Affibodies that specifically bind to CD137 and uses thereof

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 uses thereof. More specifically, the present invention 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 uses thereof for the prevention or treatment of cancer.

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

[0003] Among these, T cell-engaged bispecific antibodies such as blinatumomab and catumaxomab are currently the most advanced next-generation immune anti-cancer approaches. However, blinatumomab has only been used for hematological cancers and has safety issues, while catumaxomab is only applicable to the treatment of malignant ascites, limiting its use in the treatment of various cancers (Lakins MA, et al. Clin Cancer Res. 2020 Aug 1;26(15):4154-4167.).

[0004] Therefore, if a T cell-engaging bispecific antibody that can be applied to solid tumors in addition to blood cancers is developed, it is expected that it will be useful in the treatment of various cancers.

[0005] Numerous articles and patent documents are referenced throughout this specification and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly describe the state of the art 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 [Problem to be solved by the invention] The present inventors have made extensive research efforts to develop a T cell-engaging bispecific antibody applicable to both blood cancers and solid cancers. As a result, they developed an affibody that specifically binds to CD137, a costimulatory molecule whose expression increases on the surface of activated T cells, and used this to develop 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, thereby completing a T cell-engaging bispecific antibody applicable to both blood cancers and solid cancers.

[0008] It is therefore an object of the present invention to provide affibodies that specifically bind 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 antigen-binding fragment thereof that specifically binds to a protein on the surface of a cancer cell.

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

[0011] [Means for solving the problem] According to one aspect, the present invention provides an affibody that specifically binds to the extracellular domain of CD137, comprising an amino acid sequence shown in any one of SEQ ID NOs: 1 to 8.

[0012] The present inventors have conducted extensive research and efforts to develop a T cell-engaging bispecific antibody that can be used to treat both hematological cancers and solid cancers. As a result, they developed an affibody that specifically binds to CD137, a costimulatory molecule whose expression is increased on the surface of activated T cells, and used this to develop 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, biantibodies comprising the affibody of the present invention and an antibody against an antigen on the surface of cancer cells, i.e., T cell-engaging bispecific antibodies, can be useful for the prevention or treatment of hematological cancers and solid cancers.

[0013] As used herein, the term "CD137" refers to a member of the tumor necrosis factor receptor (TNFR) family, a costimulatory immune checkpoint molecule also known as TNFRSF9 (tumor necrosis factor receptor superfamily number 9), 4-1BB, and ILA (induced by lymphocyte activation).

[0014] CD137 (4-1BB) is a ubiquitous marker of 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 induces T cell activation, survival, and effector function upon binding to its ligand, 4-1BBL, a member of the TNF family that is expressed on mature DCs, activated B cells, and antigen-presenting cells (APCs) such as macrophages.

[0016] As used herein, the term "affibody" refers to the Z domain, which is the site of Staphylococcus aureus Protein A that has affinity for IgG (immunoglobulin G), and is also referred to as "Z body" or "Zb."

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

[0018] Furthermore, because affibodies have a very small molecular weight of 6 kDa, they diffuse throughout the body and are rapidly eliminated by renal filtration when administered to the human body, compared to IgG-type antibodies, which generally have a molecular weight of 150 kDa. Affibodies have also been developed in the form of biantibodies conjugated to standard IgG (Yu F et al., 2014, MAbs). Furthermore, affibodies are resistant to heat and alkaline conditions, and can be mass-produced using bacteria, making their production costs lower than antibodies.

[0019] An invention relating to a polypeptide scaffold based on first generation Z variants (variants of the Z domain) was previously disclosed in PCT Publication WO95 / 19374, and an invention relating to a polypeptide scaffold based on second generation Z variants was previously 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 the 58-residue immunoglobulin Fc-binding Z domain derived from Staphylococcus aureus protein A.

[0021] The 13 surface residues targeted by the 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 wild-type Z domains 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] As used herein, the term "AffiMab" refers to a bispecific antibody composed of an affibody molecule fused to the heavy or light chain of an antibody. Because AffiMab is modular, it can be easily applied to different targets (see Volk, A.L., Mebrahtu, A., Ko, B.K. et al. Drugs RD 21, 157-168, 2021). The protein complex, fusion protein, and biantibody of the present invention may be in the form of AffiMab.

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

[0024] In one embodiment of the present invention, the binding site of the affibody to the CD137 extracellular domain 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 64th to 95th amino acid residues of the CD137 extracellular domain of human (Homo sapiens) or cynomolgus monkey (Macaca fascicularis).

[0025] In another embodiment of the present invention, the affibody may be in the form of an affibody homodimer that specifically binds to the CD137 extracellular domain. The homodimer refers to a 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 to each other. The affibody homodimer may be in the form of a multimer in which affibody monomers are linked to each other via an amino acid linker. 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 chemistry methods, or indirectly, for example, via an amino acid linker.

[0026] In another embodiment of the present invention, the affibody that specifically binds to the CD137 extracellular domain may be in the form of a conjugate linked to gold nanoparticles, which may be used in nanoparticle-based immunoassays such as ELISA and immuno-PCR, which show high sensitivity and specificity for antigen-presenting cells (APCs) that express CD137, e.g., activated T cells.

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

[0028] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding an affibody according to an 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 CD137 extracellular domain.

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

[0031] In one embodiment of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding an affibody that specifically binds to the 64th to 95th amino acid residues of the amino acid sequence set forth in SEQ ID NO:27.

[0032] As used herein, the term "nucleic acid molecule" is intended to comprehensively encompass DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which the sugar or base moiety has been modified (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 is not limited to any particular nucleotide sequence, as long as it encodes the amino acid sequence that constitutes the affibody.

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

[0035] Considering the above-mentioned mutations having bioequivalent activity, the nucleic acid molecules of the present invention encoding the amino acid sequence constituting the affibody are also interpreted as including sequences showing substantial identity thereto.

[0036] The above-mentioned substantial identity means, when the above-mentioned sequence of the present invention is aligned with any other sequence so as to correspond as closely as possible, and the aligned sequences are analyzed using an algorithm commonly used in the art, at least 60% or more homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically 70% or more homology (e.g., 71%, 72%, 73%, 74%, 75%, "% homology" refers to sequences that exhibit 76%, 77%, 78%, or 79% homology, more particularly 80% or more homology (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), particularly 90% or more homology (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), and most particularly 95% or more homology (e.g., 95%, 96%, 97%, 98%, or 99%). All integers from 60% to 100% and decimals therebetween are included within the scope of the present invention in relation to percent homology.

[0037] Alignment methods for sequence comparison are well known in the art. 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 available from the National Center for Biological Information (NBCI) and can be used online in conjunction with sequence analysis programs such as blastp, blastn, blastx, tblastn, and tblastx. BLAST can be accessed through the BLAST page on the NCBI website. Methods for sequence homology comparison 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 constituting the affibody against CD137 of the present invention and the nucleotide sequence encoding the same are included in the sequence listing attached hereto.

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

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

[0041] Specifically, the present invention provides a recombinant vector comprising a nucleic acid molecule comprising 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, comprising 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 CD137 extracellular domain, comprising 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 CD137 extracellular domain, comprising the amino acid sequence set forth in SEQ ID NO: 27, which comprises the nucleic acid molecule described above.

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

[0046] As used herein, the term "vector" refers to a means for expressing a gene of interest in a host cell, and includes, but is not limited to, phagemid vectors; plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

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

[0048] As used herein, the term "operatively linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) with another nucleic acid sequence, such that the control sequence controls 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 for which are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

[0050] The vector of the present invention may typically be constructed as a vector for cloning or a vector for expression, and 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 a eukaryotic cell is used as the host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late 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 Rous sarcoma virus (RSV) promoter) may be used, which generally have a polyadenylation sequence as a transcription termination sequence.

[0052] The vectors of the present invention may be fused to other sequences to facilitate purification of the affibodies expressed therefrom, such as 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 contains an antibiotic resistance gene commonly used in the art as a selection marker, such as resistance genes to ampicillin, gentamicin, cavenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0054] Optionally, the vector can further carry genes encoding reporter molecules (eg, luciferase and glucuronidase).

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

[0056] Any host cell known in the art can be used as a host cell capable of stably and continuously cloning and expressing the vector of the present invention. For example, suitable eukaryotic host cells for the vector include, but are not limited to, yeast (Saccharomyces cerevisiae), insect cells, monkey kidney cells 7 (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, there is provided an isolated host cell comprising the above-described recombinant vector.

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

[0059] As used herein, the terms "transformed," "transduced," or "transfected" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transformed," "transduced," or "transfected" cell is a cell that has been transformed, transduced, or transfected with exogenous nucleic acid, including the cell and its progeny by subculture.

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

[0061] In the present invention, the recombinant vector injected into the host cell can express the recombinant affibody in the host cell, resulting in the production of a large amount of affibody. For example, if the expression vector contains a galactose-inducible promoter, the host cell can be treated with IPTG to induce gene expression.

