KR20210089102A - Fusion protein comprising albumin and coiled coil forming domain, and bispecfic target conjugate using the same - Google Patents

Abstract

The present invention relates to a fusion protein in which albumin or a derivative thereof, a first coiled coil forming domain and a second coiled coil forming domain are linked, and in addition, to dual target conjugate in which a target material is doubly linked to a fusion protein using self-binding properties of the coiled coil. Since the dual target conjugate is based on the albumin, which is present in large amounts in human blood, high in vivo stability and long plasma half-life of conjugate drugs can be ensured.

Description

A fusion protein in which albumin and a coiled coil-forming domain are linked, and a dual target conjugate using the same {FUSION PROTEIN COMPRISING ALBUMIN AND COILED COIL FORMING DOMAIN, AND BISPECFIC TARGET CONJUGATE USING THE SAME}

The present invention relates to a fusion protein in which albumin or a derivative thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked, and the present invention also relates to a fusion protein using the self-binding property of the coiled coil-forming domain. It relates to a dual target binding body in which two types of target binding substances are bound to a protein.

Albumin is a family of globular proteins constituting the basic material of cells and has a molecular weight of about 65 to 70 kDa, and the most common is serum albumin. Serum albumin is a soluble protein that is produced in the liver and accounts for about 55 to 60% of the total serum protein, and is a protein mainly used in the circulation. It is involved in the transport of amino acids, metals such as calcium, copper, zinc, and various drugs and metabolites, and plays an important role in the metabolism, distribution, and transport of various endogenous and exogenous ligands in the human body. It is generally known to facilitate the transport of ligands at the interface of circulation and most organs such as liver, kidney, and intestine.

Human serum albumin (HSA) is composed of 585 amino acids containing 17 disulfide bridges. It is present in large amounts in the blood at about 35 to 50 g/L and functions to maintain osmotic pressure, pH and homeostasis. Like many albumin families, human serum albumin is deeply involved in human circulatory and metabolic functions and is found in almost all human tissues.

Human serum albumin has high stability in the body, and when it is bound to a ligand or drug, it is prevented from being filtered out by the kidney due to an increase in molecular size, and similarly to an antibody, it interacts with FcRn to enter the cell and is released back out of the cell without being degraded. . Due to these characteristics, when human serum albumin is used for drug delivery, there is an effect that the half-life is longer than that of the existing ligand or drug alone. In addition, human serum albumin exhibits a phenomenon in which a large amount of albumin is selectively accumulated in cancer tissues due to its high targeting ability to cancer tissues, which is caused by the increased permeability and retention (EPR) effect around the cancer tissues. will be. Due to the effects of human serum albumin, various proteins, ligands, antibodies, and anticancer agents are chemically combined to target cancer tissues, and at the same time, studies are being conducted to increase circulation time in the blood compared to single substances.

However, due to the low yield and quality of human serum albumin when chemically binding proteins, ligands, antibodies, or anticancer drugs, manufacturing difficulties, high costs, and the absence of technology to control the amount of binding material to maintain the unique properties of albumin Commercialization is difficult.

Coiled coils are structural motifs in proteins and consist of two or more alpha helices. Coiled coils are a common method for oligomerizing proteins. The most typical coiled-coil primary sequence is repetitive, and seven residues called 'heptads' allow the two coiled-coil-forming domains to bind by hydrogen bonding, electrical bonding, hydrophobic bonding and van der Waals forces. do.

Recombinant bispecific monoclonal antibodies prepared by the dock-and-lock strategy for pretargeted radioimmunotherapy is a technique used as a drug carrier by bonding a coiled coil. To reduce dissociation of the coiled coil, a disulfide bond bridge is added to the stability of the coiled coil. Disclosed is an antibody fragment dual target drug system with increased This technology has the effect of increasing the half-life according to the increase in the molecular size, but there is a limitation in dramatically increasing the half-life of the drug because it does not have the FcRn binding ability of the antibody or albumin.

Robert et al., "Recombinant Bispecific Monoclonal Antibodies Prepared by the Dock-and-Lock Strategy for Pretargeted Radioimmunotherapy"

The present invention provides a fusion protein in which albumin or an analog thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked.

The present invention provides a nucleic acid molecule encoding the fusion protein, a vector containing the nucleic acid molecule, a host cell into which the vector is introduced, and a method for preparing a fusion protein using the host cell.

The present invention relates to albumin or an analog thereof, a fusion protein in which a first coiled coil-forming domain and a second coiled coil-forming domain are linked; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second fusion in which the second target binding material and the second 'coiled coil-forming domain are linked, wherein the first and second fusions are each bound to a fusion protein, providing a dual target binding body.

The present invention is the first fusion; or a nucleic acid molecule encoding the second fusion, a vector including the nucleic acid molecule, a host cell into which the vector is introduced, and a method for preparing a first fusion or a second fusion using the host cell.

The present invention provides a pharmaceutical composition comprising the dual target conjugate.

The present invention is in vitro albumin or an analog thereof, a fusion protein in which a first coiled coil-forming domain and a second coiled coil-forming domain are linked; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second fusion in which the second target binding material and the second 'coiled coil-forming domain are linked;

It should be understood that terms not specifically defined in this specification have meanings commonly used in the technical field to which the present invention pertains. Also, unless otherwise defined by context, the singular includes the plural and the plural includes the singular.

Terms such as first, second, etc. used in this specification may be used to describe various elements, but the elements should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from other components, and are not used to determine an order or order. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may also be referred to as a first component.

Titles of items used in this specification are only for convenience of description, and are not intended to limit the present invention.

fusion protein

One aspect of the present invention is a fusion protein in which albumin or an analog thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked. In the present invention, the first coiled coil-forming domain and the second coiled coil-forming domain may be connected to the N-terminus and C-terminus of albumin or an analog thereof, respectively, and the first coiled coil-forming domain is albumin or the second coiled coil-forming domain is linked to the C-terminus of albumin or an analogue thereof when linked to the N-terminus of the albumin or analogue thereof, and the first coiled coil-forming domain is linked to the C-terminus of the albumin or analogue thereof. The second coiled coil forming domain may be linked to the N-terminus of albumin or an analog thereof.

As used herein, the term “fusion protein” refers to a hybrid protein expressed by a nucleic acid molecule including nucleotide sequences of at least two genes. In the present invention, the fusion protein may be denoted as "X-HSA-Y", where X and Y refer to any coiled coil-forming domain assigned alphabetically herein, and HAS (Human Serum Albumin, Human Serum Albumin, Human Serum Albumin) serum albumin) means albumin or an analog thereof, and "-" means that the coiled coil forming domain is linked to HSA.

As used herein, the term "albumin" is the most abundant protein in mammalian plasma synthesized primarily in liver cells, and refers to a multifunctional plasma protein that plays an important role in maintaining blood osmotic pressure, transporting various substrates, etc. do. The albumin may be Human Serum Albumin (HSA) or any other isolated or naturally occurring albumin. Albumin has three domains, I, II and III, and each domain is composed of subdomains A and B. Albumin has a plasma half-life of 3 weeks, and the long half-life of albumin is associated with intracellular degradation of albumin by FcRn (Neonatal Fc receptor), and domains I and III of albumin play an important role in binding to FcRn. has been reported

As used herein, the term "analog" is derived from a parent protein or a parent polypeptide, and includes a portion that provides the desired properties, so that it exhibits the same or similar properties as the parent protein or polypeptide. do. In the present invention, analog is used as a term included in "variant", and variant is a protein or polypeptide in which insertions, deletions, additions and/or substitutions have occurred at one or more amino acid residues compared to the parent protein or polypeptide sequence. means In addition, although the protein has been modified by enzymatic cleavage, phosphorylation or other modifications, it may include, but is not limited to, maintaining a desired property of the fusion protein of the present invention, for example, an increase in half-life. Such a variant may be one having at least 80% homology, specifically at least 90% homology, and more specifically at least 95% homology to the sequence disclosed in the present invention. For example, an albumin analog may include substitution of some sequence in the amino acid sequence of wild-type albumin or a domain thereof to improve a desired property. For example, the half-life in blood may be extended by performing amino acid substitution to improve affinity for FcRn among the entire amino acid sequence of albumin, but is not limited thereto. For example, it includes optionally deleting amino acid(s) at a specific position, substituting one or more amino acids, or inserting one or more amino acids, see described in WO 2011051489.

In the present invention, the desired property may be a property of targeting a specific substance while maintaining a biologically required activity, increasing stability, and increasing half-life, preferably, increasing half-life. In addition, the moiety providing the desired properties may be a moiety comprising albumin domains I and III, which is known to play an important role in increasing the serum half-life of albumin, or comprising an FcRn-binding fragment of albumin.

In the present invention, the albumin analogue may be a protein or polypeptide derived from albumin and having a long half-life characteristic of albumin, but is not limited thereto.

According to an embodiment of the present invention, the albumin and its analogs may include at least one of domains I and III of albumin, but is not limited thereto.

In addition, according to another embodiment of the present invention, the albumin and its analogs may include an FcRn-binding fragment of albumin, but is not limited thereto.

The term "FcRn" used in the present invention is also referred to as neonatal Fc receptor, Brambell receptor, and the like, and refers to a protein encoded by the FCGRT gene. FcRn is an MHC class I related protein, which is expressed in vascular endothelial cells and binds to IgG and albumin, see "Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells" and "FcRn: the neonatal Fc receptor comes of age" (Molecular Biology of the Cell. 19(12): 5490-505., Nature Reviews. Immunology. 7(9): 715-25).

As used herein, the term “FcRn-binding fragment” refers to a portion of albumin that lacks some amino acids compared to the amino acid sequence of full-length albumin, but can bind to FcRn and provide an effect of increasing half-life.

As used herein, the term “homology” refers to sequence similarity determined by aligning and comparing two or more amino acid or nucleotide sequences, and such homology is generally expressed as “percent homology”.

As used herein, the terms "protein" or "polypeptide" refer to a polymer of amino acid residues and are used interchangeably in the present invention.

Albumin or an analog thereof used in the formation of the fusion protein in the present invention may be derived from any species. However, in order to avoid immunogenicity, it is preferably human serum albumin (HSA) derived from human serum, and according to an embodiment of the present invention, the albumin or an analog thereof may be human serum albumin or an analog thereof. have.

Also, in the present invention, albumin or an analog thereof may further include any molecule or substance to provide additional properties. Any molecule or substance that provides additional properties may be a therapeutic or diagnostic moiety, such as a cytotoxic or pharmaceutically active moiety, or a molecule that increases the suitability of the fusion protein for administration to a subject or other in vitro use; The present invention is not limited thereto. For example, albumin or an analog thereof may further include polyethylene glycol (PEG), thereby exhibiting an effect of improving biocompatibility without reducing binding ability to FcRn.

In the present invention, "coiled coil-forming domain" refers to a protein capable of forming a coiled coil by binding with other coiled coil-forming domains. The coiled coil may be two or more, specifically, a structural motif in which two to seven alpha helices are wound together like a strand of a rope. For example, the primary structure of the coiled coil domain may include a heptad repeat having a continuous repeating pattern of a 7 amino acid sequence. The heptane Todd repeating unit (abcdefg) _n may be expressed as, where n is the number of repetitions and, a and d has constitute the hydrophobic core of the coiled coil to a hydrophobic amino acid residue positions, e and g positions, the belt charge was Amino acid residues are positioned to allow ionic interactions. This feature enables non-covalent bonding, which is a hydrophobic and/or ionic interaction between coiled coil forming domains, allowing individual coiled coil forming domains to bind (self-bond) to each other to form a coiled coil.

According to an embodiment of the present invention, the first and second coiled coil-forming domains are an elastin-like polypeptide, a BECN (Beclin) domain, a bZIP VBP (basic leucine zipper vitellogenin-binding protein), a keratin intermediate fiber, and an AP1 (Jun) domain. /Fos), MBD2-NuRD (methyl-binding 　 domain 　 protein 2-Nucleosome Remodeling and Deacetylase) domain, GABA (γ-aminobutyric acid) receptor, zinc clamp domain, any one selected from the group consisting of leucine zippers and variants thereof However, the present invention is not limited thereto. In addition, if the first and second coiled coil forming domains are known to be capable of forming a coiled coil by combining with other coiled coil forming domains, the present invention may be applied.

According to an embodiment of the present invention, the first coiled coil-forming domain and the second coiled coil-forming domain are the amino acid sequences represented by SEQ ID NOs: 4, 5, 6, 16, 17 and 18, respectively, and the amino acid sequences of variants thereof It may include any one amino acid sequence selected from the group consisting of.

As used herein, the term "variant" may refer to a protein or polypeptide in which insertions, deletions, additions and/or substitutions have occurred at one or more amino acid residues compared to the parent protein or polypeptide sequence, and the present invention According to an embodiment, the "variant of the amino acid sequence" is the N-terminus and C of any one amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 4, 5, 6, 16, 17 and 18. -Cysteine may be added to at least one of the ends. Specifically, at least one cysteine may be added to both ends, and more specifically, one cysteine may be added to both ends, respectively.

In addition, according to an embodiment of the present invention, the variant of the amino acid sequence may be any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 42, 43, 44, 48, 49 and 50, One cysteine may be added to each of the N-terminus and C-terminus of any one amino acid sequence selected from the group consisting of the amino acid sequence represented by SEQ ID NOs: 4, 5, 6, 16, 17 and 18.

As described above, the coiled coil-forming domain in which cysteine is additionally mutated can be prepared by a technique known in the art, for example, a peptide synthesis method or a genetic engineering method. As used herein, the term “mutagenesis” means altering, deleting or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to create a variant of that sequence. Specifically, the variant of the amino acid sequence is constructed by constructing a recombinant plasmid using DNA encoding a peptide with cysteines added to both ends (ie, N-terminus and C-terminus) of the coiled coil-forming domain and transfected into a host cell. After conversion, the cysteine-added coiled coil-forming domain was expressed. The expressed cysteine-added coiled coil-forming domain may combine with other cysteine-added coiled coil-forming domains to form a coiled coil, and disulfide is formed between cysteines added to different coiled coil-forming domains. Bonds are formed to improve bond stability.

According to an embodiment of the present invention, the first coiled coil-forming domain and the second coiled coil-forming domain may be connected to the N-terminus and C-terminus of albumin or an analog thereof, respectively, and the first coiled coil When the forming domain is linked to the N-terminus of albumin or an analog thereof, the second coiled coil forming domain is linked to the C-terminus of albumin or an analog thereof, and the first coiled coil forming domain is linked to the C-terminus of albumin or an analog thereof. When linked to a terminus, the second coiled coil forming domain may be linked to the N-terminus of albumin or an analog thereof.

The terms "N-terminus" and "C-terminus" used in the present invention refer to an amino terminus and a carboxy terminus in a polypeptide, and unless otherwise specified in the present invention, as generally used, the left side of the sequence is amino Terminal, right indicates carboxy terminus. In addition, as used herein, the term "both ends" means both "N-terminal" and "C-terminal".

According to an embodiment of the present invention, the first coiled coil forming domain and the second coiled coil forming domain may not form a coiled coil through mutual coupling. If the first coiled coil-forming domain and the second coiled coil-forming domain combine with each other to form a coiled coil, a fusion protein (hereinafter, fusion protein) in which the coiled coil-forming domain is connected to both ends of albumin or an analog thereof Since the yield of albumin and the target binding material conjugate (hereinafter, double target conjugate) will decrease due to increased binding between the first and second coiled coil-forming domains, the first coiled coil-forming domain and the second coiled coil-forming domain are coupled through each other. If the yield coil cannot be formed, the binding between the fusion proteins due to the mutual binding of the first coiled coil-forming domain and the second coiled coil-forming domain is reduced and the yield of the dual target conjugate is improved.

According to an embodiment of the present invention, the amino acid sequence of the first coiled coil-forming domain and the amino acid sequence of the second coiled coil-forming domain linked to albumin or an analog thereof may be different. As in the present invention, when the first coiled coil-forming domain and the second coiled coil-forming domain include different amino acid sequences from each other, binding of two fusions with the same target-binding substance to the fusion protein is reduced and the fusion is reduced. The case where two fusions with different target binding substances are linked to a protein will increase, and when preparing a dual target binding substance, it is possible to predict the mixing ratio of fusions with different target binding substances, so that various combinations of drugs can be quickly and conveniently prepared. Not only can it be obtained, but the yield of the dual target conjugate is improved.

According to an embodiment of the present invention, the first coiled coil-forming domain is selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 4, 5, 6, 16, 17, 18, 42, 43, 44, 48, 49 and 50. It may include any one selected amino acid sequence, and the second coiled coil-forming domain is an amino acid represented by SEQ ID NOs: 4, 5, 6, 16, 17, 18, 42, 43, 44, 48, 49 and 50 It may include any one amino acid sequence selected from the group consisting of sequences. In addition, the amino acid sequence represented by SEQ ID NO: 4 and the amino acid sequence represented by SEQ ID NO: 16 or 48 can form a bond with each other, and the amino acid sequence represented by SEQ ID NO: 42 has cysteines added to both ends except that Since it can be seen as the same sequence as the amino acid sequence shown in SEQ ID NO: 4, when the first coiled coil-forming domain includes the amino acid sequence shown in SEQ ID NO: 4, the second coiled coil-forming domain is SEQ ID NO: 5, 6 , 17, 18, 43, 44, 49 and 50 may include any one amino acid sequence selected from the group consisting of amino acid sequences. Similarly, when the first coiled coil-forming domain comprises the amino acid sequence represented by SEQ ID NO: 5, the second coiled coil-forming domain is represented by SEQ ID NO: 4, 6, 16, 18, 42, 44, 48 and 50 It may include any one amino acid sequence selected from the group consisting of amino acid sequences. When the first coiled coil-forming domain includes the amino acid sequence represented by SEQ ID NO: 6, the second coiled coil-forming domain includes amino acids represented by SEQ ID NO: 4, 5, 16, 17, 42, 43, 48 and 49 It may include any one amino acid sequence selected from the group consisting of sequences. When the first coiled coil-forming domain comprises the amino acid sequence represented by SEQ ID NO: 42, the second coiled coil-forming domain includes amino acids represented by SEQ ID NO: 5, 6, 17, 18, 43, 44, 49 and 50 It may include any one amino acid sequence selected from the group consisting of sequences. When the first coiled coil-forming domain comprises the amino acid sequence represented by SEQ ID NO: 43, the second coiled coil-forming domain includes amino acids represented by SEQ ID NO: 4, 6, 16, 18, 42, 44, 48 and 50 It may include any one amino acid sequence selected from the group consisting of sequences. When the first coiled coil-forming domain comprises the amino acid sequence represented by SEQ ID NO: 44, the second coiled coil-forming domain includes amino acids represented by SEQ ID NO: 4, 5, 16, 17, 42, 43, 48 and 49 It may include any one amino acid sequence selected from the group consisting of sequences.

According to an embodiment of the present invention, the first coiled coil-forming domain and the second coiled coil-forming domain are SEQ ID NOs: 5 and 4; 43 and 42; Or it may be the amino acid sequences represented by 44 and 42, but is not limited thereto.

In addition, according to an embodiment of the present invention, the fusion protein of the present invention may include any amino acid sequence for production yield, quality control, and the like. It may include an amino acid sequence that allows the protein to be secreted out of the cell, for example, an amino acid sequence such as a signal peptide located at the N-terminus of the protein, but is not limited thereto.

According to an embodiment of the present invention, albumin or an analog thereof; and a linker between the coiled coil-forming domains. albumin or an analog thereof as in the present invention; And when the linker is included between the coiled coil-forming domains, albumin or an analog thereof, the steric hindrance that occurs when the first coiled coil-forming domain and the second coiled coil-forming domain binds together, thereby reducing coiled coil formation. has a stimulating effect.

As used herein, the term “linker” refers to an amino acid present between the N-terminus or C-terminus of albumin or an analog thereof and the N-terminus or C-terminus of a target binding substance. The linker may be any combination of amino acids, but is not limited thereto, and any linker capable of imparting flexibility to the coiled coil-forming domain may be used. In addition, the linker may have any length, but it is preferable that the linker is in a range that does not affect the biological activity while ensuring optimum stability in forming the coiled coil. The linker may have less than 30 amino acids, preferably less than 15 amino acids. In addition, the linker is preferably rich in glycine and serine, and specifically, Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser, or a combination thereof may be repeated 2 to 6 times, but limited thereto it is not going to be

In the embodiments of the present invention, albumin or an analog thereof in the fusion protein is the amino acid sequence represented by SEQ ID NO: 36, and the first and second coiled coil-forming domains are SEQ ID NO: 4, 5, 6, 16, 17, 18, 42, 43, 44, 48, 49 and 50 may be selected from the group consisting of amino acid sequences represented by, in this case, the first coiled coil-forming domain and the second coiled coil-forming domain are bound to each other Thus, a coiled coil cannot be formed, and the amino acid sequences of the first coiled coil-forming domain and the second coiled coil-forming domain are different from each other.

According to an embodiment of the present invention, albumin or an analog thereof, the first coiled coil-forming domain and the second coiled coil-forming domain are linked, the fusion protein is any one amino acid selected from SEQ ID NOs: 8, 53, 11 and 12 sequences may include, but are not limited to.

Another aspect of the present invention is albumin or an analog thereof, a nucleic acid molecule encoding a fusion protein in which a first coiled coil-forming domain and a second coiled coil-forming domain are linked, a vector comprising the nucleic acid molecule, and the vector is introduced A method for producing a fusion protein in which a host cell, albumin or an analog thereof, and a first coiled coil-forming domain and a second coiled coil-forming domain are linked using the host cell.

In another aspect of the present invention, the fusion protein is albumin or an analog thereof; And it may be a fusion protein further comprising a linker between the coiled coil-forming domains, a nucleic acid molecule encoding the fusion protein comprising the linker, a vector comprising the nucleic acid molecule, a host cell into which the vector is introduced, the host A method for producing the fusion protein using cells.

According to an embodiment of the present invention, first, the DNA of the coiled coil-forming domain is linked to the DNA sequence positions corresponding to the N-terminus and C-terminus of albumin or an analog thereof, respectively, and albumin or an analog thereof and a coiled coil-forming domain This linked, DNA encoding the fusion protein was prepared. Then, the DNA was introduced into pcDNA 3.1 vector to prepare a recombinant vector having a size of 7895 bp. Then, the vector was transformed into a host cell, specifically, a CHO cell to establish a transient expression system for producing the fusion protein in a short time.

In addition, according to an embodiment of the present invention, the DNA encoding the fusion protein is albumin or an analog thereof; and DNA encoding a linker between DNA encoding a coiled coil-forming domain.

In addition, according to an embodiment of the present invention, the DNA encoding the fusion protein is a sequence encoding cysteine at both ends of the DNA of each coiled coil-forming domain (DNA sequence positions corresponding to the N-terminus and C-terminus) This may be added. Coiled coil-forming domain of a fusion protein expressed by the DNA, comprising a coiled coil-forming domain with cysteines added to both ends; and a target binding substance linked to a coiled coil-forming domain with cysteines added to both ends When the coiled coil is formed by the bond between them, a disulfide bond is formed between the cysteines located at both ends, thereby improving the stability of the coiled coil.

