Fusion protein, conjugate, and application for same

Abstract

The present invention addresses the problem of providing a novel fusion protein that can efficiently deliver a drug such as an oligonucleotide therapeutic or a small molecule drug to a target site and can more effectively suppress side effects (systemic exposure to the drug). The present invention relates to a fusion protein that includes an antibody domain (A) that has at least one antigen binding site, a stabilization domain (B) that has an α-helix structure and is linked to the antibody domain (A) directly or by a linker, and a membrane adhesion domain (C) that is linked to the stabilization domain (B) directly or by a linker. The present invention also relates to: a conjugate that is formed by bonding the fusion protein and a particle that has an organic membrane that includes a lipid that includes an amphoteric ion; and a pharmaceutical composition that includes the conjugate.

Description

Fusion proteins, complexes and their applications

[0001] The present invention relates to a fusion protein, a complex, a pharmaceutical composition, an anticancer agent, a polynucleotide, an expression vector, a transformant, and a method for producing a fusion protein.

[0002] Currently, in addition to conventional anticancer drugs, various drugs are used in cancer treatment, such as molecular targeted drugs, hormone drugs used for prostate cancer, uterine cancer, breast cancer, etc., and immune checkpoint inhibitors. Furthermore, new cancer treatment methods have been rapidly expanding in recent years, with the practical application of antibody drug conjugates (ADCs) and treatment using chimeric antigen receptor T cells (CAR-T cell therapy), which are immune cells that have artificially incorporated antibody functions.

[0003] Antibody-drug conjugates (ADCs) are drugs that combine a small molecule drug (payload) with an antibody via an appropriate linker, and aim to effectively attack cancer by delivering a powerful chemotherapeutic agent to cancer cells via an antibody that targets cancer cells. Since many small molecule drugs have strong side effects, it is expected that by using ADCs to preferentially deliver them to the affected area, it will be possible to increase their ability to attack cancer cells (enhance their efficacy) while simultaneously reducing the side effects of the drug by suppressing off-target effects (systemic drug exposure).

[0004] For example, Patent Document 1 discloses an antibody-drug conjugate (ADC) in which a cytotoxic drug is conjugated to an antibody that binds to an antigen expressed on the surface of cancer cells and can be internalized into the cells, the antibody-drug conjugate comprising a linker having a predetermined structure, a drug, and an anti-HER2 antibody. One example of such an ADC is the anti-cancer agent "ENHERTZ®" (generic name: trastuzumab deruxtecan; recombinant anti-HER2 antibody-drug conjugate), which has been widely used in the treatment of breast cancer, gastric cancer, and other cancers since its approval in the United States in 2020. "ENHERTZ®" is an ADC in which up to eight molecules of a DNA topoisomerase I inhibitor (camptothecin derivative, DXd) are conjugated to each antibody molecule as a payload via a structurally optimized linker (Non-Patent Document 1).

[0005] As mentioned above, ADCs are sometimes used in cancer treatment, but small molecule drugs remain widely used. Research into "drug delivery systems (DDS)," a technology for delivering small molecule drugs to the desired location, is also being actively conducted. For example, liposomal irinotecan (Onivyde) is a liposomal formulation of the anticancer drug irinotecan hydrochloride hydrate (trade name: Onivyde intravenous infusion), which was approved for insurance coverage in June 2020. It is one of the DDS-based cancer treatments targeting "unresectable pancreatic cancer" (Patent Document 2). A DDS encapsulating irinotecan (a DNA topoisomerase I inhibitor) in liposomal nanoparticles is expected to reduce the side effects of irinotecan by enabling more selective delivery of small molecule anticancer drugs to tissues where cancer cells are present.

[0006] Patent No. 5998289 Patent No. 5645954

[0007] Ogitani, Y. et al., Clin. Cancer Res. 2016, 22, 5097-5108

[0008] Although several antibody-drug conjugates (ADCs) like those described above are known, they suffer from three main weaknesses. (1) The number of payloads (amount of small molecule drugs) that can be attached is determined by the linker structure, limiting the options for small molecule drugs with different chemical structures and physical properties. (2) Even if an ADC binds to a target molecule (antigen) distributed on the surface of a cancer cell membrane, it is not necessarily rapidly internalized within the cell. This forces researchers to select a powerful anticancer drug as the payload, one that exhibits cytotoxicity at low concentrations (subnanomolar or less) when cleaved from the ADC. (3) Even when structurally unstable nucleic acid drugs such as siRNA are directly conjugated to an antibody via a linker to form an ADC, they are inevitably digested by RNases present in the blood. In recent years, there has been a growing need for efficient delivery of nucleic acid drugs and small molecule drugs to target sites, and expectations for achieving this goal are rising. However, at this stage, it is extremely difficult to use nucleic acid drugs, in particular, as the payload for an ADC.

[0009] Furthermore, although several liposome-based DDSs have been approved to date, they have not been given the ability to deliver the encapsulated drug to cancer cells in specific tissues or organs, and therefore have the problem of being unable to suppress side effects (systemic exposure to the drug).

[0010] An object of the present invention is to provide a novel fusion protein that solves the problems of both ADC and DDS. Specifically, an object of the present invention is to provide a novel fusion protein that can selectively deliver drugs such as nucleic acid medicines and small molecule drugs to target sites and suppress side effects caused by systemic exposure to the drugs. Further objects of the present invention are to provide a complex, pharmaceutical composition, and anticancer agent containing the novel fusion protein, a polynucleotide encoding the novel fusion protein, an expression vector, and a transformant incorporating the expression vector, and further to provide a method for producing the novel fusion protein.

[0011] Examples of specific embodiments of the present invention are given below.