[0062] The culture is typically performed under aerobic conditions, such as by shaking or rotating on a rotor. The culture temperature is preferably between 10°C and 40°C, and the culture time is generally between 5 hours and 7 days. The pH of the medium is preferably maintained between 3.0 and 9.0 during culture. The pH of the medium can be adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, or the like. Antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline can be added during culture, if necessary, for the maintenance and expression of the recombinant vector. When culturing host cells transformed with a recombinant expression vector having an inducible promoter, an appropriate inducer can be added to the medium as needed. For example, if the expression vector contains a lac promoter or a lactose-inducible promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) can be added to the medium, and if it contains a trp promoter, indoleacrylic acid can be added to the medium.

[0063] In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an affibody according to an embodiment and a pharmaceutically acceptable carrier.

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

[0065] Since the pharmaceutical composition of the present invention uses the above-mentioned affibody of the present invention that specifically binds to CD137 as an active ingredient, the common content between the two will be omitted to avoid overcomplicating this specification.

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

[0067] In one embodiment of the present invention, the solid cancer is a brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, brain meningioma, brain lymphoma, oligodendroma, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, breast tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, The cancer may be one or more 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, colon 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 organ cancer, female urethral cancer, and skin cancer, but is not necessarily limited to these.

[0068] In one embodiment of the present invention, the hematological cancer is acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, polycythemia vera, essential thrombocytosis, myelofibrosis, monocytic leukemia, erythroleukemia, megakaryoblastic leukemia, basophilic leukemia, eosinophilic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma, B-cell lymphoma, marginal zone B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma. , extranodal marginal zone lymphoma, extranodal NK / T-cell lymphoma, peripheral T-cell lymphoma, bucket lymphoma, precursor cell tumor, mantle cell lymphoma, follicular lymphoma, immune deficiency-associated lymphoproliferative disease, nodular lymphocyte-predominant Hodgkin's disease, nodular sclerosing classical Hodgkin's disease, lymphocyte-rich classical Hodgkin's disease, mixed cellularity classical Hodgkin's disease, lymphopenic classical Hodgkin's disease, and chemotherapy-resistant hairy cell leukemia, but are not necessarily limited to these.

[0069] Pharmaceutically acceptable carriers contained in the pharmaceutical compositions of the present invention are those commonly used in pharmaceutical formulations, including, but 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 ingredients, the pharmaceutical compositions of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0070] The pharmaceutical compositions of the present invention can be administered orally or parenterally, for example, by intravenous, subcutaneous, intramuscular, intraperitoneal, intrasternal, topical, intranasal, pulmonary, and rectal administration.

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

[0072] As used herein, the term "prophylaxis" refers to the prevention or protective treatment of a disease or disease state. As used herein, the term "treatment" refers to the reduction, suppression, amelioration, or eradication of a disease state.

[0073] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients by a method readily practiced by those skilled in the art to which this invention pertains, and may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may further contain a dispersing agent or stabilizer.

[0074] According to one aspect of the present invention, there is provided 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] As used herein, the term "protein complex" refers to a group of at least two related polypeptide chains.

[0076] As used herein, the term "cancer cell surface protein" refers to a protein embedded in or spanning a cell membrane layer. The cancer cell surface protein 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 present / expressed on the surface of the cancer cell.

[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 mutation in a wild-type protein. 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 present in or produced by 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 multimeric form in which a monomer of an affibody according to one embodiment and a monomer of each antibody or antigen-binding fragment thereof that specifically binds to a protein on the surface of a cancer cell are linked.

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

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

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

[0082] Thus, the affibody and the antibody or antigen-binding fragment thereof may be linked by chemical conjugation (known as organic chemistry methods) or other means (e.g., expressing the conjugate 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 connected by at least one linker, in which case the linker may have an amino acid sequence represented by the general formula (GnSm)p or (SmGn)p:

[0084] wherein n, m, and p are independently

[0085] n is an integer from 1 to 7;

[0086] m is an integer from 0 to 7;

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

[0088] p is an integer of 1 to 7.

[0089] According to another specific embodiment of the present invention, the linker has n=1 to 5 and m=0 to 5. In a more specific embodiment, n=4 and m=1. In an even more 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 polyclonal 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 polyclonal bispecific antibody.

[0097] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to a protein on the surface of a cancer cell may be a monoclonal antibody, a polyclonal antibody, scFv, Fab, F(ab), F(ab)2, scFv-Fc, a minibody, a diabody, a triabody, a tetrabody, a bispecific antibody, a trispecific 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 is fused to the heavy or light chain of the antibody or antigen-binding fragment thereof.

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

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

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

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

[0103] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to EGFR comprises:

[0104] (a) cetuximab; or

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

[0106] 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 thereof that specifically binds to human EGFR known in the art can be used without limitation.

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

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

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

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

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

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

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

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

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

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

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

[0118] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises:

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

[0120] (d) trastuzumab; or

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

[0122] 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 thereof that specifically binds to human HER2 known in the art can be used without limitation.

[0123] In one specific embodiment of the invention, the protein complex comprises 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 comprising the amino acid sequence of SEQ ID NO: 28; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29.

[0125] More specifically, a protein complex according to one embodiment of the present invention comprises an affibody that specifically binds to the extracellular domain of CD137, the affibody comprising 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 comprising the amino acid sequence of SEQ ID NO: 28; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29.

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

[0128] (d) Includes trastuzumab.

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

[0130] (d) Includes trastuzumab.

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

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

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

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

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

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

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

[0138] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof that specifically binds to CD19 comprises:

[0139] (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising 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 known in the art can be used without limitation.

[0142] In one specific embodiment of the invention, the protein complex comprises 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 comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39.

[0144] More specifically, a protein complex according to one embodiment of the present invention comprises an affibody that specifically binds to the extracellular domain of CD137, the affibody comprising 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 comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39.

[0146] In one specific embodiment of the invention, the protein complex comprises 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 embodiment of the present invention comprises an affibody that specifically binds to the extracellular domain of CD137, the affibody comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8; and

[0149] (g) Includes FMC63.

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

[0151] In one embodiment of the present invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex comprising 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 embodiment of the invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex comprising 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 embodiment of the invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex comprising 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 invention, the nucleic acid molecule comprises a nucleotide sequence encoding a protein complex comprising 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 is not limited to any particular nucleotide sequence as long as it encodes the amino acid sequence that constitutes the protein complex.

[0156] According to another aspect of the present invention, there is provided 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, the nucleic acid molecule encoding 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 cancer cells are operatively linked to a promoter.

[0158] According to one embodiment of the present invention, in the vector of the present invention, the nucleic acid molecule encoding 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 EGFR, HER2, or CD19 are operably linked to a promoter.

[0159] According to one embodiment 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 operably linked to a promoter.

[0160] According to another embodiment of the present invention, in the vector of the present invention, the nucleic acid molecule encoding cetuximab and an affibody that specifically binds to the extracellular domain of CD137 according to one embodiment is operably linked to a promoter.

[0161] According to yet another embodiment 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

[0162] A nucleic acid molecule encoding a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 17, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 20, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 22, is operably linked to a promoter.

[0163] According to yet another embodiment 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

[0164] A nucleic acid molecule encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 24 is operably linked to a promoter.

[0165] According to one embodiment 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 nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof that specifically binds to HER2 is operably linked to a promoter.

[0167] According to yet another embodiment 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

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

[0169] (d) trastuzumab; or

[0170] (e) A nucleic acid molecule encoding a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 30, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35, is operably linked to a promoter.

[0171] According to yet another embodiment 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

[0172] A nucleic acid molecule encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 is operably linked to a promoter.

[0173] According to another embodiment 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

[0174] The nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD19 is operably linked to a promoter.

[0175] According to yet another embodiment 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

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

[0177] (g) the nucleic acid molecule encoding FMC63 is operably linked to a promoter;

[0178] As used herein, the term "operatively linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or reading of the other nucleic acid sequence.

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

[0180] Typically, the vector of the present invention may be constructed as a vector for cloning or as a vector for expression, and 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 a eukaryotic cell is used as the host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late 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 Rous sarcoma virus (RSV) promoter) may be used, which generally have a polyadenylation sequence as a transcription termination sequence.

[0182] The vectors of the present invention may be fused with other sequences to facilitate purification of antibodies expressed therefrom, such as 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 any additional sequence for purification.

[0184] On the other hand, the expression vector of the present invention contains, as a selection marker, an antibiotic resistance gene commonly used in the art, such as resistance genes to ampicillin, gentamicin, cavenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0185] Optionally, the vector can further carry genes encoding reporter molecules (eg, luciferase and glucuronidase).

[0186] According to one embodiment of the present invention, the expression vector is a vector into which a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof against EGFR, HER2, or CD19 has been inserted, and is a recombinant vector for expression in a host cell, which is operatively linked to the nucleotide sequence of the nucleic acid molecule and comprises a promoter that causes the formation of an RNA molecule in a host cell and a polyA 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, there is provided an isolated host cell comprising the recombinant vector described above.

[0188] In one embodiment of the present invention, the isolated host cell is transformed with a recombinant vector of the present invention.

[0189] Any host cell known in the art can be used as a host cell in which the vector of the present invention can be stably and continuously cloned and expressed. For example, suitable eukaryotic host cells for the vector include, but are not limited to, yeast (Saccharomyce cerevisiae), insect cells, monkey kidney cells 7 (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 include cynomolgus monkey-derived fibroblasts (CYNOM-K1, Sigma-Aldrich), rhesus monkey-derived kidney cells (ATCC, LLC-MK2 Original CCL-7 TM , or ATCC, FRhK-4, CRL-1688 TM ), or rhesus monkey-derived lymphoblast cells (ATCC, LCL8664, CRL-1805 TM ) may be.

