Coiled-coil fusion proteins

Abstract

The present invention relates to a polypeptide comprising a self-binding domain, a fusion protein comprising said polypeptide, or a nucleic acid molecule encoding the same, or a composition comprising said polypeptide, fusion protein, or nucleic acid molecule. The fusion protein or composition of the present invention can be embodied in a modular form by binding or carrying various drug moieties (proteins, antibody fragments, etc.) according to a desired purpose, and can achieve improved binding strength to a target and / or increased activity of the drug by binding via self-binding.

Description

Detailed Description of the Invention

[0001] [Technical Field] The present invention relates to a coiled-coil scaffold with improved binding stability through co-expression. The coiled-coil scaffold presented in the present invention relates to a fusion protein or drug delivery vehicle that can realize multiple complexes of various functional molecules in a modular format by binding a drug moiety (protein, antibody fragment, etc.) to an MBD2-p66α (Methyl-binding domain protein 2-Transcriptional repressor p66α) complex.

[0002] [Background technology] Drug delivery vehicles currently in use or under development are typically dual-drug loaded and are limited to one drug type. Furthermore, in order to create a delivery vehicle loaded with a new drug, the same process used to produce an existing drug delivery vehicle must be repeated, which is inefficient, and it is difficult to easily change the combination of drugs delivered.

[0003] [Prior art documents] [Patent Documents] [Patent Document 1] US2014-0073566 A1 Summary of the Invention [Problem to be solved by the invention] The present inventors have made extensive efforts to develop a fusion protein or drug delivery platform that can easily load various drug moieties (therapeutic proteins, antibody fragments, etc.) while exhibiting improved drug efficacy. As a result, they have found that when a drug moiety is attached to the N-terminus or C-terminus of a polypeptide comprising positions 360-393 of the MBD2 amino acid sequence and a polypeptide comprising positions 138-178 of the p66α amino acid sequence, the two polypeptides bind by self-binding, thereby exhibiting improved target binding strength and / or drug activity, which led to the completion of the present invention.

[0004] Therefore, an object of the present invention is to provide a fusion protein comprising polypeptide 1 comprising self-binding domain 1 and polypeptide 2 comprising self-binding domain 2.

[0005] Another object of the present invention is to provide a nucleic acid molecule encoding the fusion protein.

[0006] It is yet another object of the present invention to provide a vector containing the nucleic acid molecule.

[0007] It is yet another object of the present invention to provide an expression construct comprising nucleic acid molecule 1 encoding polypeptide 1 comprising self-binding domain 1 and nucleic acid molecule 2 encoding polypeptide 2 comprising self-binding domain 2.

[0008] It is yet another object of the present invention to provide a host cell containing or transformed with said vector or expression construct.

[0009] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, comprising the fusion protein, nucleic acid molecule, vector, expression construct or host cell.

[0010] It is still another object of the present invention to provide a kit comprising the polypeptide 1 or nucleic acid molecule 1 and the polypeptide 2 or nucleic acid molecule 2.

[0011] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims, and the drawings.

[0012] [Means for solving the problem] According to one aspect of the present invention, a fusion protein is provided comprising polypeptide 1 comprising self-binding domain 1 and polypeptide 2 comprising self-binding domain 2.

[0013] As used herein, the term "fusion protein" refers to a hybrid protein expressed by a nucleic acid molecule comprising the nucleotide sequences of at least two genes.

[0014] As used herein, the term "self-binding domain" refers to a protein that can self-bind with another self-binding domain to form a coiled coil. For example, self-binding domain 1 can self-bind with self-binding domain 2 to form a coiled coil. The self-binding domains form non-covalent bonds, such as hydrophobic and / or ionic interactions, between each other, allowing the individual domains to bind (self-bind) to each other to form a coiled coil.

[0015] The self-binding domain 1 preferably comprises the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 in which an amino acid selected from the group consisting of the 6th, 27th, and a combination thereof has been mutated to cysteine, and more preferably comprises the amino acid sequence of SEQ ID NO: 1 in which an amino acid selected from the group consisting of the 6th, 27th, and a combination thereof has been mutated to cysteine.

[0016] The self-binding domain 2 preferably comprises the amino acid sequence of SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 in which an amino acid selected from the group consisting of the 8th, 29th, and a combination thereof has been mutated to cysteine, and more preferably comprises the amino acid sequence of SEQ ID NO: 6 in which an amino acid selected from the group consisting of the 8th, 29th, and a combination thereof has been mutated to cysteine.

[0017] Mutation to cysteine ​​at this amino acid position further introduces a covalent bond (disulfide bond) between the two self-binding domains, allowing for the formation of a more stable fusion protein.

[0018] According to one embodiment of the present invention, when cysteine ​​mutations were introduced at positions other than the cysteine ​​mutation positions in self-binding domain 1 (positions 6 and 27 in SEQ ID NO: 1) and self-binding domain 2 (positions 8 and 29 in SEQ ID NO: 6) (e.g., positions 16 and 20 in SEQ ID NO: 1 and / or positions 18 and 11 in SEQ ID NO: 6), it was confirmed that a stable fusion protein could not be formed or the expression level was significantly reduced (Figures 3A and 3B).

[0019] According to a preferred embodiment, the self-binding domain 1 comprises the amino acid sequence of SEQ ID NO: 2 or 3.

[0020] According to a preferred embodiment, the self-binding domain 2 comprises the amino acid sequence of SEQ ID NO: 7 or 8.

[0021] According to a preferred embodiment of the present invention, the fusion protein comprises a self-binding domain 1 comprising the amino acid sequence of SEQ ID NO:2 and a self-binding domain 2 comprising the amino acid sequence of SEQ ID NO:7.

[0022] According to a preferred embodiment of the present invention, the fusion protein comprises a self-binding domain 1 comprising the amino acid sequence of SEQ ID NO:3 and a self-binding domain 2 comprising the amino acid sequence of SEQ ID NO:8.

[0023] The fusion proteins of the present invention may further comprise one or more drug moieties.

[0024] According to a preferred embodiment of the present invention, the fusion protein comprises a drug moiety at one or more sites selected from the group consisting of the N-terminus and C-terminus of the self-binding domain 1 or the polypeptide 1 comprising the self-binding domain 1.

[0025] According to a preferred embodiment of the present invention, the fusion protein comprises a drug moiety at one or more sites selected from the group consisting of the N-terminus and C-terminus of the self-binding domain 2 or polypeptide 2 comprising the self-binding domain 2.

[0026] The term "polypeptide 1" as used herein refers to the self-binding domain 1 or a peptide containing the self-binding domain 1, and to a peptide that has the property of self-binding to self-binding domain 2 or a peptide 2 containing the self-binding domain 2.

[0027] The term "polypeptide 2" as used herein refers to the self-binding domain 2 or a peptide containing the self-binding domain 2, and has the property of self-binding to the self-binding domain 1 or peptide 1 containing the self-binding domain 1.

