Coiled-Coil Fusion Protein

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 said, or a composition comprising said polypeptide, fusion protein or nucleic acid molecule. The fusion protein or composition of the present invention may be implemented in a modular form by combining or loading various drug moieties (proteins, antibody fragments, etc.) according to a desired purpose, and by combining them through self-binding, it is possible to achieve enhanced binding affinity to a target and / or increased drug activity.

Description

Technology Field

[0001] The present invention relates to a coiled coil scaffold with enhanced binding stability through co-expression. The coiled coil scaffold presented in the present invention relates to a fusion protein or drug delivery system capable of implementing a multi-complex of various functional molecules in a modular format by binding a drug moiety (protein, antibody fragment, etc.) to an MBD2-p66α (Methyl-binding domain protein2 - Transcriptional repressor p66α) complex. Background Technology

[0003] Drug delivery systems currently in use or under development generally have a maximum dual-drug loading capacity, and the loaded drug form is limited to a single type. Furthermore, creating a delivery system to load a new drug requires replicating the production process of existing drugs, which is inefficient and presents the disadvantage of not being able to easily change the combination of loaded drugs. Prior art literature

[0005] (Patent Document 0001) US 2014-0073566 A1 The problem to be solved

[0006] The inventors have made diligent efforts to develop a platform for a fusion protein or drug delivery system that can easily carry various types of drug moiety (therapeutic proteins, antibody fragments, etc.) while exhibiting improved pharmacological effects. As a result, the present invention was completed by confirming that when a drug moiety is attached to the N-terminus or C-terminus of a polypeptide containing positions 360-393 of the MBD2 amino acid sequence and a polypeptide containing positions 138-178 of the p66α amino acid sequence, the two polypeptides bind by self-binding, thereby exhibiting enhanced binding affinity to a target and / or drug activity.

[0007] Therefore, the object of the present invention is to provide a fusion protein comprising polypeptide 1 comprising a self-binding domain 1 and polypeptide 2 comprising a self-binding domain 2.

[0008] Another objective of the present invention is to provide a nucleic acid molecule encoding the fusion protein.

[0009] Another objective of the present invention is to provide a vector comprising the nucleic acid molecule.

[0010] Another objective of the present invention is to provide an expression construct comprising a nucleic acid molecule 1 encoding polypeptide 1 comprising a self-binding domain 1 and a nucleic acid molecule 2 encoding polypeptide 2 comprising a self-binding domain 2.

[0011] Another objective of the present invention is to provide a host cell comprising the vector or expression construct, or transformed with the vector or expression construct.

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein, nucleic acid molecule, vector, expression construct, or host cell.

[0013] Another objective of the present invention is to provide a kit comprising polypeptide 1 or nucleic acid molecule 1 and polypeptide 2 or nucleic acid molecule 2.

[0015] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0017] According to one aspect of the present invention, the present invention provides a fusion protein comprising polypeptide 1 comprising a self-binding domain 1 and polypeptide 2 comprising a self-binding domain 2.

[0019] In this specification, the term “fusion protein” means a hybrid protein expressed by a nucleic acid molecule comprising the nucleotide sequences of at least two genes.

[0020] In this specification, the term “self-binding domain” refers to a protein capable of self-binding 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 enable non-covalent bonding, which is a hydrophobic and / or ionic interaction between them, so that individual domains can bind (self-bind) to each other to form a coiled coil.

[0021] The self-binding domain 1 preferably comprises the amino acid sequence of SEQ ID NO. 1, or an amino acid sequence in which an amino acid selected from the group consisting of the 6th, 27th, and combinations thereof in SEQ ID NO. 1 is modified to cysteine, and more preferably comprises an amino acid sequence in which an amino acid selected from the group consisting of the 6th, 27th, and combinations thereof in SEQ ID NO. 1 is modified to cysteine.

[0022] The self-binding domain 2 preferably comprises the amino acid sequence of SEQ ID NO. 6, or an amino acid sequence in which an amino acid selected from the group consisting of the 8th, 29th, and combinations thereof from SEQ ID NO. 6 is modified to cysteine, and more preferably comprises an amino acid sequence in which an amino acid selected from the group consisting of the 8th, 29th, and combinations thereof from SEQ ID NO. 6 is modified to cysteine.

[0023] The mutation to cysteine ​​at the above amino acid position induces additional covalent bonding (disulfide bonding) between two self-binding domains, thereby enabling the formation of a more stable fusion protein.

[0024] According to one embodiment of the present invention, when a cysteine ​​mutation is introduced at a location other than the cysteine ​​mutation sites of the self-binding domain 1 (positions 6 and 27 in SEQ ID NO. 1) and the cysteine ​​mutation sites of the self-binding domain 2 (positions 8 and 29 in SEQ ID NO. 6), for example, at positions 16 and 20 in SEQ ID NO. 1 and / or positions 18 and 11 in SEQ ID NO. 6, etc., it can be confirmed that a stable fusion protein is not formed or the expression level is significantly reduced (Figs. 3a and b).

[0025] According to a preferred embodiment of the present invention, the self-binding domain 1 comprises the amino acid sequence of SEQ ID NO. 2 or 3.

[0026] According to a preferred embodiment of the present invention, the self-binding domain 2 comprises the amino acid sequence of SEQ ID NO. 7 or 8.

[0027] 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.

[0028] 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.

[0029] The fusion protein of the present invention may additionally include one or more drug moietys.

[0030] 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 polypeptide 1 comprising the self-binding domain 1.

[0031] 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.

[0032] The term "polypeptide 1" as used in this specification means a peptide comprising the self-binding domain 1 or the self-binding domain 1, and having the property of self-binding with the self-binding domain 2 or peptide 2 comprising the self-binding domain 2.

[0033] The term "polypeptide 2" as used in this specification means a peptide comprising the self-binding domain 2 or the self-binding domain 2, which has the property of self-binding to the self-binding domain 1 or the peptide 1 comprising the self-binding domain 1.