According to an embodiment of the present invention, a nucleic acid molecule encoding a fusion protein in which albumin or an analog thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked, is represented by SEQ ID NOs: 7, 52, 9 and 10 It may be any one nucleotide sequence selected from the group consisting of nucleotide sequences, but is not limited thereto.

As used herein, the term "nucleic acid molecule" has a meaning comprehensively including DNA and RNA molecules, and nucleotides, which are the basic structural units in the nucleic acid molecules, include natural nucleotides as well as analogs ( analogues) are also included. The sequence of the nucleic acid molecule encoding the fusion protein of the present invention may be modified, and such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

The nucleic acid molecule of the present invention is construed to include a nucleotide sequence exhibiting substantial identity to the above-described nucleotide sequence. In the present invention, the substantial identity is at least 80 when the above-described nucleotide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. % homology, specifically at least 90% homology, more specifically at least 95% homology.

As used herein, the term “vector” refers to a means for expressing a target gene in a host cell, including a plasmid vector; cozmid vector; and viral vectors such as bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, and the like, and specifically may be a plasmid vector, but is not limited thereto.

In the vector of the present invention, the nucleic acid molecule encoding the fusion protein may be operably linked to a promoter.

As used herein, the term "operably linked" refers to a functional association between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulation The sequence will control the transcription and/or translation of the other nucleic acid sequence.

The recombinant vector system of the present invention can be constructed through various methods known in the art. For example, a specific method for this is disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

Vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is a host, a strong promoter capable of propagating transcription (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter) , rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as a host cell, promoter and operator regions of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., (1984) 158: 1018-1024), and the left-handed promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., (1980) 14:399-445) can be used as regulatory regions. When Bacillus bacteria is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. (1998) 64:3932-3938; Mol. Gen. Genet. (1996) 250:734-741) ) or any promoter that can be expressed in Bacillus can be used as a regulatory region.

On the other hand, the recombinant vector of the present invention is a plasmid often used in the art (eg, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phage (eg, λgt4·λB, λ-Charon, λΔz1, and M13, etc.) or viruses (eg, SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine) promoter) or promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, The LTR promoter of HIV, the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Loose Sacoma virus (RSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence. , the recombinant vector of the present invention comprises a CMV promoter.

On the other hand, the recombinant vector of the present invention contains an antibiotic resistance gene commonly used in the art as a selection marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and a resistance gene to tetracycline.

Any host cell known in the art may be used as long as it is a host cell capable of stably and continuously cloning and expressing the vector of the present invention, for example, Escherichia coli , Bacillus subtilis ) and Bacillus Chuo ringen sheath (Bacillus thuringiensis), and Bacillus sp, Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis) or Staphylococcus including prokaryotic host cells such as (Staphylococcus), such can, but is not limited thereto.

Suitable eukaryotic host cells of the vector include fungi such as Aspergillus species , Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces . and yeast such as Neurospora crassa , other lower eukaryotic cells, cells of higher eukaryotes such as insect-derived cells, and cells derived from plants or mammals.

Specifically, host cells include COS7 cells (monkey kidney cells), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma. The cell line may be a HuT 78 cell or a 293 cell, and more specifically, a Chinese hamster ovary cell, but is not limited thereto.

In the present invention, "transformation" into a host cell includes any method of introducing a nucleic acid into an organism, cell, tissue or organ, and as is known in the art, it can be performed by selecting an appropriate standard technique according to the host cell. can Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation using silicon carbide fibers, agrobacterium-mediated transformation, PEG, dextran sulfate , lipofectamine and drying/inhibition mediated transformation methods, and the like.

In the present invention, the method for producing a fusion protein using the host cell includes the steps of: (a) culturing a host cell transformed with the recombinant vector of the present invention; and (b) expressing the fusion protein in the host cell.

The culture of the transformed host cell in the preparation of the fusion protein can be made according to an appropriate medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Such culture methods are disclosed in various literatures (eg, James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Cell culture is divided into suspension culture and adherent culture according to the cell growth method, and batch, fed-batch and continuous culture methods according to the culture method. The medium used for culture should suitably satisfy the requirements of a particular strain.

In animal cell culture, the medium contains various carbon sources, nitrogen sources and trace elements. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, and these carbon sources may be used alone or in combination.

Nitrogen sources that can be used in the present invention include, for example, organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor (CSL) and soybean wheat and urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate; The same inorganic nitrogen source may be included, and these nitrogen sources may be used alone or in combination. The medium may contain, as phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salt. It may also contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (eg, air) is injected into the culture. The temperature of the culture is usually 20°C to 45°C, preferably 25°C to 40°C.

The preparation method may further include the step of (c) recovering the fusion protein in which albumin or an analog thereof expressed in the host cell, the first coiled coil-forming domain and the second coiled coil-forming domain are linked. The fusion protein obtained by culturing the transformed host cell may be used in an unpurified state, or further purified to a high purity using various conventional methods, for example, dialysis, salt precipitation, and chromatography. can Among them, the method using chromatography is the most used, and the type and sequence of the column can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc. depending on the characteristics of the fusion protein and culture method.

concrete

In this section, the same content as described in the “fusion protein” section will be omitted to avoid complexity and excessive repetition of the specification.

One aspect of the present invention, albumin or an analog thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked, a fusion protein; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second fusion, wherein the second target material and the second 'coiled coil-forming domain are linked, wherein the first and second fusions are each bound to a fusion protein, a dual target binder. According to an embodiment of the present invention, the first and second fusions bind to different positions of the fusion protein, respectively. In FIG. 1, albumin or an analog thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked, a fusion protein; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second target material and a second 'coiled coil-forming domain connected to each other, showing a schematic schematic diagram of a second fusion.

The term "dual target binding body" used in the present invention means that two types of target binding substances are bound to albumin or an analog thereof by a coiled coil, which means a combination of albumin or an analog thereof and a target binding substance. In the present invention, they are used interchangeably.

As used herein, the term "target-binding substance" refers to a substance capable of interacting with a specific protein, for example, a protein such as an antigen, or performing a specific function upon expression. The target-binding substance may be a part of a domain or a polypeptide responsible for a function as well as a native protein capable of interacting with a specific protein, and may be a compound that does not exist in vivo. In addition, it may be a useful candidate or therapeutic agent for medicine, pharmaceuticals, etc., or a substance causing in vivo action, for example, proteins, peptides, protein domains, drugs, toxins, cytotoxic agents, contrast agents, radionuclides (radioactive isotopes), Radioactive compounds, organic polymers, inorganic polymers, biotin, antibodies, domains of antibodies, antibody fragments, single chain antibodies, domain antibodies, enzymes, ligands, receptors, binding peptides, epitope tags, recombinant polypeptide polymers, cytokines, nucleic acids or small molecules It may be a compound, but is not limited thereto.

As used herein, the term “fusion” refers to a protein expressed by a nucleic acid molecule comprising a nucleotide sequence of one or more genes or a protein expressed by a nucleic acid molecule comprising a nucleotide sequence, in which a target binding agent as defined above is linked, a fusion means material. In the present invention, the fusion may be denoted as "target binding substance-Z", where Z means the alphabet of any coiled coil-forming domain assigned alphabetically herein, and "-" means coiled-coil-forming domain. It means that the domain is linked to the target binding substance. Here, the linkage site of the target-binding substance and the coiled coil-forming domain represented by Z is selected as a site that does not interfere with the interaction in consideration of the site where the target-binding substance interacts with a receptor that recognizes the binding substance. can

According to an embodiment of the present invention, the first, second, first, and second coiled coil-forming domains included in the dual target conjugate include an elastin-like polypeptide, a BECN (Beclin) domain, and a bZIP VBP (basic) domain. leucine zipper vitellogenin-binding protein), keratin intermediate fibers, AP1 (Jun/Fos), MBD2-NuRD (methyl-binding domain domain domain 2-Nucleosome Remodeling and Deacetylase) domain, GABA (γ-aminobutyric acid) receptor, zinc clamp domain, It may be any one selected from the group consisting of leucine zippers or variants thereof, but is not limited thereto. In addition, if the first, second, first' and second' coiled coil forming domains are known to be capable of forming a coiled coil by combining with other coiled coil forming domains, the present invention may be applied.

According to an embodiment of the present invention, the first, second, first, and second coiled coil-forming domains included in the dual target binder are SEQ ID NOs: 4, 5, 6, 16, 17 and 18, respectively. It may include any one amino acid sequence selected from the group consisting of the indicated amino acid sequence and the amino acid sequence of its variants. In addition, according to an embodiment of the present invention, the "variant of the amino acid sequence" is the N of any one amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 4, 5, 6, 16, 17 and 18. A cysteine may be added to at least one of the -terminus and the C-terminus. Specifically, at least one cysteine may be added to both terminals, respectively, and more specifically, one cysteine may be added to both terminals, respectively.

According to an embodiment of the present invention, the variant of the amino acid sequence may be any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 42, 43, 44, 48, 49 and 50, and the SEQ ID NO: One cysteine may be added to each of the N-terminus and the C-terminus of any one amino acid sequence selected from the group consisting of the amino acid sequences represented by 4, 5, 6, 16, 17 and 18.

According to an embodiment of the present invention, the first coiled coil forming domain and the second coiled coil forming domain may not form a coiled coil through mutual coupling, and the first' coiled coil forming domain and the second coiled coil forming domain The 2' coiled coil forming domain may not form a coiled coil through mutual bonding. If the first 'coiled coil-forming domain and the second' coiled coil-forming domain bind to each other to form a coiled coil, the binding between the fusions increases and the yield of the dual target conjugate decreases, so as in the present invention, the first If the 'coiled coil-forming domain and the second' coiled coil-forming domain cannot form a coiled coil through mutual coupling, the mutual coupling of the first' coiled coil-forming domain and the second' coiled coil-forming domain is Binding between the fusions is decreased and the yield of the dual target conjugate is improved.

According to an embodiment of the present invention, the amino acid sequence of the first coiled coil-forming domain, the amino acid sequence of the second coiled coil-forming domain, the amino acid sequence of the first 'coiled coil-forming domain and the second The amino acid sequence of the 2' coiled coil-forming domain may be different. As in the present invention, when the first, second, first, and second coiled coil-forming domains include different amino acid sequences from each other, the same target-binding substance is linked to the fusion protein, and when two fusions are bound decreases and the number of cases where two fusions with different target binding substances are linked to the fusion protein will increase, and when preparing a dual target binding substance, it is possible to predict the mixing ratio of fusions with different target binding substances, so that various combinations of drugs can be obtained quickly and conveniently, and the yield of the dual target conjugate is improved.

According to an embodiment of the present invention, the first coiled coil-forming domain is selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 4, 5, 6, 16, 17, 18, 42, 43, 44, 48, 49 and 50. It may include any one selected amino acid sequence, and the second coiled coil-forming domain is an amino acid represented by SEQ ID NOs: 4, 5, 6, 16, 17, 18, 42, 43, 44, 48, 49 and 50 It may include any one amino acid sequence selected from the group consisting of sequences, and the first coiled coil-forming domain is SEQ ID NO: 4, 5, 6, 16, 17, 18, 42, 43, 44, 48, 49 And it may include any one amino acid sequence selected from the group consisting of the amino acid sequence represented by 50, the 2' coiled coil forming domain is SEQ ID NO: 4, 5, 6, 16, 17, 18, 42, 43 , 44, 48, 49 and 50 may include any one amino acid sequence selected from the group consisting of amino acid sequences.

According to another embodiment of the present invention, in the dual target conjugate, the first and first 'coiled coil-forming domains combine to form a first coiled coil, and the second and second' coiled coil-forming domains may be combined to form a second coiled coil. For example, when the first coiled coil-forming domain includes any one of the amino acid sequences shown in SEQ ID NOs: 4 and 42, the first coiled coil-forming domain includes amino acids shown in SEQ ID NOs: 16 and 48 It may include any one of the amino acid sequence of the sequence, and the first coiled coil-forming domain is SEQ ID NO: 5 and 43. When the first coiled coil-forming domain includes any one of the amino acid sequences of the amino acid sequence, the first coiled coil-forming domain is SEQ ID NO: It may include any one of the amino acid sequences represented by 17 and 49, and the first coiled coil-forming domain includes any one of the amino acid sequences of the amino acid sequences represented by SEQ ID NOs: 6 and 44 when the first coiled coil-forming domain includes any one of the amino acid sequences represented by SEQ ID NOs: 6 and 44. The coil-forming domain may include any one of the amino acid sequences shown in SEQ ID NOs: 18 and 50. This is also the case for the second coiled coil forming domain and the second' coiled coil forming domain. In addition, according to an embodiment of the present invention, the amino acid sequence represented by SEQ ID NO: 4 and the amino acid sequence represented by SEQ ID NO: 16 or 48 can form a bond with each other, and the amino acid sequence represented by SEQ ID NO: 42 is at both ends Since it can be seen as the same sequence as the amino acid sequence shown in SEQ ID NO: 4 except for the addition of cysteine, when the first coiled coil-forming domain includes the amino acid sequence shown in SEQ ID NO: 4, the first coiled coil The coil-forming domain may comprise one of the amino acid sequences represented by SEQ ID NO: 16 or 48, and the second coiled coil-forming domain is represented by SEQ ID NO: 5, 6, 17, 18, 43, 44, 49 and 50 It may include any one amino acid sequence selected from the group consisting of amino acid sequences. The second coiled coil-forming domain may include the amino acid sequence represented by SEQ ID NO: 17 or 49 when the second coiled coil-forming domain includes the amino acid represented by SEQ ID NO: 5, and the second coiled coil-forming domain includes the amino acid sequence represented by SEQ ID NO: 17 or 49. When the forming domain includes the amino acid represented by SEQ ID NO: 6, it may include the amino acid sequence represented by SEQ ID NO: 18 or 50, and when the second coiled coil-forming domain includes the amino acid represented by SEQ ID NO: 17 It may include the amino acid sequence represented by No. 5 or 43, and when the second coiled coil-forming domain includes the amino acid represented by SEQ ID NO: 18, it may include the amino acid sequence represented by SEQ ID NO: 6 or 44, When the second coiled coil-forming domain includes the amino acid represented by SEQ ID NO: 43, it may include the amino acid sequence represented by SEQ ID NO: 17 or 49, and the second coiled coil-forming domain includes the amino acid represented by SEQ ID NO: 44 It may include the amino acid sequence represented by SEQ ID NO: 18 or 50 when including, and when the second coiled coil-forming domain includes the amino acid represented by SEQ ID NO: 49, the amino acid sequence represented by SEQ ID NO: 5 or 43 It may include, and when the second coiled coil-forming domain includes the amino acid represented by SEQ ID NO: 50, it may include the amino acid sequence represented by SEQ ID NO: 6 or 44.

According to an embodiment of the present invention, the first coiled coil-forming domain may be any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 5, 43 and 44, and the second coiled coil-forming domain is It may be one of the amino acid sequences shown in SEQ ID NO: 4 or 42, and the first coiled coil-forming domain may be any one amino acid sequence selected from the group consisting of the amino acid sequences shown in 17, 49 and 50, The 2' coiled coil-forming domain may be one of the amino acid sequences represented by 16 or 48, but is not limited thereto.

According to an embodiment of the present invention, in the dual target conjugate, the first and second coiled coils each have a) a dissociation constant (K _d ) of 10 nM or less, b) a coiled coil-forming domain in the form of a heterodimer may include.

According to an embodiment of the present invention, the binding between the coiled coil-forming domains may be self-bonding. In addition, according to an embodiment of the present invention, the self-bonding between the coiled coil-forming domains may be a non-covalent bond.

According to an embodiment of the present invention, in the dual target conjugate, one or more cysteine residues are added at at least one of both ends of the coiled coil-forming domains, and one or more disulfide bonds are added between the added one or more cysteine residues. may be formed with In the binding between the coiled coil-forming domains as in the present invention, when a disulfide bond, which is a covalent bond, is further included in addition to the self-bond corresponding to a non-covalent bond, the binding stability between the coiled coil-forming domains is further improved.

As used herein, the term "self-bonding" refers to a non-covalent bond formed by hydrophobic or ionic interactions between peptides themselves or between peptides and domains included in the peptide. For example, in the primary structure of the coiled coil domain, hydrophobic amino acid residues are located to form the hydrophobic core of the coiled coil, and there are charged amino acid residues to enable ionic interaction, so that the domain containing the amino acid It allows the formation of a coiled coil by a non-covalent bond between hydrophobic and ionic interactions.

According to an embodiment of the present invention, the first and second target binding substances included in the dual target binding body may be different from each other. Since the dual target binding agent simultaneously contains any one target-binding substance exhibiting targeting and/or therapeutic ability capable of binding to a target molecule, etc., and the other target-binding substance exhibiting targeting and/or therapeutic activity, a specific A therapeutic agent can be delivered specifically to a tissue or cell expressing a target molecule.

According to an embodiment of the present invention, the first and second target binding substances may be any one selected from the group including proteins, antibodies, antibody fragments, hormones and peptides, respectively.

According to an embodiment of the present invention, the first or second target-binding substance may target a surface antigen on a cancer cell. For example, it may be any one selected from the group consisting of CD2, CD317, CEACAM5, CEACAM6, DR5, EphA2, gpA33, Her2, B7-H3, EGF, EGFR, VEGF and VEGFR, but is not limited thereto.

According to an embodiment of the present invention, at least one of the first and second target binding agents may be a therapeutic agent. As used herein, the term "therapeutic agent" refers to a molecule administered to a subject for a desired therapeutic effect, with a known therapeutic effect. Examples of therapeutic agents include proteins, peptides, protein domains, drugs, toxins, cytotoxic agents, contrast agents, radionuclides (radioisotopes), radioactive compounds, organic polymers, inorganic polymers, biotin, antibodies, domains of antibodies, antibody fragments, single chain antibodies, domain antibodies, enzymes, ligands, receptors, binding peptides, epitope tags, recombinant polypeptide polymers, cytokines, nucleic acids or small molecule compounds, for example, GFP, mCherry, PD-1, CD80, Insulin and GLP-1, but is not limited thereto, and the subject includes a human or non-human mammal.

According to an embodiment of the present invention, the first and second target binding substances may be any one selected from the group consisting of GFP, mCherry, PD-1, CD80, Insulin and GLP-1, respectively, and the first and the second target binding agent are different from each other.

In addition, according to an embodiment of the present invention, in the dual target binder (albumin or an analog thereof and a binder of two types of target binding substances), the two types of target binding substances are directly linked to the N-terminus and C-terminus of albumin, respectively. , the intrinsic structure and activity of each target-binding material were better maintained when compared to the fusion protein.

According to an embodiment of the present invention, the first fusion and the second fusion included in the dual target conjugate may further include a linker between the target binding material and the coiled coil-forming domain. As in the present invention, when a linker is further included between the target binding material and the coiled coil-forming domain included in the dual target binder, the first coiled coil-forming domain and the second coiled coil-forming domain of the fusion protein are each a target. The steric hindrance, etc. occurring when the first 'coiled coil-forming domain and the second' coiled coil-forming domain connected to the binding material are reduced, thereby promoting and stabilizing the formation of the coiled coil.

According to an embodiment of the present invention, the first fusion and the second fusion will each include any one amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 22, 25, 26, 28, 30, 38 and 51 can

According to an embodiment of the present invention, the dual target conjugate may be prepared in vitro . Albumin or an analog thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked, a fusion protein; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second fusion in which the second target binding substance and the second coiled coil-forming domain are linked, may be prepared by mixing in vitro. As in the present invention, when the fusion protein, the first fusion, and the second fusion constituting the dual target conjugate are separately prepared and assembled by mixing in vitro , each target is bound to each other compared to when the two types of target binding substances are expressed in-frame. The intrinsic structure and activity of the material are well maintained.

According to an embodiment of the present invention, the dual target conjugate may be prepared by mixing the fusion protein and the first fusion in a molar ratio of 1:2 to 1:10.

According to an embodiment of the present invention, the dual target conjugate may be prepared by mixing the fusion protein and the second fusion in a molar ratio of 1:2 to 1:10.

Another aspect of the present invention, the first target-binding substance and the first 'coiled coil-forming domain is linked to a first fusion; or a second fusion in which a second target binding substance and a second coiled coil-forming domain are linked; a nucleic acid molecule encoding a nucleic acid molecule, a vector comprising the nucleic acid molecule, a host cell into which the vector is introduced, and a first using the host cell A method for preparing a fusion and a second fusion.

Another aspect of the present invention is in vitro albumin or an analog thereof, a fusion protein in which a first coiled coil-forming domain and a second coiled coil-forming domain are linked; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second fusion in which the second target binding material and the second 'coiled coil-forming domain are linked, comprising the step of preparing a mixture of; The yield of the dual target conjugate of the present invention is improved when the fusion protein and each fusion with the coiled coil forming domain are prepared in vitro than when the fusion protein and the coiled coil forming domain are formed in vivo. do.

According to an embodiment of the present invention, the preparation method may additionally include the step of recovering the dual target binder from the mixture. The dual target conjugate obtained by mixing the fusion protein and the first and second fusions may be used in an unpurified state, or in addition, various conventional methods, for example, dialysis, salt precipitation and chromatography, etc. It can be used after purification with high purity. Among them, the method using chromatography is the most used, and the type and sequence of the column can be selected from ion exchange chromatography, size exclusion chromatography, affinity chromatography, etc., depending on the characteristics of the fusion protein, culture method, etc., but is not limited thereto. .

According to an embodiment of the present invention, when preparing the dual target conjugate, the fusion protein and the first fusion may be prepared by mixing in a molar ratio of 1:2 to 1:10. Preferably, it can be prepared by mixing in a molar concentration ratio of 1:4, a molar concentration ratio of 1:5, or a molar concentration ratio of 1:8.

According to an embodiment of the present invention, the fusion protein and the second fusion protein can be prepared by mixing in a molar concentration ratio of 1:2 to 1:10, preferably 1:4 in a molar concentration ratio, 1:5 in a molar ratio. It can be prepared by mixing in a concentration ratio or a molar concentration ratio of 1:8. As described above, since the first coiled coil-forming domain and the second coiled coil-forming domain according to the present invention have different amino acid sequences, the molar concentration ratio of the first fusion and the second fusion in the preparation of the dual target conjugate can be derived simply.

The method for preparing a dual-target conjugate of the present invention can obtain a desired dual-target conjugate by simply mixing the fusion protein, the first fusion, and the second fusion in a specific molar concentration ratio, as well as a dual-target conjugate including any drug combination. The target binder can be obtained in high yield. In addition, the effectiveness of the drug combination can be quickly verified using the dual target conjugate, so that the optimal drug combination to treat a specific disease can be quickly discovered, and the time and cost of proof of concept (POC) can be reduced. can save

Another aspect of the present invention is a pharmaceutical composition comprising the dual target conjugate. The pharmaceutical composition comprising the dual target conjugate of the present invention may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition comprising the dual target conjugate of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. may be administered. Since the protein or peptide is digested upon oral administration, oral compositions may be formulated to coat the active agent or to protect it from degradation in the stomach, and the composition of the present invention may be any device capable of transporting the active agent to a target cell. can be administered by

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

The albumin or its analog of the present invention, the first coiled coil-forming domain and the second coiled coil-forming domain are linked, and the dual target conjugate using a fusion protein is based on albumin present in a large amount in human blood, so the high level of the conjugate drug In vivo stability and long plasma half-life can be ensured.