[0028] As used herein, the term "drug moiety" refers to a substance that can interact with other types of proteins or substances, such as an antibody or antigen-binding fragment thereof, or a protein (e.g., a therapeutic protein, a ligand, a receptor, an Fc portion), or that can have a specific function when expressed. The protein may be a full-length protein, or a domain or portion of a polypeptide that is responsible for a specific function, and may be a naturally occurring or artificial sequence.

[0029] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor to specifically recognize an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes both polyclonal antibodies and monoclonal antibodies.

[0030] Antibodies include not only full-length antibodies but also antigen-binding fragments of antibody molecules (antibody fragments). Intact antibodies have two full-length light chains and two full-length heavy chains, with each light chain connected to a heavy chain by a disulfide bond. Heavy chain constant regions are classified into gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). Light chain constant regions are classified into kappa (κ) and lambda (λ) types.

[0031] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment that specifically recognizes an antigen within a whole antibody molecule and retains antigen-antibody binding function, and includes single-domain antibodies (sdAbs), single-chain antibodies (scFvs), Fab, F(ab'), F(ab')2, and Fv. Examples of fragments include a monovalent fragment (Fab fragment) composed of the VL, VH, Cκ (or CL), and CH1 domains; a bivalent fragment (F(ab')2 fragment) comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment composed of the VH and CH1 domains; an Fv fragment composed of the VL and VH domains of one arm of an antibody and a disulfide-linked Fv (sdFv); a dAb fragment composed of the VH domain; and a combination of separated complementarity-determining regions (CDRs) or two or more separated CDRs that can optionally be connected by a linker. Alternatively, scFvs may be linked by a linker to form a single protein chain in which the VL and VH domains pair to form a monovalent molecule. Such single-chain antibodies are also included in antibody fragments. Examples of such antibodies or antibody fragments include tetrameric antibodies containing two heavy chain molecules and two light chain molecules; antibody light chain monomers; antibody heavy chain monomers; antibody light chain dimers; antibody heavy chain dimers; intrabodies; monovalent antibodies; camelid antibodies; and single domain antibodies (sdAbs).

[0032] According to a preferred embodiment, the antigen-binding fragment of an antibody as the drug moiety is an scFv.

[0033] According to a preferred embodiment, the protein as the drug moiety is albumin binding peptide (ABP) or TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand).

[0034] As used herein, the term "albumin-binding peptide" refers to a peptide or protein that binds to albumin, a multifunctional plasma protein that plays an important role in maintaining blood osmotic pressure and transporting various substrates. Albumin has a long plasma half-life of 3 weeks due to its ability to bind to FcRn, and the inclusion of an albumin-binding peptide sequence that retains albumin-binding activity in a fusion protein can confer increased half-life properties. Various albumin-binding peptides have been disclosed in the art (e.g., Zorzi et al., Medchemcomm. 2019 Jul 1;10(7):1068-1081), which are incorporated herein by reference.

[0035] As used herein, the term "TRAIL" refers to a type of tumor necrosis factor (TNF) ligand that induces apoptosis in tumor cells. TRAIL has four receptors, of which death receptors (DR4 and DR5) are overexpressed in cancer cell lines and decoy receptors (DcR1 and DcR2) are overexpressed in normal cell lines. TRAIL can induce tumor cell death by binding to death receptors such as DR4 and DR5 on the surface of tumor cells. Decoy receptors lack a death domain at their C-terminus, preventing the intracellular transmission of apoptotic signals. Therefore, TRAIL-based therapy is a highly effective next-generation anticancer therapeutic agent because it is harmless to normal cells and induces toxicity only in various cancer cell lines. However, despite these advantages, TRAIL has limitations in its clinical application due to its poor structural stability and in vivo persistence, as the homotrimer, which is the active form of the TRAIL monomer, is linked by non-covalent bonds.

[0036] According to a preferred embodiment of the present invention, the protein as the drug moiety is a trail trimer.

[0037] The trail trimer can be bound to the N-terminus and / or C-terminus of the self-binding domain 1 or polypeptide 1 containing the self-binding domain 1, or to the N-terminus and / or C-terminus of the self-binding domain 2 or polypeptide 2 containing the self-binding domain 2, thereby constructing a trimer, hexamer, nonamer, dodecamer, etc. within the fusion protein.

[0038] According to one embodiment of the present invention, the trail hexamer formed by the self-binding of trail trimer-self-binding domain 1 and trail trimer-self-binding domain 2 was confirmed to have 2- to 5-fold increased affinity for the death receptor compared to when 1) trail trimer-self-binding domain 1 or 2) trail trimer-self-binding domain 2 were expressed alone (Figure 4).

[0039] According to yet another embodiment of the present invention, the trail hexamer formed by the self-binding of trail trimer-self-binding domain 1 and trail trimer-self-binding domain 2 was confirmed to have a cancer cell death rate that was four-fold or more higher than when 1) trail trimer-self-binding domain 1 or 2) trail trimer-self-binding domain 2 were expressed alone (FIGS. 5A and 5B).

[0040] As used herein, the terms "N-terminus" and "C-terminus" refer to the amino-terminus and carboxy-terminus of a polypeptide. The drug moiety may be attached to the N-terminus and / or C-terminus of the self-binding domain 1 or polypeptide 1 containing the self-binding domain 1 as a homogeneous protein (e.g., trail trimer + trail trimer, etc.), or to the N-terminus and / or C-terminus of the self-binding domain 2 or polypeptide 2 containing the self-binding domain 2 as a heterogeneous protein (e.g., trail trimer + ABP, or trail trimer + ABP + scFv, etc.), thereby enabling the modular realization of multiple complexes of various functional molecules.

[0041] According to a preferred embodiment, the drug moiety may be attached to the N-terminus or C-terminus of the polypeptide 1 or 2 via a linker.

[0042] The term "linker" as used herein refers to an amino acid sequence present between the N-terminus or C-terminus of the polypeptide 1 or 2 and the N-terminus or C-terminus of a drug moiety. The linker may be any amino acid combination, but is not limited thereto, and is a sequence that promotes binding and dissociation between the polypeptide 1 or 2 and the drug moiety, but may also be introduced for the purpose of imparting flexibility to the coiled-coil-forming domain or reducing steric hindrance that occurs when the coiled-coil-forming domain binds. The linker is preferably a glycine- and serine-rich sequence of any length, and may be, for example, but is not limited to, a sequence consisting of repeated Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser, or combinations thereof.

[0043] According to a preferred embodiment, the present invention relates to a coiled-coil complex comprising: (i) Polypeptide 1 in which an amino acid selected from the group consisting of positions 6, 27, and combinations thereof in SEQ ID NO: 1 is mutated to cysteine; and (ii) Polypeptide 2 in which an amino acid selected from the group consisting of positions 8, 29, and combinations thereof in SEQ ID NO: 6 is mutated to cysteine.