[0034] As used herein, the term "drug moiety" refers to a substance that interacts with other types of proteins or substances, such as antibodies or their antigen-binding fragments, or proteins (e.g., therapeutic proteins, ligands, receptors, Fc sites), or that can perform a specific function upon expression. The protein may be a full-length protein, as well as a domain responsible for a specific function or a part of a polypeptide, and may be a natural or artificial sequence.

[0035] In this specification, the term "antibody" refers to a protein molecule that acts as a receptor specifically recognizing an antigen, comprising an immunoglobulin molecule having immunological reactivity with a specific antigen, and includes both polyclonal antibodies and monoclonal antibodies.

[0036] Antibodies include not only the complete full-length antibody form but also antigen-binding fragments (antibody fragments) of the antibody molecule. A complete antibody has a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0037] In this specification, the term “antigen-binding fragment of an antibody” means a fragment within the entire antibody molecule that specifically recognizes an antigen and possesses antigen-antibody binding function, and includes single-domain antibodies (sdAb), single-strand antibodies (scFv), Fab, F(ab'), F(ab')2, and Fv, etc. Examples of fragments include a monovalent fragment (Fab fragment) composed of VL, VH, Cκ (or CL), and CH1 domains; a divalent fragment (F(ab')2 fragment) comprising two Fab fragments connected by a disulfide bridge at a hinge region; a Fd fragment composed of VH and CH1 domains; a Fv fragment composed of VL and VH domains of one arm of the antibody and a disulfide-linked Fv (sdFv); a dAb fragment composed of a VH domain; and a combination of two or more separate CDRs that may be selectively led by a separate complementarity determining region (CDR) or a linker. Additionally, scFv can be linked by a linker so that the VL and VH regions are paired to form a single protein chain that forms a monovalent molecule. Such single-strand antibodies are also included in antibody fragments. Furthermore, the antibody or antibody fragment includes a tetramer antibody comprising two heavy chain molecules and two light chain molecules; an antibody light chain monomer; an antibody heavy chain monomer; an antibody light chain dimer; an antibody heavy chain dimer; an intrabody; a monovalent antibody; a camel antibody; and a single-domain antibody (sdAb).

[0038] According to a preferred embodiment of the present invention, the antigen-binding fragment of the antibody as the drug moiety is scFv.

[0039] According to a preferred embodiment of the present invention, the protein as the drug moiety is an albumin binding peptide (ABP) or a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

[0040] 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. Since albumin possesses a binding affinity for FcRn, it has a long plasma half-life of 3 weeks, and the characteristic of increased half-life can be imparted by including the sequence of an albumin-binding peptide possessing albumin-binding activity in a fusion protein. Various types of albumin-binding peptides have been introduced in the art (Zorzi et al., Medchemcomm ... 2019 Jul 1; 10(7): 1068-1081 etc.) are incorporated herein by reference.

[0041] As used herein, the term "TRAIL" refers to a ligand that induces apoptosis in tumor cells, which is a type of Tumor Necrosis Factor (TNF). TRAIL possesses four receptors, among which death receptors (DR4, DR5) are overexpressed in cancer cell lines, while decoy receptors (DcR1, DcR2) are overexpressed in normal cell lines. TRAIL can induce tumor cell death by binding to death receptors, such as DR4 and DR5, located on the surface of tumor cells. In the case of the decoy receptors, they do not possess a death domain at their c-terminus, so apoptosis signaling is not transmitted into the cell. Therefore, TRAIL-based therapy is a highly effective next-generation anticancer agent characterized by being harmless to normal cells while inducing toxicity only in various cancer cell lines. However, despite the above advantages, the active form of TRAIL monomer, the trimer, is connected by non-covalent bonds, resulting in poor structural stability and persistence in the body, which limits its clinical application.

[0042] According to a preferred embodiment of the present invention, the protein as the drug moiety is a timer.

[0043] The above trail trimer can be formed within the fusion protein by being bound to the N-terminus and / or C-terminus of polypeptide 1 containing the self-binding domain 1 or polypeptide 1 containing the self-binding domain 1, or by being bound to the N-terminus and / or C-terminus of polypeptide 2 containing the self-binding domain 2 or polypeptide 2 containing the self-binding domain 2.

[0044] According to one embodiment of the present invention, a trail hexamer formed by the self-bonding of trail trimer-self-bonding domain 1 and trail trimer-self-bonding domain 2 can be seen to have an affinity for a death receptor increased by 2-5 times compared to the case where 1) trail trimer-self-bonding domain 1 or 2) trail trimer-self-bonding domain 2 is expressed alone (Fig. 4).

[0045] According to another embodiment of the present invention, a trail hexamer formed by the self-bonding of trail trimer-self-bonding domain 1 and trail trimer-self-bonding domain 2 can be confirmed to have a cancer cell death rate increased by more than four times compared to the case where 1) trail trimer-self-bonding domain 1 or 2) trail trimer-self-bonding domain 2 is expressed alone (Figs. 5a and b).

[0046] The terms "N-terminus" and "C-terminus" as used in this specification refer to the amino terminus and the carboxy terminus of a polypeptide. The drug moiety may be formed such that proteins of the same type (e.g., Trail Trimer + Trail Trimer, etc.) are 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, or may be formed such that proteins of different types (e.g., Trail Trimer + ABP or Trail Trimer + ABP + scFv, etc.) are bound to their respective N-terminus and / or C-terminus, and may be implemented as a modular complex of multiple functional molecules.

[0047] According to a preferred embodiment of the present invention, the drug moiety is linked to the N-terminus or C-terminus of polypeptide 1 or 2 by a linker.