[0191] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a protein complex according to an embodiment and a pharmaceutically acceptable carrier.

[0192] Since the pharmaceutical composition of the present invention utilizes the above-mentioned protein complex as an active ingredient, the description of what is common to both of them will be omitted to avoid overcomplicating this specification.

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

[0194] In one embodiment of the present invention, the solid cancer is a brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, brain meningioma, brain lymphoma, oligodendroma, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, breast tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, The cancer may be one or more 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, colon 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 organ cancer, female urethral cancer, and skin cancer, but is not necessarily limited to these.

[0195] In one embodiment of the present invention, the hematological cancer is acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasm, polycythemia vera, essential thrombocytosis, myelofibrosis, monocytic leukemia, erythroleukemia, megakaryoblastic leukemia, basophilic leukemia, eosinophilic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma, B-cell lymphoma, marginal zone B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma. , extranodal marginal zone lymphoma, extranodal NK / T-cell lymphoma, peripheral T-cell lymphoma, bucket lymphoma, precursor cell tumor, mantle cell lymphoma, follicular lymphoma, immune deficiency-associated lymphoproliferative disease, nodular lymphocyte-predominant Hodgkin's disease, nodular sclerosing classical Hodgkin's disease, lymphocyte-rich classical Hodgkin's disease, mixed cellularity classical Hodgkin's disease, lymphopenic classical Hodgkin's disease, and chemotherapy-resistant hairy cell leukemia, but are not necessarily limited to these.

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

[0197] Specifically, since the protein complex of the present invention specifically binds to a protein on the surface of cancer cells and simultaneously specifically binds to CD137 on T cells, the protein complex acts as an agonist for T cells to induce T cell activation, thereby reducing the number of cancer cells or suppressing an increase in the number of cancer cells, and is therefore useful for the prevention or treatment of cancer. That is, the protein complex of the present invention induces T cell activation depending on the protein on the surface of cancer cells.

[0198] More specifically, since 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, the protein complex acts as an agonist for T cells to induce T cell activation, thereby reducing the number of cancer cells expressing EGFR or suppressing an increase in the number of cancer cells expressing EGFR, and is therefore useful for the prevention or treatment of cancer. That is, the protein complex of the present invention induces T cell activation in an EGFR-dependent manner.

[0199] The cancer may be EGFR positive.

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

[0201] The cancer may be, but is not necessarily limited to, non-small cell lung cancer (NSCLC), lung cancer, colorectal cancer, brain tumor, astrocytoma, esophageal cancer, cervical cancer, or synovial sarcoma.

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

[0203] More specifically, since 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, the protein complex acts as an agonist for T cells to induce T cell activation, thereby reducing the number of HER2-expressing cancer cells or suppressing an increase in the number of HER2-expressing cancer cells, and is therefore useful for the prevention or treatment of cancer. That is, the biantibody of the present invention induces T cell activation in a HER2-dependent manner.

[0204] The cancer may be HER2 positive.

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

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

[0207] More specifically, since 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, the protein complex acts as an agonist for T cells to induce T cell activation, thereby reducing the number of cancer cells expressing CD19 or suppressing an increase in the number of cancer cells expressing CD19, and is therefore useful for the prevention or treatment of cancer. That is, the protein complex of the present invention induces T cell activation in a CD19-dependent manner.

[0208] The cancer may be CD19 positive.

[0209] The 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 necessarily limited to, marginal zone lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), myeloid leukemia, or chemotherapy-resistant hairy cell leukemia.

[0211] In one embodiment of the present invention, activation of the T cells results in an increase in 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 embodiment of the present invention, the pharmaceutical composition of the present invention increases interferon gamma in an EGFR protein-dependent manner, i.e., when the composition contacts EGFR-positive or EGFR-overexpressing cancer cells, it activates T cells and increases the concentration of interferon gamma.

[0214] In another embodiment of the present invention, the pharmaceutical composition of the present invention increases interferon gamma in a HER2 protein-dependent manner, i.e., when the composition contacts HER2-positive or HER2-overexpressing cancer cells, it activates T cells and increases the concentration of interferon gamma.

[0215] In yet another embodiment of the present invention, the pharmaceutical composition of the present invention increases interferon gamma in a CD19 protein-dependent manner, i.e., when the composition contacts CD19-positive or CD19-overexpressing cancer cells, 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 embodiment of the present invention, the pharmaceutical composition of the present invention induces T cell activation in an EGFR protein-dependent manner, i.e., when the composition contacts EGFR-positive or EGFR-overexpressing cancer cells, it induces T cell activation.

[0218] In another embodiment of the present invention, the pharmaceutical composition of the present invention induces T cell activation in a HER2 protein-dependent manner, i.e., when the composition contacts HER2-positive or HER2-overexpressing cancer cells, it induces T cell activation.

[0219] In yet another embodiment of the present invention, the pharmaceutical composition of the present invention induces T cell activation in a CD19 protein-dependent manner, i.e., when the composition contacts CD19-positive or CD19-overexpressing cancer cells, it induces T cell activation.

[0220] Pharmaceutically acceptable carriers contained in the pharmaceutical compositions of the present invention are those commonly used in pharmaceutical formulations, including, but 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 ingredients, the pharmaceutical compositions of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0221] The pharmaceutical compositions of the present invention can be administered orally or parenterally, for example, by intravenous, subcutaneous, intramuscular, intraperitoneal, intrasternal, topical, intranasal, pulmonary, and rectal administration.

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

[0223] As used herein, the term "prophylaxis" refers to the prevention or protective treatment of a disease or disease state. As used herein, the term "treatment" refers to the reduction, suppression, amelioration, or eradication of a disease state.

[0224] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients in a manner easily practiced by those skilled in the art to which this invention pertains, and may be in the form of a solution, suspension, or emulsion in an oily or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may further contain a dispersing agent or stabilizer.

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

[0226] The present invention provides affibodies that specifically bind to the extracellular domain of CD137.

[0227] The present invention provides a protein complex comprising 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.

[0228] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above-described affibody or protein complex and a pharmaceutically acceptable carrier.

[0229] The affibodies of the present invention that specifically bind to CD137 can be used to develop protein complexes that specifically bind to T cells and also specifically bind to antigens on the surface of various cancer cells, and can be useful in the prevention or treatment of blood cancers and solid cancers.

[0230] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results of identifying affibodies that bind to CD137 protein in the form of periplasmic extracts.

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

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

[0233] FIG. 3A is a diagram confirming that a dual antibody based on the cetuximab (CET) antibody and composed of a CD137 affibody binds to the EGFR protein.

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

[0235] FIG. 3C is an illustration confirming that the dual antibody composed of the 15E3 antibody and the CD137 affibody binds to the EGFR protein.

[0236] FIG. 3D is an image confirming that the dual antibody composed of the 15E3 antibody and a CD137 affibody binds to the CD137 protein.

[0237] FIG. 4A shows that a dual antibody based on the cetuximab (CET) antibody and composed of a CD137 affibody binds to activated CEMT cells expressing CD137.

[0238] FIG. 4B shows that the dual antibody composed of the 15E3 antibody and a CD137 affibody binds to activated CEMT cells expressing CD137.

[0239] FIG. 5A shows that a dual antibody based on the cetuximab (CET) antibody and composed of a CD137 affibody binds to HT29 cells expressing EGFR.

[0240] FIG. 5B is an image confirming that the dual antibody composed of the 15E3 antibody and a CD137 affibody binds to HT29 cells expressing EGFR.

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

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

[0243] 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 different ECD domains 1 to 5. The amino acid sequence of human CD137 ECD composed of domains 1 to 5 is shown in SEQ ID NO: 27.

[0244] FIG. 7B is an illustration confirming that urelumab binds to domain 1 of CD137 ECD.

[0245] FIG. 7C is a diagram confirming that utomilumab binds to domain 3 of CD137 ECD.

[0246] FIG. 7D is a diagram confirming that cetuximab (negative Ab) does not bind to any domain of CD137 ECD.

[0247] FIG. 7E is an illustration confirming that affibody ZAAD01 binds to domain 3 of CD137 ECD.

[0248] FIG. 7F is an illustration confirming that affibody ZAAD05 binds to domain 3 of CD137 ECD.

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

[0250] FIG. 8A is a diagram confirming that affibody ZAAD01 specifically binds only to CD137.

[0251] FIG. 8B is a diagram confirming that affibody ZAA0D5 specifically binds only to CD137.

[0252] FIG. 9 shows the cross-species reactivity of the two selected CD137 affibodies with CD137 protein in humans, mice, and cynomolgus monkeys.

[0253] FIG. 9A is a diagram confirming that affibody ZAAD01 binds to human and cynomolgus monkey CD137 proteins.

[0254] FIG. 9B is a diagram confirming that affibody ZAAD05 binds to human and cynomolgus monkey CD137 proteins.