[0044] According to another aspect of the invention, there is provided a nucleic acid molecule encoding said fusion protein or coiled-coil complex.

[0045] As used herein, the term "nucleic acid molecule" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogs with modified sugar or base moieties (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of a nucleic acid molecule encoding a fusion protein of the present invention may be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0046] The nucleic acid molecules of the present invention are also understood to include nucleotide sequences that exhibit substantial identity to the above-described nucleotide sequences, where "substantial identity" refers to a nucleotide sequence that exhibits at least 80% homology, in one specific example, at least 90% homology, and in another specific example, at least 95% homology, when the above-described nucleotide sequences of the present invention are aligned with any other sequence to the greatest possible extent and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0047] According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the polypeptide 1 (nucleic acid molecule 1) and / or the nucleic acid molecule encoding the polypeptide 2 (nucleic acid molecule 2) may contain a nucleotide sequence encoding a drug moiety at a position selected from the group consisting of the 5' end and the 3' end.

[0048] According to yet another aspect of the present invention, there is provided a vector comprising the nucleic acid molecule.

[0049] As used herein, the term "vector" refers to a vehicle into which a polynucleotide sequence can be inserted for introduction into a cell capable of replicating the nucleic acid molecule sequence. The polynucleotide sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmid vectors, and viral vectors (such as retroviruses, adenoviruses, and adeno-associated virus vectors). Those skilled in the art can construct vectors using standard recombinant techniques (e.g., Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, 1994).

[0050] As used herein, the term "expression construct" refers to a vector containing a nucleotide sequence encoding at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various regulatory sequences. In addition to regulatory sequences regulating transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that provide other functions. The expression construct may contain nucleic acid molecule 1 encoding polypeptide 1 and nucleic acid molecule 2 encoding polypeptide 2 simultaneously in the same vector, or in different vectors. Nucleic acid molecules 1 and 2 may self-associate in vitro or in vivo to form a fusion protein when polypeptides 1 and 2 are expressed from the same vector or different vectors.

[0051] According to yet another aspect, the present invention provides a host cell comprising or transformed with a vector or expression construct as described above.

[0052] As used herein, the term "cell" includes eukaryotic and prokaryotic cells and refers to any transformable cell that can replicate the vector or express a gene encoded by the vector. A cell may be transfected, transduced, or transformed by the vector, which refers to the process by which an exogenous polynucleotide (nucleic acid molecule) is transferred or introduced into a host cell. As used herein, the term "transformation" encompasses both transfected and transduced.

[0053] The (host) cells of the present invention are not limited, but are preferably insect cells or mammalian cells, more preferably Sf9 insect cells, and HEK293 cells, HeLa cells, ARPE-19 cells, RPE-1 cells, HepG2 cells, Hep3B cells, Huh-7 cells, C8D1a cells, Neuro2A cells, CHO cells, MES13 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, MCF-7 cells, HC70 cells, HCC1428 cells, BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, and MIA cells. PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells, PC-12 cells, NIH3T3 cells, etc. can be used.

[0054] The host cells of the present invention are preferably isolated host cells.

[0055] According to yet another aspect, the present invention provides a composition comprising the fusion protein, the nucleic acid molecule, the vector, the expression construct, or the cell described above.

[0056] In yet another aspect, the present invention provides a composition comprising: i) polypeptide 1 comprising the amino acid sequence of SEQ ID NO: 2 or 3 as self-binding domain 1, or nucleic acid molecule 1 encoding said polypeptide 1; and ii) polypeptide 2 comprising the amino acid sequence of SEQ ID NO: 7 or 8 as self-binding domain 2, or nucleic acid molecule 2 encoding said polypeptide 2.

[0057] In the present invention, the same contents as those described above, such as the "polypeptide," "nucleic acid molecule," and "drug moiety," will be omitted to avoid the complexity and repetition of the specification.

[0058] A drug moiety may be bound to one or more sites selected from the group consisting of the N-terminus and C-terminus of polypeptide 1 comprising the amino acid sequence of SEQ ID NO: 2 or 3 and polypeptide 2 comprising the amino acid sequence of SEQ ID NO: 7 or 8 contained in the composition.

[0059] Thus, the composition may comprise i) polypeptide 1-drug moiety, drug moiety-polypeptide 1, or drug moiety-polypeptide 1-drug moiety, and ii) polypeptide 2-drug moiety, drug moiety-polypeptide 2, or drug moiety-polypeptide 2-drug moiety.

[0060] Furthermore, a nucleotide sequence encoding a drug moiety may be bound to a position selected from the group consisting of the 5' end or the 3' end of nucleic acid molecule 1 encoding polypeptide 1 comprising the amino acid sequence of SEQ ID NO: 2 or 3 and nucleic acid molecule 2 encoding polypeptide 2 comprising the amino acid sequence of SEQ ID NO: 7 or 8, both of which are contained in the composition.

[0061] Furthermore, the nucleotide sequences encoding nucleic acid molecule 1, nucleic acid molecule 2, and the drug moieties bound thereto contained in the composition may be present in the composition in the form of being inserted into one or more vectors.

[0062] According to a preferred embodiment, the composition is a pharmaceutical composition for preventing or treating cancer.

[0063] According to yet another aspect of the present invention, there is provided a method for preventing or treating cancer, comprising administering to a subject a pharmaceutically effective amount of the above-mentioned fusion protein, the nucleic acid molecule, the vector, the expression construct, the cell, or the composition.

[0064] According to yet another aspect, the present invention provides the above-mentioned fusion protein, said nucleic acid molecule, said vector, said expression construct, said cell or said composition for use in therapy.

[0065] According to a preferred embodiment of the present invention, the therapeutic use is the treatment of cancer.

[0066] As used herein, the term "prevention" means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having, but is susceptible to, such disease or condition.

[0067] As used herein, the term "treatment" means (a) inhibiting the development of a disease, disorder, or condition; (b) alleviating a disease, disorder, or condition; or (c) eliminating a disease, disorder, or condition.

[0068] Therefore, the composition of the present invention may be a composition for cancer treatment by itself, or may be administered in combination with other pharmacological ingredients to be used as a therapeutic adjuvant that improves the therapeutic response. Thus, as used herein, the terms "treatment" or "therapeutic agent" encompass the meaning of "therapeutic adjuvant" or "therapeutic adjuvant."

[0069] As used herein, the term "administration" or "administering" refers to administering a therapeutically effective amount of a composition of the present invention directly to a subject, thereby causing the same amount to be formed in the subject's body.

[0070] In the present invention, the term "therapeutically effective amount" means the content of the composition of the pharmacological component of the composition sufficient to provide a therapeutic or prophylactic effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and thus includes a "prophylactically effective amount."

[0071] As used herein, the term "subject" includes, but is not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus monkey. Specifically, the subject of the present invention is a human.