[0048] As used herein, the term "linker" refers to an amino acid sequence existing between the N-terminus or C-terminus of polypeptide 1 or 2 and the N-terminus or C-terminus of the drug moiety. The linker may be any combination of amino acids, but is not limited thereto, and may be introduced for the purpose of promoting the binding and dissociation of polypeptide 1 or 2 and the drug moiety, providing 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, a repeating sequence of Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser, or a combination thereof, but is not limited thereto.

[0049] According to a preferred embodiment of the present invention, the present invention relates to a coiled-coil composite comprising the following configuration:

[0050] (i) polypeptide 1 in which an amino acid selected from the group consisting of the 6th, 27th, and combinations thereof from SEQ ID NOs 1 is modified to cysteine; and

[0051] (ii) polypeptide 2 in which an amino acid selected from the group consisting of sequence numbers 6 through 8, 29 and combinations thereof is modified to cysteine.

[0053] According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the fusion protein or the coiled-coil complex.

[0055] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes 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 sugar or base sites are modified (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, (1990) 90:543-584). The sequence of the nucleic acid molecule encoding the fusion protein of the present invention may be modified. Such modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0056] The nucleic acid molecule of the present invention is interpreted to include a nucleotide sequence that exhibits substantial identity with respect to the nucleotide sequence described above. The substantial identity described above refers to a nucleotide sequence that exhibits at least 80% homology, at least 90% homology in one specific example, and at least 95% homology in another specific example when any other sequence is aligned to correspond as much as possible with the nucleotide sequence of the present invention described above and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0057] According to a preferred embodiment of the present invention, a nucleotide sequence encoding a drug moiety may be included at a position selected from the group consisting of the 5' end or the 3' end of a nucleic acid molecule encoding polypeptide 1 (nucleic acid molecule 1) and / or a nucleic acid molecule encoding polypeptide 2 (nucleic acid molecule 2).

[0059] According to another aspect of the present invention, the present invention provides a vector comprising the nucleic acid molecule.

[0061] In this specification, the term "vector" refers to a carrier capable of inserting a polynucleotide sequence for introduction into a cell capable of replicating the nucleic acid molecular sequence. The polynucleotide sequence may be exogenous or heterologous. Examples of vectors include, but are not limited to, plasmids, cosmid vectors, and viral vectors (retroviruses, adenoviruses, and adeno-associated virus vectors, etc.). Those skilled in the art may construct vectors using standard recombinant techniques (Maniatis, et al., *Molecular Cloning, A Laboratory Manual*, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., *Current Protocols in Molecular Biology*, John, Wiley & Sons, Inc, NY, 1994, etc.).

[0062] In this specification, the term “expression construct” means a vector containing a nucleotide sequence encoding at least a portion of a gene product being transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. The expression vector may include various regulatory sequences. Along with regulatory sequences that regulate transcription and translation, the vector and the expression vector may also include nucleic acid sequences that provide other functions. The expression construct may simultaneously contain nucleic acid molecule 1 encoding polypeptide 1 and nucleic acid molecule 2 encoding polypeptide 2 within the same vector, or may contain them within different vectors. In vitro or in vivo, polypeptide 1 and 2 expressed from the same vector or different vectors may self-couple to form a fusion protein.

[0064] According to another aspect of the present invention, the present invention provides a host cell comprising the aforementioned vector or expression construct or transformed with said vector or expression construct.

[0066] In this specification, the term "cell" includes eukaryotic and prokaryotic cells and refers to any transgenic cell capable of replicating the vector or expressing the gene encoded by the vector. The cell may be transfected, transduced, or transformed by the vector, which refers to the process in which an exogenous polynucleotide (nucleic acid molecule) is delivered or introduced into a host cell. In this specification, the term "transformation" is used to include the meanings of transfected and transduced.

[0067] The (host) cells of the present invention are not limited, but preferably insect cells or mammalian cells, more preferably Sf9 in the case of 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, MIA 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.

[0068] The host cell of the present invention is preferably an isolated host cell.

[0070] According to another aspect of the present invention, the present invention provides a composition comprising the aforementioned fusion protein, the nucleic acid molecule, the vector, the expression construct, or the cell.

[0072] According to another aspect of the present invention, the present invention provides a composition comprising i) a polypeptide 1 having an amino acid sequence of SEQ ID NO. 2 or 3 as a self-binding domain 1, or a nucleic acid molecule 1 encoding said polypeptide 1, and ii) a polypeptide 2 having an amino acid sequence of SEQ ID NO. 7 or 8 as a self-binding domain 2, or a nucleic acid molecule 2 encoding said polypeptide 2.

[0074] In the present invention, details identical to the items described above, such as "polypeptide," "nucleic acid molecule," and "drug moiety," are to be omitted to avoid complexity and repetition in the specification.

[0075] A drug moiety may be attached to one or more sites selected from the group consisting of the N-terminus and C-terminus of polypeptide 1 having the amino acid sequence of SEQ ID NO. 2 or 3 and polypeptide 2 having the amino acid sequence of SEQ ID NO. 7 or 8, which are included in the above composition.

[0076] Accordingly, the above composition may include 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.

[0077] In addition, a nucleotide sequence encoding a drug moiety may be attached to a position selected from the group consisting of a nucleic acid molecule 1 encoding polypeptide 1 containing the amino acid sequence of SEQ ID NO. 2 or 3 and a nucleic acid molecule 2 encoding polypeptide 2 containing the amino acid sequence of SEQ ID NO. 7 or 8, which are included in the above composition.

[0078] In addition, the nucleotide sequence encoding nucleic acid molecule 1, nucleic acid molecule 2, and the drug moiety bound thereto, which is included in the above composition, may exist within the composition in a form inserted into one or more vectors.

[0079] According to a preferred embodiment of the present invention, the composition is a pharmaceutical composition for the prevention or treatment of cancer.

[0081] According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer comprising the step of administering to a subject a pharmaceutically effective amount of the aforementioned fusion protein, the nucleic acid molecule, the vector, the expression construct, the cell, or the composition.