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

[0256] FIG. 11 shows the level of EGFR expression in each cell type of EGFR-expressing cells as determined by flow cytometry, and confirms that the level of T cell activation by the double antibody based on the level of EGFR expression correlates with the amount of EGFR expression.

[0257] FIG. 11A is a diagram confirming EGFR expression in EGFR-positive cells, A431 cells, HT29 cells, and SW403 cells, and EGFR-negative cells, MOLT4 cells.

[0258] FIG. 11B is a graph confirming the EGFR-dependent T cell activation of the 15E3-ZAAD01 bibody depending on the level of EGFR expression by IFN-gamma secretion analysis.

[0259] FIG. 12 is a diagram confirming that the dual antibodies produced using a CD137 affibody and an HER2 antibody or a CD19 antibody bind to CD137 and HER2 or CD19, respectively.

[0260] FIG. 12A is a diagram confirming that the trastuzumab-ZAAD01 dual antibody (anti-HER2 Ab1_ZAAD01) and the hz1E11.10-ZAAD01 dual antibody (anti-HER2 Ab2_ZAAD01) bind to CD137.

[0261] FIG. 12B is a diagram confirming that the trastuzumab-ZAAD01 dual antibody and the hz1E11.10-ZAAD01 dual antibody bind to HER2.

[0262] FIG. 12C is an illustration confirming that the FMC63-ZAAD01 double antibody (anti-CD19 Ab_ZAAD01) binds to CD137.

[0263] FIG. 12D is an image confirming that the FMC63-ZAAD01 dual antibody binds to CD19.

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

[0265] FIG. 13A is a diagram confirming, by IFN-gamma secretion analysis, that the trastuzumab-ZAAD01 biantibody and the hz1E11.10-ZAAD01 biantibody activated T cells in a HER2-dependent manner compared to utomirumab.

[0266] FIG. 13B is a diagram confirming that the FMC63-ZAAD01 dual antibody activates T cells in an EGFR-dependent manner compared to utomirumab, as determined by IFN-gamma secretion analysis.

[0267] FIG. 14A shows that activation of immune cells by dual antibodies (CET_ZAAD01 and CET_ZAAD05) results in the death of DLD1-luc cells.

[0268] FIG. 14B shows that activation of immune cells by dual antibodies (CET_ZAAD01 and CET_ZAAD05) increases IFN-gamma secretion.

[0269] FIG. 15 shows the survival benefit of the anti-cancer effect of dual antibodies (CET_ZAAD01 and CET_ZAAD05) in a humanized CD137 model.

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

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

[0272] FIG. 16C shows tumor size in the dual antibody (CET_ZAAD01) treatment group in the humanized CD137 model.

[0273] FIG. 16D shows tumor size in the dual antibody (CET_ZAAD05) treatment group in the humanized CD137 model.

[0274] [Mode for Carrying Out the Invention] The present invention will be described in more detail below with reference to examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0275] Example Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise specified.

[0276] Example 1. Affibody development against CD137

[0277] 1-1: Screening of 10 affibody clones that specifically bind to human CD137

[0278] Clones that specifically bind to CD137 were selected by panning from an affibody (Zb) library using CD137 protein. The selected clones were then produced as AffiMabs (double antibodies composed of monoclonal antibody-based affibodies) using animal cells, and clones that bind to CD137-expressing cells were selected.

[0279] Panning - affinity maturation

[0280] The affibody library was rescued in phage form using VSCM13 helper phage and used for panning. The number of library phages initially bound to the antigen was 10 13 Since the number of phage was more than 100, four rounds of panning were performed. As the number of panning rounds increased, the amount of antigen was gradually decreased (10 μg, 5 μg, 2 μg, and 1 μg), and the number of washes was gradually increased (3, 5, 7, and 10), to selectively select phages with high affinity. Binder phages obtained from each round of panning were infected into E. coli ER2537 (New England Biolabs® Inc.), and the resulting colonies were examined for antigen binding by ELISA.

[0281] Periplasmic extraction - Affibody isolation

[0282] Colonies obtained by infection with binder phage were inoculated into SB medium (MOPS 10 g / L, Bacto yeast extract 20 g / L, and tryptone 30 g / L), and then the OD 600 After culturing until the optical density at 600 nm reached 0.8, 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside, LPS Solutions) was added and the cells were incubated with shaking at 30°C to allow overexpression of affibody (Zb). Periplasmic extraction (PE) of affibody was performed using BBS buffer (200 mM boric acid, 150 mM NaCl, and 1 mM EDTA). Binders were screened using ELISA.

[0283] ELISA Method - Binder Screening

[0284] ELISA was performed by treating plates coated with hCD137-ECD-Fc (human CD137-extracellular domain-fragment crystallizable region) protein at a concentration of 2 μg / mL with affibody periplasmic extracts and then treating with a secondary antibody (anti-HA-HRP, Roche, 12013819001, human influenza hemagglutinin (HA) protein), followed by TMB (3,3',5,5'-tetramethylbenzidine, BioFX). TM The color reaction was developed using an ELISA reader (Victor X3, PerkinElmer, Inc.), and the OD was measured. 450 The values ​​were measured, and the results are shown in Figure 1. For controls, plates coated with mouse CD137 ECD Fc protein (mouse CD137) or no protein (negative protein) were also used. Negative PE was an extract of E. coli ER2537 without the affibody plasmid.

[0285] ELISA results - 10 affibody clones selected

[0286] As shown in Figure 1, in negative PE, OD 450 Almost no values ​​were measured, and the OD 450 Almost no OD values ​​were measured, whereas none of the 10 affibodies bound to it, but a high OD 450 The values ​​confirmed that all 10 affibodies bound to human CD137 protein.

[0287] Based on the results of the ELISA, 10 affibodies, ZAAD01 to ZAAD10, that bind to human CD137 protein were selected. After confirming the affibody sequences, 10 unique clones (cloned specific cell lines) that bind to human CD137 were identified.

[0288] 1-2: Selection of 8 types of affibodies

[0289] The 10 unique clones described above were cloned as Zb-Fc and produced in mammalian cells. Cell binding (fusion) was then 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 10 mL of each sample was prepared and treated with 50 ng / mL of PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich®, P1585) and 1 μg / mL of ionomycin (Sigma-Aldrich®, I9657), followed by 16 hours of incubation for activation.

[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 activating T cells, thereby inducing 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, while unstimulated naive T cells do not express CD137, stimulated activated T cells are known to transiently express CD137 at high levels.

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

[0294] The cells were then treated with 1 μg / mL of anti-human Fc-FITC (Life Technologies®, A11013) and incubated on ice for 45 minutes in the dark. After washing the cells three times with 200 μL of 5% FBS in PBS by centrifugation at 1,200 rpm for 3 minutes, the fluorescence intensity was measured using a Beckman Coulter (Beckman Coulter, Inc.) FACS (Fluorescence-Activated Cell Sorting) instrument. The results are shown in Figure 2. In the FACS graph, the peak shifts to the right, indicating stronger binding to the cells. FITC only represents the case where only buffer was present during Zb-Fc binding, and negative Ab represents the case where hIgG (human IgG) was used.

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

[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 FITC peak in activated CEMT cells, confirming that the eight affibodies did not bind to non-activated CEMT cells but did bind to activated CEMT cells.

[0297] However, affibodies ZAAD06 and ZAAD08 were not selected because there was no difference in peak activity between inactivated and activated CEMT cells compared to the control group (negative Ab) to which hIgG was added, and they do not specifically bind to cells expressing CD137, i.e., activated CEMT cells.

[0298] Example 2. Affibody selection against CD137

[0299] 2-1: Production of 16 types of double antibodies

[0300] Based on two antibodies, cetuximab (CET) and the proprietary 15E3 antibody (see Korean Patent Publication No. 10-2017-0012754), which specifically bind to EGFR (Epidermal Growth Factor Receptor), the 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 were cloned in the form of a dual antibody with cetuximab (CET), they were designated 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 dual antibody with the 15E3 antibody, they were designated 15E3-ZAAD01, 15E3-ZAAD02, 15E3-ZAAD03, 15E3-ZAAD04, 15E3-ZAAD05, 15E3-ZAAD07, 15E3-ZAAD09, and 15E3-ZAAD010, respectively.

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

[0302] 2-2: Confirmation of binding of dual antibodies to CD137 and EGFR proteins

[0303] Using purified forms of the dual antibody, we screened for CD137 affibodies that bind to both EGFR and CD137 proteins by protein binding, expressing cell binding, and T cell activation tests.

[0304] The binding of each of the 16 double antibodies (eight Cetuximab-Zb and eight 15E3-Zb) produced in animal cells to the CD137 and EGFR proteins was confirmed by ELISA. For ELISA, the purified double antibodies were applied to plates coated with hCD137-ECD-his and hEGFR-ECD-his proteins at a concentration of 2 μg / mL, respectively, at 7 dilutions starting from 60 nM and diluted 1 / 5. After treatment with a secondary antibody (Goat anti-human IgG-F(ab')2-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097), the antibodies were incubated with TMB (BioFX). TM The color reaction was developed using an ELISA reader (Victor X3, PerkinElmer, Inc.), and the OD was measured using an ELISA reader (Victor X3, PerkinElmer, Inc.). 450 The EC values ​​were measured using GraphPad Prism software. 50 The half maximal effective concentration (HALC) values ​​were calculated, and the results are shown in FIGS. 3A to 3D.

