC to CD8 + T cell isolation kit (CD8 + CD8 according to the protocol of the T cell isolation kit (Miltenyi Biotec Inc., 130-096-495) + T cells were prepared. CD8 + The number of T cells was measured and 1.4 × 10 6 After dissolving the biantibody in R10 medium to the desired concentration ( / mL), 50 μL of the solution was applied to plates treated with the biantibody. Three days after treatment, the plates were centrifuged at 2,000 rpm for 10 minutes, and the supernatant was collected. The IFN-γ concentration was measured using this supernatant according to the protocol of the IFN-γ measurement kit (BD Biosciences, 555142), and the results are shown in Figures 13A and 13B. Utomirumab, a monoclonal antibody against CD137, was used as the negative control group.

[0393] Test results

[0394] As shown in Figures 13A and 13B, the biantibodies produced using the CD137 affibody and either the HER2 antibody or the CD19 antibody (anti-HER2 Ab1-ZADD1, anti-HER2 Ab1-ZADD1, and anti-CD19 Ab-ZADD1) activated T cells in a HER2 or CD19 protein-dependent manner, as confirmed by the increased secretion of IFN-γ.

[0395] Specifically, as shown in Figure 13A, when treated with utomirumab, a monoclonal antibody against CD137, IFN-γ was affected regardless of the presence or absence of HER2. + Almost no secretion was produced from T cells. When treated with the biantibodies anti-HER2 Ab1-ZAAD01 (trastuzumab-ZAAD01) and anti-HER2 Ab2-ZAAD01 (hz1E11.10-ZAAD01), CD8 was secreted more when HER2 was treated (HER2) compared to when HER2 was not treated (no HER2). + T cells showed a significantly increased secretion of IFN-γ.

[0396] Furthermore, Figure 13B shows the difference in CD8 levels when treated with utomirumab, a monoclonal antibody against CD137, depending on the presence or absence of CD19. + There was no significant difference in the amount of IFN-γ secreted by T cells. When treated with the biantibody anti-CD19 Ab-ZAAD01 (FMC63-ZAAD01), CD8 was treated with HER2 (CD19) compared to when CD19 was not treated (no CD19). + T cells showed a significantly increased secretion of IFN-γ.

[0397] The inventors confirmed, by IFN-gamma secretion analysis, that a biantibody composed of an anti-HER2 antibody or an anti-CD19 antibody and a ZAAD01 affibody activated T cells in a manner dependent on their respective target antibodies, HER2 or CD19.

[0398] In particular, these results confirm the presence or absence of T cell activation by other extended target proteins, such as HER2 or CD19, through IFN-γ measurement. This demonstrates that when the CD137 affibody according to the present invention is used in a modular format, it can be applied not only to the target proteins disclosed in the present invention—EGFR, HER2, and CD19—but also to various other target proteins that are different from those disclosed.

[0399] Therefore, from the disclosures of the present invention, those skilled in the art will understand that by constructing a biantibody composed of an antibody against a target protein specifically expressed / present in specific cancer cells and the CD137 affibody of the present invention, CD137 can be usefully used for the prevention or treatment of various types of cancer through an immunomodulatory approach.

[0400] As a result, the CD137 affibody of the present invention, or the biantibody composed of the CD137 affibody and an antibody against a cancer cell-specific target protein, can be usefully used for the prevention or treatment of various types of cancer by using various antigen-presenting cells (APCs) that express CD137, for example, by ADCC (antibody-dependent cell-mediated cytotoxicity) of NK (natural killer) cells or T cell activation of DC (dendritic cells).

[0401] Example 6. Confirmation of T cell activity and cell death by EGFR-CD137-targeted biantibody.

[0402] The inventors conducted the following tests to confirm the T-cell activity and cell death effects of a biantibody that simultaneously targets EGFR and CD137. The biantibodies used in this example (CET_ZAAD01 and CET-ZAAD05) are fusion proteins of cetuximab, which targets EGFR, and the affibody ZAAD01 or ZAAD05 of the present invention, which targets CD137.