[0072] Disclosed herein are compositions of the present invention having the desired degree of purity in a pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and may include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, zeaxanthin, erythritol ... amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0073] In some embodiments, the pharmaceutical composition comprises a fusion protein, nucleic acid molecule, vector, expression construct, or cell disclosed herein in a pharmaceutically acceptable carrier. In particular embodiments, the pharmaceutical composition comprises an effective amount of a fusion protein, nucleic acid molecule, vector, expression construct, or cell disclosed herein, and, optionally, one or more additional prophylactic or therapeutic agents, in a pharmaceutically acceptable carrier. In some embodiments, the fusion protein, nucleic acid molecule, vector, expression construct, or cell is the only active ingredient contained in the pharmaceutical composition.

[0074] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonicity agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents for metal ions, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, intravenous infusion injection, isotonic dextrose injection, sterile water injection, and dextrose and lactate intravenous infusion injection. Non-aqueous vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antibacterial agents in bacteriostatic or fungistatic concentrations may be added to parenteral preparations packaged in multi-dose containers, including phenols or cresols, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonicity agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Topical anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifying agents include polysorbate 80 (TWEEN® 80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0075] Pharmaceutical compositions may be formulated for any route of administration to a subject. Specific examples of administration routes include intranasal, oral, parenteral, intrathecal, intraventricular, pulmonary, subcutaneous, or intraventricular routes. Parenteral administration, characterized by subcutaneous, intramuscular, or intravenous injection, is also contemplated. Injectables may be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the administered pharmaceutical composition may also contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other formulating agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0076] Preparations for parenteral administration of pharmaceutical compositions include sterile dry soluble products ready for combination with a solvent immediately prior to use, including sterile solutions ready for injection, tablets for hypodermic injection, such as lyophilized powders, sterile suspensions ready for injection, sterile dry insoluble products ready for combination with a vehicle immediately prior to use, and sterile emulsions. The solutions may be aqueous or non-aqueous.

[0077] The pharmaceutical compositions disclosed herein may also be formulated to target specific tissues, receptors, or other body regions of the subject to be treated. Various targeting methods are known to those skilled in the art. Such targeting methods are contemplated for use with the present compositions. For non-limiting examples of targeting methods, see U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. In certain embodiments, the pharmaceutical compositions disclosed herein may be used to treat cancer.

[0078] Compositions to be used for in vivo administration may be sterilized, for example, by filtration through sterile filtration membranes.

[0079] According to yet another aspect, the present invention provides a kit comprising: (i) a polypeptide 1 comprising an amino acid sequence in which an amino acid selected from the group consisting of positions 6, 27, and combinations thereof in SEQ ID NO: 1 is mutated to cysteine, or a nucleic acid molecule 1 encoding the same; (ii) a polypeptide 2 comprising an amino acid sequence in which an amino acid selected from the group consisting of positions 8, 29, and combinations thereof in SEQ ID NO: 6 is mutated to cysteine, or a nucleic acid molecule 2 encoding the same; and (iii) a drug moiety bound to one or more sites selected from the group consisting of the N-terminus and C-terminus of said polypeptides 1 and 2, or a drug moiety-encoding nucleotide sequence (nucleic acid encoding a drug moiety) bound to a site selected from the group consisting of the 5'-terminus or 3'-terminus of said nucleic acid molecules 1 and 2.

[0080] In the present invention, the same contents as those described above, such as "polypeptide," "nucleic acid molecule," and "drug moiety," will be omitted to avoid complexity and repetition in the specification.

[0081] In some embodiments, the present specification discloses a pharmaceutical pack or kit comprising one or more containers filled with one or more of (i) polypeptide 1 or nucleic acid molecule 1 encoding same, (ii) polypeptide 2 or nucleic acid molecule 2 encoding same, and (iii) a drug moiety bound to one or more sites selected from the group consisting of the N-terminus and C-terminus of polypeptides 1 and 2, or a drug moiety-encoding nucleotide sequence bound to a site selected from the group consisting of the 5'-end or 3'-end of nucleic acid molecules 1 and 2; and optional instructions for use. In some embodiments, the kit contains a pharmaceutical composition disclosed herein and any of the prophylactic or therapeutic agents disclosed herein. In some embodiments, the kit discloses a composition for preventing or treating cancer, the kit including instructions for the composition disclosed herein.

[0082] [Effects of the Invention] The features and advantages of the present invention can be summarized as follows: (i) The present invention provides a polypeptide comprising a self-binding domain, a fusion protein comprising the polypeptide, or a nucleic acid molecule encoding the same, or a composition comprising the polypeptide, fusion protein, or nucleic acid molecule.

[0083] (ii) The fusion proteins of the present invention can be modularly embodied by binding or carrying various drug moieties (proteins, antibody fragments, etc.) according to the desired purpose, and can be linked by self-binding to achieve improved binding strength to the target and / or increased drug activity.

[0084] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] The structure predictions of coiled C'-GFP-6xHis and coiled C-mCherry-6xHis using Alphafold2.

[0085] [Figure 1B] The molecular weights of purified C'-GFP and C-mCherry were measured by SDS-PAGE.

[0086] [Fig. 2A] Native PAGE was performed to confirm whether the coil-fused fluorescent protein was correctly folded.

[0087] [Figure 2B] Thermodynamics between C'-GFP and C-mCherry was investigated using isothermal thermoscopy (ITC) to determine binding parameters.

[0088] [FIG. 3A] Results of confirming whether Ala27-Ile8, Thr6-Arg29, coiled coil_F1, and coiled coil_F2 form stable complexes.

[0089] [Fig. 3B] Comparison of the relative expression levels of Ala27-Ile8, Thr6-Arg29, coiled coil_F1, and coiled coil_F2.

[0090] [Figure 3C] The FRET efficiency of Thr6-Arg29 and C'-GFP / C-mCherry was compared relative to the FRET efficiency of Ala27-Ile8, which was set at 100%.

[0091] Figure 4 shows the results of comparing the affinity of the TRAIL hexamer for death receptors (DR and scTRAIL-C + scTRAIL-C') with that of the TRAIL trimer-C or TRAIL trimer-C' for death receptors (DR and scTRAIL-C or DR and scTRAIL-C').

[0092] [Figure 5A] Flow cytometry (FACS) was used to observe glioblastoma cell lines treated with the positive control group (R&D TRAIL), TRAIL trimer-C (I) test group, TRAIL trimer-C' (II) test group, and TRAIL hexamer test group (I + II).

[0093] [Figure 5B] Comparison of cancer cell death rates among the positive control group (PC), the Trail Trimer-C test group (T1), the Trail Trimer-C' (T2) test group, and the Trail Hexamer test group (T3).

[0094] [Mode for Carrying Out the Invention] The present invention will be described in more detail below using 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.