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

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

[0307] 2-3: Confirmation of binding of the double antibody format to CD137-expressing cells

[0308] To confirm the binding of the 16 dual antibody forms to CD137-expressing cells, 1 × 10 CEMT cells were 7 The cells were treated with 50 ng / mL PMA and 1 μg / mL ionomycin, and then incubated for 16 hours for activation. 5 × 10 activated CEMT cells were cultured in a 5 × 10 5 After preparation in a tube, cells were collected by centrifugation at 1,200 rpm for 3 minutes. After washing with 5% FBS in PBS, the cells were treated with 5 μg / mL of double antibody and incubated on ice for 1 hour. The cells were washed three times with 200 μL of 5% FBS in PBS by centrifugation at 1,200 rpm for 3 minutes. Anti-human Fc-FITC (Life Technologies®, A11013) was then treated at 1 μg / mL and incubated on ice for 45 minutes in the dark. The cells were washed three times with 200 μL of 5% FBS in PBS by centrifugation at 1,200 rpm for 3 minutes. The fluorescence intensity was measured using a Beckman Coulter FACS instrument, and the results are shown in Figures 4A and 4B. Utomilumab was used as a positive control, and human IgG (hIgG) was used as a negative control.

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

[0310] Figure 4B shows the FACS results for the 15E3-based CD137 affibody biantibodies, which showed 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 above results, it was confirmed that all 16 types of bibodies of the present invention specifically bind to cells expressing CD137.

[0312] 2-4: Confirmation of binding of the double antibody to EGFR-expressing cells

[0313] Similarly, to confirm the binding of the 16 types of dual antibody forms in EGFR-expressing cells, HT29 cells (Korean Cell Line Bank, KCLB, 30038, human colon cancer cell line) known to exhibit high EGFR expression were cultured at 5 × 10 5 After preparing the cells in a tube, the cells were collected by centrifugation at 1,200 rpm for 3 minutes. After washing with 5% FBS in PBS, the cells were treated with 5 μg / mL of the 16 dual antibodies and incubated on ice for 1 hour. The cells were washed three times with 200 μL of 5% FBS in PBS by centrifugation at 1,200 rpm for 3 minutes. The cells were then treated with 1 μg / mL of anti-human-Fc-FITC (Life Technologies A11013) and incubated on ice for 45 minutes in the dark. The cells were washed three times with 200 μL of 5% FBS in PBS by centrifugation at 1,200 rpm for 3 minutes. Fluorescence intensity was measured using a Beckman Coulter FACS instrument, and the results are shown in Figures 5A and 5B. In addition, as a positive control group (Positive Ab), CET-IgG form was used in the CET-based evaluation, and 15E3-IgG form was used in the 15E3-based evaluation, and hIgG was used as a negative control group.

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

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

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

[0317] 2-5: EGFR-dependent T cell activation test - Affibodies ZAAD01 and ZAAD05 selected

[0318] A total of 16 dual antibodies were produced and subjected to affibody selection using an EGFR-dependent T cell activation test.

[0319] Testing Methodology

[0320] One day before the test, anti-CD3 antibody (Invitrogen) was administered at a concentration of 3 μg / mL. TM50 μL of the antibody (16-0037-85) was coated onto a 96-well round plate. The next day, the plate was washed three times with 100 μL of sterile PBS. 50 μL of 5 μg / mL hEGFR-ECD-Fc was again coated onto the plate. Control wells were treated with PBS alone and incubated at 37°C for 3 hours. The plate was washed three times with 100 μL of R10 medium (RPMI 1640, 10% FBS, and 10 mM HEPES), and then blocked with 100 μL of R10 medium at 37°C for 1 hour. The R10 medium was completely removed from the plate, and the plate was treated with 50 μL of 16 dual antibodies (8 Cetuximab-Zb and 8 15E3-Zb) at 0.1 nM or 1 nM for 30 minutes.

[0321] Meanwhile, blood from two donors was diluted 1 / 2 with PBS and placed in a Leucosep tube (Greiner Bio-One, 227290) containing floating Ficoll (Ficoll®, GE Healthcare, 17-1440-12), followed by centrifugation at 1,000 g for 30 minutes. Only the layer of peripheral blood mononuclear cells (PBMCs) located on top of the Ficoll was collected. After two washes using 2% PBMCs in PBS and centrifugation at 300 g for 10 minutes, the number of PBMCs was measured. PBMCs were then counted as CD8 + T cell isolation kit (CD8 + CD8 T cell isolation kit (Miltenyi Biotec Inc., 130-096-495) was used according to the protocol. + T cells were prepared.

[0322] CD8 + The number of T cells was measured to be 1.4 × 10 6The solution was dissolved in R10 medium at a concentration of 1 / mL, and 50 μL of each solution was applied to the plates treated with the 16 types of double antibodies. Three days after treatment, the plates were centrifuged at 2,000 rpm for 10 minutes, and the supernatant was collected and used to measure interferon gamma (IFN-gamma, IFN-γ) levels according to the protocol of the human interferon gamma ELISA kit (IFN-gamma ELISA set, BD Biosciences, 555142). The results are shown in Figure 6.

[0323] Test Results

[0324] As shown in Figure 6, the degree of T cell activation in the presence or absence of EGFR was confirmed by measuring IFN-γ in a T cell activation assay. The results showed that treatment with the CET-based ZAAD01 affibody bibody, the CET-based ZAAD05 affibody bibody, the 15E3-based ZAAD01 affibody bibody, and the 15E3-based ZAAD05 affibody bibody at 1 nM significantly increased IFN-γ levels. Therefore, among the eight affibodies, the affibodies ZAAD01 and ZAAD05, which increased IFN-γ levels, were selected.

[0325] Specifically, in the EGFR-dependent T cell activation test, Cetuximab-Zb or 15E3-Zb binds to hEGFR-ECD-Fc coated on a plate, and CD8 expressing CD3 is bound to anti-CD3 antibody coated on the plate. + T cells bind to the hEGFR-ECD-Fc-bound Cetuximab-Zb or 15E3-Zb affibody, which binds to the anti-CD3 antibody. + By binding to CD137 on the surface of T cells, CD8 + T cells are activated and activated CD8 + It is possible to measure IFN-γ secreted by T cells. It is known that T cells are activated by the agonism of CD137.

[0326] The affibodies ZAAD01 and ZAAD05 of the present invention bind to CD8 + It activates CD137 by binding to the CD137 ECD on the surface of T cells and binds to CD8 + T cells were made to secrete IFN-γ.

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

[0328] Based on the above results, the inventors confirmed that the dual antibodies Cetuximab-ZAAD01, 15E3-ZAAD01, Cetuximab-ZAAD01, and 15E3-ZAAD01 activate T cells in an EGFR-dependent manner, and final selection was made of affibodies ZAAD01 and ZAAD05.

[0329] Example 3. Characterization of selected affibodies

[0330] 3-1: Confirmation of affibody binding properties using CD137 ECD domain mapping

[0331] The binding characteristics of the two selected affibodies, ZAAD01 and ZAAD05, were confirmed by CD137 domain mapping. The 15E3-based ZAAD01 and ZAAD05 affibody biantibodies are hereafter referred to as 15E3ZAAD01 and 15E3ZAAD05, respectively. The binding domains of the CD137-specific monoclonal antibodies 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, we divided the CD137 ECD into five domains and cloned domain mutants. A schematic diagram of the domain mutants is shown in Figure 7A.

[0333] As shown in Figure 7A, the cloned domain mutants included a signal peptide and different combinations of ECD domains 1 to 5, specifically: hCD137-ECD, which contains the signal peptide and all of domains 1 to 5; hCD137-ECD-ΔD1, which lacks domain 1; hCD137-ECD-ΔD12, which lacks domains 1 and 2; hCD137-ECD-ΔD123p, which lacks all of domains 1 and 2 and part of domain 3 (deleted up to the 104th amino acid); hCD137-ECD-ΔD123, which lacks domains 1 to 3; and hCD137-ECD-ΔD3 and hCD137-ECD-ΔD5, which lack domain 5.

[0334] FIG. 7A is a schematic diagram of hCD137 ECD containing a signal peptide and domains 1 to 5, and SEQ ID NO: 27 shows the amino acid sequences for domains 1 to 5, excluding the amino acid sequence for the signal peptide.

[0335] The cloned domain mutants were produced using animal cells. Purified urelumab, utomilumab, 15E3, 15E3ZAAD01, and 15E3ZAAD05 were treated at 10 μg / mL, 2 μg / mL, and 0.4 μg / mL, respectively, on a plate coated with the produced domain mutants at a concentration of 2 μg / mL. After treatment with a secondary antibody (anti-hIgG-Fab-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097), TMB (BioFX) was added. TMThe color reaction was developed using an ELISA reader (Victor X3, PerkinElmer, Inc.), and the OD was measured. 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 hCD137ECD ΔD1, hCD137ECD ΔD12, hCD137ECD ΔD123P, and hCD137ECD ΔD123 among the domain mutations, confirming that urelumab binds to domain 1 of CD137 ECD.