[0403] One day prior to double antibody treatment, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) was dispensed per well into 96-well round plates at a concentration of 5 μg / mL and coated. The following day, 100 μL of R10 medium was added, followed by blocking by incubation at 37°C for 1 hour. DLD-1-Luc cells were placed in each plate at a rate of 1.0 x 10⁴ 5After adding 50 μL each to achieve a concentration of / mL, 50 μL of biantibodies (CET_ZAAD01 and CET-ZAAD05), prepared by diluting from a 10 nM concentration to 1 / 10, were applied to each well. Meanwhile, blood provided by the donor was diluted 1 / 2 with PBS, placed in a Leucosep tube (greiner bio-one, 227290) with Ficol (GE healthcare, 17-1440-12) floating on it, and centrifuged at 1000 x g for 30 minutes. After separating only the PBMC layer located on the Ficol, 10 mL of PBMC was mixed with 40 mL of PBS containing 2% FBS, and the washing process was repeated twice by centrifuging at 300 x g for 10 minutes, after which the number of PBMCs was measured. 5 After dissolving the cells in R10 medium to a concentration of / mL, 50 μL of the solution was added to a plate treated with the biantibody. Three days after treatment, the plate was centrifuged at 2000 RPM for 10 minutes, and the supernatant was collected. The amount of IFN-gamma was measured using an IFN-gamma analyzer kit (BD, 555142). 50 μL of lysis buffer (75 mM Tris (pH 8.0), 30% glycerol, 3% Triton X-100) was added to the wells containing the remaining cells and medium and mixed well. The mixture was then left at room temperature for 15 minutes. 50 μL of the cell and lysis buffer mixture was transferred to a 96-well white plate, and the same amount of 5'-fluoroluciferin (Bio-Glo) was added. TM After placing the Luciferase Assay System (promea / G7940) in the ELISA and allowing it to stand at room temperature for 15 minutes, the luminescence was measured using an ELISA reader.

[0404] The results are shown in Figures 14A and 14B.

[0405] As shown in Figures 14A and 14B, the activation of immune cells by the biantibody confirmed the death of DLD1-Luc cells, as well as an increase in IFN-gamma concentration.

[0406] Example 7: Confirmation of anticancer efficacy of EGFR-CD137-targeted biantibody using an animal model.

[0407] The efficacy of biantibodies targeting EGFR and CD137 (CET_ZAAD01 and CET-ZAAD05) was confirmed using a humanized CD137 mouse model.

[0408] EGFR / MC38 cells, 5.0 x 10 5 The mice were prepared in PBS to a concentration of 1 / mL and artificially induced to develop cancer by subcutaneous injection into the flank of humanized CD137 mice. The tumor size was approximately 100 mm. 3 At that time, the animals were randomly grouped into groups of five as designed. Cetuximab or EGFR-CD137-targeted biantibodies (CET_ZAAD01 and CET-ZAAD05) were diluted in PBS to a concentration of 5 mpk and administered intraperitoneally twice a week for a total of five times. Tumor size and weight were measured three times a week, and tumor size was measured using a digital vernier caliper. The tumor size was 2500 mm. 3 When the limit was exceeded, they were euthanized.

[0409] The results are shown in Figures 15 and 16A to 16D.

[0410] As shown in Figure 15, the two types of biantibodies of the present invention (CET_ZAAD01 and CET-ZAAD05) showed a survival-extending effect in mice.

[0411] Furthermore, as shown in Figures 16A to 16D, the biantibody of the present invention was confirmed to reduce the size of tumors.

[0412] In contrast, the vehicle and cetuximab treatment groups showed a tendency for cancer size to continue increasing, while the two types of dual antibodies (CET_ZAAD01 and CET-ZAAD05) suppressed cancer size and resulted in tumor death.

[0413] Although specific parts of the present invention have been described in detail above, it is clear to those with ordinary skill in the art that such specific descriptions are merely preferred examples and do not limit the scope of the present invention. [Brief explanation of the drawing]