[0083] According to another aspect of the present invention, the present invention provides a therapeutic use of the aforementioned fusion protein, the nucleic acid molecule, the vector, the expression construct, the cell, or the composition.

[0085] According to a preferred embodiment of the present invention, the therapeutic use is for the treatment of cancer.

[0086] In this specification, the term "prevention" means suppressing the occurrence of a disease or illness in subjects who have not been diagnosed with having such a disease or illness but are at risk of developing such a disease or illness.

[0087] In this specification, the term “treatment” means (a) inhibition of the development of a disease, illness, or symptom; (b) alleviation of a disease, illness, or symptom; or (c) elimination of a disease, illness, or symptom.

[0088] Accordingly, the composition of the present invention may serve as a composition for treating cancer itself, or it may be applied as a therapeutic adjuvant to enhance therapeutic responsiveness when administered together with other pharmacological components. Accordingly, in this specification, the terms "treatment" or "therapeutic agent" include the meanings of "therapeutic aid" or "therapeutic adjuvant."

[0089] In this specification, the terms "administration" or "to administer" refer to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that an equal amount is formed within the subject's body.

[0090] In the present invention, the term "therapeutic effective amount" refers to the content of a composition in which the pharmacological component within the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to an individual to whom the pharmaceutical composition of the present invention is to be administered, and includes the meaning of "preventive effective amount."

[0091] In this specification, the term "object" includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys. Specifically, the object of the present invention is a human.

[0092] Compositions disclosed herein are disclosed among pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA) having a desired degree of purity. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers, e.g., phosphates, citrates, 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, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulin; Hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counter-ions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, e.g., TWEEN ® , PLURONICS ® or includes polyethylene glycol (PEG).

[0093] In some embodiments, the pharmaceutical composition comprises the fusion protein, nucleic acid molecule, vector, expression construct, or cell disclosed herein in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of the 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 included in the pharmaceutical composition.

[0094] Pharmaceutically acceptable carriers used in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, metal ion blocking or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous vehicles include sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, and dextrose and lactate Ringer's injection. Non-aqueous vehicles include fixed oils of plant origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations may be added to parenteral formulations packaged in multi-dose containers, and these include phenol or cresol, mercury-containing substances, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspension and dispersants include sodium carboxymethylcellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN ®80) includes. The metal ion blocking or chelating agent includes EDTA. The pharmaceutical carrier also includes ethyl alcohol, polyethylene glycol, and propylene glycol for water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0095] The pharmaceutical composition may be formulated for any route of administration to the subject. Specific examples of routes of administration include intranasal, oral, parenteral, intravertebral, intraventricular, pulmonary, subcutaneous, or intraventricular routes. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also considered. The injectable substance may be prepared in conventional forms, as a liquid solution or suspension, as a solid form suitable for becoming a solution or suspension in liquid before injection, or as an emulsion. The injectable substance, solution, and emulsion also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other agents, such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrin.

[0096] A pharmaceutical composition prepared for parenteral administration comprises a sterile solution ready for injection, a sterile dry soluble product that can be combined with a solvent immediately before use including a tablet for subcutaneous injection, such as a lyophilized powder, a sterile suspension ready for injection, a sterile dry insoluble product that can be combined with a vehicle immediately before use, and a sterile emulsion. The solution may be aqueous or non-aqueous.

[0097] The pharmaceutical compositions disclosed herein may also be formulated to target specific tissues, receptors, or other body regions of the target to be treated. Various targeting methods are known to those skilled in the art. The use of such targeting methods in the compositions is considered. For non-limiting examples of targeting methods, see U.S. Patents No. 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 composition disclosed herein may be used to treat cancer.

[0098] The composition used for in vivo administration can be sterilized. For example, it can be sterilized by filtration through a sterile filter membrane.

[0100] According to another aspect of the present invention, the present invention provides a kit comprising the following configuration:

[0101] (i) polypeptide 1 comprising an amino acid sequence in which an amino acid selected from the group consisting of sequence numbers 1 to 6, 27, and combinations thereof is modified to cysteine, or nucleic acid molecule 1 encoding the same;

[0102] (ii) polypeptide 2 comprising an amino acid sequence in which an amino acid selected from the group consisting of SEQ ID NOs 6 through 8, 29, and combinations thereof is modified to cysteine, or nucleic acid molecule 2 encoding the same; and

[0103] (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 nucleic acid encoding a drug moiety bound to a site selected from the group consisting of the 5' terminus or 3' terminus of nucleic acid molecules 1 and 2.

[0105] In the present invention, details identical to the items described above, such as "polypeptide," "nucleic acid molecule," and "drug moiety," are to be omitted to avoid complexity and repetition in the specification.

[0106] In some embodiments, a pharmaceutical pack or kit is disclosed herein comprising one or more containers filled with one or more of the following: (i) polypeptide 1 or a nucleic acid molecule 1 encoding thereof, (ii) polypeptide 2 or a nucleic acid molecule 2 encoding thereof, and (iii) a drug moiety coupled 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-coding nucleotide sequence coupled to a position 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 the pharmaceutical composition disclosed herein and any preventive or therapeutic agent disclosed herein. In some embodiments, the kit discloses a composition for the prevention or treatment of cancer comprising instructions for the composition disclosed herein. Effects of the invention

[0108] The features and advantages of the present invention are summarized as follows:

[0109] (i) The present invention provides a polypeptide comprising a self-binding domain, a fusion protein comprising said polypeptide or a nucleic acid molecule encoding said polypeptide, or a composition comprising said polypeptide, fusion protein or nucleic acid molecule.