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

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

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

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

[0341] From the above results, the present 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 selected affibodies and TNFRSF

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

[0344] Plates coated with TNFRSF proteins CD137 (TNFRSF9), CD27 (TNFRSF7), CD30 (TNFRSF8), CD134 (TNFRSF4), CD270 (TNFRSF14), CD357 (TNFRSF18), and TNF-alpha protein 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) followed by TMB (BioFX). TM The color reaction was developed using an ELISA reader (Victor X3, PerkinElmer, Inc.) and the OD was measured. 450 The values ​​were measured and the results are shown in Figures 8A and 8B.

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

[0346] From the above results, the present 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-reactivity of selected affibodies

[0348] The interspecies cross-reactivity of the 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 5 μg / mL, and then treated with a secondary antibody (anti-hIgG-Fab-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097) and TMB (BioFX). TM The color reaction was developed using an ELISA reader (Victor X3, PerkinElmer, Inc.) and the OD was measured. 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 above results confirmed that both of the selected CD137 affibodies exhibited species cross-reactivity with CD137 protein in humans and cynomolgus monkeys.

[0352] Example 4. Confirmation of target protein binding and EGFR-dependent efficacy of dual antibodies

[0353] 4.1: Check whether the dual antibody binds to both targets simultaneously

[0354] We used BLI (BioLayer Interferometry) to confirm whether four dual antibodies, CET-ZAAD01, CET-ZAAD05, 15E3-ZAAD01, and 15E3-ZAAD05, composed of cetuximab and 15E3, antibodies that bind to EGFR, and ZAAD01 and ZAAD05, affibodies that bind to CD137, simultaneously bind to their respective target proteins.

[0355] EGFR-ECD-Fc protein was immobilized at a concentration of 5 μg / mL on an AR2G sensor chip (Amine Reactive Second-Generation Biosensor, SATORIUS Co., Ltd., 18-5092) using the amine coupling method with EDC / NHS. The four biantibodies were bound to the sensor chip with EGFR-ECD-Fc immobilized thereon at a concentration of 10 μg / mL for 5 minutes and then allowed to stabilize for 5 minutes. CD137-ECD-his protein was added at a concentration of 30 μg / mL to the sensor chip with the biantibodies bound to the immobilized EGFR protein, allowed to bind for 5 minutes, and then allowed to stabilize for 5 minutes. The results are shown in Figures 10A to 10D. The results for the control groups treated with CET or 15E3 are shown in red, and the results for the four biantibodies of the present invention are shown in blue.

[0356] As shown in Figure 10A, when CD137-ECD-Fc protein was added to the sensor chip after CET treatment as a control, the binding signal remained unchanged (red line). However, when CD137-ECD-Fc protein was added to the sensor chip after CET-ZAAD01 dual antibody treatment, the binding signal increased (blue line). This indicates that the CET-ZAAD01 dual antibody simultaneously binds to the EGFR protein immobilized on the sensor chip in advance and the CD137-ECD-Fc protein added later.

[0357] Similar to the results for the CET-ZAAD01 double antibody, the CET-ZAAD05 double antibody (Figure 10B), 15E3-ZAAD01 double antibody (Figure 10C), and 15E3-ZAAD05 double antibody (Figure 10D) also showed increased binding signals when CD137-ECD-his protein was added to the sensor chip, confirming that these double antibodies also simultaneously bind to EGFR protein and CD137 protein.

[0358] 4.2: Dual antibody T cell activation according to EGFR expression level

[0359] We investigated whether a dual antibody 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 in 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), and EGFR-negative cells, MOLT4 (KCLB, 21582, adult T acute lymphoblastic leukemia), were cultured at 5 × 10 5 / Prepared in a tube.

[0362] The cells were then collected by centrifugation at 1,200 rpm for 3 minutes, washed with 5% FBS in PBS, and treated with 5 μg / mL of 15E3ZAAD01 bilayer antibody. The cells were then incubated on ice for 1 hour. The cells were washed three times with 200 μL of 5% FBS in PBS by centrifugation at 1,200 rpm for 3 minutes. The cells were then treated with 1 μg / mL of anti-human Fc FITC and incubated on ice for 45 minutes in the dark. The cells were washed three times with 200 μL of 5% FBS in PBS 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) values, and the results are shown in Figure 11A. The MFI value increased with increasing cell binding of the 15E3ZAAD01 bilayer antibody, indicating high EGFR expression levels.

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

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

[0365] T cell assay was performed using the cells with different levels of EGFR expression. The day before, anti-CD3 antibody (Invitrogen) was added at a concentration of 3 μg / mL. TM 50 μL of the PBS solution (16-0037-85) was coated onto a 96-well round plate. The next day, the plate was washed three times with 100 μL of sterilized PBS. The plate was then washed three times with 100 μL of R10 medium, and blocked with 100 μL of R10 medium at 37°C for 1 hour. Four cell lines, A431, H29, SW403, and MOLT4, were plated at 2.4 × 10 550 μL of 15E3ZAAD01 double antibody was added at a concentration of 5 μg / mL for 30 minutes.

[0366] Meanwhile, blood from two donors was diluted 1 / 2 with PBS, placed in Leucosep tubes (Greiner Bio-One LLC, 227290) containing Ficoll (FicollGE Healthcare, 17-1440-12), and centrifuged at 1,000 g for 30 minutes.

[0367] Only the layer of PBMCs located on the Ficoll was collected. After two washes with 2% PBMCs in PBS and centrifugation at 300 g for 10 minutes, the number of PBMCs was determined. PBMCs were then isolated as CD8 + T cell isolation kit (CD8 + CD8 T cell isolation kit (Miltenyi Biotec Inc., 130-096-495) was used according to the protocol. + T cells were prepared.

[0368] CD8 + The number of T cells was measured to be 1.4 × 10 6 The antibody was dissolved in R10 medium at a concentration of 1 / mL and then applied to the 15E3ZAAD01 biantibody-treated plate in a volume of 50 μL. Three days after treatment, the plate was centrifuged at 2,000 rpm for 10 minutes to collect the supernatant, which was then used to measure the IFN-γ concentration according to the protocol of the IFN-γ measurement kit (BD Biosciences, 555142). Whether the biantibody of the present invention activates T cells through EGFR expression levels was confirmed by measuring the amount of IFN-γ secreted by T cells, and the results are shown in Figure 11B.

[0369] As shown in FIG. 11B, T cell activation analysis confirmed that increased EGFR expression increased T cell activity, as the concentration of IFN-γ increased with increasing EGFR expression.

[0370] Specifically, the order of increasing IFN-γ secretion corresponded to the order of increasing EGFR expression levels described above: the 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 the IFN-γ concentrations increased in this order.

[0371] From the above results, the inventors confirmed that the EGFR-dependent T cell activation of the 15E3-ZAAD01 bibody is dependent on the level of EGFR expression, indicating that the degree of T cell activation by the bibody is correlated with the amount of EGFR expression.

[0372] When using the EGFR antibody-based CD137 affibody biantibody of the present invention, CD137-expressing T cells can be located in close proximity to, bind to, and be activated by cancer cells and tissues that overexpress EGFR, making it useful for the prevention or treatment of various types of cancer that overexpress EGFR.

[0373] Example 5. Development Potential of CD137 Affibody-Based Dual Antibody Targeting Various Antigens—HER2 and CD19

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

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

[0376] The selected CD137 affibody and antibodies that bind to HER2 and CD19, proteins other than EGFR, were cloned into a doublet using ZAAD01. The doublets composed of a HER2 antibody and a CD137 affibody were designated anti-HER2 Ab1-ZAAD01 and anti-HER2 Ab2-ZAAD01, respectively, and the doublet composed of a CD19 antibody and a CD137 affibody was designated 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 the independently developed hz1E11.10 antibody, which specifically binds to human HER2 (see Korean Patent Publication No. 10-2017-0117330).

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

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

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

[0381] The above-described biantibodies were produced using animal cells, and protein binding and T cell activation tests were performed in the form of purified biantibodies to determine whether the CD137 affibodies of the present invention could be developed and used in a modular fashion as biantibodies targeting various other antigens.

[0382] ELISA was performed on a plate coated with CD137 protein at a concentration of 2 μg / mL and hHer2-ECD-Fc protein and hCD19-ECD-Fc protein, which correspond to the target proteins of each antibody. The purified double antibodies were treated at 1 / 5 dilutions starting from 60 nM for 7 points. After treatment with a secondary antibody (anti-hIgG-Fab-HRP, Jackson ImmunoResearch Laboratories, Inc., JAC-109-035-097), TMB (BioFX) was added. TM The color reaction was developed using an ELISA reader (Victor X3, PerkinElmer, Inc.), and the OD was measured. 450 The values ​​were measured, and the results are shown in Figures 12A to 12D.

[0383] As shown in Figures 12A to 12D, the dual antibodies produced using the CD137 affibody and the HER2 antibody or 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 dual antibodies, composed of an anti-HER2 antibody and a ZAAD01 affibody, showed concentration-dependent increases 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 double antibody, composed of an anti-CD19 antibody and a ZAAD01 affibody, to its target proteins, CD137 (Figure 12C) and CD19 (Figure 12D), respectively, increased in a concentration-dependent manner.