[0414] [Figure 1] The results of identifying affibodies that bind to the CD137 protein in the form of a periplasmic extract are shown. [Figure 2] This paper shows the results of identifying CD137-binding affibodies expressed on CD137 using activated CEMT cells, which are CD137-positive cells, based on the morphology of the antibody-affibodies that bind to CD137. [Figure 3A] This figure shows the results of testing whether a biantibody composed of CD137 affibodies, produced based on two types of EGFR-binding antibodies (CET or 15E3), binds to the CD137 protein and the EGFR protein. It confirms that a biantibody composed of CD137 affibodies based on cetuximab (CET) antibody binds to the EGFR protein. [Figure 3B] This figure shows the results of testing whether a biantibody composed of CD137 affibodies, produced based on two types of EGFR-binding antibodies (CET or 15E3), binds to the CD137 protein and the EGFR protein. It confirms that a biantibody composed of CD137 affibodies based on cetuximab (CET) antibody binds to the CD137 protein. [Figure 3C] This figure shows the results of testing whether a biantibody composed of CD137 affibodies, produced based on two types of EGFR-binding antibodies (CET or 15E3), binds to the CD137 protein and the EGFR protein. The figure confirms that a biantibody composed of CD137 affibodies based on the 15E3 antibody binds to the EGFR protein. [Figure 3D] This figure shows the results of testing whether a biantibody composed of CD137 affibodies, produced based on two types of EGFR-binding antibodies (CET or 15E3), binds to the CD137 protein and the EGFR protein. It also shows confirmation that a biantibody composed of CD137 affibodies based on the 15E3 antibody binds to the CD137 protein. [Figure 4A] This figure confirms that a biantibody composed of a cetuximab (CET) antibody-based CD137 affibody binds to activated CEMT cells expressing CD137. [Figure 4B] This figure confirms that a biantibody composed of a 15E3 antibody-based CD137 affibody binds to activated CEMT cells expressing CD137. [Figure 5A] This figure confirms that a biantibody composed of a cetuximab (CET) antibody-based CD137 affibody binds to HT29 cells that express EGFR. [Figure 5B] This figure confirms that a biantibody composed of a 15E3 antibody-based CD137 affibody binds to HT29 cells that express EGFR. [Figure 6] This figure shows the confirmation of EGFR-dependent T cell activation by IFN-gamma secretion analysis for the selection of CD137 affibodies. [Figure 7A]This figure shows which extracellular domain (ECD) of CD137 each of the two selected CD137 affibodies binds to. Schematic diagrams of hCD137-ECD, hCD137-ECD-ΔD1, hCD137-ECD-ΔD12, hCD137-ECD-ΔD123p, hCD137-ECD-ΔD123, hCD137-ECD-ΔD3, and hCD137-ECD-ΔD5 are composed of signal peptides and different ECD domains 1-5. The amino acid sequences for human CD137 ECD composed of domains 1-5 are shown in Sequence ID No. 27. [Figure 7B] This figure shows which extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to. This figure also shows that urelumab binds to domain 1 of the CD137 ECD. [Figure 7C] This figure shows which extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to. This figure also shows that utomilumab binds to domain 3 of the CD137 ECD. [Figure 7D] This figure shows which extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to. This figure also shows that cetuximab (Negative Ab) does not bind to any domain of the CD137 ECD. [Figure 7E] This figure shows which extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to. This figure shows that affibo ZAAD01 binds to domain 3 of the CD137 ECD. [Figure 7F]This figure shows which extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to. This figure shows that affibo ZAAD05 binds to domain 3 of CD137 ECD. [Figure 8A] This figure shows the cross-reactivity between two selected CD137 affibodies and other proteins belonging to the TNFR superfamily: CD27, CD30, CD134, CD270, CD357, and TNF-α. This figure confirms that affibodies ZAAD01 specifically bind only to CD137. [Figure 8B] This figure shows the cross-reactivity between two selected CD137 affibodies and other proteins belonging to the TNFR superfamily: CD27, CD30, CD134, CD270, CD357, and TNF-α. This figure confirms that affibodies ZAA0D5 specifically bind only to CD137. [Figure 9A] This figure shows the interspecies cross-reactivity between two selected CD137 affibodies and the CD137 protein in humans, mice, and cynomolgus monkeys. This figure confirms that affibodies ZAAD01 bind to the CD137 proteins of humans and cynomolgus monkeys. [Figure 9B] This figure shows the interspecies cross-reactivity between two selected CD137 affibodies and the CD137 protein in humans, mice, and cynomolgus monkeys. This figure