In addition, the dual target conjugate of the present invention can contain various combinations of drugs, and can be quickly manufactured in high yield, shortening the time required to verify the effectiveness of the drug combination, thereby providing a proof of concept (POC, Proof of Concept) Time and cost can be saved, and thus the optimal drug combination can be quickly discovered.

In addition, since the dual target conjugate of the present invention is prepared by self-binding between the coiled coil-forming domains in vitro after separately expressing the fusion of the albumin fusion protein and two types of target binding substances, the problems with the existing technology, That is, when two types of target-binding substances are expressed in-frame, it is difficult to maintain the original structure and activity of each target-binding substance.

1 is a schematic diagram showing the forms of a fusion protein, a first fusion, and a second fusion.
Figure 2 shows a recombinant plasmid comprising a nucleic acid encoding a fusion protein, to which human serum albumin (HSA) and a coiled coil-forming domain are linked.
3 shows the results of confirming the expression level of the fusion protein in which human serum albumin (HSA) and the coiled coil-forming domain are linked in a temporary expression system using SDS-PAGE.
4 shows the results of histidine affinity chromatography, and FIG. 4a shows the chromatogram of a fusion protein in which human serum albumin (HSA) purified by histidine affinity chromatography and a coiled coil-forming domain are linked, FIG. 4b shows the results of qualitative and quantitative confirmation of the protein sample of the fraction identified as a peak after HSA affinity chromatography using SE-HPLC.
Figure 5 shows the anion exchange resin chromatography results, Figure 5a shows the chromatogram of the fusion protein, in which human serum albumin (HSA) purified by anion exchange resin chromatography and a coiled coil-forming domain are linked, Figure 5b shows the results of qualitative and quantitative confirmation of the protein sample of the fraction identified as a peak after performing anion exchange resin chromatography using SE-HPLC.
6 shows the results of confirming the purity of the fusion, and shows the results of qualitatively and quantitatively confirming the protein sample of the fraction identified as a peak after performing anion exchange resin chromatography using SDS-PAGE.
7 shows a recombinant plasmid, FIG. 7a shows a recombinant plasmid in which GFP and a coiled coil-forming domain are linked, DNA encoding the fusion is inserted, and FIG. 7b is mCherry and a coiled coil-forming domain are linked, the fusion shows a recombinant plasmid into which DNA encoding the
8 shows the result of purifying the fusion by histidine affinity chromatography, FIG. 8a shows the chromatogram of the fusion in which GFP and coiled coil-forming domains are linked, and FIG. 8b shows mCherry and coiled coil-forming domains Chromatograms of linked, fusions are shown.
Figure 9 shows the result of purifying the fusion by histidine affinity chromatography, Figure 9a is a fusion sample in which GFP and coiled coil-forming domains of the fraction identified as a peak are connected after histidine affinity chromatography is SE - It shows the results confirmed qualitatively and quantitatively by using HPLC, and FIG. 9b is a fusion sample in which mCherry and coiled coil-forming domains of the fraction identified as a peak are connected after histidine affinity chromatography is performed by SE-HPLC Shows the results confirmed qualitatively and quantitatively using
Figure 10 shows the result of purifying the fusion by anion exchange resin chromatography, Figure 10a shows the chromatogram of the fusion, in which GFP and coiled coil-forming domains purified by anion exchange resin chromatography are linked, Figure 10b is Shows a chromatogram of a fusion in which mCherry purified by anion exchange resin chromatography and a coiled coil-forming domain are linked.
11 shows the result of confirming the purity of the fusion, FIG. 11a is a protein sample of the fraction identified as a peak after purification of the fusion, in which GFP and the coiled coil-forming domain are linked, using histidine affinity chromatography SE-HPLC shows the results confirmed qualitatively and quantitatively using , and FIG. 11b is a protein sample of the fraction identified as a peak after purifying the fusion, in which mCherry and the coiled coil-forming domain are linked, using histidine affinity chromatography, SE-HPLC Shows the results confirmed qualitatively and quantitatively using
Figure 12 shows the result of confirming whether the recovered protein is a desired protein, Figure 12a is a fraction identified as a peak after purifying the fusion, in which GFP and coiled coil-forming domains are linked using anion exchange resin chromatography Shows the results of qualitative and quantitative confirmation of the protein sample using SDS-PAGE, and FIG. 12b shows the fraction identified as peak after purifying the fusion, in which mCherry and coiled coil-forming domains are linked using anion exchange resin chromatography. Shows the results of qualitative and quantitative confirmation of protein samples using SDS-PAGE.
13 shows a recombinant plasmid comprising a nucleic acid encoding a fusion, in which PD-1 and a coiled coil-forming domain are linked.
14 shows the results of confirming the expression level of the fusion, in which PD-1 and the coiled coil-forming domain are linked, in the temporary expression system using SDS-PAGE.
Figure 15 shows the result of purifying the fusion by Flag affinity chromatography, Figure 15a shows the chromatogram of the fusion, in which PD-1 and coiled coil-forming domains purified by Flag affinity chromatography are linked, Fig. 15b shows the results of qualitative and quantitative confirmation of the protein sample of the fraction identified as a peak after performing Flag affinity chromatography on the fusion, in which PD-1 and the coiled coil-forming domain are linked, using SE-HPLC.
Figure 16 shows the result of confirming the purity of the fusion, Figure 16a shows the chromatogram of the fusion, in which PD-1 purified by anion exchange resin chromatography and the coiled coil-forming domain are linked, Figure 16b is PD-1 and the results of qualitatively and quantitatively confirming the protein sample of the fraction identified as a peak after performing anion exchange resin chromatography on the fusion, in which the coiled coil-forming domain is linked, using SE-HPLC.
Figure 17 shows the result of confirming whether the recovered protein is the desired protein, PD-1 and the coiled coil-forming domain are linked, the protein sample of the fraction identified as a peak after performing anion exchange resin chromatography on the fusion shows the results of qualitative and quantitative confirmation using SDS-PAGE.
18 shows a recombinant plasmid comprising a nucleic acid encoding a fusion, in which CD80 and a coiled coil forming domain are linked.
19 shows the results of confirming the expression level of the fusion, in which CD80 and the coiled coil-forming domain are linked, in the temporary expression system using SDS-PAGE.
20 shows the result of purifying the fusion by histidine affinity chromatography, and FIG. 20a shows the chromatogram of the fusion, in which CD80 purified by histidine affinity chromatography and the coiled coil-forming domain are linked, and FIG. 20b is The results of qualitative and quantitative confirmation of the protein sample of the fraction identified as a peak after performing histidine affinity chromatography on the fusion, in which CD80 and the coiled coil-forming domain are linked, are shown using SE-HPLC.
Figure 21 shows the result of purifying the fusion by anion exchange resin chromatography, Figure 21a shows the chromatogram of the fusion, in which CD80 and coiled coil-forming domains purified by anion exchange resin chromatography are linked, Figure 21b is Shows the results of qualitative and quantitative confirmation of the protein sample of the fraction identified as a peak after performing anion exchange resin chromatography on the fusion, in which CD80 and the coiled coil-forming domain are linked, using SE-HPLC.
Figure 22 shows the result of confirming whether the recovered protein is a desired protein. After performing anion exchange resin chromatography on the fusion, in which CD80 and the coiled coil-forming domain are connected, the protein sample of the fraction identified as a peak SDS Shows the results confirmed qualitatively and quantitatively using -PAGE.
23 shows a recombinant plasmid comprising a nucleic acid encoding a fusion in which insulin and a coiled coil-forming domain are linked.
24 shows a chromatogram of a fusion in which insulin purified by histidine affinity chromatography and a coiled coil-forming domain are linked.
25 shows the results of qualitative and quantitative confirmation of a protein sample of a fraction identified as a peak after performing histidine affinity chromatography on a fusion, in which insulin and a coiled coil-forming domain are linked, using SE-HPLC.
26 shows a chromatogram of a dual target conjugate (target binding material: mCherry/GFP) purified using anion exchange resin chromatography.
27 shows the results of quantification of a double target binder (target binding material: mCherry/GFP) using BCA, Lowry quantification method and UV spectrometer.
Figure 28 shows the results of purification of the dual target conjugate by histidine affinity chromatography, and Figure 28a shows the dual target conjugate purified by histidine affinity chromatography (target binding material: PD-1/CD80, covalent bond added) shows the chromatogram of, Figure 28b is a protein sample of the fraction identified as a peak purified by histidine affinity chromatography of a double target binder (target binding material: PD-1/CD80, covalent bond added) RP-HPLC Shows the results confirmed qualitatively and quantitatively using
Figure 29 shows a recombinant plasmid, Figure 29a shows a recombinant plasmid in which a DNA encoding a protein in which PD-1 is fused to the N-terminus and CD80 to the C-terminus is inserted, and Figure 29b is insulin to the N-terminus. , shows a recombinant plasmid in which DNA encoding a protein in which GLP-1 is fused to the C-terminus is inserted.
30 shows the results of purifying the dual target conjugate by histidine affinity chromatography, and FIG. 30A is a fusion dual protein in which PD-1 is fused to the N-terminus and CD80 to the C-terminus purified by histidine affinity chromatography. shows the chromatogram of, and FIG. 30b shows the chromatogram of the fusion double protein in which insulin is fused to the N-terminus and GLP-1 to the C-terminus purified by histidine affinity chromatography.
Figure 31 shows the result of confirming the purity of the fusion duplex protein, Figure 31a is a fusion duplex protein in which PD-1 at the N-terminus and CD80 at the C-terminus are purified using histidine affinity chromatography to peak Shows the results of qualitative and quantitative confirmation of protein samples of the identified fractions using SE-HPLC, and FIG. 31b shows histidine affinity chromatography of a fusion double protein in which insulin is fused to the N-terminus and GLP-1 is fused to the C-terminus. Shows the results of qualitative and quantitative confirmation of the protein sample of the fraction identified as a peak after purification using a graph using SE-HPLC.
Figure 32 shows the result of purifying the fusion by anion exchange resin chromatography, Figure 32a is a chromatography of a fusion double protein in which PD-1 at the N-terminus and CD80 at the C-terminus are fused to the C-terminus purified by anion exchange resin chromatography. grams, and FIG. 32B shows a chromatogram of a fusion double protein in which insulin is fused to the N-terminus and GLP-1 to the C-terminus purified by anion exchange resin chromatography.
Figure 33 shows the result of confirming the purity of the fusion duplex protein. Figure 33a shows the fusion duplex protein in which PD-1 at the N-terminus and CD80 at the C-terminus are fused to the N-terminal and CD80 fractions recovered after anion exchange chromatography SE. -HPLC shows the results identified, and FIG. 33b shows a fusion double protein in which insulin and C-terminus are fused to insulin and GLP-1 to the C-terminus, which are fractionated and recovered after anion exchange chromatography is performed by SE-HPLC. The results of the analysis and identification are shown.
Figure 34 shows the result of confirming whether the fusion protein and the two types of fusions are self-associated, and Figure 34a is a fusion protein and different target binding materials are linked through SE-HPLC peak shift observation, the fusions are double through self-binding It shows the result of confirming the formation of the target conjugate (coiled coil: D/D' and C/C', complex: D-HSA-C, D'-GFP and mCherry-C'), Figure 34b is SE- It shows the results of confirming that the fusion protein and different target binding substances are linked through HPLC peak shift observation, and that the fusions form a double target binding body through self-binding (coiled coil: K/K' and C/C', complex: K-HSA-C, CD80-K' and PD1-C'), FIG. 34c is a fusion protein and two types of target binding materials linked through SE-HPLC peak shift observation, the fusion is a dual target through self-binding The results confirming the formation of a conjugate are shown (coiled coil: K/K' and C/C', complex: K-HSA-C, insuln-K' and GLP-1-C').
35 shows the results of measuring the dissociation constants of the coiled coil-forming domains bound to HSA or the coiled coil-forming domains C/C' and D/D' bound to a target binding substance, FIG. 35a is a double target binder It shows the result of calculating the dissociation constant of the coiled coil through the measurement of the binding force between the coiled coil-forming domains in (coiled coil: C/C'), and FIG. 35b is the binding force measurement between the coiled coil-forming domains in the dual target binder. shows the result of calculating the dissociation constant of the coiled coil (coiled coil: D/D').
36 shows the results of measuring the binding stability of the dual target conjugate according to the pH change using SE-HPLC.
37 shows the results of confirming the stability of the dual target conjugate (PD1/CD80, including covalent bond) in rat plasma using ELISA (here, complex represents the double binding conjugate in which the target binding substances are PD1 and CD80).
Figure 38 shows the results of confirming the stability in rat plasma compared to wild type human serum albumin (wild type HSA) as a control of the dual target conjugate using ELISA, Figure 38a is a dual target conjugate (GLP-1 / Insulin, including covalent bond) ) and the control group, wild-type human serum albumin (wild type HSA), shows the results of comparison of the stability in rat plasma, and FIG. 38b shows the stability of the double target conjugate in rat plasma (here, the complex is the target binding substance GLP). -1 and Insulin represents a double bond complex).
Figure 39 shows the CD80 in vitro ELISA competitive assay results of the dual target conjugate (here, the complex is a double binding conjugate in which the target binding substances are CD80 and PD-1, and the fusion is PD-1 at the N-terminus, C-terminus represents a fusion duplex protein fused to CD80).
Figure 40 shows the PD-1 in vitro ELISA competitive assay results of the dual target conjugate (here, the complex is a double binding conjugate in which the target binding substances are CD80 and PD-1, and the fusion is PD-1, C at the N-terminus. -represents a fusion duplex protein with CD80 fused at the end).
Figure 41 shows the results of the competitive assay of GLP-1 in vitro ELISA of the dual target conjugate (here, the complex is a double binding conjugate of which the target binding substances are GLP-1 and insulin, Insulin at the N-terminus, and GLP at the C-terminus. -1 indicates the fused duplex protein).
Figure 42 shows the CD80 in vitro cell blockade assay results of the dual target conjugate (here, the complex is a double binding conjugate in which the target binding substances are CD80 and PD-1, and fusion is PD-1 at the N-terminus, and the C-terminus at the C-terminus. represents a fusion duplex protein fused to CD80).
43 shows the results of PD-1 in vitro cell blockade assay of the dual target conjugate (here, the complex is a double binding conjugate of which the target binding substances are CD80 and PD-1, and the fusion is PD-1, C at the N-terminus. -represents a fusion duplex protein with CD80 fused at the end).

Hereinafter, the present invention will be described in more detail by way of Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example 1. A fusion protein in which albumin and coiled coil-forming domains are linked

1-1. Construction of a recombinant plasmid comprising a nucleic acid encoding a fusion protein in which human serum albumin (HSA) and a coiled coil-forming domain are linked

After codon optimization (codon optimization) of DNA of HSA and coiled coil-forming domain for gene cloning, DNA of coiled coil-forming domain through overlap PCR is connected to the N-terminus and C-terminus of HSA, respectively, HSA and DNA encoding the fusion protein to which the coiled coil-forming domain is linked was prepared. The primers used for the overlap PCR are shown in Table 1, the primer concentration was 500 nM and the template concentration was 50 ng (the same primer was used for DNA encoding the cysteine-introduced fusion protein).

Specifically, pcDNA 3.1 vector was used for gene construction, and the insert DNA and vector were treated with DpnI restriction enzyme after overlap PCR, respectively, and the nucleic acid (DNA) encoding the fusion protein, in which the HSA and coiled coil forming domains are linked, is inserted. A vector with a size of 7895 bp was prepared. 2 shows the finally constructed recombinant plasmid. After inserting the vector into Stella ^® competent cell (manufacturer: Clontech), it was plated on an agar plate to which an ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and colonies were selected, followed by an Applied biosystems 3730xl DNA analyzer (manufacturer). : The nucleotide sequence was confirmed through DNA sequencing using Thermo Fisher Scientific).

The nucleotide sequence and amino acid sequence of the DNA encoding the coiled coil-forming domain confirmed through DNA sequencing are shown in Table 2, and the DNA nucleotide sequence and amino acid sequence are assigned alphabets according to the type. In addition, the amino acid sequence with cysteine introduced at both ends of the coiled coil-forming domain and the nucleotide sequence encoding the amino acid sequence are shown in Table 2. In addition, the nucleotide sequence and amino acid sequence of DNA encoding human serum albumin are shown in Table 3, and as an example of the present invention, HSA and coiled coil-forming domains are linked, the DNA nucleotide sequence encoding the fusion protein and the fusion protein The amino acid sequence is shown in Table 4.

In addition, the coiled coil-forming domain and HSA introduced cysteines at both ends (ie, N-terminal and C-terminus) of the coiled coil-forming domain in order to increase the stability of the bond between the coiled coil-forming domains through a covalent bond. The linked DNA encoding the fusion protein was constructed as a recombinant plasmid through the same method as above, and was expressed after transformation into a host cell. As an example of the present invention, the cysteine mutation introduced by DNA sequencing, the coiled coil-forming domain and the HSA are linked, the DNA nucleotide sequence encoding the fusion protein and the amino acid sequence of the fusion protein are shown in Table 5.

(The superscript “C” indicates that a cysteine has been introduced into the coiled-coil-forming domain, and cysteines in the sequence are indicated in bold and underlined.)

1-2. Human serum albumin (HSA) and coiled coil-forming domains are linked, establishment of a temporary expression system of a fusion protein

(1) transformation

The final vector obtained through gene cloning in Example 1-1 ( ^{DNA encoding K C} -HSA-C ^C is inserted) was introduced into Expi CHO animal cells to obtain the fusion protein of the present invention in a short time. A transient expression system was established.

Specifically, one day before transformation, cells were seeded in a flask at a ^{concentration of 3.0 x 10 6} cells/mL and cultured for 24 hours. On the day of transduction, ExpiCHO ^® expression medium was added and diluted to a concentration of ^{6.0 x 10 6 cells/mL.} Then, 1 μg/mL of DNA and Opti-PRO ^® medium were mixed, and ExpiFectamine ^® reagent and Opti-PRO ^® medium were mixed according to volume and left at room temperature for 5 minutes. Next, DNA and Expifectamine ^® reagent were reacted for 5 minutes and added to the flask, treated with feed and enhancer after 16 to 22 hours, and additionally fed after 5 days. On the 8th day, it was recovered by checking whether the viability was 75% or more.

(2) Analysis of transiently expressed proteins using SDS-PAGE

After adding 4 μL of 5x reducing-PAGE sample buffer to 16 μL of the recovered culture, it was heated at 100° C. for 5 minutes, and then left at room temperature for 20 minutes. After inserting polyamide gel (12-20%, Invitrogen) into the gel kit, loading the sample and size standard ladder, electrophoresis at 125V for 2 hours, the gel was separated from the kit, coomassie blue staining solution was added, and left for 1 hour. . The left gel was transferred to destaining buffer (70% tertiary distilled water, 20% methanol, 10% acetic acid) and incubated overnight.

As a result, as shown in FIG. 3, it was confirmed that the fusion protein, in which HSA and the coiled coil-forming domain are linked, was overexpressed at a suitable molecular weight position, through which HSA and the coiled coil-forming domain were linked in the temporary expression system. expression was confirmed.

1-3. Purification and concentration of fusion protein in which human serum albumin (HSA) and coiled coil-forming domains are linked

(1) Purification using HSA affinity chromatography

The culture solution obtained from the temporary expression system of Example 1-2 was filtered through a PES filtration membrane having pores having a size of 0.2 μm to remove impurities. Sartocon ^® Slice 200 Hydrosart ^® Cassette 30 KD (Sartorius) was added to the filtered culture medium in a concentrator / dialyzer, concentrated, dialyzed against 20 mM sodium phosphate, pH 7.0 buffer, and then recovered.

Next, the recovered dialysate ^{was loaded onto an albumin affinity matrix Albupure ®} (Prometic Bioseparations Ltd) to purify the fusion protein, in which HSA and coiled coil-forming domains were linked, and the results are shown in FIG. 4A . The conditions of the HSA affinity chromatography used for the purification are as follows.

<Chromatography Conditions>

- Resin: Albupure ^®

- Flow rate: 480 cm/h

- Equilibrium: 20 mM sodium phosphate, pH 7.0 buffer

- Loading: up to 35 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 20 mM sodium phosphate, 20 mM sodium octanonate, pH 7.0 buffer

- Storage: 20% EtOH

After HSA affinity chromatography, only protein molecules having a peak eluted before wild-type HSA as a control were recovered. The fractionated and recovered protein was analyzed by SE-HPLC.

As a result, as shown in FIG. 4b, it was confirmed that the recovered HSA and the coiled coil-forming domain were linked, and the fusion protein had a purity of 90% or more.

(2) Purification using anion exchange resin chromatography

The protein solution obtained through HSA affinity chromatography was loaded into Q impres (GE healthcare) to purify the fusion protein in which HSA and the coiled coil-forming domain were connected, and the results as shown in FIG. 5a were obtained. The anion exchange resin chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Resin: Q impres

- Flow rate: 120 cm/h

- Equilibrium: 20 mM sodium phosphate, pH 7.0 buffer

- Loading: max 10 g protein/L resin volume

- Reequilibration: 5 mM sodium phosphate, pH 5.8 buffer

- Wash: 5 mM sodium phosphate, 100 mM sodium chloride pH 5.8 buffer

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 20 mM sodium phosphate, 200 mM sodium chloride, pH 7.0 buffer

- Storage: 0.01 M NaOH solution

After anion exchange chromatography, the fractionated and recovered protein was analyzed by SE-HPLC for identification. As a result of SE-HPLC analysis, the peak from the washing buffer was HSA in which the coiled coil was not expressed or the coiled coil bond was broken during purification, and it was confirmed that the peak from the eluate was a fusion protein in which HSA and the coiled coil forming domain were linked. could

In addition, as shown in FIG. 5b, the recovered HSA and the coiled coil-forming domain were linked, and it was confirmed that the fusion protein had a purity of 98% or more.

In addition, as shown in FIG. 6 , it was confirmed that a high-purity band appeared at an appropriate molecular weight position through SDS-PAGE analysis, and it was confirmed that the recovered protein was a fusion protein in which HSA and coiled coil-forming domains were connected.

Thereafter, the recovered HSA and coiled coil-forming domains were linked, and the amount of the fusion protein was measured using a UV spectrometer, concentrated using an ultrafiltration method, and then dialyzed with PBS and stored at -20°C.

Example 2. Target binding substance (GFP or mCherry) and coiled coil-forming domain linked, fusion

2-1. Construction of a recombinant plasmid encoding the fusion, in which GFP/mCherry and coiled-coil forming domains are linked

After codon optimization of DNA of GFP / mCherry and coiled coil-forming domain for gene cloning, DNA of coiled coil-forming domain is located at the N-terminus of GFP and C-terminus of mCherry through overlap PCR, respectively. DNA encoding the fusion, in which GFP and coiled coil-forming domains are linked to each other; And mCherry and coiled coil-forming domains were linked, DNA encoding a fusion was prepared, respectively (this is referred to as a fusion, in which GFP/mCherry and coiled coil-forming domains are linked).