[0386] The above results confirmed that the dual antibodies anti-HER2 Ab1-ZAAD01 and anti-HER2 Ab2-ZAAD01 of the present invention bind to both CD137 and HER2 proteins, and that the dual antibody anti-CD19 Ab-ZAAD01 of the present invention binds to both CD137 and CD19 proteins.

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

[0388] As described above, the produced dual antibodies were confirmed to bind to each target protein, and the efficacy of the dual antibodies was confirmed by performing expanded T cell activation tests dependent on other proteins, namely HER2 or CD19.

[0389] Testing Methodology

[0390] One day before the treatment, 50 μL of anti-CD3 antibody (Invitrogen, 16-0037-85) was coated onto a 96-well round plate at a concentration of 3 μg / mL. The following day, the plate was washed three times with 100 μL of sterile PBS. 50 μL of HER2 or CD19 was again coated onto the plate at a concentration of 5 μg / mL. Control wells (no HER2 or CD19 treatment) were treated with PBS alone and then incubated at 37°C for 3 hours. The plate was washed three times with 100 μL of R10 medium, and then blocked with 100 μL of R10 medium at 37°C for 1 hour. The R10 medium was completely removed from the plate, and the plate was treated with 50 μL of the dual antibody at a concentration of 5 μg / mL for 30 minutes.

[0391] Meanwhile, blood from two donors was diluted 1 / 2 with PBS, placed in Leucosep tubes (Greiner Bio-One, 227290) containing Ficoll (FicollGE Healthcare, 17-1440-12), and centrifuged at 1,000 g for 30 minutes. Only the PBMC layer located on top of the Ficoll was collected. After two washes using 2% PBMC in PBS and centrifugation at 300 g for 10 minutes, the number of PBMCs was measured.

[0392] PBMCs to CD8 + T cell isolation kit (CD8 + CD8 T cell isolation kit (Miltenyi Biotec Inc., 130-096-495) was used according to the protocol. + T cells were prepared. CD8 + The number of T cells was measured to be 1.4 × 10 6 The solution was dissolved in R10 medium at a concentration of 1 / mL and then applied to the double antibody-treated plate in a volume of 50 μL. Three days after treatment, the plate was centrifuged at 2,000 rpm for 10 minutes to collect the supernatant, which was used to measure the IFN-γ concentration according to the protocol of the IFN-γ measurement kit (BD Biosciences, 555142). The results are shown in Figures 13A and 13B. Utomilumab, a monoclonal antibody against CD137, was used as a negative control.

[0393] Test Results

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

[0395] Specifically, in Figure 13A, IFN-γ was upregulated in CD8 cells treated with utomirumab, a monoclonal antibody against CD137, regardless of the presence or absence of HER2. + When treated with the dual antibody anti-HER2 Ab1-ZAAD01 (trastuzumab-ZAAD01) and anti-HER2 Ab2-ZAAD01 (hz1E11.10-ZAAD01), the CD8 + T cells showed significantly increased IFN-γ secretion.

[0396] In addition, in Figure 13B, when utomirumab, a monoclonal antibody against CD137, was treated, the CD8 + There was no significant difference in the amount of IFN-γ secreted by T cells. Treatment with the double antibody anti-CD19 Ab-ZAAD01 (FMC63-ZAAD01) resulted in a significant increase in CD8 T cells secreted by HER2 (CD19) compared to treatment without CD19 (no CD19). + T cells showed significantly increased IFN-γ secretion.

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

[0398] In particular, these results confirmed the presence or absence of T cell activation by the expanded target proteins HER2 or CD19 through the measurement of IFN-γ, and suggest that when the CD137 affibody of the present invention is used in a modular format, it can be applied to various target proteins different from the target proteins disclosed in the present invention, such as EGFR, HER2, and CD19.

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

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

[0401] Example 6. Confirmation of T cell activation and cell killing by EGFR-CD137 targeting dual antibody

[0402] The present inventors conducted the following tests to confirm the T cell activation and cell killing effects of a dual antibody that simultaneously targets EGFR and CD137. The dual antibodies 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] Anti-CD3 antibody (Invitrogen, 16-0037-85) was coated onto a 96-well round plate at a concentration of 5 μg / mL, 50 μL per well, one day before double antibody treatment. The next day, 100 μL of R10 medium was added, followed by blocking by incubation at 37°C for 1 hour. DLD-1-Luc cells were plated at 1.0x10 550 μL of each antibody was added to each well to achieve a concentration of 1.0 × 10 / mL. Then, 50 μL of double antibodies (CET_ZAAD01 and CET-ZAAD05), diluted 1 / 10 starting from 10 nM, were added per well. Meanwhile, donor blood was diluted 1 / 2 with PBS and placed in a Leucosep tube (greiner bio-one, 227290) containing Ficoll (GE Healthcare, 17-1440-12). The tube was centrifuged at 1000 × g for 30 minutes. After isolating the PBMC layer on top of the Ficoll, 10 mL of PBMC was mixed with 40 mL of 2% FBS-containing PBS and centrifuged twice at 300 × g for 10 minutes. The number of PBMCs was then counted. The PBMCs were diluted to 1.0 × 10 5 The cells were dissolved in R10 medium at a concentration of 1 / mL and then 50 μL of the solution was applied to the double antibody-treated plate. Three days after treatment, the plate was centrifuged at 2000 RPM for 10 minutes to remove the supernatant, which was then used to measure IFN-gamma levels using an IFN-gamma measurement kit (BD, 555142). 50 μL of lysis buffer (75 mM Tris (pH 8.0), 30% glycerol, 3% Triton X-100) was added to the well containing the remaining cells and medium, mixed well, and 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 mixed with an equal amount of 5'-fluoroluciferin (Bio-Glo TM Luciferase Assay System (promea / G7940) was added and left to stand at room temperature for 15 minutes, after which 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 dual antibody resulted in the death of DLD1-Luc cells and an increase in the concentration of IFN-gamma.

[0406] Example 7: Confirmation of anti-cancer efficacy of EGFR-CD137 targeting dual antibody using animal models

[0407] Using a humanized CD137 mouse model, we confirmed the efficacy of dual antibodies targeting EGFR and CD137 (CET_ZAAD01 and CET-ZAAD05).

[0408] EGFR / MC38 cells, 5.0x10 5 The tumors were prepared in PBS at a concentration of 1 / mL and injected subcutaneously into the flanks of humanized CD137 mice to artificially induce tumor formation. 3 When tumor size reached 2500mm, the animals were randomly grouped into groups of 5 as designed. Cetuximab or EGFR-CD137 targeting dual antibodies (CET_ZAAD01 and CET-ZAAD05) were diluted with PBS to a concentration of 5mpk and administered intraperitoneally twice a week for a total of 5 doses. The size and weight of the tumor were measured 3 times a week, and tumor size was measured using a digital Vernier caliper. When tumor size reached 2500mm, tumor size increased to 2500mm. 3 When it exceeded this limit, the animals were euthanized.

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

[0410] As shown in FIG. 15, the two dual antibodies of the present invention (CET_ZAAD01 and CET-ZAAD05) showed a survival-prolonging effect in mice.

[0411] Furthermore, as shown in Figures 16A to 16D, it was confirmed that the double antibody of the present invention reduced the size of the cancer.

[0412] In contrast, the vehicle and cetuximab treatment groups showed a tendency for the tumor size to continue to increase, while the two types of double antibodies (CET_ZAAD01 and CET-ZAAD05) were found to suppress the tumor size and kill it.

[0413] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. [Brief explanation of the drawings]

[0414] [Figure 1] 1 shows the results of confirming that affibodies that bind to CD137 protein are in the form of periplasmic extracts. [Figure 2] Based on the form of the antibody-affibody that binds to CD137, the results of confirming the binding of affibodies to CD137 expressed on cells were obtained using activated CEMT cells, which are CD137-positive cells. [Figure 3A] Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on two EGFR-binding antibodies (CET or 15E3) binds to both CD137 and EGFR proteins. Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on the cetuximab (CET) antibody binds to EGFR protein. [Figure 3B] Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on two EGFR-binding antibodies (CET or 15E3) binds to both CD137 and EGFR proteins after producing the bibody. Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on the cetuximab (CET) antibody binds to CD137 protein. [Figure 3C] Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on two EGFR-binding antibodies (CET or 15E3) binds to both CD137 and EGFR proteins after producing the bibody. Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on the 15E3 antibody binds to EGFR protein. [Figure 3D] Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on two EGFR-binding antibodies (CET or 15E3) binds to both CD137 and EGFR proteins after producing the bibody. Figure 1 shows the results of testing whether a bibody composed of a CD137 affibody based on the 15E3 antibody binds to CD137 protein. [Figure 4A] This figure confirms that a dual antibody based on the cetuximab (CET) antibody and composed of a CD137 affibody binds to activated CEMT cells that express CD137. [Figure 4B] This figure confirms that a dual antibody based on the 15E3 antibody and composed of a CD137 affibody binds to activated CEMT cells that express CD137. [Figure 5A] This figure confirms that a dual antibody based on the cetuximab (CET) antibody and composed of a CD137 affibody binds to HT29 cells expressing EGFR. [Figure 5B] This figure confirms that a dual antibody based on the 15E3 antibody and composed of a CD137 affibody binds to HT29 cells expressing EGFR. [Figure 6] FIG. 1 shows that EGFR-dependent T cell activation was confirmed by IFN-gamma secretion analysis for the selection of CD137 affibodies. [Figure 7A]