confirms that affibodies ZAAD05 bind to the CD137 proteins of humans and cynomolgus monkeys. [Figure 10A]This figure shows the confirmation, using BioLayer Interferometry (BLI), that four types of biantibodies in antibody-affibody form simultaneously bind to both the CD137 antigen and the EGFR antigen. Figure 10A shows the BLI results for the CET-ZAAD01 biantibody. [Figure 10B] This figure shows the confirmation, using BLI (BioLayer Interferometry), that four types of biantibodies in antibody-affibody form simultaneously bind to both the CD137 antigen and the EGFR antigen targets. Figure 10B shows the BLI results for the CET-ZAAD05 biantibody. [Figure 10C] This figure shows the confirmation, using BLI (BioLayer Interferometry), that four types of biantibodies in antibody-affibody form simultaneously bind to both the CD137 antigen and the EGFR antigen targets. Figure 10C shows the BLI results for the 15E3-ZAAD01 biantibody. [Figure 10D] This figure shows the confirmation, using BLI (BioLayer Interferometry), that four types of biantibodies in antibody-affibody form simultaneously bind to both the CD137 antigen and the EGFR antigen targets. Figure 10D shows the BLI results for the 15E3-ZAAD05 biantibody. [Figure 11A] This figure shows the cell-specific expression levels of EGFR-expressing cells, confirmed by flow cytometry, and confirming that the degree of T cell activation by the biantibody correlates with EGFR expression levels. The figure also shows EGFR expression in EGFR-positive cells (A431, HT29, and SW403) and EGFR-negative cells (MOLT4). [Figure 11B] This figure shows the cell-specific expression levels of EGFR-expressing cells, confirmed by flow cytometry, and confirming that the degree of T cell activation by the biantibody correlates with EGFR expression levels. This figure also shows the EGFR-dependent T cell activation by the 15E3-ZAAD01 biantibody, confirmed by IFN-gamma secretion analysis. [Figure 12A]This figure shows that biantibodies produced using a CD137 affibody and a HER2 antibody or CD19 antibody bind to CD137 and HER2 or CD19, respectively. This figure also shows that trastuzumab-ZAAD01 biantibody (anti-HER2 Ab1_ZAAD01) and hz1E11.10-ZAAD01 biantibody (anti-HER2 Ab2_ZAAD01) bind to CD137. [Figure 12B] This figure shows that biantibodies produced using a CD137 affibody and a HER2 antibody or CD19 antibody bind to CD137 and HER2 or CD19, respectively. This figure also shows that trastuzumab-ZAAD01 biantibody and hz1E11.10-ZAAD01 biantibody bind to HER2. [Figure 12C] This figure shows that a biantibody produced using a CD137 affibody and either a HER2 antibody or a CD19 antibody binds to CD137 and HER2 or CD19, respectively. This figure also shows that the FMC63-ZAAD01 biantibody (anti-CD19 Ab_ZAAD01) binds to CD137. [Figure 12D] This figure shows that biantibodies produced using a CD137 affibody and either a HER2 antibody or a CD19 antibody bind to CD137 and HER2 or CD19, respectively. This figure also shows that the FMC63-ZAAD01 biantibody binds to CD19. [Figure 13A] This figure shows that biantibodies produced using a CD137 affibody and a HER2 antibody or CD19 antibody activate T cells in a HER2 or CD19-dependent manner, as confirmed by IFN-gamma secretion analysis. This figure also shows that trastuzumab-ZAAD01 biantibody and hz1E11.10-ZAAD01 biantibody activate T cells in a HER2-dependent manner compared to utomirumab, as confirmed by IFN-gamma secretion analysis. [Figure 13B]This figure shows that biantibodies produced using a CD137 affibody and a HER2 antibody or CD19 antibody activate T cells in a HER2 or CD19-dependent manner, as confirmed by IFN-gamma secretion analysis. This figure also shows that the FMC63-ZAAD01 biantibody activates T cells in an EGFR-dependent manner compared to utomirumab, as confirmed by IFN-gamma secretion analysis. [Figure 14A] This study demonstrates that DLD1-luc cells are killed by the activation of immune cells using a dual antibody (CET_ZAAD01 and CET_ZAAD05). [Figure 14B] This study demonstrates that activation of immune cells by the biantibodies (CET_ZAAD01 and CET_ZAAD05) increases IFN-gamma secretion. [Figure 15] In a humanized CD137 model, the biantibody (CET_ZAAD01 and CET_ZAAD05) demonstrated an extended survival period due to its anticancer effect. [Figure 16A] This figure shows the tumor size in the vehicle-treated group in a humanized CD137 model. [Figure 16B] This figure shows the tumor size in the cetuximab-treated group in a humanized CD137 model. [Figure 16C] This figure shows the tumor size in the group treated with the biantibody (CET_ZAAD01) in a humanized CD137 model. [Figure 16D] This figure shows the tumor size in the group treated with the biantibody (CET_ZAAD05) in a humanized CD137 model.