[0095] <Example> Materials and Methods <1. Materials> NaCl (sodium chloride), imidazole, NaOH (sodium hydroxide), tris(hydroxymethyl)aminomethane, glycerol (99.8%), methyl alcohol (99.8%), acetic acid (99.7%), and H2PO4 (dihydrogen phosphate) were purchased from SAMCHUN. Yeast extract, agar, 6N-HCl (6N-hydrochloric acid), and bromophenol blue were purchased from DAEJUNG. BL21(DE3) chemically competent E. coli was purchased from Enzynomics. 10% SDS solution, DTT (DL-Dithiothreitol), and Coomassie bright blue G-250 staining solution were purchased from Biosesang. Bacto tryptone was purchased from BD DIFCO. Ampicillin sodium salt and lysozyme were purchased from SIGMA. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Bioneer. Dulbecco's Phosphate Buffered Saline (DPBS) was purchased from Welgene. Glycine was purchased from Duksan. Tris / Glycine SDS pre-cast gel 8-16% and Tris / Glycine non-SDS pre-cast gel 12% were purchased from Komabiotech. Polypropylene and PD10 columns were purchased from Qiagen and GE Healthcare, respectively. Unstained protein MW markers from Pierce Biotechnology Inc. were used for SDS-PAGE analysis.

[0096] 2. Plasmid construction and bacterial strains Inserts encoding the coiled-coil region of human MBD2 or its mutants (amino acids 360-393, SEQ ID NOS: 1-5) or p66 or its mutants (amino acids 138-178, SEQ ID NOS: 6-10) were inserted into pET21a, which confers ampicillin resistance. pET-GFP-p66α was generated by cloning GFP-p66α between the NdeI and XhoI sites of pET21a, and mCherry-MBD2 was also cloned between the NdeI and XhoI sites of pET21a. The experiments were performed at BIONICS.

[0097] [Table 1]

[0098] Human MBD2 or its mutant, and p66α or its mutant sequence 3. Protein expression and purification - Culture BL21[DE3] (chemically competent cells) were thawed on ice, and 50 μl of each was placed in a microcentrifuge tube, followed by 1 μl of expression vector. Two negative control microcentrifuge tubes were prepared, one containing the expression vector and the other without. The tubes were then placed on ice for 30 minutes, heat-shocked at 42°C for 30 seconds, and then placed on ice for 2 minutes. 200 μl of Luria Bertani medium was then added, and 70 μl of each tube was absorbed onto Luria Bertani agar plates. Finally, colonies were harvested in an incubator set at 37°C for approximately 12 hours, transferred to 5 mL of Luria Bertani medium, and cultured overnight at 37°C and 200 rpm in a shaking incubator. 400 mL of Luria Bertani medium was placed in a 1 L Erlenmeyer flask and placed in a shaking incubator set at 37°C and 200 rpm. Once the temperature was adjusted, 400 μl of ampicillin was added, and the tube culture was induced with isopropyl β-D-1-thiogalactopyranoside at an A600 of 0.6. Cells were harvested when the A600 reached 2. All absorbance readings were measured on a Biodrop Duo Micro Volume Spectrophotometer (BIODROP).

[0099] - Cell harvesting / lysis For centrifugation, the expressed medium was divided into halves and placed in Oak Ridge Centrifuge Tubes. After centrifugation at 4,000 rcf for 10 minutes at 4°C, the supernatant was discarded and the precipitated cells were stored at -20°C. 20 mL of lysis buffer was added to each cell pellet to dissolve the pellet. The lysed solution was then transferred to a 50 mL conical tube, and 400 μl of lysozyme was added. The mixture was mixed on a rotator for 30 minutes at 4°C. The well-mixed solution was sonicated on ice, then sonicated for 7 minutes. The mixture was then centrifuged at 10,000 rcf for 20 minutes at 4°C. The precipitate was discarded and the supernatant was collected.

[0100] - Affinity Refinement After adding 1.5 mL of Ni-NTA agarose to the solution, the mixture was mixed on a rotator for 10 minutes. To obtain the protein alone, the solution was added to a 5 mL polypropylene column. Approximately 15 mL of the solution was passed over the beads to remove impurities with a wash buffer. Five 500 μl portions of elution buffer were added to the beads, and the solution exiting the column was placed in a sterile microtube. Since the purified GFP-p66alpha and mCherry-MBD2 were in the elution buffer, the buffer was replaced with PBS. After filling a PD10 column with PBS, 2.5 mL of the protein-containing solution was added, followed by a 3 mL PBS buffer change to change the protein buffer.

[0101] - Size Exclusion Chromatography (FPLC) Size-exclusion chromatography (SEC) was performed on a ProteoSEC Dynamic 11 / 303-70 HR SEC column (Protein Ark, UK) equilibrated with PBS. The Ni-NTA eluted sample was concentrated using a centrifugal filter, filtered through a 0.22 μm syringe filter, and loaded onto the column. Chromatography was performed on an AKTAprime plus column using PBS at a flow rate of 1 ml / min, and the eluted protein fractions were collected separately.

[0102] 4. Protein Electrophoresis Samples collected at various stages of protein purification were analyzed on a 12% discontinuous polyacrylamide gel containing sodium dodecyl sulfate (SDS). Protein samples were mixed with pure distilled water and incubated at 98°C for 7 minutes before separation by SDS-PAGE. To visualize proteins, the gel was stained with Coomassie Brilliant Blue. Native PAGE was run on the 12% polyacrylamide gel for 2 hours at a maximum voltage of 125V. Protein fluorescence was then observed under UV irradiation.

[0103] <5.Isothermal titration calorimeter> ITC analysis was performed at the Korea Institute for Basic Science (Ochang, Korea) using a MicroCal Auto-iTC200 (Malvern Panalytical). Binding affinity was measured using 40 μl of C'-GFP and 200 μl of C-mCherry in PBS buffer (pH 7.5). For titration experiments, 2 μl of C'-GFP was injected into C-mCherry 19 times for 4 seconds at 150-second intervals at 25°C. Data were analyzed using MicroCal Origin 7.0 software. N is the stoichiometric number, KD is the binding affinity, and ΔH and ΔS are the changes in enthalpy and entropy of binding, respectively.

[0104] 6. Size Exclusion Chromatography (HPLC) To analyze protein-protein interactions, a Cytiva Superdex 200 increase 10 / 300 GL column equilibrated with PBS (pH 7.5) was used on a YL HPLC system (semi-prep) (YL9100S HPLC). Samples (C'-GFP and C-mCherry or C'-GFP / C-mCherry) were loaded onto the column alone or in an equimolar mixture. The eluent was monitored by absorbance at 280 nm.