[0039] Figure 1 confirms which extracellular domain (ECD) of CD137 the two selected CD137 affibodies bind to. Figure 2 shows 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, each of which is composed of a combination of a signal peptide and different ECD domains 1 to 5. The amino acid sequence of human CD137 ECD, which is composed of domains 1 to 5, is shown in SEQ ID NO: 27. [Figure 7B] Figure 1 shows the binding of two selected CD137 affibodies to the extracellular domain (ECD) of CD137. Figure 2 shows the binding of urelumab to domain 1 of the CD137 ECD. [Figure 7C] Figure 1 shows the binding of two selected CD137 affibodies to the extracellular domain (ECD) of CD137. Figure 2 shows the binding of utomilumab to domain 3 of the CD137 ECD. [Figure 7D] Figure 1 shows the binding of two selected CD137 affibodies to the extracellular domain (ECD) of CD137. Figure 2 shows the binding of cetuximab (negative Ab) to any domain of the CD137 ECD. [Figure 7E] Figure 1 shows the binding of two selected CD137 affibodies to the extracellular domain (ECD) of CD137. Figure 2 shows the binding of affibody ZAAD01 to domain 3 of the CD137 ECD. [Figure 7F]Figure 1 shows the binding of two selected CD137 affibodies to the extracellular domain (ECD) of CD137. Figure 2 confirms that affibody ZAAD05 binds to domain 3 of the CD137 ECD. [Figure 8A] This figure confirms the cross-reactivity of the two selected CD137 affibodies with other proteins in the TNFR superfamily, including CD27, CD30, CD134, CD270, CD357, and TNF-α. This figure confirms that the affibody ZAAD01 specifically binds only to CD137. [Figure 8B] This figure confirms the cross-reactivity of the two selected CD137 affibodies with other proteins belonging to the TNFR superfamily, including CD27, CD30, CD134, CD270, CD357, and TNF-α. This figure confirms that the affibody ZAA0D5 specifically binds only to CD137. [Figure 9A] Figure 1 shows cross-species reactivity of two selected CD137 affibodies with CD137 proteins in humans, mice, and cynomolgus monkeys. Figure 1 confirms that affibody ZAAD01 binds to CD137 proteins in humans and cynomolgus monkeys. [Figure 9B] Figure 1 shows cross-species reactivity of two selected CD137 affibodies with CD137 proteins in humans, mice, and cynomolgus monkeys, confirming that affibody ZAAD05 binds to CD137 proteins in humans and cynomolgus monkeys. [Figure 10A]Figure 10A shows the BLI results for the CET-ZAAD01 biantibody, demonstrating that four antibody-affibody biantibodies simultaneously bind to both CD137 and EGFR antigens. [Figure 10B] Figure 10B shows the BLI results for the CET-ZAAD05 biantibody, demonstrating that four antibody-affibody biantibodies simultaneously bind to both CD137 and EGFR antigens. [Figure 10C] Figure 10B shows the BLI results for the 15E3-ZAAD01 biantibody, demonstrating that four antibody-affibody biantibodies simultaneously bind to both CD137 and EGFR antigens. [Figure 10D] Figure 10B shows the BLI results for the 15E3-ZAAD05 biantibody, demonstrating that four antibody-affibody biantibodies simultaneously bind to both CD137 and EGFR antigens. [Figure 11A] The expression level of EGFR-expressing cells was determined by flow cytometry, and it was confirmed that the degree of T cell activation by the double antibody depending on the level of EGFR expression correlated with the amount of EGFR expression. EGFR expression was confirmed in EGFR-positive cells A431 cells, HT29 cells, and SW403 cells, and EGFR-negative cells MOLT4 cells. [Figure 11B] The expression level of EGFR-expressing cells was determined by flow cytometry, and it was confirmed that the degree of T cell activation by the bibody depending on the level of EGFR expression correlated with the amount of EGFR expression. This figure also shows that the EGFR-dependent T cell activation by the 15E3-ZAAD01 bibody depending on the level of EGFR expression was confirmed by IFN-gamma secretion analysis. [Figure 12A]Figure 1 shows that bibodies produced using a CD137 affibody and an HER2 antibody or a CD19 antibody bind to CD137 and HER2 or CD19, respectively. Figure 1 shows that trastuzumab-ZAAD01 bibodies (anti-HER2 Ab1_ZAAD01) and hz1E11.10-ZAAD01 bibodies (anti-HER2 Ab2_ZAAD01) bind to CD137. [Figure 12B] Figure 10 shows that bibodies produced using a CD137 affibody and an HER2 antibody or a CD19 antibody bind to CD137 and HER2 or CD19, respectively. Figure 10 shows that trastuzumab-ZAAD01 bibodies and hz1E11.10-ZAAD01 bibodies bind to HER2. [Figure 12C] Figure 10 shows that bibodies produced using a CD137 affibody and an HER2 antibody or a CD19 antibody bind to CD137 and HER2 or CD19, respectively. Figure 10 shows that the FMC63-ZAAD01 bibodies (anti-CD19 Ab_ZAAD01) bind to CD137. [Figure 12D] Figure 10 shows a diagram confirming that bibodies produced using a CD137 affibody and an HER2 antibody or a CD19 antibody bind to CD137 and HER2 or CD19, respectively. Figure 10 shows a diagram confirming that the FMC63-ZAAD01 bibodies bind to CD19. [Figure 13A] Figure 1 shows IFN-gamma secretion analysis confirming that biantibodies produced using a CD137 affibody and a HER2 antibody or a CD19 antibody activate T cells in a HER2- or CD19-dependent manner. Figure 1 shows IFN-gamma secretion analysis confirming that the trastuzumab-ZAAD01 biantibody and hz1E11.10-ZAAD01 biantibody activate T cells in a HER2-dependent manner compared to utomilumab. [Figure 13B]Figure 1 shows IFN-gamma secretion analysis confirming that biantibodies produced using a CD137 affibody and a HER2 antibody or a CD19 antibody activate T cells in a HER2- or CD19-dependent manner. Figure 1 shows IFN-gamma secretion analysis confirming that the FMC63-ZAAD01 biantibody activates T cells in an EGFR-dependent manner compared to utomilumab. [Figure 14A] 1 shows that activation of immune cells by dual antibodies (CET_ZAAD01 and CET_ZAAD05) results in the death of DLD1-luc cells. [Figure 14B] Figure 1 shows that activation of immune cells by dual antibodies (CET_ZAAD01 and CET_ZAAD05) increases IFN-gamma secretion. [Figure 15] Figure 1 shows the anti-cancer effect of dual antibodies (CET_ZAAD01 and CET_ZAAD05) on survival in a humanized CD137 model. [Figure 16A] FIG. 1 shows tumor size in the vehicle-treated group in the humanized CD137 model. [Figure 16B] FIG. 1 shows tumor size in the cetuximab-treated group in the humanized CD137 model. [Figure 16C] FIG. 1 shows tumor size in the double antibody (CET_ZAAD01) treatment group in the humanized CD137 model. [Figure 16D] FIG. 1 shows tumor size in the double antibody (CET_ZAAD05) treatment group in the humanized CD137 model.

Claims

1. An affibody that specifically binds to the extracellular domain of CD137, comprising an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8.

2. The affibody of claim 1 , wherein the CD137 is CD137 derived from a human or a cynomolgus monkey.

3. The affibody of claim 1, wherein the binding site of the affibody for the CD137 extracellular domain is located between amino acid residues 64 and 95 of the amino acid sequence of SEQ ID NO:

27.

4. A nucleic acid molecule comprising a nucleotide sequence encoding the affibody of claim 1.

5. The nucleic acid molecule according to claim 4, wherein the nucleotide sequence comprises any one of the nucleotide sequences set forth in SEQ ID NOs: 9 to 16.

6. A recombinant vector comprising the nucleic acid molecule of claim 4.

7. An isolated host cell comprising the recombinant vector of claim 6.

8. A pharmaceutical composition for preventing or treating cancer, comprising the affibody of claim 1 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, wherein the cancer is a blood cancer or a solid cancer.

10. A protein complex comprising: An affibody that specifically binds to the extracellular domain of CD137 according to claim 1; and An antibody or antigen-binding fragment thereof that specifically binds to a protein on the surface of a cancer cell.

11. The protein complex according to claim 10, 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 differentiation 19).

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

22.

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

24.

14. The protein complex of claim 11 , wherein the antibody or antigen-binding fragment thereof that specifically binds to HER2 comprises: (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 28; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 29; (d) trastuzumab; or (e) a heavy chain variable region comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 30, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 32; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

35.

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

37.

16. The protein complex of claim 11 , wherein the monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD19 comprises: (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 39; or (g) FMC63

17. A nucleic acid molecule comprising a nucleotide sequence encoding the protein complex of any one of claims 10 to 16.

18. A recombinant vector comprising the nucleic acid molecule of claim 17.

19. 20. An isolated host cell comprising the recombinant vector of claim 18.

20. A pharmaceutical composition for preventing or treating cancer, comprising the protein complex according to any one of claims 10 to 16 and a pharmaceutically acceptable carrier.

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