The pQE80 vector was used for gene construction, and the insert DNA and the vector were treated with DpnI restriction enzyme after overlap PCR, respectively, the GFP/mCherry and coiled coil forming domains were linked, DNA encoding the fusion was inserted, 5544 and 5571, respectively. A vector of bp size was constructed. Figure 7a shows a recombinant plasmid in which GFP and a coiled coil-forming domain are linked, DNA encoding the fusion is inserted, and Figure 7b is a recombinant plasmid in which mCherry and a coiled coil-forming domain are linked, DNA encoding the fusion is inserted. . Finally, the constructed recombinant plasmid is shown. After inserting the vector into Stella ^® competent cell (manufacturer: Clontech), it was plated on an agar plate to which ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and colonies were selected, followed by an Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Fisher) Scientific) was used to confirm the nucleotide sequence through DNA sequencing.

The DNA nucleotide sequence encoding the coiled coil-forming domain confirmed through DNA sequencing is shown in Table 6, and the DNA nucleotide sequence was assigned an alphabet according to the type (here, "'" written next to the alphabet is a code having the corresponding alphabet. It means that a coiled coil forming domain and a coiled coil can be formed. For example, C and C' are coiled coil forming domains capable of forming a coiled coil). In addition, the amino acid sequence in which cysteine was introduced at both ends of the coiled coil-forming domain of Table 6 and the DNA nucleotide sequence encoding the amino acid sequence are shown. In addition, as an example of the present invention, the DNA base sequence encoding the fusion and the amino acid sequence of the fusion, in which GFP / mCherry and the coiled coil forming domain are linked, are shown in Table 7.

In addition, GFP/mCherry and coiled coil formation by introducing cysteines at both ends (ie, N-terminus and C-terminus) of the coiled coil-forming domains to increase the stability of bonding between the coiled coil-forming domains through covalent bonding. The domain-linked DNA encoding the fusion was constructed as a recombinant plasmid through the same method as above, and was then transformed and expressed in a host cell. As an example of the present invention, the cysteine mutation confirmed through DNA sequencing is introduced, the GFP/mCherry and the coiled coil-forming domain are linked, the DNA nucleotide sequence encoding the fusion and the amino acid sequence of the fusion are shown in Table 8.

(The superscript “C” indicates that a cysteine has been introduced into the coiled-coil-forming domain, and cysteines in the sequence are indicated in bold and underlined.)

2-2. Establishment of a transient expression system of fusion, in which GFP/mCherry and coiled coil forming domains are linked

After transforming the final vector obtained through gene cloning in Example 2-1 to BL21 cells through heat shock method, spread it on an agar plate to which an ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and single colonies were obtained. was selected. Colonies were cultured overnight in 2xYT medium containing 5 mL of ampicillin for seed culture. The next day, the main culture was transferred to 1 L of 2xYT medium containing ampicillin. After measuring whether the logarithmic growth phase of OD 0.6 to 0.8 section was reached at 600 nm using a UV spectrometer, IPTG was put in to induce protein, and after 4 hours, GFP/mCherry and the coiled coil-forming domain were connected, and the fusion was recovered.

When the recovered GFP/mCherry and the coiled coil-forming domain are linked, the fusions show green and pink fluorescence when expressed, respectively, so the expression of the fusions was checked according to the presence or absence of each fluorescence. As a result, it was confirmed that the fusion was expressed in which GFP/mCherry and the coiled coil-forming domain were linked in the temporary expression system by confirming that the fluorescence was observed.

2-3. Purification and enrichment of fusions in which GFP/mCherry and coiled coil-forming domains are linked

(1) Purification using histidine affinity chromatography

The culture solution obtained from BL21 cells was filtered through a PES filtration membrane having pores of 0.2 μm in size to remove impurities. The filtrate was loaded into Histrap HP (GE healthcare) to purify the fusion, in which GFP/mCherry and coiled coil-forming domains were linked. The results are as shown in FIGS. 8A and 8B. The conditions of histidine affinity chromatography used for the purification are as follows.

<Chromatography Conditions>

- Resin: Histrap HP

- Flow rate: 180 cm/h

- Equilibrium: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole pH 8.0 buffer

- Loading: up to 30 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 50 mM sodium phosphate, 300 mM sodium chloride, 500 mM imidazole pH 8.0 buffer

- Storage: 20% EtOH

While flowing the equilibration buffer and elution buffer with a concentration gradient, the peak eluted with GFP/mCherry and the coiled coil-forming domain connected to the fusion was analyzed using SE-HPLC. A fusion having a peak eluted before wild-type HSA as a control. Only molecules were recovered. The results are as shown in FIGS. 9A and 9B.

(2) Purification using anion exchange resin chromatography

The solution obtained through histidine affinity chromatography ^{was put into a concentrator/dialyzer equipped with a Sartocon ®} Slice 200 Hydrosart ^® Cassette 10 KD (Sartorius), concentrated and dialyzed against 10 mM sodium phosphate, pH 8.0 buffer, and then recovered. The recovered solution was loaded into Q impres (GE healthcare) to purify the fusion, in which GFP/mCherry and the coiled coil-forming domain were linked. The results are shown in FIGS. 10A and 10B . The anion exchange resin chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Resin: Q impres

- Flow rate: 120 cm/h

- Equilibrium: 10 mM sodium phosphate, pH 8.0 buffer

- Loading: max 10 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 10 mM sodium phosphate, 500 mM sodium chloride, pH 8.0 buffer

- Storage: 0.01 M NaOH solution

After anion exchange chromatography, the fractionated fusion was identified by SE-HPLC analysis. As NaCl entered the concentration gradient, it was confirmed as a result of SE-HPLC analysis that a fusion with GFP/mCherry and coiled coil-forming domains was connected in a low section, and an aggregation form appeared in a high section.

In addition, as shown in FIGS. 11a and 11b, it was confirmed that the recovered GFP/mCherry and the coiled coil-forming domain were linked, and the purity of the fusion was 95% or more.

In addition, as shown in FIGS. 12A and 12B , it was confirmed that a band appeared at an appropriate molecular weight position through SDS-PAGE analysis, and it was confirmed that the recovered fusion was a fusion in which GFP/mCherry and a coiled coil-forming domain were connected.

Thereafter, the recovered GFP/mCherry and the coiled coil-forming domain were linked, and the amount of the fusion was measured using a UV spectrometer, concentrated using an ultrafiltration method, and then dialyzed against PBS and stored at -20°C.

Example 3. Target binding substance (PD-1) and coiled coil-forming domains are linked, fusion

3-1. Construction of a recombinant plasmid encoding the fusion, in which PD-1 and coiled coil-forming domains are linked

After codon optimization of the DNA of PD-1 and the coiled coil forming domain for gene cloning, the DNA of the coiled coil forming domain is linked to the C-terminus of PD-1 through overlap PCR, PD- 1 and coiled coil-forming domains were linked, DNA encoding the fusion was prepared.

For gene construction, pcDNA 3.1 vector was used, and the insert DNA and vector were respectively overlapped by PCR and then treated with DpnI restriction enzyme to connect the coiled coil forming domain to the C-terminus of PD-1. DNA encoding the fusion was inserted. A vector of 6473 bp size was constructed. 13 shows the finally constructed recombinant plasmid. After inserting the vector into Stella ^® competent cell (manufacturer: Clontech), it is plated on an agar plate containing an ampicillin antibiotic suitable for the antibiotic resistance gene in the vector, colonies are selected, and the nucleotide sequence is confirmed through DNA sequencing. did.

The nucleotide sequence of the DNA encoding the synthesized coiled coil-forming domain confirmed through DNA sequencing is shown in Table 9, and as an example of the present invention, PD-1 and the coiled coil-forming domain are linked, the DNA nucleotide sequence encoding the fusion And the amino acid sequence of the fusion is shown in Table 9.

(The superscript “C” indicates that a cysteine has been introduced into the coiled-coil-forming domain, and cysteines in the sequence are indicated in bold and underlined.)

3-2. Establishment of a temporary expression system for fusions, in which PD-1 and coiled coil-forming domains are linked

(1) transformation

The final vector obtained through gene cloning in Example 3-1 was introduced into Expi CHO animal cells to establish a temporary expression system for quickly securing the fusion of the present invention.

Specifically, one day before transformation, cells were aliquoted in a flask at a ^{concentration of 3.0 x 10 6} cells/mL and cultured for 24 hours. On the day of transduction, ExpiCHO ^® expression medium was added and diluted to a concentration of ^{6.0 x 10 6 cells/mL.} Thereafter, DNA 1 μg/mL and Opti-PRO ^® medium were mixed, and ExpiFectamine ^® reagent and Opti-PRO ^® medium were mixed according to volume and left at room temperature for 5 minutes. Then, DNA and Expifectamine ^® reagent were reacted for 5 minutes and added to the flask, treated with feed and enhancer after 16 to 22 hours, and additionally fed after 5 days. On the 8th day, it was checked whether the viability was 75% or more and recovered.

(2) Analysis of transient expression fusions through SDS-PAGE

After adding 4 μL of 5x reducing-PAGE sample buffer to 16 μL of the recovered culture, it was heated at 100° C. for 5 minutes, and then left at room temperature for 20 minutes. After inserting polyacrylamide gel (12~20%, Invitrogen) into the gel kit, loading the sample and size standard ladder, electrophoresis at 125V for 2 hours, separate the gel from the kit, add coomassie blue staining solution, and leave for 1 hour did. The left gel was transferred to destaining buffer (70% tertiary distilled water, 20% methanol, 10% acetic acid) and incubated overnight. texture

As a result, as shown in FIG. 14, it was confirmed that the fusion, in which PD-1 and the coiled coil-forming domain were linked, was overexpressed at a suitable molecular weight position, through which PD-1 and the coiled coil-forming domain were linked in the temporary expression system, It was confirmed that the fusion was expressed.

3-3. Purification and concentration of the fusion, in which PD-1 and coiled coil-forming domains are linked

(1) Purification using Flag affinity chromatography

The culture solution obtained from the temporary expression system of Example 3-2 was filtered through a PES filtration membrane having pores of 0.2 μm in size to remove impurities. The filtrate was loaded into Anti-DYKDDDDK (Thermo fisher) to purify the fusion, in which PD-1 and coiled coil-forming domains were linked. The results are as shown in FIG. 15A. Flag affinity chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Resin: Anti-DYKDDDDK

- Flow rate: 300 cm/h

- Equilibrium: Phosphate-buffered saline

- Loading: up to 3 g protein/L resin volume

- Regeneration and sterilization: 0.01 M NaOH solution

- Elution: 0.1 M glycine-Hcl pH 2.7 buffer

- Storage: 20% EtOH

After flag affinity chromatography, the fractionated fusions were neutralized with 0.2 M sodium phosphate, pH 7.0 buffer, and analyzed by SE-HPLC to recover only fusion molecules having a peak eluted before wild-type HSA as a control. The fractionated and recovered fusions were analyzed by SE-HPLC.

As a result, as shown in FIG. 15b, it was confirmed that the fusion, in which the fractionated and recovered PD-1 and the coiled coil-forming domain were linked, had a purity of 80% or more.

(2) Purification using anion exchange resin chromatography

The fusion solution obtained through Flag affinity chromatography ^{was put into a concentrator/dialyzer equipped with Sartocon ®} Slice 200 Hydrosart ^® Cassette 10 KD (Sartorius), concentrated and dialyzed against 10 mM sodium phosphate, pH 8.0 buffer, and then recovered. The recovered dialysate was loaded into Q impres (GE healthcare) to purify the fusion, in which PD-1 and coiled coil-forming domains were connected. The results are as shown in FIG. 16A. The anion exchange resin chromatography conditions used for purification are as follows.

<Chromatography Conditions>

- Resin: Q impres

- Flow rate: 120 cm/h

- Equilibrium: 10 mM sodium phosphate, pH 8.0 buffer

- Loading: max 10 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 10 mM sodium phosphate, 500 mM sodium chloride, pH 8.0 buffer

- Storage: 0.01 M NaOH solution

After the anion exchange chromatography was performed, the fractionated and recovered fusions were identified by SE-HPLC analysis. As NaCl entered the concentration gradient, a fusion in which PD-1 and coiled coil-forming domains were connected in a low section came out, and an aggregation form appeared in a high section, SE-HPLC analysis was performed.

As a result, as shown in FIG. 16b, it was confirmed that the recovered PD-1 and the coiled coil-forming domain were linked, and the fusion had a purity of 98% or more.

In addition, as shown in FIG. 17 , as a result of SDS-PAGE analysis, it was confirmed that a band appeared at a suitable molecular weight position, thereby confirming that the purified fusion was a fusion in which PD-1 and the coiled coil forming domain were connected.

Thereafter, the amount of the purified PD-1 and the fusion to which the coiled coil-forming domain was linked was measured using a UV spectrometer, concentrated using an ultrafiltration method, and then dialyzed with PBS and stored at -20°C.

Example 4. Target binding substance (CD80) and coiled coil domains linked, fusion

4-1. Construction of a recombinant plasmid encoding the fusion, in which CD80 and coiled-coil forming domains are linked

After codon optimization of the DNA of CD80 and the coiled coil-forming domain for gene cloning, the DNA of the coiled coil-forming domain is linked to the C-terminus of CD80 through overlap PCR to form CD80 and coiled coil DNA encoding the fusion, in which the domains were linked, was prepared.

For gene construction, pcDNA 3.1 vector was used, and the insert DNA and vector were treated with DpnI restriction enzyme after overlap PCR, respectively, and the CD80 and coiled coil forming domains were linked, the fusion-encoding DNA was inserted into a 6641 bp vector. produced. 18 shows the finally constructed recombinant plasmid. After inserting the vector into Stella ^® competent cell (manufacturer: Clontech), it was plated on an agar plate to which ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and colonies were selected, followed by an Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Fisher) Scientific) was used to confirm the nucleotide sequence through DNA sequencing.

The nucleotide sequence of the synthesized coiled coil protein-encoding DNA confirmed through DNA sequencing is shown in Table 10. As an example of the present invention, the DNA nucleotide sequence encoding the fusion in which CD80 and the coiled coil forming domain are linked and the amino acid sequence of the fusion are shown in Table 10.

(The superscript “C” indicates that a cysteine has been introduced into the coiled-coil-forming domain, and cysteines in the sequence are indicated in bold and underlined.)

4-2. CD80 and coiled coil-forming domains are linked, establishment of a transient expression system of the fusion

(1) transformation

The final vector obtained through gene cloning in Example 4-1 was introduced into Expi 293 animal cells to establish a temporary expression system for quickly securing the fusion of the present invention.

Specifically, one day before transformation, cells were seeded in a flask at a ^{concentration of 2.5 to 3.0 x 10 6} cells/mL and then cultured for 24 hours. On the day of transduction, ExpiCHO ^® expression medium was added and diluted to a concentration of ^{3.0 x 10 6 cells/mL.} Then, DNA 1 μg/mL and Opti-MEM ^® medium were mixed, and ExpiFectamine ^® reagent and Opti-MEM ^® medium were mixed according to the volume and left at room temperature for 5 minutes. Then, DNA and Expifectamine ^® reagent were reacted for 20 minutes and added to the flask, treated with enhancers 1 and 2 after 16 to 22 hours, and recovered by checking whether viability is 50% or more after 5 days.

(2) Analysis of transiently expressed proteins through SDS-PAGE

After adding 4 μL of 5x reducing-PAGE sample buffer to 16 μL of the recovered culture, it was heated at 100° C. for 5 minutes, and then left at room temperature for 20 minutes. After inserting polyacrylamide gel (12~20%, Invitrogen) into the gel kit, loading the sample and size standard ladder, electrophoresis at 125V for 2 hours, separating the gel from the kit, adding coomassie blue staining solution, and leaving it for 1 hour . The left gel was transferred to destaining buffer (70% tertiary distilled water, 20% methanol, 10% acetic acid) and incubated overnight.

As a result, as shown in FIG. 19 , it was confirmed that the fusion, in which CD80 and the coiled coil-forming domain are linked, was overexpressed at a suitable molecular weight position, through which CD80 and the coiled coil-forming domain were linked, and the fusion was expressed in the transient expression system. Confirmed.

4-3. Purification and concentration of fusions in which CD80 and coiled coil-forming domains are linked

(1) Purification using histidine affinity chromatography

The culture solution obtained in the temporary expression system was filtered through a PES filtration membrane having pores of 0.2 μm in size to remove impurities. The filtrate ^{was put into a concentrator/dialysis machine equipped with a Sartocon ®} Slice 200 Hydrosart ^® Cassette 10 KD (Sartorius), concentrated, and then dialyzed against 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 8.0 buffer and recovered. The recovered dialysate was loaded into Histrap HP (GE healthcare) to purify the fusion, in which CD80 and coiled coil-forming domains were linked. The results are as shown in FIG. 20A. Histidine affinity chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Resin: Histrap HP

- Flow rate: 180 cm/h

- Equilibrium: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole pH 8.0 buffer

- Loading: up to 30 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 50 mM sodium phosphate, 300 mM sodium chloride, 500 mM imidazole pH 8.0 buffer

- Storage: 20% EtOH

After histidine affinity chromatography, the peak of the fusion eluted with CD80 and coiled coil-forming domains linked to CD80 and coiled coil-forming domain was analyzed by SE-HPLC while flowing equilibration buffer and elution buffer with a concentration gradient, and eluted before wild-type CD80 as a control. Only fusion molecules with peaks were recovered. The fractionated and recovered fusions were analyzed by SE-HPLC.

As a result, as shown in FIG. 20b, it was confirmed that the recovered CD80 and the coiled coil-forming domain were linked, and the fusion had a purity of 80% or more.

(2) Purification using anion exchange resin chromatography

The solution obtained through histidine affinity chromatography ^{was put into a concentrator/dialyzer equipped with a Sartocon ®} Slice 200 Hydrosart ^® Cassette 10 KD (Sartorius), concentrated and dialyzed against 10 mM sodium phosphate, pH 8.0 buffer, and then recovered. The recovered dialysate was loaded into Q impres (GE healthcare) to purify the fusion in which the CD80 and coiled coil-forming domains were linked. The results are as shown in FIG. 21A. The anion exchange resin chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Resin: Q impres

- Flow rate: 120 cm/h

- Equilibrium: 10 mM sodium phosphate, pH 8.0 buffer

- Loading: max 10 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 10 mM sodium phosphate, 500 mM sodium chloride, pH 8.0 buffer

- Storage: 0.01 M NaOH solution

After anion exchange chromatography, the fractionated fusion was identified by SE-HPLC analysis. As NaCl entered the concentration gradient, it was confirmed as a result of SE-HPLC analysis that a fusion with CD80 and coiled coil-forming domains were connected in the low section, and that the aggregated form appeared in the high section. As a result, as shown in FIG. 21B , it was confirmed that the fusion in which the recovered CD80 and coiled coil-forming domains were linked had a purity of 98% or more.

In addition, as shown in FIG. 22 , it was confirmed that a band appeared at a suitable molecular weight position through SDS-PAGE analysis, and it was confirmed that the recovered fusion was a fusion in which CD80 and coiled coil forming domains were connected.

Thereafter, the recovered CD80 and coiled coil-forming domains were linked, and the amount of the fusion was measured using a UV spectrometer, concentrated using an ultrafiltration method, and then dialyzed with PBS and stored at -20°C.

Example 5. Target binding substance (Insulin, insulin) and coiled coil-forming domain linked, fusion

5-1. Construction of a recombinant plasmid encoding a fusion in which Insulin and coiled coil-forming domains are linked

After codon optimization of DNA of insulin and coiled coil forming domain for gene cloning, DNA of coiled coil forming domain is linked to C-terminus of insulin through overlap PCR to form insulin and coiled coil DNA encoding the fusion, in which the domains were linked, was prepared.

The pQE80 vector was used for gene construction, and the insert DNA and vector were treated with DpnI restriction enzyme after overlap PCR, respectively, and the insulin and coiled coil forming domains were linked, and the DNA encoding the fusion was inserted into a vector of 5013 bp size. did. 23 shows the finally constructed recombinant plasmid. After inserting the vector into Stella ^® competent cell (manufacturer: Clontech), it was plated on an agar plate to which ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and colonies were selected, followed by an Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Fisher) Scientific) was used to confirm the nucleotide sequence through DNA sequencing.

The nucleotide sequence of the synthesized coiled coil protein-encoding DNA confirmed through DNA sequencing is shown in Table 11, and as an example of the present invention, insulin and the coiled coil forming domain are linked, the DNA nucleotide sequence encoding the fusion, and the amino acid of the fusion The sequence is shown in Table 11.

(The superscript “C” indicates that a cysteine has been introduced into the coiled-coil-forming domain, and cysteines in the sequence are indicated in bold and underlined.)

5-2. Establishment of a temporary expression system for fusions in which Insulin and coiled coil-forming domains are linked

After transforming the final vector obtained through gene cloning in Example 5-1 to BL21 cells through heat shock method, spread it on an agar plate to which an ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and a single colony was obtained. was selected. Colonies were cultured overnight in 2xYT medium containing 5 mL of ampicillin for seed culture. The next day, the main culture was transferred to 1 L of 2xYT medium containing ampicillin. After measuring whether the logarithmic growth phase of the OD 0.6 to 0.8 section was reached at 600 nm using a UV spectrometer, the protein was induced by adding IPTG, and after 4 hours, the insulin and the coiled coil-forming domain were connected, and the fusion was recovered.

5-3. Purification and concentration of fusions in which Insulin and coiled coil-forming domains are linked

(1) cell disruption

50 mM sodium phosphate, 300 mM sodium chloride, and 10 mM imidazole pH 8.0 (Histrap HP Equilibration buffer) buffer solution was added to the cell pellet recovered from the culture medium for resuspension, cooled with ice, and crushed for 30 minutes using an ultrasonicator. The crushed culture medium was centrifuged at 11,000 x g, 4°C, 30 minutes to obtain a pellet, and then washed twice with HisTrap HP Equilibration buffer.

(2) denaturation step

Denaturation buffer (50 mmol/L sodium phosphate, 300 mmol/L sodium chloride, 8 mol/L urea, 0.1% Triton X-100, 5 mmol/L DTT, pH 8.0) was added to the pellet of (1) for resuspension. After blocking the light and fixing it on a rocker, it was rocked at 100 rpm at RT for 3 hours. Next, it was moved to 4°C, rocked slowly, and incubated overnight.

(3) Purification using histidine affinity chromatography

The solution obtained in (2) was centrifuged at 11,000 x g, 4°C, 30 minutes, and the supernatant was filtered through a PES filtration membrane having pores of 0.2 μm in size to remove impurities. The filtrate was loaded into Histrap HP (GE healthcare) to purify the fusion in which insulin and coiled coil-forming domains were linked. The results are as shown in FIG. 24 . The conditions of histidine affinity chromatography used for the purification are as follows.