[0110] (ii) The fusion protein of the present invention may be implemented in a modular form by combining or loading various drug moieties (proteins, antibody fragments, etc.) according to the desired purpose, and by combining them through self-binding, it may achieve enhanced binding affinity to a target and / or increased drug activity. Brief explanation of the drawing

[0112] Figure 1a shows the results of predicting the structures of coil C'-GFP-6xHis and coil C-mCherry-6xHis using Alphafold2. Figure 1b shows the results of measuring the molecular weight of purified C'-GFP and C-mCherry by SDS-PAGE. Figure 2a is the result of performing Native PAGE to check whether the coiled fusion fluorescent protein was folded correctly. Figure 2b shows the results of determining the binding parameters by investigating the thermodynamics between C'-GFP and C-mCherry using an isothermal titration calorimeter (ITC). Figure 3a shows the results of confirming whether Ala27-Ile8, Thr6-Arg29, Coiled coil_F1, and Coiled coil_F2 form a stable complex. Figure 3b shows the results of comparing the relative expression levels of Ala27-Ile8, Thr6-Arg29, Coiled coil_F1, and Coiled coil_F2. Figure 3c is the result of a relative comparison of the FRET efficiencies of Thr6-Arg29 and C'-GFP / C-mCherry when the FRET efficiencies of Ala27-Ile8 are set to 100%. Figure 4 shows the results of comparing the affinity of the trail hexamer and the death receptor (DR and scTRAIL-C+scTRAIL-C') with the affinity of the trail trimer-C or trail trimer-C' with the death receptor (DR and scTRAIL-C or DR and scTRAIL-C'). Figure 5a shows the results of observing glioblastoma cell lines treated with a positive control (R&D TRAIL), a TRAIL trimer-C (I) or TRAIL trimer-C' (II) test group, and a TRAIL hexamer test group (I+II) using a flow cytometry analyzer (FACS). Figure 5b shows the results of comparing the cancer cell death rates of the positive control group (PC) and the Trail Triomer-C (T1) or Trail Triomer-C' (T2) test group with the Trail Hexomer test group (T3). Specific details for implementing the invention

[0113] The present invention will be described in more detail below through embodiments. These embodiments are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these embodiments according to the gist of the invention.

[0115] Examples

[0116] Materials and Methods

[0117] 1. Ingredients

[0118] 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.coliIt 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. DPBS (Dulbecco's Phosphate Buffered Saline) was purchased from WELKENE. Glycine was purchased from DUKSAN. Tris / Gycine SDS Pre-cast gel 8-16% and Tris / Glycine non-SDS Pre-cast gel 12% were purchased from KOMABIOTECH. Polypropylene columns 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.

[0120] 2. Plasmid Construction and Bacterial Strains

[0121] Insertions encoding the coiled-coil region of human MBD2 or its variants (amino acids 360-393, SEQ ID NOs 1 to 5) or p66 or its variants (amino acids 138-178, SEQ ID NOs 6 to 10) were inserted into ampicillin-resistant pET21a. pET-GFP-p66α was generated by cloning GFP-p66α between NdeI and XhoI of pET21a, and mCherry-MBD2 was also cloned between NdeI and XhoI of pET21a. The above experiments were performed on BIONICS.

[0122] Human MBD2 or its variants and p66α or its variants sequences Peptide Mutation SEQ ID NO Sequence MBD2 Wild type 1 KAFIVTDEDIRKQEERVQQVRKKLEEALMADILS A27C 2 KAFIVTDEDIRKQEERVQQVRKKLEE C LMADILS T6C 3 KAFIF C DEDIRKQEERVQQVRKKLEEALMADILS R16C 4 KAFIVTDEDIRKQEE C VQQVRKKLEEALMADILS V20C 5 KAFIVTDEDIRKQEERVQQ C RKKLEEALMADILS p66α Wild type 6 PEERERMIKQLKEELRLEEAKLVLLKKLRQSQIQKEATAQK I8C 7 PEERERM C KQLKEELRLEEAKLVLLKKLRQSQIQKEATAQK R29C 8 PEEERMIKQLKEELRLEEAKLVLLKKL C QSQIQKEATAQK E18C 9 PEERERMIKQLKEELRL C EAKLVLLKKLRQSQIQKEATAQK L11C 10 PEERERMIKQ C KEELRLEEAKLVLLKKLRQSQIQKEATAQK

[0124] 3. Protein Expression and Purification

[0125] - culture

[0126] BL21[DE3] (chemically competent cells) were thawed on ice, 50 µl of each was taken and placed into a microcentrifuge tube, and 1 µl of expression vector was added. At this time, two negative control microcentrifuge tubes were prepared; the procedure was performed by adding the expression vector to one tube and not adding it to the other. The cells were placed on ice for 30 minutes, subjected to a heat shock at 42°C for 30 seconds, and then placed on ice for 2 minutes. Subsequently, 200 µl of Luria Bertani medium was added, and 70 µl of each was taken and absorbed onto Luria Bertani agar plates. Finally, colonies were collected in an incubator set to 37°C for at least 12 hours, placed in 5 ml of Luria Bertani medium, and cultured overnight in a shaking incubator at 37°C and 200 rpm. 400 mL of Luria Bertani medium was taken and placed in a 1 L Erlenmeyer flask, then placed in a shaking incubator set to 37°C and 200 rpm. Once the temperature was regulated, 400 µl of ampicillin was added, followed by the tube culture, and the cells were induced with isopropyl β-D-1-thiogalactopyranoside at an A600 of 0.6. Cells were collected when the A600 was measured to reach 2. All absorbances were measured using a Biodrop Duo Micro Volume Spectrophotometer (BIODROP).

[0127] - Cell harvesting / lysis

[0128] The weight of the medium labeled for centrifugation was divided exactly in half and placed in Oak Ridge Centrifuge Tubes. After centrifugation at 4000 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 lyse the pellet. Subsequently, the lysed solution was transferred to a 50 mL conical tube, 400 µl of lysozyme was added, and the mixture was stirred using a rotator at 4°C for 30 minutes. The well-mixed solution was sonicated over ice for 7 minutes, centrifuged at 10000 rcf for 20 minutes at 4°C, the precipitate was discarded, and the supernatant was collected.