[0105] 7. Fluorescence Resonance Energy Transfer (FRET) Analysis Samples containing wild-type GFP alone or an equimolar mixture of C'-GFP / C-mCherry, Ala27-Ile8, and Thr6-Arg29 were appropriately diluted in phosphate-buffered saline (PBS) to a total volume of 200 μL and a concentration of 4 μM. The samples were transferred to a 96-well plate, and the absorbance at 488 nm and the emission (fluorescence) at 510 nm of each well were measured using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). The emission per unit absorbance (A) of the wild-type GFP alone sample and the emission per unit absorbance (B) of each sample were calculated, and the Förster resonance energy transfer (FER) efficiency was calculated using the following equation:

[0106]

number

[0107] <Experimental Results> <1. Generation and Characterization of Fluorescent Protein-Fused Wild-Type Coils> To construct non-natural heterodimeric protein complexes using coiled coils as scaffolds, we used the complementary self-assembling coiled coil regions 138–178 (coil C') and 360–393 (coil C) of p66alpha and MBD2. Subsequently, we visually monitored coil expression and complex formation using green fluorescent protein (GFP) and a red fluorescent protein, mCherry. GFP and mCherry were fused to the C-termini of coil C' and coil C, respectively. A hexa-histidine tag (6xHis) was further fused to the C-terminus of each fluorescent protein, and the proteins were purified using nickel-nitrilotriacetic acid agarose resin. Alphafold2 predicted the complex structures of the coil C'-GFP-6xHis fusion protein (hereafter referred to as C'-GFP) and coil C-mCherry-6xHis fusion protein (hereafter referred to as C-mCherry) (Figure 1A). pET-C'-GFP and pET-C-mCherry were expressed in the E. coli BL21 system and purified by affinity chromatography followed by FPLC. Purification yielded highly purified protein at a yield of approximately 4 mg per liter of culture. After purification, the collected protein samples were separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and visualized by bleaching (Figure 1B). The predicted molecular weights of the proteins were 33.64 kDa and 32.65 kDa for C'-GFP and C-mCherry, respectively, and SDS-PAGE provided molecular weight estimates near the 35 kDa marker, consistent with our results.

[0108] After purification, we performed native PAGE to confirm the correct folding of the coil-fused fluorescent proteins. Native PAGE confirmed that C'-GFP and C-mCherry, when present alone, exhibited green and red fluorescence (Figure 2A). However, when C'-GFP and C-mCherry were mixed at a 1:1 ratio, the green and red fluorescence combined to produce yellow fluorescence. This indicates that each fluorescent protein was correctly folded and the fused coils self-assembled in a complementary manner. We also determined the thermodynamics of the interaction between C'-GFP and C-mCherry using isothermal titration calorimetry (ITC). The measured parameters between C'-GFP and C-mCherry were N = 0.7, KD = 10.7 nM, ΔH = -2.6 kcal / mol, and ΔS = -52.4 cal / mol / deg. The reaction between the two molecules was spontaneously driven, demonstrating high binding affinity (Figure 2B). Furthermore, C'-GFP and C-mCherry reacted at a ratio of 0.7:1, lower than the theoretical 1:1 ratio. This may be due to the formation of homodimers between coils or to differences in the concentrations used to fit the isotherms to the actual concentrations. In summary, we expressed C'-GFP and C-mCherry that are capable of correct folding and can bind with high affinity to form heterodimeric complexes.

[0109] 2. Design and stability evaluation of coil-type cysteine ​​mutants Disulfide bridges formed between the thiol groups of intramolecular or intermolecular cysteine ​​(Cys) residues can serve as important components for correct protein folding and structural stability. To provide improved resistance to dissociation during self-association, we sought to generate disulfide-linked coiled-coil complexes. To achieve this, we identified pairs of residues that could form disulfide bonds while minimizing the disruption of existing coiled-coil interactions. C'_Ile8-C_Ala27 (I8C-A27C) (SEQ ID NO:7 + SEQ ID NO:2) and C'_Arg29-C_Thr6 (R29C-T6C) (SEQ ID NO:8 + SEQ ID NO:3) were determined to be optimal cross-linking sites for disulfide bond formation while maintaining favorable electrostatic interactions. To examine the extent to which the mutations suppressed coiled-coil interactions, the favorable electrostatic interaction residue pairs C'_Glu18-C_Arg16 (E18C-R16C) (SEQ ID NO: 9 + SEQ ID NO: 4) and C'_Leu11-C_Val20 (L11C-V20C) (SEQ ID NO: 10 + SEQ ID NO: 5) were selected and compared. The coiled-coil interactions between wild-type GFP-C' and wild-type mCherry-C (SEQ ID NO:6 + SEQ ID NO:1) (GFP-C' + mCherry-C) with or without DTT and mutations were analyzed by native PAGE. Results showed that C'_Ile8-C_Ala27 (I8C-A27C) (SEQ ID NO:7 + SEQ ID NO:2) (Ala27-Ile8) and C'_Arg29-C_Thr6 (R29C-T6C) (SEQ ID NO:8 + SEQ ID NO:3) (Thr6-Arg29) formed more stable complexes than C'_Glu18-C_Arg16 (E18C-R16C) (SEQ ID NO:9 + SEQ ID NO:4) (Coiled coil_F1) and C'_Leu11-C_Val20 (L11C-V20C) (SEQ ID NO:10 + SEQ ID NO:5) (Coiled coil_F2) (Figure 3A). Furthermore, a comparison of the relative expression levels confirmed that Ala27-Ile8 and Thr6-Arg29 were more than twice as high as those of Coiled coil_F1 and Coiled coil_F2 (Fig. 3B).Furthermore, quantitative analysis of the efficiency of fluorescence resonance energy transfer (FRET) between GFP and mCherry molecules, which is due to the stable coiled-coil interactions between Ala27-Ile8 and Thr6-Arg29, confirmed that the efficiency was more than twice as high as that of the wild-type (GFP-C' + mCherry-C) (Fig. 3C).

[0110] 3. Preparation of Trailing Trimer-Coil and Trailing Hexamer 3.1. Cell Expression and Purification Two proteins (trail trimer-MBD2 (scTRAIL-C) and trail trimer-p66α (scTRAIL-C')) were expressed by culturing transformed E. coli and purified using affinity chromatography. More specifically, trail trimer-MBD and trail trimer-p66α were expressed and purified by the following immobilization: Transformed BL21(DE3) E. coli cells were cultured for 12 hours in solid LB medium containing 0.1 mg / mL ampicillin. Strains containing confirmed vector insertions were then inoculated into 2xYT medium (ampicillin 0.1 mg / mL, hereafter the same) and cultured at 37°C for 12 hours. The resulting cells were then diluted 1:100 and re-inoculated into 2xYT medium. The cells were then cultured at 30°C with stirring at 200 rpm. When the absorbance at 600 nm reached 0.4-0.5, IPTG was added to a final concentration of 1.0 mM to induce protein expression, and the temperature was lowered to 16-18°C. After 20-40 hours of culture, the cells were centrifuged at 4000 g for 10 minutes to collect cell pellets, which were then stored frozen at -20°C.