<Chromatography Conditions>

- Resin: Histrap HP

- Flow rate: 180 cm/h

- Equilibrium: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole pH 8.0 buffer

- Loading: up to 30 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 50 mM sodium phosphate, 300 mM sodium chloride, 500 mM

imidazole pH 8.0 buffer

- Storage: 20% EtOH

While the equilibration buffer and the elution buffer were flowed in a concentration gradient, the peaks in which the insulin and coiled coil-forming domains were connected and the fusion was eluted were collected and recovered.

(4) regeneration step

After injecting 12 mL each of the eluate of (3) into two 3.5 kDa dialysis cassettes (Thermo scientific), add 0.1 mmol/L glycine, 1 mmol/L DTT, pH 10.5, 5L, and dialysis overnight at 4℃ with slow stirring. to proceed with refolding.

After recovering the refolded sample, 5% (v/v) phosphoric acid was added to bring the pH to ~ 2.0, and 5 mM GSH (oxidized) was additionally added to terminate the oxidation reaction. Next, after concentration using ultrafiltration method, dialyzed against sodium acetate buffer pH4.5 and stored at -20 ℃.

As a result, as shown in FIG. 25 , it was confirmed using SE-HPLC that the recovered fusion had a purity of 90% or more.

Example 6. Target binding substance (GLP-1) and coiled coil-forming domain linked, fusion

The fusion, in which GLP-1 and the coiled coil-forming domain were linked, was synthesized by request of GL Biochem (Shanghai) Ltd., and the amino acid sequence of the fusion is shown in Table 12 below.

(The superscript “C” indicates that a cysteine has been introduced into the coiled-coil-forming domain, and cysteines in the sequence are indicated in bold and underlined.)

Example 7. Purification and concentration of dual target conjugates

7-1. Purification and concentration of dual target conjugates

A fusion protein in which the HSA purified in the above example and the coiled coil-forming domain are linked; And in order to bind the fusion in which the two types of target binding substances and the coiled coil forming domain are linked to form a double target conjugate, a process of purifying the fusion protein and the two types of fusions is required after binding.

The required amount (molar concentration) of the fusion protein and the fusion for the preparation of the dual target conjugate is as shown in Table 13 below.

Each concentration of the fusion protein and the two types of fusions were mixed and reacted at room temperature for 30 minutes. Thereafter, the reaction solution was loaded into Q impres (GE healthcare) to purify the double target conjugate. The results are as shown in FIG. 26 . The chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Resin: Q impres

- Flow rate: 120 cm/h

- Equilibrium: 20 mM sodium phosphate, pH 7.0

- Loading: max 10 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Wash: 20 mM sodium phosphate, 60 mM sodium chloride, pH 7.0

- Elution: 20 mM sodium phosphate, 200 mM sodium chloride, pH 7.0

- Storage: 20% EtOH

The double target binder was quantified using BCA and Lowry quantification method and UV spectrometer.

As a result, as shown in FIG. 27 , it was confirmed that the values measured using a UV spectrometer match the extinction coefficient values obtained from the total protein quantification method and the amino acid sequence, and through this, a quantification method of the double target binder was constructed.

Thereafter, the amount of the purified double target conjugate was measured using a UV spectrometer, concentrated using an ultrafiltration method, dialyzed against PBS, and stored at -20°C.

7-2. Purification and enrichment of dual target conjugates including covalent bonds between coiled coil-forming domains (introduction of cysteine mutations)

In order to prepare a double target conjugate by combining the fusion protein and the two types of fusions, including the coiled coil-forming domain into which the cysteine mutation is introduced, prepared in the Examples, reduction/oxidation process of each material is required prior to purification.

The required amount (molar concentration) of the fusion protein and the two types of fusions required for the preparation of the dual target conjugate and the concentration conditions of TCEP for reduction/oxidation are shown in Tables 14 to 16 below.

The fusion proteins and two types of fusion proteins at each concentration listed in Tables 14 to 16 were treated with TCEP at each concentration and reacted for 25 minutes. Then, the fusion protein and the two types of fusions were mixed and reacted for an additional 5 minutes. The reaction solution was mixed with 2 mM oxidized glutathione and allowed to form a disulfide bond by cysteine in the coiled coil-forming domain through air oxidation at 4°C for 3 days.

Thereafter, the reaction product obtained from the fusion protein of Table 14 and the two fusions was purified and concentrated by the same method as in Example 8-1.

In addition, the reaction product obtained from the fusion protein of Table 15 and the two fusions was loaded into Capture select human albumin (Thermo fisher) to purify the dual target conjugate. The results are as shown in FIG. 28A. The HSA affinity chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Suzy: Capture select human albumin

- Flow rate: 480 cm/h

- Equilibration: PBS buffer

- Loading: max 10 g protein/L resin volume

- Regeneration and sterilization: 0.01 M NaOH solution

- Elution: 0.1 M glycine-Hcl

- Storage: 20% EtOH

After HSA affinity chromatography, the fractionated and recovered dual target conjugate was neutralized with 0.2 M sodium phosphate, pH 7.0. It was analyzed by RP-HPLC to confirm the formation of covalent bonds.

As a result, as shown in FIG. 28b, it was confirmed that the dual target conjugate had a purity of 98% or more, and a covalent bond was formed by disulfide bond.

Thereafter, the amount of the purified double target conjugate was measured using a UV spectrometer, concentrated using an ultrafiltration method, dialyzed against PBS, and stored at -20°C.

Example 8. Fusion Duplex Proteins

8-1. Construction of a recombinant plasmid encoding a fusion duplex protein in which albumin and two proteins are expressed in one polypeptide

For gene cloning, after codon optimization of the DNA of the fusion duplex protein in which albumin and two proteins are expressed in one polypeptide, the DNA of the protein is transferred to the N-terminus through overlap PCR, PD-1, C- DNA encoding a protein fused to CD80 at the end, insulin at the N-terminus and DNA encoding a protein fused to GLP-1 at the C-terminus were prepared.

For gene construction, pcDNA 3.1 vector was used, and after overlap PCR of insert DNA and vector, DpnI restriction enzyme was treated to insert the DNA encoding the protein fused to PD-1 at the N-terminus and CD80 at the C-terminus. A vector of 8738 bp in size, insulin at the N-terminus, and DNA encoding a protein in which GLP-1 is fused at the C-terminus were inserted into a 7988 bp vector, and the finally constructed recombinant plasmid is shown in FIGS. 29A and 29B. shown in After inserting the vector into Stella ^® competent cell (manufacturer: Clontech), it was plated on an agar plate to which ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and colonies were selected, followed by an Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Fisher) Scientific) was used to confirm the nucleotide sequence through DNA sequencing.

As an example of the present invention, a DNA sequence and a fusion protein encoding a protein in which PD-1 is fused to the N-terminus and CD80 to the C-terminus, insulin is fused to the N-terminus and GLP-1 is fused to the C-terminus. The amino acid sequences of are shown in Tables 17 and 18.

7-2. Establishment of a transient expression system for fusion double proteins

The final vector obtained through gene cloning in Example 7-1 was introduced into Expi CHO animal cells to establish a temporary expression system for quickly securing the fusion protein of the present invention.

Specifically, the day before transformation ^{, cells at a concentration of 3.0 x 10 6} cells/mL were dispensed into the flask and cultured for 24 hours. On the day of transduction, ExpiCHO ^® expression medium was added and diluted to a concentration of ^{6.0 x 10 6 cells/mL.} Thereafter, DNA 1 μg/mL and Opti-PRO ^® medium were mixed, and ExpiFectamine ^® reagent and Opti-PRO ^® medium were mixed according to volume and left at room temperature for 5 minutes. Then, DNA and Expifectamine ^® reagent were reacted for 5 minutes and added to the flask, treated with feed and enhancer after 16 to 22 hours, and additionally fed after 5 days. On the 8th day, it was checked whether the viability was more than 75% and recovered.

7-3. Purification and concentration of fusion duplex proteins

(1) Purification using HSA affinity chromatography

The culture solution obtained from the transient expression system of Example 7-2 was filtered through a PES filtration membrane having a pore size of 0.2 μm to remove impurities. Sartocon ^® Slice 200 Hydrosart ^® Cassette 30 KD (Sartorius) was added to the filtered culture medium in a concentrator / dialyzer, concentrated, dialyzed against 20 mM sodium phosphate, pH 7.0 buffer, and then recovered. The recovered dialysate was loaded into an albumin affinity matrix Albupure ^® (Prometic Bioseparations Ltd), and as shown in FIG. 30a, PD-1 at the N-terminus and CD80 at the C-terminus were fused to the fusion double protein and as shown in FIG. 30b. A fusion double protein in which insulin and GLP-1 were fused to the same N-terminus was purified. The conditions of the HSA affinity chromatography used for the purification are as follows.

<Chromatography Conditions>

- Resin: Albupure ^®

- Flow rate: 480 cm/h

- Equilibrium: 20 mM sodium phosphate, pH 7.0 buffer

- Loading: up to 35 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 20 mM sodium phosphate, 20 mM sodium octanonate, pH 7.0 buffer

- Storage: 20% EtOH

After HSA affinity chromatography, a peak in which a protein in which PD-1 is fused to the N-terminus and CD80 is fused to the C-terminus, and a protein fused to insulin and GLP-1 at the C-terminus are eluted was recovered. . The fractionated and recovered protein was analyzed by SE-HPLC.

As a result, as can be seen in FIGS. 31A and 31B , the recovered protein in which PD-1 and CD80 are fused to the N-terminus and insulin to the N-terminus and GLP-1 to the C-terminus are fused to the recovered N-terminus. It was confirmed that the protein had a purity of 80% or more.

(2) Purification using anion exchange resin chromatography

The protein solution obtained through HSA affinity chromatography was loaded into Q impres (GE healthcare), and a protein fused to PD-1 at the N-terminus and CD80 at the C-terminus, insulin at the N-terminus, and GLP- at the C-terminus The protein fused to 1 was purified. The results are as shown in FIGS. 32A and 32B. The anion exchange resin chromatography conditions used for the purification are as follows.

<Chromatography Conditions>

- Resin: Q impres

- Flow rate: 120 cm/h

- Equilibrium: 20 mM sodium phosphate, pH 7.0 buffer

- Loading: max 10 g protein/L resin volume

- Regeneration and sterilization: 1 M NaOH solution

- Elution: 20 mM sodium phosphate, 200 mM sodium chloride, pH 7.0 buffer

- Storage: 0.01 M NaOH solution

After anion exchange chromatography, the fractionated and recovered protein was analyzed by SE-HPLC and identified.

As a result, as shown in FIGS. 33a and 33b, the peaks from the eluate are a fusion double protein in which PD-1 at the N-terminus and CD80 at the C-terminus are fused, insulin at the N-terminus, and GLP-1 at the C-terminus. It was confirmed through SE-HPLC that the fusion duplex protein was fused, and the purity of the fusion duplex protein was confirmed to be 95% or higher.

After measuring the amount of the recovered protein in which PD-1 and CD80 are fused to the N-terminus, insulin to the N-terminus, and GLP-1 to the C-terminus, recovered using a UV spectrometer, ultrafiltration method was used. After concentration, it was dialyzed against PBS and stored at -20°C.

Experimental Example 1. Confirmation of formation of dual target conjugate

It was measured by SE-HPLC whether the double-target binder forms a hetero complex due to self-binding between the coiled coil-forming domains, resulting in an increase in size. The fusion protein and two types of fusion proteins used to form the double target conjugate were reduced and then combined because cysteine was engineered into the end of the coiled coil-forming domain. Specifically, the cysteine of the fusion protein and two types of fusions was reduced using TCEP (tris(2-carboxyethyl)phosphine), which is one of the substances used as a reducing agent. The concentration of TCEP was adjusted to reduce only the disulfide bonds around the external coiled coil-forming domain without reducing the disulfide bond inside each fusion protein and the two fusion proteins, and HSA and the coiled coil-forming domain were linked to the fusion protein. After reacting for 30 minutes with 15 times the concentration of TCEP, 7.5 times the concentration of the fusion to which each target binding substance and the coiled coil-forming domain are linked, the reaction was performed by SE-HPLC.

As a result, as shown in FIGS. 34a to 34c, as a result of SE-HPLC analysis, it was confirmed that the peaks shifted to the left in both of the two dual target binders. This means that the size of the fusion protein and the two types of fusions becomes larger than when the fusion protein and the two types of fusions form a double target complex by self-binding, respectively. Specifically, FIG. 34a relates to a double conjugate into which GFP and mCherry are introduced as target binding substances, FIG. 34b relates to a double conjugate into which CD80 and PD-1 are introduced as target binding substances, and FIG. 34c is a target binding substance. It relates to a double conjugate into which Insulin and GPL-1 are introduced. Therefore, it was confirmed from the result of the shift of the peak that a dual target conjugate (a conjugate of HSA and a target binding substance) was formed through self-binding of the fusion protein and two types of fusions.

Experimental Example 2. Measurement of binding force of dual target binders

To compare the binding force due to self-binding between coiled coil-forming domains, surface plasma resonance (SPR) values were measured using a Biacore T-100 (GE healthcare) instrument.

A CM5 chip was mounted on the Biacore T-100 device and HBS (HEPES buffered saline, HBS) buffer was flowed thereinto. After confirming that the base line of the graph is kept constant, 1-ethyl-3-(dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (N-hydroxy succinimide) , NHS) was flowed to the chip to activate the amine group. Then, the fusion protein in which the HSA and coiled coil-forming domains were connected was injected into the chip to form a covalent bond with the activated amine group, followed by immobilization, and then completely fixed with ethanolamine. A fusion that can be bound by forming a coiled coil with a fusion protein in which the immobilized HSA and the coiled coil-forming domain are linked (a fusion with a target binding material (mCherry or GFP) and a coiled coil-forming domain linked) is flowed to the chip. After confirming the degree of binding as a resonance value (resonance unit, RU), the dissociation constant (K _d ) was calculated using a program.

As a result, as shown in FIGS. 35A and 35B, the dissociation constant of the coiled coil-forming domain bound to the HSA of the present invention or the coiled coil-forming domain C/C' bound to the target binding substance was 6.85 nM, D/D The dissociation constant of ' was measured to be 1.32 nM, and the measured dissociation constant is a dissociation constant similar to that of a coiled coil-forming domain to which HSA or a target binding material is not bound (C/C': 42, D/D': 1 nM ) was found to have a similar value to Through this, it was confirmed that there is no problem in coiled coil formation by self-binding even when HSA or a target binding material is bound to the coiled coil forming domain.

Experimental Example 3. Measurement of pH stability of dual target conjugates

Whether the binding stability of the dual target conjugate was maintained according to the pH change was confirmed using SE-HPLC.

Specifically, each dual target conjugate was prepared through dialysis with buffers of pH 4, 6 and 7.4, and the SE-HPLC mobile phase was also set with buffers of each pH.

As a result, as shown in FIG. 36 , an increase in size due to the formation of the dual target conjugate was observed in the pH range of 4 to 7.4, confirming that the self-binding between the coiled coil-forming domains was made regardless of whether the pH was changed.

Experimental Example 4. Rat plasma stability of dual target conjugate

In order to determine whether the binding force between the coiled coil-forming domains of the dual target conjugate is maintained in rat plasma at 37° C., a dual target conjugate by non-covalent bonding; And the stability test in rat plasma of the dual target conjugate with improved binding stability between the coiled coil-forming domains by an additional covalent bond (disulfide bond) was conducted.

Specifically, at the same concentration to conduct the pharmacokinetic test in rats, the double target conjugate and the control wild-type HSA in an amount equivalent to 5 mg/kg were added to 200 μL of rat plasma, and reacted at 37 ° C. Days 0 and 1 and the concentrations of the double-target conjugate and the control wild-type HSA on day 2 were measured, and the concentrations of the dual-target conjugate and the wild-type HSA were measured using ELISA. 100 μL of each of mCherry antibody or Human CD80 (B7-1) antibody diluted in the coating solution was added to a 96 well plate and left overnight at 4°C. The plate was washed 5 times with PBST (Phosphate buffered saline, Tween-200) washing solution, and then 200 μL of blocking buffer containing 3% skim milk was added per well and blocked at 37° C. for 2 hours. After washing the plate 5 times with PBST, add 100 μL of standard solution diluted by 1/2 to 128 ng/mL to 0.25 ng/mL or rat plasma sample diluted at an appropriate ratio, and incubate at 37°C for 2 hours with PBST It was washed 5 times with a washing solution. After that, 1/100 of the biotinylated anti-PD-1 polyclonal antibody included in the GFP antibody or PD-1 human ELISA kit was diluted 1/100, put into each well, and incubated at 37°C for 1.5 hours. After washing the plate 5 times with PBST washing solution, 1/200 dilution of anti-mouse IGG-HRP or Streptavidin-HRP was put into each well, and incubated at 37°C for 1 hour. Wash 5 times with PBST washing solution, add 100 μL of TMB substrate solution per well, incubate for 10 minutes at dark room temperature, terminate the reaction with 100 μL of 1N sulfuric acid per well, and measure the absorbance at 450 nm with a plate reader measured. The concentration of the protein was calculated by calculating the dilution factor to the amount calculated from ELISA.

As a result, as shown in FIG. 37 , it was confirmed that the dual target conjugate (PD1/CD80) further including an additional covalent bond (disulfide bond) was maintained for 2 days in rat plasma similar to the control wild-type HSA.

In addition, as shown in FIGS. 38a and 38b, the dual target conjugate (GLP-1/insulin) further comprising an additional covalent bond (disulfide bond) is stable for 2 days in rat plasma similar to the control wild-type HSA. Confirmed.

Thus, it was confirmed that the dual target binder of the present invention maintains the binding force between the coiled coil-forming domains in rat plasma.

Experimental Example 5. Dual target conjugate in vitro efficacy test

(1) CD80 in vitro ELISA competitive assay

CD28, a ligand protein of CD80, was diluted to 1 µg/mL in antigen coating buffer and then coated on an EIA plate at 100 µL at 4°C overnight. After removing the sample from the antibody coating plate, 200 μL of 3% skim milk blocking solution was loaded, followed by incubation at RT for 2 hours for blocking. After removing 3% skim milk blocking solution, CD80-K' was diluted to 100 nmol/L and loaded in duplicate by 50 μL. In a well loaded with CD80-K', in serial dilution of 10 concentration intervals, double target conjugate (complex, albumin or its analog and target binding substance, target binding substance (PD-1, CD80)) sample in duplicate 50 μL After loading each, it was incubated for 2 hours at RT. After removing the sample, it was washed 3 times with 0.05% PBST. Anti-HSA-antibody-HRP was diluted 1:5000 in 3% skim milk blocking solution, loaded into each well, and incubated at RT for 1 hour. After removing Anti-HSA-antibody-HRP, it was washed 5 times with 0.05% PBST. 100 μL of TMB solution was loaded to develop color. After stopping the color reaction by adding 100 μL of 1 N Sulfuric acid, absorbance was measured at 450 nm using a microplate reader.

As a result, as shown in FIG. 39 , the in vitro efficacy of the dual target conjugate (complex, a conjugate of albumin or an analog thereof and a target binding substance, target binding substance (PD-1, CD80)) was confirmed, and specifically, the N-terminal It was confirmed that the in vitro efficacy of the dual target conjugate was higher than that of the fusion dual protein in which PD-1 and CD80 were fused to the C-terminus.

(2) PD-1 in vitro ELISA competitive assay

PDL1, a ligand protein of PD1, was diluted to 1 μg/mL in antigen coating buffer and then coated on an EIA plate at 100 μL at 4°C overnight. After removing the sample from the antibody coating plate, 200 μL of 3% skim milk blocking solution was loaded and incubated at RT for 2 hours for blocking. After removing 3% skim milk blocking solution, PD1-C' was diluted to 100 nmol/L and loaded in duplicate by 50 μL. In a well loaded with PD1-C', double target conjugate (complex, albumin or its analogue and target binding substance, target binding substance (PD-1, CD80)) sample in serial dilution of 10 concentration intervals in duplicate 50 μL After loading each, it was incubated for 2 hours at RT. After removing the sample, it was washed 3 times with 0.05% PBST. After removing the sample, it was washed 3 times with 0.05% PBST. Anti-HSA-antibody-HRP was diluted 1:5000 in 3% skim milk blocking solution, loaded into each well, and incubated at RT for 1 hour. After removing Anti-HSA-antibody-HRP, it was washed 5 times with 0.05% PBST. 100 μL of TMB solution was loaded to develop color. After stopping the color reaction by adding 100 μL of 1 N sulfuric acid, absorbance was measured at 450 nm using a microplate reader.

As a result, as shown in FIG. 40 , the in vitro efficacy of the dual target conjugate (complex, a conjugate of albumin or an analog thereof and a target binding substance, target binding substance (PD-1, CD80)) was confirmed, and specifically, the N-terminal It was confirmed that the in vitro efficacy of the dual target conjugate was higher than that of the fusion dual protein in which PD-1 and CD80 were fused to the C-terminus.

(3) GLP-1 in vitro ELISA competitive assay

GLP-1 receptor GLP-1 receptor was diluted to 1 μg/mL in antigen coating buffer and then coated on an EIA plate at 100 μL at 4°C overnight. After removing the sample from the antibody coating plate, 200 μL of 3% skim milk blocking solution was loaded lo, followed by incubation at RT for 2 hours for blocking. After removing 3% skim milk blocking solution, PD1-C' was diluted to 100 nmol/L and loaded in duplicate by 50 μL. In a well loaded with GLP-1-C', serial dilution of 10 concentration sections was performed to duplicate the sample of the double target conjugate (complex, albumin or its analog and target binding substance, target binding substance (GLP-1, Insulin)). After loading 50 μL each, it was incubated at RT for 2 hours. After removing the sample, it was washed 3 times with 0.05% PBST. After removing the sample, it was washed 3 times with 0.05% PBST. Anti-HSA-antibody-HRP was diluted 1:5000 in 3% skim milk blocking solution, loaded into each well, and incubated at RT for 1 hour. After removing Anti-HSA-antibody-HRP, it was washed 5 times with 0.05% PBST. 100 μL of TMB solution was loaded to develop color. After stopping the color reaction by adding 100 μL of 1 N Sulfuric acid, absorbance was measured at 450 nm using a microplate reader.

As a result, as shown in FIG. 41 , the in vitro efficacy of the dual target conjugate (complex, a conjugate of albumin or an analog thereof and a target binding substance, target binding substance (GLP-1, Insulin)) was confirmed, and specifically, the N-terminal Insulin, it was confirmed that the in vitro efficacy of the dual target conjugate was higher than that of the fusion dual protein in which GLP-1 was fused to the C-terminus.

(4) CD80 in vitro cell blockade assay

In order to confirm the activity of the dual target binder, cell blockade assay was performed using the CTLA-4 blockade assay kit (Promega). After diluting the double target conjugate (albumin or an analog thereof and the target binding substance, target binding substance (PD-1, CD80)) to 10 μmol/L, serial dilution was performed 3 times to prepare 9 concentrations of samples. The effector cells, samples (duplicates), and APC cells in the kit were sequentially placed in a 96 well plate, and then _{incubated at 37°C, 5% CO 2 in an} incubator for 6 hours. After 10 minutes of putting the reagent inside the kit, luminescence was measured using a microplate reader.