[0129] - Affinity Refining

[0130] 1.5 mL of Ni-NTA Agarose was added to the solution and mixed with a rotator for 10 minutes. To obtain only the protein from the solution, the solution was added to a polypropylene column (5 mL). Approximately 15 mL of washing buffer was flowed over the beads to remove impurities. 500 µl of elution buffer was added to the beads five times, and the solution exiting the column was transferred to a sterile micro tube. Since purified GFP-p66alpha and mCherry-MBD2 were present in the elution buffer, the buffer was changed to PBS. After filling the PD10 column with PBS, 2.5 mL of the protein-containing solution was added, and 3 mL of PBS was added to change the protein buffer.

[0131] - Size Exclusion Chromatography (FPLC)

[0132] Size exclusion chromatography (SEC) was performed on a ProteoSEC Dynamic 11 / 30 3-70 HR SEC column (Protein Ark, UK) equilibrated with PBS. Samples eluted from Ni-NTA were 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 using PBS at a flow rate of 1 ml / min, and the eluted protein fractions were collected separately.

[0134] 4. Protein Electrophoresis

[0135] 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 prior to separation by SDS-PAGE. The gel was stained with Coomassie Brilliant Blue to visualize the proteins. Native PAGE was run on the 12% polyacrylamide gel for 2 hours at a voltage of up to 125 V. Subsequently, UV irradiation was performed to observe the fluorescence of the proteins.

[0137] 5. Isothermal Titration Calorimeter

[0138] ITC analysis was performed at the Institute for Basic Science (Ochang, Korea) using the 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 the titration experiment, 2 µl of C'-GFP was injected into C-mCherry 19 times at 150-second intervals for 4 seconds at 25°C, and the data were analyzed using MicroCal Origin 7.0 software. N is the stoichiometric number, K D ε is the binding affinity, and ΔH and ΔS are the changes in enthalpy and binding entropy, respectively.

[0140] 6. Size Exclusion Chromatography (HPLC)

[0141] To analyze protein-protein interactions, a Cytiva Superdex 200 Increase 10 / 300 GL column equilibrated with PBS (pH 7.5) was used in a YL HPLC system (Semi-prep) (YL9100S HPLC). Samples (C'-GFP and C-mCherry or C'-GFP / C-mCherry) were loaded onto the column either individually or in equimolar ratios. The eluent was monitored by detecting absorbance at 280 nm.

[0143] 7. Fluorescence Resonance Energy Transfer Analysis (FRET Analysis)

[0144] Samples of wild-type GFP alone or a mixture of C'-GFP / C-mCherry, Ala27-Ile8, and Thr6-Arg29 in equal molar amounts were appropriately diluted with 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 488 nm absorbance and 510 nm luminescence (fluorescence) of each well were measured using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). After calculating the luminescence per unit absorbance of the wild-type GFP-only sample (A) and the luminescence per unit absorbance of each sample (B), the fluorescence resonance energy transfer efficiency was calculated using the following equation.

[0145]

[0147] Experimental results

[0148] 1. Generation and Characterization of Fluorescent Protein Fusion Wild-Type Coils

[0149] To construct a non-natural heteromeric protein complex using a coiled coil as a scaffold, amino acids 138–178 (coil C') and 360–393 (coil C), which are the coiled coil regions of p66alpha and MBD2 that self-assemble complementarily, were intended to be used. Then, the expression of the coil and complex formation were visually tracked using green fluorescent protein (GFP) and mCherry, one of the red fluorescent proteins. GFP and mCherry were fused to the C-terminuses of coil C' and coil C, respectively. A 6-histidine tag (6xHis) was additionally fused to the C-terminuses of each fluorescent protein, and the proteins were purified using nickel-nitrilotriacetic acid agarose resin. The complex structures of the coiled C'-GFP-6xHis fusion protein (hereinafter referred to as C'-GFP) and the coiled C-mCherry-6xHis fusion protein (hereinafter referred to as C-mCherry) were predicted using Alphafold2 (Fig. 1a). pET-C'-GFP and pET-C-mCherry are E. coli The proteins were expressed in the BL21 system and purified by FPLC following affinity chromatography. As a result of purification, high-purity proteins were obtained in a yield of approximately 4 mg or more per liter of culture medium. Protein samples collected after purification were separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and then destained for visualization (Fig. 1b). The estimated 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 similar to the results.

[0150] After purification, Native PAGE was performed to verify whether the coiled fusion fluorescent proteins were correctly folded. The results of Native PAGE confirmed that C'-GFP and C-mCherry exhibited green and red fluorescence when present alone (Fig. 2a). However, when C'-GFP and C-mCherry were mixed in a 1:1 ratio, the green and red fluorescences combined to produce yellow fluorescence. This indicates that each fluorescent protein is correctly folded and the fused coils self-assemble complementarily. The thermodynamics between C'-GFP and C-mCherry were investigated using an isothermal titration calorimeter (ITC) to determine the binding parameters. The measured parameters between C'-GFP and C-mCherry were N = 0.7, K D = 10.7 nM, ΔH = -2.6 kcal / mol, and ΔS = -52.4 cal / mol / deg; the reaction between the two molecules was driven spontaneously and exhibited high binding affinity (Fig. 2b). Additionally, C'-GFP and C-mCherry reacted at a ratio of 0.7:1, which was lower than the theoretical ratio of 1:1. This could be due to homomer formation between the coils or an error caused by the difference between the actual concentration and the concentration used to match the isotherm. In summary, we expressed C'-GFP and C-mCherry capable of folding correctly and binding with high affinity to form heteromer complexes.