[0111] To purify proteins from the cells, 40 mL of lysis buffer (pH 8.0, 50 mM sodium phosphate, 0.3 M NaCl, and 10 mM imidazole) was added to the pellet to resuspend the frozen cells. Lysozyme was added to a final concentration of 1 mg / mL and mixed by rotation at 4°C for approximately 30 minutes. The mixture was then sonicated for 8 minutes at 10-second intervals and centrifuged again at 10,000 g for 30 minutes to separate the protein and cell debris. The supernatant containing the protein was separated, and 1.5 mL of Ni-NTA agarose beads was added. The mixture was mixed by rotation at 4°C for 30 minutes. After immobilizing a gravity-flow column, the supernatant mixed with Ni-NTA was allowed to flow down the column, separating the beads from the flow-through. The flow-through was removed, and the beads were washed with washing buffer (pH 8.0, 50 mM sodium phosphate, 0.3 M NaCl, and 20 mM imidazole) until the absorbance of the solution flowing through the flow-through reached 0.00 at 280 nm to remove impurities from the beads. To separate the proteins bound to the beads, 500 μL of elution buffer (50 mM sodium phosphate (pH 8.0), 0.3 M NaCl, and 250 mM imidazole) was added and the flow-through was dispensed into microtubes. The concentration of each dispensed solution was measured at 280 nm using a Biodrop and stored refrigerated at 4°C.

[0112] <3.2. Characterization> Using binding assays (MicroScale Thermophoresis, MST), we measured 1) the uptake affinity of scTRAIL-C and scTRAIL-C', 2) the affinity of scTRAIL-C or scTRAIL-C' to death receptors (DRs), and 3) the affinity of scTRAIL-hexamer (scTRAIL-C + scTRAIL-C') to death receptors (DRs). DRs are the cognate receptors for TRAIL.

[0113] More specifically, it was measured by the following method: To prepare the samples prior to analysis, the death receptor (Biolegend) and Flamma® 648 Sulfo-NHS ester dissolved in DMSO were mixed at a 1:5 molar ratio and incubated at room temperature for 1 hour. A Zeba™ Spin Desalting Column (40K MWCO, 0.5 mL) was centrifuged at 1500 g for 1 minute to remove the storage solution before sample injection. The column was then filled with 450 μL of 150 mM sodium carbonate solution and centrifuged again at 1500 g for 1 minute. Then, 100–150 μL of the reacted DR mixture was injected and centrifuged at 1500 g for 2 minutes to remove traces of DMSO from the sample and replace it with sodium carbonate solution, simultaneously separating the non-labeled and labeled DR.

[0114] Before analyzing the samples using a Monolith NT.115 (Germany) MST instrument, the measurement intensity was adjusted using labeled DR. After filling four capillaries with the same concentration of labeled DR, the excitation power was adjusted while measuring, and the fluorescence intensity value was confirmed to be between 800 and 1000.

[0115] The recombinant proteins used as ligands were monomolecular TRAIL, TRAIL trimer-C, TRAIL trimer-C, and TRAIL hexamer, respectively. The receptor-ligand mixing method was as follows: First, 10 μL of the receptor protein, the death receptor, was dispensed into each of the 15 racks except for the first rack. Next, 20 μL of TRAIL trimer-C, which served as the ligand, was added to the first rack. Sequentially, 10 μL was taken from the first rack and diluted into the second rack, and another 10 μL was taken and diluted into the third rack. This procedure was repeated 16 times. As a result, the final 16th sample was diluted 216-fold. Samples of different concentrations were injected into 16 different capillaries, which were then attached to the detection device and measurements began. The excitation output was increased by 2x from the initial setting. Then, trail trimer-C' and trail hexamer are prepared in the same manner.

[0116] The experimental results confirmed that the affinity of the trail hexamer with the death receptor (163 nM) was approximately 2 to 5 times higher than that of the trail trimer-C or trail trimer-C' with the death receptor (337 nM and 787 nM, respectively) (Figure 4).

[0117] 3.3. In vitro cell assay Glioblastoma cell line U87-luc cells (PerkinElmer, Akron, Ohio, USA) treated with a positive control (R&D TRAIL, R&D Systems, Minneapolis, MN, USA), TRAIL trimer-C (I), TRAIL trimer-C' (II), or TRAIL hexamer (I + II) for 4 hours were observed using a flow cytometer (FACS).

[0118] As a result of this analysis, in contrast to the TRAIL trimer-C (I) or TRAIL trimer-C' (II) test groups, which showed a mortality rate of less than 5%, the TRAIL hexamer test group (I+II) showed a mortality rate of approximately 30%, which was approximately four times higher than the mortality rate of 8% for the positive control group (R&D TRAIL) (Figures 5A and 5B).

[0119] The above describes an embodiment of the present invention, but a person having ordinary knowledge in the field of technology would be able to modify and change the present invention in various ways by adding, changing, deleting or adding components, etc., within the scope of the concept of the present invention as set forth in the claims, and it can be said that such modifications and changes are also included in the scope of the present invention. [Brief explanation of the drawings]

[0120] [Figure 1A] The results of predicting the structures of coil C'-GFP-6xHis and coil C-mCherry-6xHis using Alphafold2. [Figure 1B] The molecular weights of purified C'-GFP and C-mCherry were measured by SDS-PAGE. [Figure 2A] This is the result of Native PAGE performed to confirm whether the coil-fused fluorescent protein was correctly folded. [Figure 2B] The thermodynamics between C'-GFP and C-mCherry was investigated using isothermal thermocalorimetry (ITC) to determine the binding parameters. [Figure 3A] This shows the results of confirming whether Ala27-Ile8, Thr6-Arg29, coiled coil_F1, and coiled coil_F2 form stable complexes. [Figure 3B] The results show a comparison of the relative expression levels of Ala27-Ile8, Thr6-Arg29, Coiled coil_F1, and Coiled coil_F2. [Figure 3C]The FRET efficiency of Ala27-Ile8 is set to 100%, and the results show a relative comparison of the FRET efficiencies of Thr6-Arg29 and C'-GFP / C-mCherry. [Figure 4] The affinity of the trail hexamer for death receptors (DR and scTRAIL-C + scTRAIL-C') was compared with the affinity of the trail trimer-C or trail trimer-C' for death receptors (DR and scTRAIL-C or DR and scTRAIL-C'). [Figure 5A] These are the results of observations using a flow cytometer (FACS) for glioblastoma cell lines treated with the positive control group (R&D TRAIL), the TRAIL trimer-C (I) test group, the TRAIL trimer-C' (II) test group, and the TRAIL hexamer test group (I+II). [Figure 5B] The results show a comparison of cancer cell death rates among the positive control group (PC), the Trail Trimer-C test group (T1), the Trail Trimer-C' (T2) test group, and the Trail Hexamer test group (T3).