As a result, as shown in FIG. 42 , the in vitro cell blockade efficacy of the dual target conjugate (complex, a conjugate of albumin or an analog thereof and a target binding substance, target binding substance (PD-1, CD80)) was confirmed, and specifically, N - It was confirmed that the in vitro cell blockade efficacy of the dual target conjugate was higher than that of the fusion dual protein in which PD-1 at the terminal and CD80 at the C-terminus were fused.

(5) PD-1 in vitro cell blockade assay

In order to confirm the activity of the dual target binder, cell blockade assay was performed using the PD1-PDL1 blockade assay kit (Promega). The APC cells inside the kit were put in a 96 well plate, and then incubated overnight at _{37°C, 5% CO 2 in an incubator.} After diluting the double target conjugate (complex, a conjugate of albumin or an analog thereof and a target binding substance, target binding substance (PD-1, CD80)) to 10 μmol/L, serial dilution by 2.5 times to prepare 9 concentrations of samples did. The sample and the effector cell inside the kit were sequentially placed in a 96 well plate, and then incubated for 6 hours at _{37°C, 5% CO 2 in an incubator.} After putting the regent inside the kit, luminescence was measured using a microplate reader after 10 minutes.

As a result, as shown in FIG. 43, the in vitro cell blockade efficacy of the dual target conjugate (complex, a conjugate of albumin or an analog thereof and a target binding substance, target binding substance (PD-1, CD80)) was confirmed, and specifically, N - It was confirmed that the in vitro cell blockade efficacy of the dual target conjugate was higher than that of the fusion dual protein in which PD-1 at the terminal and CD80 at the C-terminus were fused.