[0152] 2. Design and Stability Evaluation of Coiled Cysteine ​​Mutants

[0153] Disulfide bridges formed between two thiol groups of intramolecular or intermolecular cysteine ​​(Cys) residues can serve as important components for proper protein folding and structural stability. To provide enhanced resistance to dissociation upon self-coupling, we sought to generate disulfide-coiled coiled complexes. To this end, we identified the locations of paired residues capable of forming disulfide bonds while minimizing inhibition of existing coiled coil interactions. C'_Ile8-C_Ala27(I8C-A27C)(SEQ No. 7 + SEQ No. 2) and C'_Arg29-C_Thr6(R29C-T6C)(SEQ No. 8 + SEQ No. 3) were determined to be optimal cross-linking sites where disulfide bonds are formed while maintaining good electrostatic interactions. To investigate the degree of inhibition of coiled-coil interactions by mutation, good electrostatic interaction residue pairs C'_Glu18-C_Arg16(E18C-R16C)(SEQ No. 9 + SEQ No. 4) and C'_Leu11-C_Val20(L11C-V20C)(SEQ No. 10 + SEQ No. 5) were selected and compared. Native PAGE analysis of the coiled-coil interaction (SEQ No. 6 + SEQ No. 1)(GFP-C' + mCherry-C) between wild-type GFP-C' and wild-type mCherry-C with and without DTT and mutations revealed that C'_Ile8-C_Ala27(I8C-A27C)(SEQ No. 7 + SEQ No. 2)(Ala27-Ile8) and C'_Arg29-C_Thr6(R29C-T6C)(SEQ No. 8 + SEQ No. 3)(Thr6-Arg29) were compared with C'_Glu18-C_Arg16(E18C-R16C)(SEQ No. 9 + SEQ No. 4)(Coiled coil_F1) and C'_Leu11-C_Val20(L11C-V20C)(SEQ No. 10 + SEQ No. 5) (Coiled coil_F2) It was found that a more stable complex was formed by comparison (Fig. 3a).In addition, a comparison of relative expression levels revealed that Ala27-Ile8 and Thr6-Arg29 were more than twice as high as Coiled coil_F1 and Coiled coil_F2 (Fig. 3b). Furthermore, quantitative analysis (FRET analysis) of the fluorescence resonance energy transfer efficiency between GFP and mCherry molecules, attributed to the stable coiled-coil interactions of Ala27-Ile8 and Thr6-Arg29, showed an efficiency more than twice as high as that of the wild type (GFP-C' + mCherry-C) (Fig. 3c).

[0155] 3. Manufacture of Trail Triomer-Coil and Trail Hexomer

[0156] 3.1. Cell Expression and Purification

[0157] Two proteins (trail trimer-MBD2 (scTRAIL-C) and trail trimer-p66α (scTRAIL-C')) were expressed in cultured transformed E. coli and purified using affinity chromatography. More specifically, trail trimer-MBD and trail trimer-p66α were expressed and purified through the following fixation:

[0158] Transformed BL21 (DE3) E. coli cells were cultured for 12 hours in solid LB medium adjusted to an ampicillin concentration of 0.1 mg / mL. Subsequently, strains confirmed to have vector insertion were inoculated into 2xYT medium (ampicillin 0.1 mg / mL, hereinafter the same) and cultured at 37°C for 12 hours. Afterward, the strains were inoculated again into 2xYT medium diluted 1:100 and cultured at 30°C with stirring at 200 rpm. When the absorbance reached 0.4–0.5 at 600 nm, 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 the cell pellet, which was then frozen and stored at -20°C.

[0159] 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 the mixing process was carried out by rotating at 4°C for approximately 30 minutes. Subsequently, sonication was performed at 10-second intervals for 8 minutes, followed by centrifugation at 10,000 g for 30 minutes to separate the proteins from the cellular residue. The supernatant containing the proteins was transferred separately, 1.5 mL of Ni-NTA agarose beads were added, and the mixture was stirred at 4°C for 30 minutes. After fixing the gravity-flow column, the supernatant mixed with Ni-NTA was fed down the column to separate the beads from the flow-through. After removing the flow-through, wash buffer (pH 8.0, 50 mM sodium phosphate, 0.3 M NaCl, and 20 mM imidazole) was sufficiently run through the beads to wash them and remove impurities attached to the beads until the absorbance of the solution flowing through the flow-through became 0.00 at 280 nm. To separate the proteins bound to the beads, the flow-through was dispensed into microtubes while running 500 µL of elution buffer (50 mM sodium phosphate (pH 8.0), 0.3 M NaCl, and 250 mM imidazole). The concentration of the dispensed solution was measured at 280 nm using a Biodrop, and the samples were refrigerated at 4°C.

[0161] 3.2. Characteristic Evaluation

[0162] Using a binding assay (MicroScale Thermophoresis, MST), 1) the affinity between trail-C (scTRAIL-C) and trail-C' (scTRAIL-C'), 2) the affinity between trail-C or trail-C' and the death receptor (DR), and 3) the affinity between trail hexamer (scTRAIL-C+scTRAIL-C') and the death receptor (DR) were measured. DR is a cognate receptor of trail.

[0163] More specifically, it was measured according to the following method:

[0164] For sample pretreatment prior to analysis, the death receptor (Biolegend) and Flamma® 648 Sulfo-NHS ester dissolved in DMSO were mixed at a molar ratio of 1:5 and reacted at room temperature for 1 hour. Before injecting the sample, the preservation solution was removed by centrifuging at 1500 g for 1 minute on a Zeba® Spin Desalting column (40K MWCO, 0.5 mL). Then, 450 µL of a 150 mM sodium carbonate solution was filled into the column, followed by centrifugation at 1500 g for 1 minute. Subsequently, 100-150 µL of the reacted DR mixture was injected, followed by centrifugation at 1500 g for 2 minutes to remove trace amounts of DMSO contained in the sample and to replace the solution with sodium carbonate, while simultaneously separating the non-labeled and labeled DR.