Claims

1. A fusion protein comprising: (a) (i) Polypeptide 1 comprising an amino acid sequence in which the sixth amino acid in SEQ ID NO: 1 is mutated to cysteine; (ii) Polypeptide 2 comprising an amino acid sequence in which the 29th amino acid in SEQ ID NO: 6 is mutated to cysteine; and (iii) a drug moiety attached to one or more sites selected from the group consisting of the N-terminus and C-terminus of said polypeptides 1 and 2; The polypeptides 1 and 2 bind to each other via self-binding. (b) (i) Polypeptide 1 comprising an amino acid sequence in which the 27th amino acid in SEQ ID NO: 1 is mutated to cysteine; (ii) Polypeptide 2 comprising an amino acid sequence in which the 8th amino acid in SEQ ID NO: 6 is mutated to cysteine; and (iii) a drug moiety attached to one or more sites selected from the group consisting of the N-terminus and C-terminus of said polypeptides 1 and 2; The polypeptides 1 and 2 bind together via self-binding, or (c) (i) Polypeptide 1 comprising an amino acid sequence in which the amino acids at positions 6 and 27 in SEQ ID NO: 1 are mutated to cysteine; (ii) Polypeptide 2 comprising an amino acid sequence in which the amino acids at positions 29 and 8 in SEQ ID NO: 6 are mutated to cysteine; and (iii) a drug moiety attached to one or more sites selected from the group consisting of the N-terminus and C-terminus of said polypeptides 1 and 2; The polypeptides 1 and 2 bind to each other via self-binding.

2. The fusion protein of claim 1 , wherein the drug moiety is an antibody or an antigen-binding fragment thereof, or a protein.

3. The fusion protein according to claim 2 , wherein the antigen-binding fragment of the antibody is an scFv.

4. The fusion protein according to claim 2, wherein the protein is an albumin binding peptide (ABP) or TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand).

5. The fusion protein according to claim 4, wherein the protein is a trailing trimer.

6. The fusion protein of claim 1, wherein the drug moiety is linked to the N-terminus or C-terminus of the polypeptide 1 or 2 via a linker.

7. A nucleic acid molecule encoding the fusion protein of claim 1.

8. A vector comprising the nucleic acid molecule of claim 7.

9. An expression construct comprising: (a) (i) a nucleic acid molecule 1 encoding a polypeptide 1 comprising an amino acid sequence in which the sixth amino acid in SEQ ID NO: 1 is mutated to cysteine; and (ii) a nucleic acid molecule 2 encoding a polypeptide 2 comprising an amino acid sequence in which the 29th amino acid in SEQ ID NO: 6 has been mutated to cysteine; (b) (i) a nucleic acid molecule 1 encoding a polypeptide 1 comprising an amino acid sequence in which the 27th amino acid in SEQ ID NO: 1 is mutated to cysteine; and (ii) a nucleic acid molecule 2 encoding a polypeptide 2 comprising an amino acid sequence in which the eighth amino acid in SEQ ID NO: 6 has been mutated to cysteine; or (c) (i) a nucleic acid molecule 1 encoding a polypeptide 1 comprising an amino acid sequence in which the amino acids at positions 6 and 27 in SEQ ID NO: 1 are mutated to cysteine; and (ii) a nucleic acid molecule 2 encoding a polypeptide 2 comprising an amino acid sequence in which the amino acids at positions 29 and 8 in SEQ ID NO: 6 are mutated to cysteine; an expression construct comprising: An expression construct, wherein the nucleic acid molecules 1 and 2 are expressed in the same vector or different vectors, and polypeptides 1 and 2 can be self-associated.

10. A host cell comprising the vector of claim 8 or the expression construct of claim 9.

11. A composition comprising: (a) i) a polypeptide 1 comprising the amino acid sequence of SEQ ID NO: 3 as a self-binding domain 1, or a nucleic acid molecule 1 encoding said polypeptide 1; and ii) a polypeptide 2 comprising the amino acid sequence of SEQ ID NO: 8 as the self-binding domain 2, or a nucleic acid molecule 2 encoding said polypeptide 2; or (b) i) a polypeptide 1 comprising the amino acid sequence of SEQ ID NO: 2 as a self-binding domain 1, or a nucleic acid molecule 1 encoding said polypeptide 1; and ii) A polypeptide 2 comprising the amino acid sequence of SEQ ID NO: 7 as a self-binding domain 2, or a nucleic acid molecule 2 encoding said polypeptide 2.

12. A pharmaceutical composition for preventing or treating cancer, comprising the fusion protein of claim 1, the nucleic acid molecule of claim 7, the vector of claim 8, the expression construct of claim 9, or the composition of claim 11.

13. A pharmaceutical composition for preventing or treating cancer, comprising the host cell of claim 10.

14. A kit containing: (a) (i) a polypeptide 1 comprising an amino acid sequence in which the sixth amino acid in SEQ ID NO: 1 has been mutated to cysteine, or a nucleic acid molecule 1 encoding the polypeptide 1; (ii) a polypeptide 2 comprising an amino acid sequence in which the 29th amino acid in SEQ ID NO: 6 is mutated to cysteine, or a nucleic acid molecule 2 encoding the polypeptide 2; and (iii) a drug moiety bound to one or more sites selected from the group consisting of the N-terminus and C-terminus of said polypeptides 1 and 2, or a drug moiety-encoding nucleotide sequence bound to a position selected from the group consisting of the 5'-terminus or 3'-terminus of said nucleic acid molecules 1 and 2; (b) (i) a polypeptide 1 comprising an amino acid sequence in which the 27th amino acid in SEQ ID NO: 1 has been mutated to cysteine, or a nucleic acid molecule 1 encoding the polypeptide 1; (ii) a polypeptide 2 comprising an amino acid sequence in which the eighth amino acid in SEQ ID NO: 6 is mutated to cysteine, or a nucleic acid molecule 2 encoding the polypeptide 2; and (iii) a drug moiety bound to one or more sites selected from the group consisting of the N-terminus and C-terminus of said polypeptides 1 and 2, or a drug moiety-encoding nucleotide sequence bound to a position selected from the group consisting of the 5'-terminus or 3'-terminus of said nucleic acid molecules 1 and 2; or (c) (i) a polypeptide 1 comprising an amino acid sequence in which the amino acids at positions 6 and 27 in SEQ ID NO: 1 are mutated to cysteine, or a nucleic acid molecule 1 encoding the polypeptide 1; (ii) a polypeptide 2 comprising an amino acid sequence in which the amino acids at positions 29 and 8 in SEQ ID NO: 6 are mutated to cysteine, or a nucleic acid molecule 2 encoding the polypeptide 2; and (iii) a drug moiety bound to one or more sites selected from the group consisting of the N-terminus and C-terminus of said polypeptides 1 and 2, or a drug moiety-encoding nucleotide sequence (nucleic acid encoding a drug moiety) bound to a position selected from the group consisting of the 5'-terminus or 3'-terminus of said nucleic acid molecules 1 and 2.

15. 15. The kit of claim 14, wherein the drug moiety is TRAIL.

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