<110> HK Inno.N <120> FUSION PROTEIN COMPRISING ALBUMIN AND COILED COIL FORMING DOMAIN, AND ALBUMIN AND TARGET SUBSTANCE CONJUGATE USING THE SAME <130> P19-114-REA-HKI <150> KR 10-2020-0002238 <151> 2020-01-07 <160> 57 <170> KoPatentIn 3.0 <210> 1 <211> 102 <212> DNA <213> Homo sapiens <400> 1 aaggctttca ttgtcacgga tgaggacatt agaaaacaag aagaaagagt tcagcaggtg 60 aggaaaaagt tggaagaagc attgatggct gacatattga gt 102 <210> 2 <211> 84 <212> DNA <213> Homo sapiens <400> 2 gaaattgcgg cactcgaaaa agagattgcc gcgcttgaaa aagagaatgc tgcacttgaa 60 tgggagatag ccgcattgga aaaa 84 <210> 3 <211> 81 <212> DNA <213> Homo sapiens <400> 3 ggacttgagc aagagattgc ggcgctggaa aaggagaacg cagccttgga atgggaaatt 60 gccgcacttg aacagggcgg a 81 <210> 4 <211> 34 <212> PRT <213> Homo sapiens <400> 4 Lys Ala Phe Ile Val Thr Asp Glu Asp Ile Arg Lys Gln Glu Glu Arg 1 5 10 15 Val Gln Gln Val Arg Lys Lys Leu Glu Glu Ala Leu Met Ala Asp Ile 20 25 30 Leu Ser <210> 5 <211> 28 <212> PRT <213> Homo sapiens <400> 5 Glu Ile Ala Ala Leu Glu Lys Glu Ile Ala Ala Leu Glu Lys Glu Asn 1 5 10 15 Ala Ala Leu Glu Trp Glu Ile Ala Ala Leu Glu Lys 20 25 <210> 6 <211> 27 <212> PRT <213> Homo sapiens <400> 6 Gly Leu Glu Gln Glu Ile Ala Ala Leu Glu Lys Glu Asn Ala Ala Leu 1 5 10 15 Glu Trp Glu Ile Ala Ala Leu Glu Gln Gly Gly 20 25 <210> 7 <211> 2115 <212> DNA <213> Artificial Sequence <220> <223> Albumin and coiled coil forming domain <400> 7 atgaagtggg ttaccttcat ctcccttttg tttctgttta gcagtgcgta cagcagggga 60 gtattcagac gcggaggcgg ggaaattgcg gcactcgaaa aagagattgc cgcgcttgaa 120 aaagagaatg ctgcacttga atgggagata gccgcattgg aaaaaggggg tgggggaagt 180 ggtggtggtg ggagtggagg cggtggatca gacgcccaca agagcgaggt agctcaccgc 240 ttcaaagacc tgggggagga aaactttaag gcactcgtgc ttatcgcttt cgcgcagtat 300 ttgcagcagt gtcctttcga ggaccatgtg aaattggtaa atgaagtaac tgagtttgca 360 aagacgtgtg tcgcggacga atccgccgaa aactgtgaca agtccttgca cacgcttttc 420 ggggataaat tgtgtaccgt cgcaaccctc cgcgaaacat acggagaaat ggcggattgc 480 tgtgccaaac aagaaccgga gcgcaacgag tgctttctcc aacataagga cgataatcca 540 aatctccccc gcctggtccg cccggaagtc gatgtcatgt gtacagcatt ccatgataat 600 gaagaaacct ttctgaaaaa gtatttgtat gaaatagcca gaaggcaccc gtacttttac 660 gccccagaac tgctgttttt tgcgaagagg tacaaggccg cgtttaccga gtgctgtcag 720 gccgcggaca aagcggcctg cctgctccct aaattggacg agttgaggga tgaagggaag 780 gcttcttctg ctaaacagcg cctcaagtgc gcgtctttgc agaagttcgg agagagagcc 840 tttaaagcat gggctgttgc tcgcctgtca cagaggtttc caaaagcaga gtttgctgaa 900 gtcagcaaac tggtcacaga ccttaccaaa gtacatacgg aatgctgtca tggtgatctt 960 ttggaatgcg cagatgaccg agccgacctt gcgaagtata tctgtgaaaa tcaggacagt 1020 atttcctcaa aattgaagga gtgttgcgag aaaccacttc tcgagaagag tcactgcatc 1080 gcagaagttg agaatgacga gatgccggcg gacttgccta gcctggctgc tgattttgtt 1140 gaaagtaagg acgtgtgcaa gaactacgct gaagctaaag acgtctttct tgggatgttc 1200 ctgtatgagt acgcacgaag acatcctgat tactccgttg tactgttgct tagactggca 1260 aaaacgtatg agaccacctt ggagaaatgt tgtgcggcgg cagaccccca tgaatgttac 1320 gccaaagtat tcgacgagtt taagcccctt gtagaagagc ctcagaacct gataaagcaa 1380 aactgtgagc ttttcgagca gctgggggaa tataaattcc aaaacgctct tctcgtgcgc 1440 tacactaaga aagttccgca ggtttccacc cccacccttg tagaagtgtc taggaacctt 1500 ggtaaagttg gcagcaaatg ctgtaaacat cctgaagcaa agcggatgcc gtgcgccgag 1560 gactatcttt ctgtagttct gaatcagctc tgtgttctcc acgaaaaaac gccggtaagt 1620 gatcgcgtta caaaatgctg tacagagagc ctggtcaacc ggcgaccctg tttctccgct 1680 cttgaagttg acgagactta cgtcccgaaa gaattcaacg ccgaaacatt cacatttcac 1740 gccgatatct gtaccttgtc tgaaaaggaa cgccaaatta aaaagcaaac cgccctggtg 1800 gagctggtta aacacaagcc gaaggcaact aaagagcagc tgaaggctgt tatggacgat 1860 ttcgctgcgt tcgtggagaa atgttgcaag gccgacgaca aagagacgtg tttcgcagag 1920 gaaggaaaga aacttgtggc tgcttctcaa gccgcacttg gactgggggg aggcggcagt 1980 ggaggagggg ggtcaggagg tgggggtggg aaggctttca ttgtcacgga tgaggacatt 2040 agaaaacaag aagaaagagt tcagcaggtg aggaaaaagt tggaagaagc attgatggct 2100 gacatattga gtggg 2115 <210> 8 <211> 705 <212> PRT <213> Artificial Sequence <220> <223> Albumin and coiled coil forming domain <400> 8 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Gly Gly Gly Glu Ile Ala Ala Leu 20 25 30 Glu Lys Glu Ile Ala Ala Leu Glu Lys Glu Asn Ala Ala Leu Glu Trp 35 40 45 Glu Ile Ala Ala Leu Glu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 50 55 60 Ser Gly Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg 65 70 75 80 Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala 85 90 95 Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 100 105 110 Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 115 120 125 Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 130 135 140 Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 145 150 155 160 Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 165 170 175 Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 180 185 190 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr 195 200 205 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu 210 215 220 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln 225 230 235 240 Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg 245 250 255 Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 260 265 270 Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 275 280 285 Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 290 295 300 Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu 305 310 315 320 Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu 325 330 335 Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 340 345 350 Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met 355 360 365 Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp 370 375 380 Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 385 390 395 400 Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 405 410 415 Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 420 425 430 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys 435 440 445 Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 450 455 460 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg 465 470 475 480 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 485 490 495 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 500 505 510 Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 515 520 525 Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 530 535 540 Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 545 550 555 560 Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr 565 570 575 Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 580 585 590 Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys 595 600 605 Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 610 615 620 Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 625 630 635 640 Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly 645 650 655 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Lys Ala 660 665 670 Phe Ile Val Thr Asp Glu Asp Ile Arg Lys Gln Glu Glu Arg Val Gln 675 680 685 Gln Val Arg Lys Lys Leu Glu Glu Ala Leu Met Ala Asp Ile Leu Ser 690 695 700 Gly 705 <210> 9 <211> 2115 <212> DNA <213> Artificial Sequence <220> <223> Albumin and coiled coil forming domain <400> 9 atgaagtggg ttaccttcat ctcccttttg tttctgttta gcagtgcgta cagcagggga 60 gtattcagac gcggaggctg tgaaattgcg gcactcgaaa aagagattgc cgcgcttgaa 120 aaagagaatg ctgcacttga atgggagata gccgcattgg aaaaatgtgg tgggggaagt 180 ggtggtggtg ggagtggagg cggtggatca gacgcccaca agagcgaggt agctcaccgc 240 ttcaaagacc tgggggagga aaactttaag gcactcgtgc ttatcgcttt cgcgcagtat 300 ttgcagcagt gtcctttcga ggaccatgtg aaattggtaa atgaagtaac tgagtttgca 360 aagacgtgtg tcgcggacga atccgccgaa aactgtgaca agtccttgca cacgcttttc 420 ggggataaat tgtgtaccgt cgcaaccctc cgcgaaacat acggagaaat ggcggattgc 480 tgtgccaaac aagaaccgga gcgcaacgag tgctttctcc aacataagga cgataatcca 540 aatctccccc gcctggtccg cccggaagtc gatgtcatgt gtacagcatt ccatgataat 600 gaagaaacct ttctgaaaaa gtatttgtat gaaatagcca gaaggcaccc gtacttttac 660 gccccagaac tgctgttttt tgcgaagagg tacaaggccg cgtttaccga gtgctgtcag 720 gccgcggaca aagcggcctg cctgctccct aaattggacg agttgaggga tgaagggaag 780 gcttcttctg ctaaacagcg cctcaagtgc gcgtctttgc agaagttcgg agagagagcc 840 tttaaagcat gggctgttgc tcgcctgtca cagaggtttc caaaagcaga gtttgctgaa 900 gtcagcaaac tggtcacaga ccttaccaaa gtacatacgg aatgctgtca tggtgatctt 960 ttggaatgcg cagatgaccg agccgacctt gcgaagtata tctgtgaaaa tcaggacagt 1020 atttcctcaa aattgaagga gtgttgcgag aaaccacttc tcgagaagag tcactgcatc 1080 gcagaagttg agaatgacga gatgccggcg gacttgccta gcctggctgc tgattttgtt 1140 gaaagtaagg acgtgtgcaa gaactacgct gaagctaaag acgtctttct tgggatgttc 1200 ctgtatgagt acgcacgaag acatcctgat tactccgttg tactgttgct tagactggca 1260 aaaacgtatg agaccacctt ggagaaatgt tgtgcggcgg cagaccccca tgaatgttac 1320 gccaaagtat tcgacgagtt taagcccctt gtagaagagc ctcagaacct gataaagcaa 1380 aactgtgagc ttttcgagca gctgggggaa tataaattcc aaaacgctct tctcgtgcgc 1440 tacactaaga aagttccgca ggtttccacc cccacccttg tagaagtgtc taggaacctt 1500 ggtaaagttg gcagcaaatg ctgtaaacat cctgaagcaa agcggatgcc gtgcgccgag 1560 gactatcttt ctgtagttct gaatcagctc tgtgttctcc acgaaaaaac gccggtaagt 1620 gatcgcgtta caaaatgctg tacagagagc ctggtcaacc ggcgaccctg tttctccgct 1680 cttgaagttg acgagactta cgtcccgaaa gaattcaacg ccgaaacatt cacatttcac 1740 gccgatatct gtaccttgtc tgaaaaggaa cgccaaatta aaaagcaaac cgccctggtg 1800 gagctggtta aacacaagcc gaaggcaact aaagagcagc tgaaggctgt tatggacgat 1860 ttcgctgcgt tcgtggagaa atgttgcaag gccgacgaca aagagacgtg tttcgcagag 1920 gaaggaaaga aacttgtggc tgcttctcaa gccgcacttg gactgggggg aggcggcagt 1980 ggaggagggg ggtcaggagg tgggggttgt aaggctttca ttgtcacgga tgaggacatt 2040 agaaaacaag aagaaagagt tcagcaggtg aggaaaaagt tggaagaagc attgatggct 2100 gacatattga gttgt 2115 <210> 10 <211> 2112 <212> DNA <213> Artificial Sequence <220> <223> Albumin and coiled coil forming domain <400> 10 atgaagtggg ttaccttcat ctcccttttg tttctgttta gcagtgcgta cagcagggga 60 gtattcagac gcggaggctg tggacttgag caagagattg cggcgctgga aaaggagaac 120 gcagccttgg aatgggaaat tgccgcactt gaacagggcg gatgtggtgg gggaagtggt 180 ggtggtggga gtggaggcgg tggatcagac gcccacaaga gcgaggtagc tcaccgcttc 240 aaagacctgg gggaggaaaa ctttaaggca ctcgtgctta tcgctttcgc gcagtatttg 300 cagcagtgtc ctttcgagga ccatgtgaaa ttggtaaatg aagtaactga gtttgcaaag 360 acgtgtgtcg cggacgaatc cgccgaaaac tgtgacaagt ccttgcacac gcttttcggg 420 gataaattgt gtaccgtcgc aaccctccgc gaaacatacg gagaaatggc ggattgctgt 480 gccaaacaag aaccggagcg caacgagtgc tttctccaac ataaggacga taatccaaat 540 ctccccccgcc tggtccgccc ggaagtcgat gtcatgtgta cagcattcca tgataatgaa 600 gaaacctttc tgaaaaagta tttgtatgaa atagccagaa ggcacccgta cttttacgcc 660 ccagaactgc tgttttttgc gaagaggtac aaggccgcgt ttaccgagtg ctgtcaggcc 720 gcggacaaag cggcctgcct gctccctaaa ttggacgagt tgagggatga agggaaggct 780 tcttctgcta aacagcgcct caagtgcgcg tctttgcaga agttcggaga gagagccttt 840 aaagcatggg ctgttgctcg cctgtcacag aggtttccaa aagcagagtt tgctgaagtc 900 agcaaactgg tcacagacct taccaaagta catacggaat gctgtcatgg tgatcttttg 960 gaatgcgcag atgaccgagc cgaccttgcg aagtatatct gtgaaaatca ggacagtatt 1020 tcctcaaaat tgaaggagtg ttgcgagaaa ccacttctcg agaagagtca ctgcatcgca 1080 gaagttgaga atgacgagat gccggcggac ttgcctagcc tggctgctga ttttgttgaa 1140 agtaaggacg tgtgcaagaa ctacgctgaa gctaaagacg tctttcttgg gatgttcctg 1200 tatgagtacg cacgaagaca tcctgattac tccgttgtac tgttgcttag actggcaaaa 1260 acgtatgaga ccaccttgga gaaatgttgt gcggcggcag acccccatga atgttacgcc 1320 aaagtattcg acgagtttaa gccccttgta gaagagcctc agaacctgat aaagcaaaac 1380 tgtgagcttt tcgagcagct gggggaatat aaattccaaa acgctcttct cgtgcgctac 1440 actaagaaag ttccgcaggt ttccaccccc acccttgtag aagtgtctag gaaccttggt 1500 aaagttggca gcaaatgctg taaacatcct gaagcaaagc ggatgccgtg cgccgaggac 1560 tatctttctg tagttctgaa tcagctctgt gttctccacg aaaaaacgcc ggtaagtgat 1620 cgcgttacaa aatgctgtac agagagcctg gtcaaccggc gaccctgttt ctccgctctt 1680 gaagttgacg agacttacgt cccgaaagaa ttcaacgccg aaacattcac atttcacgcc 1740 gatatctgta ccttgtctga aaaggaacgc caaattaaaa agcaaaccgc cctggtggag 1800 ctggttaaac acaagccgaa ggcaactaaa gagcagctga aggctgttat ggacgatttc 1860 gctgcgttcg tggagaaatg ttgcaaggcc gacgacaaag agacgtgttt cgcagaggaa 1920 ggaaagaaac ttgtggctgc ttctcaagcc gcacttggac tgggggggagg cggcagtgga 1980 ggaggggggt caggaggtgg gggttgtaag gctttcattg tcacggatga ggacattaga 2040 aaacaagaag aaagagttca gcaggtgagg aaaaagttgg aagaagcatt gatggctgac 2100 atattgagtt gt 2112 <210> 11 <211> 705 <212> PRT <213> Artificial Sequence <220> <223> Albumin and coiled coil forming domain <400> 11 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Gly Gly Cys Glu Ile Ala Ala Leu 20 25 30 Glu Lys Glu Ile Ala Ala Leu Glu Lys Glu Asn Ala Ala Leu Glu Trp 35 40 45 Glu Ile Ala Ala Leu Glu Lys Cys Gly Gly Gly Ser Gly Gly Gly Gly 50 55 60 Ser Gly Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg 65 70 75 80 Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala 85 90 95 Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 100 105 110 Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 115 120 125 Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 130 135 140 Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 145 150 155 160 Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 165 170 175 Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 180 185 190 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr 195 200 205 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu 210 215 220 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln 225 230 235 240 Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg 245 250 255 Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 260 265 270 Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 275 280 285 Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 290 295 300 Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu 305 310 315 320 Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu 325 330 335 Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 340 345 350 Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met 355 360 365 Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp 370 375 380 Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 385 390 395 400 Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 405 410 415 Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 420 425 430 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys 435 440 445 Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 450 455 460 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg 465 470 475 480 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 485 490 495 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 500 505 510 Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 515 520 525 Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 530 535 540 Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 545 550 555 560 Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr 565 570 575 Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 580 585 590 Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys 595 600 605 Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 610 615 620 Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 625 630 635 640 Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly 645 650 655 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Cys Lys Ala 660 665 670 Phe Ile Val Thr Asp Glu Asp Ile Arg Lys Gln Glu Glu Arg Val Gln 675 680 685 Gln Val Arg Lys Lys Leu Glu Glu Ala Leu Met Ala Asp Ile Leu Ser 690 695 700 Cys 705 <210> 12 <211> 704 <212> PRT <213> Artificial Sequence <220> <223> Albumin and coiled coil forming domain <400> 12 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Gly Gly Cys Gly Leu Glu Gln Glu 20 25 30 Ile Ala Ala Leu Glu Lys Glu Asn Ala Ala Leu Glu Trp Glu Ile Ala 35 40 45 Ala Leu Glu Gln Gly Gly Cys Gly Gly Gly Ser Gly Gly Gly Gly Ser 50 55 60 Gly Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe 65 70 75 80 Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe 85 90 95 Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val 100 105 110 Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala 115 120 125 Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys 130 135 140 Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys 145 150 155 160 Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp 165 170 175 Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met 180 185 190 Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu 195 200 205 Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu 210 215 220 Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala 225 230 235 240 Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp 245 250 255 Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu 260 265 270 Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu 275 280 285 Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val 290 295 300 Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu 305 310 315 320 Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn 325 330 335 Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu 340 345 350 Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro 355 360 365 Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val 370 375 380 Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu 385 390 395 400 Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu 405 410 415 Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala 420 425 430 Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro 435 440 445 Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe 450 455 460 Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr 465 470 475 480 Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser 485 490 495 Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala 500 505 510 Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln 515 520 525 Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys 530 535 540 Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu 545 550 555 560 Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe 565 570 575 Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile 580 585 590 Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala 595 600 605 Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val 610 615 620 Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu 625 630 635 640 Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly 645 650 655 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Cys Lys Ala Phe 660 665 670 Ile Val Thr Asp Glu Asp Ile Arg Lys Gln Glu Glu Arg Val Gln Gln 675 680 685 Val Arg Lys Lys Leu Glu Glu Ala Leu Met Ala Asp Ile Leu Ser Cys 690 695 700 <210> 13 <211> 123 <212> DNA <213> Homo sapiens <400> 13 cccgaagaaa gggagaggat gattaaacag cttaaagaag agctgcgcct tgaggaagcg 60 aaactggtac tgctcaaaaa acttaggcag tcacaaatcc aaaaagaggc gacggcccaa 120 aaa 123 <210> 14 <211> 84 <212> DNA <213> Homo sapiens <400> 14 aagattgcag cactcaagga aaaaatcgca gctctcaagg aaaagaacgc ggcgctcaaa 60 tggaagatcg cggcgctgaa agaa 84 <210> 15 <211> 90 <212> DNA <213> Homo sapiens <400> 15 ggaggctgtg ggctgaagca aaagatagct gccttgaagt ataagaacgc cgcattaaag 60 aaaaaaatcg cagctcttaa acagggcggc 90 <210> 16 <211> 41 <212> PRT <213> Homo sapiens <400> 16 Pro Glu Glu Arg Glu Arg Met Ile Lys Gln Leu Lys Glu Glu Leu Arg 1 5 10 15 Leu Glu Glu Ala Lys Leu Val Leu Leu Lys Lys Leu Arg Gln Ser Gln 20 25 30 Ile Gln Lys Glu Ala Thr Ala Gln Lys 35 40 <210> 17 <211> 28 <212> PRT <213> Homo sapiens <400> 17 Lys Ile Ala Ala Leu Lys Glu Lys Ile Ala Ala Leu Lys Glu Lys Asn 1 5 10 15 Ala Ala Leu Lys Trp Lys Ile Ala Ala Leu Lys Glu 20 25 <210> 18 <211> 27 <212> PRT <213> Homo sapiens <400> 18 Gly Leu Lys Gln Lys Ile Ala Ala Leu Lys Tyr Lys Asn Ala Ala Leu 1 5 10 15 Lys Lys Lys Ile Ala Ala Leu Lys Gln Gly Gly 20 25 <210> 19 <211> 869 <212> DNA <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 19 ggaggcggaa agattgcagc actcaaggaa aaaatcgcag ctctcaagga aaagaacgcg 60 gcgctcaaat ggaagatcgc ggcgctgaaa gaaggaggag gcggcagtgg aggagggggg 120 tcaggaggtg ggggttcaag taaaggagaa gaacttttca ctggagttgt cccaattctt 180 gttgaattag atggtgatgt taatgggcac aaattttctg tccgtggaga gggtgaaggt 240 gatgcaacaa acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt 300 ccatggccaa cacttgtcac tactctgacc tatggtgttc aatgcttctc ccgttatccg 360 gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa 420 cgcactataa gcttcaaaga tgacgggacc tacaagacgc gtgctgaagt caagtttgaa 480 ggtgataccc ttgttaatcg tatcgagtta aaaggtattg attttaaaga agatggaaac 540 attctcggac acaaactcga gtacaacttt aactcacaca atgtatacat caccgcagac 600 aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacgttga agatggatcc 660 gttcaactag cagaccata tcaacaaaat actccaattg gcgatggccc tgtcctttta 720 ccagacaacc attacctgtc gacacaatct gtcctttcga aagatcccaa cgaaaagcgt 780 gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg catggatgag 840 ctctacaaac atcaccatca ccatcactg 869 <210> 20 <211> 894 <212> DNA <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 20 catcaccatc accatcacgt ttcaaaaggc gaagaagaca atatggcaat catcaaagaa 60 tttatgcgtt ttaaagtcca tatggaaggc agcgtgaacg gtcacgaatt cgaaattgaa 120 ggtgaaggcg aaggtcgccc gtatgaaggc acccagacgg caaaactgaa agttaccaaa 180 ggcggtccgc tgccgtttgc ttgggatatt ctgagcccgc aattcatgta tggctctaaa 240 gcgtacgtca aacatccggc cgatatcccg gactatctga aactgagctt tccggaaggt 300 ttcaaatggg aacgtgtgat gaactttgaa gatggcggtg tggttaccgt tacgcaggat 360 agctctctgc aagacggcga atttatttat aaagtcaaac tgcgcggcac caatttcccg 420 tctgatggtc cggtgatgca gaagaaaacc atgggttggg aagcgagttc cgaacgtatg 480 tacccggaag acggcgccct gaaaggtgaa atcaaacagc gcctgaaact gaaagatggc 540 ggtcactatg acgcagaagt gaaaaccacg tacaaagcta aaaaaccggt tcaactgccg 600 ggtgcataca acgtcaacat caaactggat atcaccagtc ataacgaaga ctatacgatc 660 gttgaacagt acgaacgcgc ggaaggccgc cactccaccg gcggtatgga tgaactgtac 720 aaagggggag gcggcagtgg aggagggggg tcaggaggtg ggggtggtcc cgaagaaagg 780 gagaggatga ttaaacagct taaagaagag ctgcgccttg aggaagcgaa actggtactg 840 ctcaaaaaac ttaggcagtc acaaatccaa aaagaggcga cggcccaaaa aggt 894 <210> 21 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 21 Gly Gly Gly Lys Ile Ala Ala Leu Lys Glu Lys Ile Ala Ala Leu Lys 1 5 10 15 Glu Lys Asn Ala Ala Leu Lys Trp Lys Ile Ala Ala Leu Lys Glu Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Ser Lys 35 40 45 Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp 50 55 60 Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly 65 70 75 80 Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly 85 90 95 Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly 100 105 110 Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe 115 120 125 Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser 130 135 140 Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu 145 150 155 160 Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys 165 170 175 Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn Ser 180 185 190 His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala 195 200 205 Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala 210 215 220 Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu 225 230 235 240 Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Val Leu Ser Lys Asp Pro 245 250 255 Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala 260 265 270 Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 275 280 <210> 22 <211> 298 <212> PRT <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 22 His His His His His His Val Ser Lys Gly Glu Glu Asp Asn Met Ala 1 5 10 15 Ile Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val 20 25 30 Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr 35 40 45 Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu 50 55 60 Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys 65 70 75 80 Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser 85 90 95 Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly 100 105 110 Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe 115 120 125 Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro 130 135 140 Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met 145 150 155 160 Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys 165 170 175 Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys 180 185 190 Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys 195 200 205 Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr 210 215 220 Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly 245 250 255 Pro Glu Glu Arg Glu Arg Met Ile Lys Gln Leu Lys Glu Glu Leu Arg 260 265 270 Leu Glu Glu Ala Lys Leu Val Leu Leu Lys Lys Leu Arg Gln Ser Gln 275 280 285 Ile Gln Lys Glu Ala Thr Ala Gln Lys Gly 290 295 <210> 23 <211> 869 <212> DNA <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 23 ggaggctgta agattgcagc actcaaggaa aaaatcgcag ctctcaagga aaagaacgcg 60 gcgctcaaat ggaagatcgc ggcgctgaaa gaatgtggag gcggcagtgg aggagggggg 120 tcaggaggtg ggggttcaag taaaggagaa gaacttttca ctggagttgt cccaattctt 180 gttgaattag atggtgatgt taatgggcac aaattttctg tccgtggaga gggtgaaggt 240 gatgcaacaa acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt 300 ccatggccaa cacttgtcac tactctgacc tatggtgttc aatgcttctc ccgttatccg 360 gatcatatga aacggcatga ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa 420 cgcactataa gcttcaaaga tgacgggacc tacaagacgc gtgctgaagt caagtttgaa 480 ggtgataccc ttgttaatcg tatcgagtta aaaggtattg attttaaaga agatggaaac 540 attctcggac acaaactcga gtacaacttt aactcacaca atgtatacat caccgcagac 600 aaacaaaaga atggaatcaa agctaacttc aaaattcgcc acaacgttga agatggatcc 660 gttcaactag cagaccata tcaacaaaat actccaattg gcgatggccc tgtcctttta 720 ccagacaacc attacctgtc gacacaatct gtcctttcga aagatcccaa cgaaaagcgt 780 gaccacatgg tccttcttga gtttgtaact gctgctggga ttacacatgg catggatgag 840 ctctacaaac atcaccatca ccatcactg 869 <210> 24 <211> 894 <212> DNA <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 24 catcaccatc accatcacgt ttcaaaaggc gaagaagaca atatggcaat catcaaagaa 60 tttatgcgtt ttaaagtcca tatggaaggc agcgtgaacg gtcacgaatt cgaaattgaa 120 ggtgaaggcg aaggtcgccc gtatgaaggc acccagacgg caaaactgaa agttaccaaa 180 ggcggtccgc tgccgtttgc ttgggatatt ctgagcccgc aattcatgta tggctctaaa 240 gcgtacgtca aacatccggc cgatatcccg gactatctga aactgagctt tccggaaggt 300 ttcaaatggg aacgtgtgat gaactttgaa gatggcggtg tggttaccgt tacgcaggat 360 agctctctgc aagacggcga atttatttat aaagtcaaac tgcgcggcac caatttcccg 420 tctgatggtc cggtgatgca gaagaaaacc atgggttggg aagcgagttc cgaacgtatg 480 tacccggaag acggcgccct gaaaggtgaa atcaaacagc gcctgaaact gaaagatggc 540 ggtcactatg acgcagaagt gaaaaccacg tacaaagcta aaaaaccggt tcaactgccg 600 ggtgcataca acgtcaacat caaactggat atcaccagtc ataacgaaga ctatacgatc 660 gttgaacagt acgaacgcgc ggaaggccgc cactccaccg gcggtatgga tgaactgtac 720 aaagggggag gcggcagtgg aggagggggg tcaggaggtg ggggttgtcc cgaagaaagg 780 gagaggatga ttaaacagct taaagaagag ctgcgccttg aggaagcgaa actggtactg 840 ctcaaaaaac ttaggcagtc acaaatccaa aaagaggcga cggcccaaaa atgt 894 <210> 25 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 25 Gly Gly Cys Lys Ile Ala Ala Leu Lys Glu Lys Ile Ala Ala Leu Lys 1 5 10 15 Glu Lys Asn Ala Ala Leu Lys Trp Lys Ile Ala Ala Leu Lys Glu Cys 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Ser Lys 35 40 45 Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp 50 55 60 Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu Gly 65 70 75 80 Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly 85 90 95 Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly 100 105 110 Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe 115 120 125 Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Ser 130 135 140 Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu 145 150 155 160 Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys 165 170 175 Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn Ser 180 185 190 His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala 195 200 205 Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu Ala 210 215 220 Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu 225 230 235 240 Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Val Leu Ser Lys Asp Pro 245 250 255 Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala 260 265 270 Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys 275 280 <210> 26 <211> 298 <212> PRT <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 26 His His His His His His Val Ser Lys Gly Glu Glu Asp Asn Met Ala 1 5 10 15 Ile Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val 20 25 30 Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr 35 40 45 Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu 50 55 60 Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys 65 70 75 80 Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser 85 90 95 Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly 100 105 110 Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe 115 120 125 Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro 130 135 140 Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met 145 150 155 160 Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys 165 170 175 Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys 180 185 190 Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys 195 200 205 Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr 210 215 220 Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr 225 230 235 240 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Cys 245 250 255 Pro Glu Glu Arg Glu Arg Met Ile Lys Gln Leu Lys Glu Glu Leu Arg 260 265 270 Leu Glu Glu Ala Lys Leu Val Leu Leu Lys Lys Leu Arg Gln Ser Gln 275 280 285 Ile Gln Lys Glu Ala Thr Ala Gln Lys Cys 290 295 <210> 27 <211> 708 <212> DNA <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 27 atgaagtggg ttaccttcat ctcccttttg tttctgttta gcagtgcgta cagcagggga 60 gtattcagac gcctcgactc ccctgatcga ccctggaacc cacctacttt ttctccagct 120 ctgctggttg ttaccgaagg agataatgcc actttcactt gctcatttag caataccagt 180 gagagcttcg ttctgaactg gtacagaatg agtccctcta atcaaacaga taagttggct 240 gcatttccag aagatcgttc acaaccaggt caggactgca ggtttcgcgt aacccagctc 300 cctaatggac gagactttca tatgagtgtc gttagggcac gccgaaacga tagcgggacc 360 tacctctgtg gagctatttc tcttgctccc aaagcacaaa ttaaagaatc cctgagggct 420 gaactgagag tcaccgagag aagggctgag gtgcctacag cccacccctc cccaagccca 480 cgccccgctg gacaatttca aggaggtggt ggcagcggcg gcggaggttc tggcggcgga 540 ggatgtcccg aagaaaggga gaggatgatt aaacagctta aagaagagct gcgccttgag 600 gaagcgaaac tggtactgct caaaaaactt aggcagtcac aaatccaaaa agaggcgacg 660 gcccaaaaat gttgtggggg gggcgactac aaagacgatg acgacaag 708 <210> 28 <211> 235 <212> PRT <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 28 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly 165 170 175 Ser Gly Gly Gly Gly Cys Pro Glu Glu Arg Glu Arg Met Ile Lys Gln 180 185 190 Leu Lys Glu Glu Leu Arg Leu Glu Glu Ala Lys Leu Val Leu Leu Lys 195 200 205 Lys Leu Arg Gln Ser Gln Ile Gln Lys Glu Ala Thr Ala Gln Lys Cys 210 215 220 Gly Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys 225 230 235 <210> 29 <211> 867 <212> DNA <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 29 atgaagtggg ttaccttcat ctcccttttg tttctgttta gcagtgcgta cagcagggga 60 gtattcagac gcgtaataca tgtaacaaaa gaggtcaaag aagtagcaac acttagttgt 120 ggccacaacg tatctgttga agaactggcc cagacccgga tttactggca aaaagaaaag 180 aagatggtgc tgactatgat gagcggtgac atgaacatat ggcccgaata taagaaccgg 240 accatctttg acataaccaa caacctttct attgttatct tggcactgcg gccttctgat 300 gagggcactt acgaatgtgt cgttctgaag tatgagaaag atgcatttaa gcgtgagcat 360 ctggccgaag taactttgtc tgtgaaagcc gattttccta ctccctctat tagcgatttt 420 gaaattccca cttctaacat cagacgcata atatgctcta ccagcggcgg atttcctgag 480 ccacaccttt catggctcga aaatggggaa gaattgaacg ctatcaatac tactgtttcc 540 caagatccag aaacagaatt gtacgccgta agtagcaagc tcgatttcaa tatgacaact 600 aatcacagct ttatgtgtct tattaaatat ggacatctgc gggtcaacca aaccttcaac 660 tggaacacaa ctaagcagga acattttcca gataacggcg gtggggggtc cggtggggga 720 ggttctggag gagggggctg tggaggctgt gggctgaagc aaaagatagc tgccttgaag 780 tataagaacg ccgcattaaa gaaaaaaatc gcagctctta aacagggcgg ctgtggagga 840 ggacaccatc atcaccacca tcaccac 867 <210> 30 <211> 286 <212> PRT <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 30 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Val Ile His Val Thr Lys Glu Val 20 25 30 Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu 35 40 45 Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu 50 55 60 Thr Met Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg 65 70 75 80 Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu 85 90 95 Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu 100 105 110 Lys Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val 115 120 125 Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr 130 135 140 Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu 145 150 155 160 Pro His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn 165 170 175 Thr Thr Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser 180 185 190 Lys Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile 195 200 205 Lys Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr Thr 210 215 220 Lys Gln Glu His Phe Pro Asp Asn Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Gly Cys Gly Leu Lys Gln Lys Ile Ala Ala Leu 245 250 255 Lys Tyr Lys Asn Ala Ala Leu Lys Lys Lys Ile Ala Ala Leu Lys Gln 260 265 270 Gly Gly Cys Gly Gly Gly His His His His His His His His 275 280 285 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 31 ttcagacgcg gaggctgtg 19 <210> 32 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 32 ggatcgaagg atccttaaca actcaatatg tca 33 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 33 gggagtattc agacgcggag g 21 <210> 34 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 34 ggatcgaagg atccttaaca actcaatatg tca 33 <210> 35 <211> 1755 <212> DNA <213> Homo sapiens <400> 35 gacgcccaca agagcgaggt agctcaccgc ttcaaagacc tgggggagga aaactttaag 60 gcactcgtgc ttatcgcttt cgcgcagtat ttgcagcagt gtcctttcga ggaccatgtg 120 aaattggtaa atgaagtaac tgagtttgca aagacgtgtg tcgcggacga atccgccgaa 180 aactgtgaca agtccttgca cacgcttttc ggggataaat tgtgtaccgt cgcaaccctc 240 cgcgaaacat acggagaaat ggcggattgc tgtgccaaac aagaaccgga gcgcaacgag 300 tgctttctcc aacataagga cgataatcca aatctccccc gcctggtccg cccggaagtc 360 gatgtcatgt gtacagcatt ccatgataat gaagaaacct ttctgaaaaa gtatttgtat 420 gaaatagcca gaaggcaccc gtacttttac gccccagaac tgctgttttt tgcgaagagg 480 tacaaggccg cgtttaccga gtgctgtcag gccgcggaca aagcggcctg cctgctccct 540 aaattggacg agttgaggga tgaagggaag gcttcttctg ctaaacagcg cctcaagtgc 600 gcgtctttgc agaagttcgg agagagagcc tttaaagcat gggctgttgc tcgcctgtca 660 cagaggtttc caaaagcaga gtttgctgaa gtcagcaaac tggtcacaga ccttaccaaa 720 gtacatacgg aatgctgtca tggtgatctt ttggaatgcg cagatgaccg agccgacctt 780 gcgaagtata tctgtgaaaa tcaggacagt atttcctcaa aattgaagga gtgttgcgag 840 aaaccacttc tcgagaagag tcactgcatc gcagaagttg agaatgacga gatgccggcg 900 gacttgccta gcctggctgc tgattttgtt gaaagtaagg acgtgtgcaa gaactacgct 960 gaagctaaag acgtctttct tgggatgttc ctgtatgagt acgcacgaag acatcctgat 1020 tactccgttg tactgttgct tagactggca aaaacgtatg agaccacctt ggagaaatgt 1080 tgtgcggcgg cagaccccca tgaatgttac gccaaagtat tcgacgagtt taagcccctt 1140 gtagaagagc ctcagaacct gataaagcaa aactgtgagc ttttcgagca gctgggggaa 1200 tataaattcc aaaacgctct tctcgtgcgc tacactaaga aagttccgca ggtttccacc 1260 cccacccttg tagaagtgtc taggaacctt ggtaaagttg gcagcaaatg ctgtaaacat 1320 cctgaagcaa agcggatgcc gtgcgccgag gactatcttt ctgtagttct gaatcagctc 1380 tgtgttctcc acgaaaaaac gccggtaagt gatcgcgtta caaaatgctg tacagagagc 1440 ctggtcaacc ggcgaccctg tttctccgct cttgaagttg acgagactta cgtcccgaaa 1500 gaattcaacg ccgaaacatt cacatttcac gccgatatct gtaccttgtc tgaaaaggaa 1560 cgccaaatta aaaagcaaac cgccctggtg gagctggtta aacacaagcc gaaggcaact 1620 aaagagcagc tgaaggctgt tatggacgat ttcgctgcgt tcgtggagaa atgttgcaag 1680 gccgacgaca aagagacgtg tttcgcagag gaaggaaaga aacttgtggc tgcttctcaa 1740 gccgcacttg gactg 1755 <210> 36 <211> 585 <212> PRT <213> Homo sapiens <400> 36 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 <210> 37 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 37 atgttcgtca accaacacct ttgtggtagt catttggtcg aggccctcta ccttgtgtgc 60 ggggagcggg gcttctttta tacgccgaag acccaacgcg gcggtggttc aggtggtgga 120 cagaaaagag gcattgtaga gcagtgctgc acctccatat gctcccttta tcagctcgaa 180 aactactgta acggcggtgg ggggtccggt gggggaggtt ctggaggagg gggctgtggg 240 ctgaagcaaa agatagctgc cttgaagtat aagaacgccg cattaaagaa aaaaatcgca 300 gctcttaaac agggcggctg tcatcaccat caccatcac 339 <210> 38 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Target substance and coiled coil forming domain <400> 38 Met Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu 1 5 10 15 Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Gln 20 25 30 Arg Gly Gly Gly Ser Gly Gly Gly Gln Lys Arg Gly Ile Val Glu Gln 35 40 45 Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn 50 55 60 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Cys Gly 65 70 75 80 Leu Lys Gln Lys Ile Ala Ala Leu Lys Tyr Lys Asn Ala Ala Leu Lys 85 90 95 Lys Lys Ile Ala Ala Leu Lys Gin Gly Gly Cys His His His His His 100 105 110 His <210> 39 <211> 105 <212> DNA <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_CC <400> 39 aaggctttca ttgtcacgga tgaggacatt agaaaacaag aagaaagagt tcagcaggtg 60 aggaaaaagt tggaagaagc attgatggct gacatattga gtggg 105 <210> 40 <211> 132 <212> DNA <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_DC <400> 40 tgtgaaattg cggcactcga aaaagagatt gccgcgcttg aaaaagagaa tgctgcactt 60 gaatgggaga tagccgcatt ggaaaaatgt ggtgggggaa gtggtggtgg tgggagtgga 120 ggcggtggat ca 132 <210> 41 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_KC <400> 41 tgtggacttg agcaagagat tgcggcgctg gaaaaggaga acgcagcctt ggaatgggaa 60 attgccgcac ttgaacaggg cggatgtggt gggggaagtg gtggtggtgg gagtggaggc 120 ggtggatca 129 <210> 42 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_CC <400> 42 Cys Lys Ala Phe Ile Val Thr Asp Glu Asp Ile Arg Lys Gln Glu Glu 1 5 10 15 Arg Val Gln Gln Val Arg Lys Lys Leu Glu Glu Ala Leu Met Ala Asp 20 25 30 Ile Leu Ser Cys 35 <210> 43 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_DC <400> 43 Cys Glu Ile Ala Ala Leu Glu Lys Glu Ile Ala Ala Leu Glu Lys Glu 1 5 10 15 Asn Ala Ala Leu Glu Trp Glu Ile Ala Ala Leu Glu Lys Cys 20 25 30 <210> 44 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_KC <400> 44 Cys Gly Leu Glu Gln Glu Ile Ala Ala Leu Glu Lys Glu Asn Ala Ala 1 5 10 15 Leu Glu Trp Glu Ile Ala Ala Leu Glu Gln Gly Gly Cys 20 25 <210> 45 <211> 129 <212> DNA <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_CC_ <400> 45 tgtcccgaag aaagggagag gatgattaaa cagcttaaag aagagctgcg ccttgaggaa 60 gcgaaactgg tactgctcaa aaaacttagg cagtcacaaa tccaaaaaga ggcgacggcc 120 caaaaatgt 129 <210> 46 <211> 90 <212> DNA <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_DC_ <400> 46 tgtaagattg cagcactcaa ggaaaaaatc gcagctctca aggaaaagaa cgcggcgctc 60 aaatggaaga tcgcggcgct gaaagaatgt 90 <210> 47 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_KC_ <400> 47 tgtggaggct gtgggctgaa gcaaaagata gctgccttga agtataagaa cgccgcatta 60 aagaaaaaaa tcgcagctct taaacagggc ggctgt 96 <210> 48 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_CC_ <400> 48 Cys Pro Glu Glu Arg Glu Arg Met Ile Lys Gln Leu Lys Glu Glu Leu 1 5 10 15 Arg Leu Glu Glu Ala Lys Leu Val Leu Leu Lys Lys Leu Arg Gln Ser 20 25 30 Gln Ile Gln Lys Glu Ala Thr Ala Gln Lys Cys 35 40 <210> 49 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_DC_ <400> 49 Cys Lys Ile Ala Ala Leu Lys Glu Lys Ile Ala Ala Leu Lys Glu Lys 1 5 10 15 Asn Ala Ala Leu Lys Trp Lys Ile Ala Ala Leu Lys Glu Cys 20 25 30 <210> 50 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> COILED COIL FORMING DOMAIN_KC_ <400> 50 Cys Gly Leu Lys Gln Lys Ile Ala Ala Leu Lys Tyr Lys Asn Ala Ala 1 5 10 15 Leu Lys Lys Lys Ile Ala Ala Leu Lys Gln Gly Gly Cys 20 25 <210> 51 <211> 84 <212> PRT <213> Artificial Sequence <220> <223> GLP-1-CC_ <400> 51 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly 20 25 30 Gly Ser Gly Gly Gly Ser Gly Gly Gly Cys Pro Glu Glu Arg Glu Arg 35 40 45 Met Ile Lys Gln Leu Lys Glu Glu Leu Arg Leu Glu Glu Ala Lys Leu 50 55 60 Val Leu Leu Lys Lys Leu Arg Gln Ser Gln Ile Gln Lys Glu Ala Thr 65 70 75 80 Ala Gln Lys Cys <210> 52 <211> 2100 <212> DNA <213> Artificial Sequence <220> <223> K-HSA-C <400> 52 atgaagtggg ttaccttcat ctcccttttg tttctgttta gcagtgcgta cagcagggga 60 gtattcagac gcggaggcgg acttgagcaa gagattgcgg cgctggaaaa ggagaacgca 120 gccttggaat gggaaattgc cgcacttgaa cagggcggag gtgggggaag tggtggtggt 180 gggagtggag gcggtggatc agacgcccac aagagcgagg tagctcaccg cttcaaagac 240 ctgggggagg aaaactttaa ggcactcgtg cttatcgctt tcgcgcagta tttgcagcag 300 tgtcctttcg aggaccatgt gaaattggta aatgaagtaa ctgagtttgc aaagacgtgt 360 gtcgcggacg aatccgccga aaactgtgac aagtccttgc acacgctttt cggggataaa 420 ttgtgtaccg tcgcaaccct ccgcgaaaca tacggagaaa tggcggattg ctgtgccaaa 480 caagaaccgg agcgcaacga gtgctttctc caacataagg acgataatcc aaatctcccc 540 cgcctggtcc gcccggaagt cgatgtcatg tgtacagcat tccatgataa tgaagaaacc 600 tttctgaaaa agtatttgta tgaaatagcc agaaggcacc cgtactttta cgccccagaa 660 ctgctgtttt ttgcgaagag gtacaaggcc gcgtttaccg agtgctgtca ggccgcggac 720 aaagcggcct gcctgctccc taaattggac gagttgaggg atgaagggaa ggcttcttct 780 gctaaacagc gcctcaagtg cgcgtctttg cagaagttcg gagagagagc ctttaaagca 840 tgggctgttg ctcgcctgtc acagaggttt ccaaaagcag agtttgctga agtcagcaaa 900 ctggtcacag accttaccaa agtacatacg gaatgctgtc atggtgatct tttggaatgc 960 gcagatgacc gagccgacct tgcgaagtat atctgtgaaa atcaggacag tatttcctca 1020 aaattgaagg agtgttgcga gaaaccactt ctcgagaaga gtcactgcat cgcagaagtt 1080 gagaatgacg agatgccggc ggacttgcct agcctggctg ctgattttgt tgaaagtaag 1140 gacgtgtgca agaactacgc tgaagctaaa gacgtctttc ttgggatgtt cctgtatgag 1200 tacgcacgaa gacatcctga ttactccgtt gtactgttgc ttagactggc aaaaacgtat 1260 gagaccacct tggagaaatg ttgtgcggcg gcagaccccc atgaatgtta cgccaaagta 1320 ttcgacgagt ttaagcccct tgtagaagag cctcagaacc tgataaagca aaactgtgag 1380 cttttcgagc agctggggga atataaattc caaaacgctc ttctcgtgcg ctacactaag 1440 aaagttccgc aggtttccac ccccaccctt gtagaagtgt ctaggaacct tggtaaagtt 1500 ggcagcaaat gctgtaaaca tcctgaagca aagcggatgc cgtgcgccga ggactatctt 1560 tctgtagttc tgaatcagct ctgtgttctc cacgaaaaaa cgccggtaag tgatcgcgtt 1620 acaaaatgct gtacagagag cctggtcaac cggcgaccct gtttctccgc tcttgaagtt 1680 gacgagactt acgtcccgaa agaattcaac gccgaaacat tcacatttca cgccgatatc 1740 tgtaccttgt ctgaaaagga acgccaaatt aaaaagcaaa ccgccctggt ggagctggtt 1800 aaacacaagc cgaaggcaac taaagagcag ctgaaggctg ttatggacga tttcgctgcg 1860 ttcgtggaga aatgttgcaa ggccgacgac aaagagacgt gtttcgcaga ggaaggaaag 1920 aaacttgtgg ctgcttctca agccgcactt ggactggggg gaggcggcag tggaggaggg 1980 gggtcaggag gtgggggtaa ggctttcatt gtcacggatg aggacattag aaaacaagaa 2040 gaaagagttc agcaggtgag gaaaaagttg gaagaagcat tgatggctga catattgagt 2100 2100 <210> 53 <211> 700 <212> PRT <213> Artificial Sequence <220> <223> K-HSA-C <400> 53 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Gly Gly Gly Leu Glu Gln Glu Ile 20 25 30 Ala Ala Leu Glu Lys Glu Asn Ala Ala Leu Glu Trp Glu Ile Ala Ala 35 40 45 Leu Glu Gln Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 50 55 60 Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp 65 70 75 80 Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln 85 90 95 Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu 100 105 110 Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn 115 120 125 Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val 130 135 140 Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys 145 150 155 160 Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn 165 170 175 Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr 180 185 190 Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu 195 200 205 Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe 210 215 220 Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp 225 230 235 240 Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly 245 250 255 Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys 260 265 270 Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln 275 280 285 Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp 290 295 300 Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys 305 310 315 320 Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp 325 330 335 Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu 340 345 350 Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp 355 360 365 Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys 370 375 380 Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu 385 390 395 400 Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu 405 410 415 Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp 420 425 430 Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val 435 440 445 Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln 450 455 460 Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys 465 470 475 480 Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn 485 490 495 Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg 500 505 510 Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys 515 520 525 Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys 530 535 540 Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val 545 550 555 560 Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe 565 570 575 His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys 580 585 590 Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys 595 600 605 Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys 610 615 620 Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys 625 630 635 640 Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Gly Gly Gly 645 650 655 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Lys Ala Phe Ile Val Thr 660 665 670 Asp Glu Asp Ile Arg Lys Gln Glu Glu Arg Val Gln Gln Val Arg Lys 675 680 685 Lys Leu Glu Glu Ala Leu Met Ala Asp Ile Leu Ser 690 695 700 <210> 54 <211> 2970 <212> DNA <213> Artificial Sequence <220> <223> PD-1_HSA_CD80 <400> 54 atgaagtggg ttaccttcat ctcccttttg tttctgttta gcagtgcgta cagcagggga 60 gtattcagac gcctcgactc ccctgatcga ccctggaacc cacctacttt ttctccagct 120 ctgctggttg ttaccgaagg agataatgcc actttcactt gctcatttag caataccagt 180 gagagcttcg ttctgaactg gtacagaatg agtccctcta atcaaacaga taagttggct 240 gcatttccag aagatcgttc acaaccaggt caggactgca ggtttcgcgt aacccagctc 300 cctaatggac gagactttca tatgagtgtc gttagggcac gccgaaacga tagcgggacc 360 tacctctgtg gagctatttc tcttgctccc aaagcacaaa ttaaagaatc cctgagggct 420 gaactgagag tcaccgagag aagggctgag gtgcctacag cccacccctc cccaagccca 480 cgccccgctg gacaatttca agggggtggg ggaagcggtg gtggtgggag tggaggcggt 540 ggatcagacg cccacaagag cgaggtagct caccgcttca aagacctggg ggaggaaaac 600 tttaaggcac tcgtgcttat cgctttcgcg cagtatttgc agcagtgtcc tttcgaggac 660 catgtgaaat tggtaaatga agtaactgag tttgcaaaga cgtgtgtcgc ggacgaatcc 720 gccgaaaact gtgacaagtc cttgcacacg cttttcgggg ataaattgtg taccgtcgca 780 accctccgcg aaacatacgg agaaatggcg gattgctgtg ccaaacaaga accggagcgc 840 aacgagtgct ttctccaaca taaggacgat aatccaaatc tccccccgcct ggtccgcccg 900 gaagtcgatg tcatgtgtac agcattccat gataatgaag aaacctttct gaaaaagtat 960 ttgtatgaaa tagccagaag gcacccgtac ttttacgccc cagaactgct gttttttgcg 1020 aagaggtaca aggccgcgtt taccgagtgc tgtcaggccg cggacaaagc ggcctgcctg 1080 ctccctaaat tggacgagtt gagggatgaa gggaaggctt cttctgctaa acagcgcctc 1140 aagtgcgcgt ctttgcagaa gttcggagag agagccttta aagcatgggc tgttgctcgc 1200 ctgtcacaga ggtttccaaa agcagagttt gctgaagtca gcaaactggt cacagacctt 1260 accaaagtac atacggaatg ctgtcatggt gatcttttgg aatgcgcaga tgaccgagcc 1320 gaccttgcga agtatatctg tgaaaatcag gacagtattt cctcaaaatt gaaggagtgt 1380 tgcgagaaac cacttctcga gaagagtcac tgcatcgcag aagttgagaa tgacgagatg 1440 ccggcggact tgcctagcct ggctgctgat tttgttgaaa gtaaggacgt gtgcaagaac 1500 tacgctgaag ctaaagacgt ctttcttggg atgttcctgt atgagtacgc acgaagacat 1560 cctgattact ccgttgtact gttgcttaga ctggcaaaaa cgtatgagac caccttggag 1620 aaatgttgtg cggcggcaga cccccatgaa tgttacgcca aagtattcga cgagtttaag 1680 ccccttgtag aagagcctca gaacctgata aagcaaaact gtgagctttt cgagcagctg 1740 ggggaatata aattccaaaa cgctcttctc gtgcgctaca ctaagaaagt tccgcaggtt 1800 tccaccccca cccttgtaga agtgtctagg aaccttggta aagttggcag caaatgctgt 1860 aaacatcctg aagcaaagcg gatgccgtgc gccgaggact atctttctgt agttctgaat 1920 cagctctgtg ttctccacga aaaaacgccg gtaagtgatc gcgttacaaa atgctgtaca 1980 gagagcctgg tcaaccggcg accctgtttc tccgctcttg aagttgacga gacttacgtc 2040 ccgaaagaat tcaacgccga aacattcaca tttcacgccg atatctgtac cttgtctgaa 2100 aaggaacgcc aaattaaaaa gcaaaccgcc ctggtggagc tggttaaaca caagccgaag 2160 gcaactaaag agcagctgaa ggctgttatg gacgatttcg ctgcgttcgt ggagaaatgt 2220 tgcaaggccg acgacaaaga gacgtgtttc gcagaggaag gaaagaaact tgtggctgct 2280 tctcaagccg cacttggact ggggggaggc ggcagtggag gaggggggtc aggaggtggg 2340 ggttcagtaa tacatgtaac aaaagaggtc aaagaagtag caacacttag ttgtggccac 2400 aacgtatctg ttgaagaact ggcccagacc cggatttact ggcaaaaaga aaagaagatg 2460 gtgctgacta tgatgagcgg tgacatgaac atatggcccg aatataagaa ccggaccatc 2520 tttgacataa ccaacaacct ttctattgtt atcttggcac tgcggccttc tgatgagggc 2580 acttacgaat gtgtcgttct gaagtatgag aaagatgcat ttaagcgtga gcatctggcc 2640 gaagtaactt tgtctgtgaa agccgatttt cctactccct ctattagcga ttttgaaatt 2700 cccacttcta acatcagacg cataatatgc tctaccagcg gcggatttcc tgagccacac 2760 ctttcatggc tcgaaaatgg ggaagaattg aacgctatca atactactgt ttcccaagat 2820 ccagaaacag aattgtacgc cgtaagtagc aagctcgatt tcaatatgac aactaatcac 2880 agctttatgt gtcttattaa atatggacat ctgcgggtca accaaacctt caactggaac 2940 acaactaagc aggaacattt tccagataac 2970 <210> 55 <211> 990 <212> PRT <213> Artificial Sequence <220> <223> PD-1_HSA_CD80 <400> 55 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Gly Gly Gly Gly Ser Gly Gly Gly Gly 165 170 175 Ser Gly Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg 180 185 190 Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala 195 200 205 Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 210 215 220 Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 225 230 235 240 Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 245 250 255 Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 260 265 270 Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 275 280 285 Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 290 295 300 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr 305 310 315 320 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu 325 330 335 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln 340 345 350 Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg 355 360 365 Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 370 375 380 Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 385 390 395 400 Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 405 410 415 Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu 420 425 430 Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu 435 440 445 Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 450 455 460 Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met 465 470 475 480 Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp 485 490 495 Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 500 505 510 Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 515 520 525 Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 530 535 540 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys 545 550 555 560 Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 565 570 575 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg 580 585 590 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 595 600 605 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 610 615 620 Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 625 630 635 640 Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 645 650 655 Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 660 665 670 Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr 675 680 685 Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 690 695 700 Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys 705 710 715 720 Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 725 730 735 Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 740 745 750 Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly 755 760 765 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Val Ile 770 775 780 His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys Gly His 785 790 795 800 Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys 805 810 815 Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp 820 825 830 Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser 835 840 845 Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys 850 855 860 Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala 865 870 875 880 Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser 885 890 895 Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr 900 905 910 Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu 915 920 925 Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu Thr Glu 930 935 940 Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His 945 950 955 960 Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr 965 970 975 Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn 980 985 990 <210> 56 <211> 2220 <212> DNA <213> Artificial Sequence <220> <223> Insulin_HSA_GLP-1 <400> 56 atggcgctgt ggatgaggct gctccctctt ctcgccctct tggctctctg ggggcctgac 60 ccagccgcgg catttgtaaa ccagcatctg tgtggctccc acctcgtaga agcgctgtat 120 ttggtctgtg gggaaagggg gttcttctat acccctaaga cacagcgggg tgggggctct 180 ggcggtggtc agaagagggg tatcgtggaa caatgttgca cttcaatctg ctccctttac 240 cagctggaga attattgcaa tggtggcggt ggtagcggtg gggggggttc tgggggagga 300 ggatctgatg cacacaaaag tgaagttgcg caccgcttta aagacctggg agaagagaac 360 tttaaggcgc tcgttttgat agcgttcgcg caatatcttc agcaatgtcc ctttgaagac 420 cacgtgaagc tcgtcaacga ggtgaccgag ttcgcaaaga catgtgtcgc agacgaatcc 480 gcggagaact gtgacaaatc attgcatact ctttttggcg acaaactctg tacagtggca 540 actttgcgcg aaacttacgg agaaatggct gactgttgtg ctaagcagga accagagcgc 600 aacgaatgct ttttgcaaca caaggatgat aatcccaacc tcccgaggct cgtaagaccc 660 gaagtggacg tcatgtgcac ggcgtttcac gacaatgagg agaccttttt gaaaaagtat 720 ttgtatgaga tagcacgaag gcatccatac ttttacgcac ccgagctgct ctttttcgct 780 aaacggtata aggccgcgtt taccgagtgt tgtcaggcag ccgacaaagc ggcttgtctc 840 cttcctaagc tcgacgagct gagagacgaa gggaaggcaa gtagcgccaa gcagcgcctt 900 aagtgcgctt cattgcaaaa gtttggggaa agggccttta aagcttgggc tgtggcacgc 960 ttgtcacaaa ggttccctaa agcggagttc gctgaagtaa gtaagttggt aacagatctt 1020 accaaagttc atactgaatg ctgtcatgga gatctgctcg aatgcgctga cgaccgggcc 1080 gatttggcta agtacatttg cgaaaatcaa gatagtattt catcaaaact gaaagaatgt 1140 tgcgagaagc cactcctcga gaaatctcac tgcatagcgg aagtggaaaa cgacgaaatg 1200 cctgctgacc ttccgagctt ggcagctgac ttcgtcgaaa gtaaagatgt ctgtaaaaat 1260 tatgctgagg caaaggatgt atttttgggc atgtttctct acgagtatgc caggcgacac 1320 ccagattata gcgtagttct cctgttgcgg ctggctaaga cttacgaaac gactttggag 1380 aagtgttgcg ccgcagctga ccctcatgag tgttatgcca aagtcttcga tgagtttaaa 1440 cctcttgtgg aggaaccaca gaatcttatt aagcagaatt gtgagctttt tgagcaattg 1500 ggggagtaca aatttcagaa cgcgctgctg gtgcggtaca cgaaaaaggt tccgcaggtg 1560 tcaaccccta cccttgtgga ggtctctcgg aatctcggga aagtaggatc caaatgctgt 1620 aagcatccgg aagctaaacg gatgccctgt gccgaagact atctttcagt tgtcttgaat 1680 cagctttgcg ttttgcatga aaagacccct gtaagcgatc gcgttaccaa gtgttgtaca 1740 gaatccctgg taaaccgacg gccgtgtttc tcagcccttg aagtcgacga aacctacgtt 1800 ccaaaggaat ttaatgccga gacatttacg ttccatgcag acatatgtac gctctccgaa 1860 aaggagagac aaattaaaaa gcaaaccgct ttggtggaac tggtaaagca taagccgaag 1920 gcgaccaagg agcaactgaa ggcggttatg gatgactttg ctgcatttgt ggaaaagtgt 1980 tgtaaggccg atgacaagga aacatgcttt gctgaagagg gcaaaaagct tgtagctgct 2040 tctcaggccg cattgggact cggaggggga ggctcaggtg gcggaggtag tggaggtgga 2100 ggcagtcatg gcgagggcac ttttacttct gatgtgtcta gttacttgga agggcaggcg 2160 gcgaaggaat ttattgcatg gctcgtaaaa ggccgccacc accaccatca ccatcatcat 2220 2220 <210> 57 <211> 740 <212> PRT <213> Artificial Sequence <220> <223> Insulin_HSA_GLP-1 <400> 57 Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 1 5 10 15 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly 20 25 30 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe 35 40 45 Phe Tyr Thr Pro Lys Thr Gln Arg Gly Gly Gly Ser Gly Gly Gly Gln 50 55 60 Lys Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr 65 70 75 80 Gln Leu Glu Asn Tyr Cys Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly 85 90 95 Ser Gly Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg 100 105 110 Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala 115 120 125 Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 130 135 140 Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 145 150 155 160 Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu 165 170 175 Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys 180 185 190 Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 195 200 205 Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 210 215 220 Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr 225 230 235 240 Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu 245 250 255 Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln 260 265 270 Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg 275 280 285 Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 290 295 300 Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 305 310 315 320 Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu 325 330 335 Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu 340 345 350 Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu 355 360 365 Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 370 375 380 Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met 385 390 395 400 Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp 405 410 415 Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 420 425 430 Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 435 440 445 Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 450 455 460 Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys 465 470 475 480 Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu 485 490 495 Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg 500 505 510 Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 515 520 525 Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 530 535 540 Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn 545 550 555 560 Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr 565 570 575 Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 580 585 590 Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr 595 600 605 Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 610 615 620 Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys 625 630 635 640 Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe 645 650 655 Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 660 665 670 Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly 675 680 685 Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser His Gly 690 695 700 Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala 705 710 715 720 Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg His His His His 725 730 735 His His His His 740