[0165] Before analyzing the sample using the Monolith NT.115 (Germany) MST instrument, the measurement intensity was adjusted using labeled DR. After filling four capillaries with labeled DR of the same concentration, measurements were taken and the excitation power was adjusted to confirm that the fluorescence intensity value appeared between 800 and 1000.

[0166] The recombinant proteins used as ligands were MonoTRAIL, TRAIL Trimer-C and TRAIL Trimer-C, or TRAIL Hexomer, respectively. The receptor-ligand mixing method is as follows: First, 10 µL of the death receptor protein is dispensed into 15 racks, excluding the first rack. Next, 20 µL of TRAIL Trimer-C, which acts as the ligand, is placed in the first rack. Then, 10 µL is drawn from the first rack and diluted in the second rack, and another 10 µL is drawn and diluted in the third rack; this process is repeated 16 times. Consequently, a sample diluted 216-fold becomes the final 16th sample. Samples of different concentrations are injected into 16 different capillaries, fixed to the detection device, and measurement is started. During this process, the excitation power is set to twice the initial setting value. Subsequently, the same procedure is followed for both TRAIL Trimer-C' and TRAIL Hexomer.

[0167] Experimental results confirmed that the affinity between the trail hexamer and the death receptor (163 nM) was about 2-5 times higher than the affinity between the trail trimer-C or trail trimer-C' and the death receptor (337 nM and 787 nM, respectively) (Fig. 4).

[0169] 3.3. In vitro cell assay

[0170] Glioblastoma cell line U87-luc (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), and TRAIL Hexomer (I+II) for 4 hours each was observed using a flow cytometry analyzer (FACS).

[0171] As a result of the analysis above, in contrast to the Trail Triomer-C (I) or Trail Triomer-C' (II) test groups which showed a mortality rate of less than 5%, the Trail Hexomer test group (I+II) showed a mortality rate of approximately 30%, which was confirmed to be about 4 times higher than the mortality rate of 8% of the positive control (R&D TRAIL) (Figs. 5a and b).

[0173] Although embodiments of the present invention have been described above, those skilled in the art may modify and change the present invention in various ways by adding, changing, deleting, or adding components, etc., without departing from the spirit of the invention as described in the claims, and such modifications and changes are also to be included within the scope of the rights of the present invention.

Claims

Claim 1 A fusion protein comprising the following composition: (a) (i) polypeptide 1 having an amino acid sequence in which the amino acid from SEQ ID NO. 1 to 27 is mutated to cysteine; and (ii) polypeptide 2 having an amino acid sequence in which the amino acid from SEQ ID NO. 6 to 8 is mutated to cysteine, or (b) (i) polypeptide 1 having an amino acid sequence in which the amino acid from SEQ ID NO. 1 to 6 is mutated to cysteine; and (ii) polypeptide 2 having an amino acid sequence in which the amino acid from SEQ ID NO. 6 to 29 is mutated to cysteine, wherein polypeptide 1 and 2 are combined by self-junction. Claim 2 A fusion protein according to claim 1, characterized in that (a) polypeptide 1 is composed of the amino acid sequence of SEQ ID NO. 2 and polypeptide 2 is composed of the amino acid sequence of SEQ ID NO. 7; or (b) polypeptide 1 is composed of the amino acid sequence of SEQ ID NO. 3 and polypeptide 2 is composed of the amino acid sequence of SEQ ID NO.

8. Claim 3 The fusion protein according to claim 1, characterized in that the fusion protein has a drug moiety additionally bound to one or more sites selected from the group consisting of the N-terminus and C-terminus of polypeptides 1 and 2. Claim 4 A fusion protein according to claim 3, characterized in that the drug moiety is an antibody or an antigen-binding fragment thereof, or a protein. Claim 5 A fusion protein according to claim 4, characterized in that the protein is an albumin binding peptide (ABP) or a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Claim 6 A fusion protein according to claim 5, characterized in that the protein is a timer. Claim 7 A fusion protein according to claim 3, wherein the drug moiety is linked to the N-terminus or C-terminus of polypeptide 1 or 2 by a linker. Claim 8 A nucleic acid molecule encoding the fusion protein of claim 1. Claim 9 A vector containing a nucleic acid molecule of claim 8. Claim 10 An expression construct comprising the following composition: (a) (i) a nucleic acid molecule 1 encoding polypeptide 1, wherein the amino acid at the 27th position of SEQ ID NO. 1 is mutated to cysteine; and (ii) a nucleic acid molecule 2 encoding polypeptide 2, wherein the amino acid at the 6th position of SEQ ID NO. 6 is mutated to cysteine; or (b) an expression construct comprising (i) a nucleic acid molecule 1 encoding polypeptide 1, wherein the amino acid at the 6th position of SEQ ID NO. 1 is mutated to cysteine; and (ii) a nucleic acid molecule 2 encoding polypeptide 2, wherein the nucleic acid molecules 1 and 2 are expressed in the same vector or different vectors, such that polypeptide 1 and 2 can be self-coupled. Claim 11 The expression construct according to claim 10, characterized in that the expression construct further has a nucleic acid encoding a drug moiety sequence additionally attached to a position selected from the group consisting of the 5' end or 3' end of the nucleic acid molecules 1 and 2. Claim 12 Isolated host cells comprising the vector of claim 9 or the expression construct of claim 10. Claim 13 An expression construct according to claim 10, characterized in that (a) the nucleic acid molecule 1 codes for the amino acid sequence of SEQ ID NO. 2 and the nucleic acid molecule 2 codes for the amino acid sequence of SEQ ID NO. 7; or (b) the nucleic acid molecule 1 codes for the amino acid sequence of SEQ ID NO. 3 and the nucleic acid molecule 2 codes for the amino acid sequence of SEQ ID NO.

8. Claim 14 An expression construct according to claim 11, characterized in that the drug moiety is an antibody or an antigen-binding fragment thereof, or a protein. Claim 15 An expression construct according to claim 14, characterized in that the protein is an albumin binding peptide (ABP) or a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

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