Claims

1. A polypeptide scaffold based on a variant of the Z-domain that specifically binds to the extracellular domain of CD137 in human or cynomolgus monkey, containing the amino acid sequence represented by one of SEQ ID NOs: 1 to 8.

2. The polypeptide scaffold based on the Z-domain variant according to claim 1, wherein the binding site for the extracellular domain of the CD137 of the polypeptide scaffold based on the Z-domain variant is located between amino acid residues 64 to 95 of the amino acid sequence of SEQ ID NO:

27.

3. A nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide scaffold based on the Z-domain variant described in claim 1.

4. The nucleic acid molecule according to claim 3, wherein the nucleotide sequence includes one nucleotide sequence from sequence numbers 9 to 16.

5. A recombinant vector comprising the nucleic acid molecule described in claim 3.

6. Isolated host cells comprising the recombinant vector described in claim 5.

7. A pharmaceutical composition for the prevention or treatment of cancer, comprising a polypeptide scaffold based on a Z-domain variant as described in claim 1 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein the cancer is a hematological cancer or a solid tumor.

9. Protein complex containing the following: A polypeptide scaffold based on a variant of the Z-domain that specifically binds to the extracellular domain of human or cynomorgus monkey CD137 as described in claim 1; and An antibody or its antigen-binding fragment that specifically binds to proteins on the surface of cancer cells.

10. The protein complex according to claim 9, wherein the protein on the surface of the cancer cell is EGFR (epidermal growth factor receptor), HER2 (human epidermal growth factor receptor 2), or CD19 (cluster of difference 19).

11. The protein complex according to claim 10 comprises the following: an antibody or antigen-binding fragment thereof that specifically binds to the EGFR: (a) cetuximab; or (b) Heavy chain variable region including CDR-H1 containing the amino acid sequence of SEQ ID NO: 17, CDR-H2 containing the amino acid sequence of SEQ ID NO: 18, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 19; and light chain variable region including CDR-L1 containing the amino acid sequence of SEQ ID NO: 20, CDR-L2 containing the amino acid sequence of SEQ ID NO: 21, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

22.

12. The protein complex according to claim 11, wherein (b) comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 23; and a light chain variable region containing the amino acid sequence of SEQ ID NO:

24.

13. The antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises the following protein complex according to claim 10: (c) Heavy chain variable region containing the amino acid sequence of SEQ ID NO: 28; and light chain variable region containing the amino acid sequence of SEQ ID NO: 29; (d) Trastuzumab; or (e) Heavy chain variable regions including CDR-H1 containing the amino acid sequence of SEQ ID NO: 30, CDR-H2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 32; and light chain variable regions including CDR-L1 containing the amino acid sequence of SEQ ID NO: 33, CDR-L2 containing the amino acid sequence of SEQ ID NO: 34, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

35.

14. The protein complex according to claim 13, wherein (e) comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 36; and a light chain variable region containing the amino acid sequence of SEQ ID NO:

37.

15. The single clone antibody or antigen-binding fragment thereof that specifically binds to the CD19 comprises the protein complex according to claim 10: (f) A heavy chain variable region containing the amino acid sequence of SEQ ID NO: 38; and a light chain variable region containing the amino acid sequence of SEQ ID NO:

39.

16. A nucleic acid molecule comprising a nucleotide sequence encoding a protein complex according to any one of claims 9 to 15.

17. A recombinant vector comprising the nucleic acid molecule described in claim 16.

18. Isolated host cells comprising the recombinant vector according to claim 17.

19. A pharmaceutical composition for the prevention or treatment of cancer, comprising a protein complex according to any one of claims 9 to 15 and a pharmaceutically acceptable carrier.

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