Claims (36)

Albumin or an analog thereof, a first coiled coil-forming domain and a second coiled coil-forming domain are linked, a fusion protein. The fusion protein according to claim 1, wherein the albumin or its analog comprises any one or more of domains I and III of albumin. The fusion protein according to claim 1, wherein the albumin or its analog comprises an FcRn-binding fragment. The method of claim 1, wherein the first and second coiled coil forming domains are each an elastin-like polypeptide, BECN domain, bZIP VBP, keratin intermediate fiber, AP1 (Jun/Fos), MBD2-NuRD domain, GABA receptor, zinc Any one selected from the group consisting of a tong domain, a leucine zipper, and a variant thereof, a fusion protein. The fusion protein according to claim 1, wherein the first and second coiled coil-forming domains cannot form a coiled coil through mutual binding. The fusion protein according to claim 1, wherein the first and second coiled coil-forming domains have different amino acid sequences from each other. The method according to claim 1, wherein the first and second coiled coil-forming domains each comprise any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 4, 5, 6, 16, 17 and 18. that is, a fusion protein. According to claim 1, wherein said albumin or an analog thereof; and a coiled coil forming domain; Which further comprises a linker between, the fusion protein. The fusion protein according to claim 1, wherein the fusion protein comprises any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 8, 53, 11 and 12. A nucleic acid molecule encoding the fusion protein of claim 1 or 8. A vector comprising the nucleic acid molecule of claim 10 . A host cell into which a vector containing a nucleic acid molecule encoding the fusion protein of claim 1 is introduced. A host cell into which a vector containing a nucleic acid molecule encoding the fusion protein of claim 8 is introduced. albumin or an analog thereof, a fusion protein in which a first coiled coil-forming domain and a second coiled coil-forming domain are linked; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second fusion, wherein the second target binding substance and the second 'coiled coil-forming domain are linked, wherein the first and second fusions are each bound to the fusion protein. 15. The method of claim 14, wherein the first, second, first' and second coiled coil forming domains are each an elastin-like polypeptide, BECN domain, bZIP VBP, keratin intermediate fiber, AP1 (Jun/Fos), MBD2 -NuRD domain, GABA receptor, zinc clamp domain, leucine zipper, and any one selected from the group consisting of variants thereof, the dual target binder. The method according to claim 14, wherein the first and second coiled coil-forming domains cannot form a coiled coil through mutual coupling, and the first 'and second' coiled coil-forming domains cannot form a coiled coil through mutual coupling. A dual target conjugate that cannot form a yield coil. 15. The dual-target binder of claim 14, wherein the first, second, first, and second coiled coil-forming domains are different from each other in amino acid sequence. 15. The method according to claim 14, wherein the first, second, first, and second coiled coil-forming domains are selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 4, 5, 6, 16, 17 and 18, respectively. Any one of the amino acid sequences to be included, the dual target conjugate. 15. The method of claim 14, wherein the first and first 'coiled coil forming domains combine to form a first coiled coil, and the second and second' coiled coil forming domains combine to form a second coil Which will form a yield coil, a dual target conjugate. 20. The method of claim 19, wherein the first and second coiled coil, respectively,
a) the dissociation constant (K _d ) is 10 nM or less,
b) which comprises a coiled coil-forming domain in the form of a heterodimer, a dual target binder. The dual target binder of claim 19, wherein the binding is self-binding. The dual target conjugate of claim 21, wherein the self-binding is non-covalent bonding. The dual target conjugate of claim 19, wherein the bond further comprises a disulfide bond, and the disulfide bond is formed in at least one or more of both ends of the coiled coil-forming domain. 15. The method of claim 14, wherein the first and the second target binding agent is different from each other, dual target binding body. 15. The method of claim 14, wherein the first and the second target binding material is each independently any one selected from the group comprising a protein, an antibody, an antibody fragment, a hormone, and a peptide, the dual target conjugate. The dual target binding body according to claim 14, wherein the first or second target binding agent targets a surface antigen on a cancer cell. 15. The method of claim 14, wherein at least one of the first and second target binding agent is a therapeutic agent, the dual target binding body. 15. The method of claim 14, wherein the first and second target binding material is any one selected from the group consisting of GFP, mCherry, PD-1, CD80, Insulin, and GLP-1, respectively, the dual-target binder. 15. The method of claim 14, wherein the first fusion and the second fusion is a target binding agent; and a coiled coil forming domain; The dual target conjugate further comprising a linker between them. 15. The method of claim 14, wherein the first fusion and the second fusion is any one amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 21, 22, 25, 26, 28, 30, 38 and 51, respectively That comprising, a dual target binder. The dual-target conjugate according to claim 14, wherein the binding of the fusion protein, the first fusion, and the second fusion is formed in vitro in the dual-target conjugate. a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; Or a second target binding substance and a second 'coiled coil-forming domain is linked, a second fusion; encoding, a nucleic acid molecule. A vector comprising the nucleic acid molecule of claim 32 . A pharmaceutical composition comprising the dual target conjugate of claim 14 . in vitro albumin or an analog thereof, a fusion protein in which a first coiled coil-forming domain and a second coiled coil-forming domain are linked; a first fusion in which a first target binding substance and a first 'coiled coil-forming domain are linked; and a second fusion, wherein the second target binding material and the second 'coiled coil-forming domain are linked, comprising the step of preparing a mixture; 36. The method of claim 35,
Further comprising the step of recovering the dual target conjugate from the mixture, the method for producing a dual target conjugate.

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