[0012] [1] A fusion protein comprising: an antibody domain (A) having at least one antigen-binding site; a stabilizing domain (B) having an α-helical structure linked to the antibody domain (A) directly or via a linker; and a membrane-binding domain (C) linked to the stabilizing domain (B) directly or via a linker. [2] The antibody domain (A) comprises a peptide chain (a1) comprising an immunoglobulin heavy chain variable region (VH) domain and a peptide chain (a2) comprising an immunoglobulin light chain variable region (VL) domain; the stabilization domain (B) comprises a first peptide chain (b1) and a second peptide chain (b2), and the first peptide chain (b1) and the second peptide chain (b2) have a coiled-coil structure; the membrane-binding domain (C) comprises a first membrane-binding peptide chain (c1) and a second membrane-binding peptide chain (c2), and the C-terminus of the peptide chain (a1) and the N-terminus of the first peptide chain (b1) are linked directly or via a linker, and the C-terminus of the peptide chain (a2) and the N-terminus of the second peptide chain (b2) are linked directly or via a linker; The fusion protein according to [1], wherein the C-terminus of the first peptide chain (b1) is linked to the N-terminus of the first membrane-binding peptide chain (c1) directly or via a linker, and the C-terminus of the second peptide chain (b2) is linked to the N-terminus of the second membrane-binding peptide chain (c2) directly or via a linker. [3] The fusion protein according to [1] or [2], wherein the antigen-binding site is a binding site for an epitope. [4] The fusion protein according to any of [1] to [3], wherein the antigen-binding site is a binding site for an epitope present on the surface of a tumor cell. [5] The fusion protein according to any of [1] to [4], wherein the stabilizing domain (B) consists of a SARAH domain derived from human Mst1 kinase. [6] The fusion protein according to any of [1] to [5], wherein the membrane-binding domain (C) has the property of adhering to an organic membrane containing a lipid having a zwitterion. [7] The fusion protein according to any one of [1] to [6], wherein the membrane-binding domain (C) is amphipathic and consists of a basic peptide chain.[8] The fusion protein according to any one of [1] to [7], wherein the membrane-binding domain (C) has the property of adhering to a phospholipid membrane. [9] The fusion protein according to any one of [1] to [8], wherein the membrane-binding domain (C) consists of a C-terminal polypeptide of α1,3 / α1,4-fucosyltransferase derived from Helicobacter pylori.

[10] The fusion protein according to any one of [1] to [9], wherein the membrane-binding domain (C) has an amino acid sequence selected from the following (i) to (vi): (i) SKIYRKAYQKSLPLLRAIRRWVKK (SEQ ID NO: 105) (ii) FKIYRKAYQKSLPLLRAIRRWVRK (SEQ ID NO: 106) (iii) FKIYRKIYQKSLPLLRVIRRWVKK (SEQ ID NO: 107) (iv) FKIYRKAYQKSLPLLRAVRKLVKK (SEQ ID NO: 108) (v) FKIYRKAYQKSLPLLRTIRRWVKK (SEQ ID NO: 109) (vi) FKIYRKAYQKSLPLLRAIRRWVKK (SEQ ID NO: 110)

[11] A complex comprising the fusion protein according to any one of [1] to

[10] and a particle having an organic membrane containing a lipid having a zwitterion.

[12] The complex according to

[11] , wherein the particle encapsulates a drug.

[13] The complex according to

[11] or

[12] , wherein the particle is a liposome.

[14] A pharmaceutical composition comprising the complex according to any one of

[11] to

[13] .

[15] An anticancer agent comprising the complex according to any one of

[11] to

[13] .

[16] A polynucleotide encoding the fusion protein according to any one of [1] to

[10] .

[17] An expression vector containing the polynucleotide according to

[16] .

[18] A transformant transformed with the expression vector according to

[17] .

[19] A method for producing a fusion protein, comprising: preparing an expression vector containing a polynucleotide encoding the fusion protein according to any one of [1] to

[10] ; and culturing a transformant obtained by introducing the expression vector into a host cell.

[20] The antibody domain (A) comprises a peptide chain (a1) comprising an immunoglobulin heavy chain variable region (VH) domain and a peptide chain (a2) comprising an immunoglobulin light chain variable region (VL) domain; the stabilizing domain (B) comprises a first peptide chain (b1) having an α-helical structure and a second peptide chain (b2) having an α-helical structure, and the first peptide chain (b1) and the second peptide chain (b2) have a coiled-coil structure; the membrane-binding domain (C) comprises a first membrane-binding peptide chain (c1) and a second membrane-binding peptide chain (c2); and a method for producing a first fusion protein by preparing a first expression vector having a polynucleotide encoding the peptide chain (a1), the first peptide chain (b1), and the first membrane-binding peptide chain (c1), and culturing a transformant obtained by introducing the first expression vector into a host cell; The method for producing the fusion protein according to

[19] , comprising the steps of: preparing a second expression vector having polynucleotides encoding the peptide chain (a2), the second peptide chain (b2), and the second membrane-binding peptide chain (c2); introducing the second expression vector into a host cell to obtain a transformant, thereby culturing the transformant, to obtain a second fusion protein; and folding the first fusion protein and the second fusion protein.

[0013] According to the present invention, it is possible to provide a novel fusion protein that can selectively deliver drugs such as nucleic acid medicines and small molecule drugs to target sites and suppress side effects caused by systemic exposure to the drugs. The novel fusion protein of the present invention can solve the problems of both ADC and DDS, and is a novel fusion protein with high applicability and versatility.

[0014] FIG. 1 is a schematic diagram illustrating the three-dimensional structure of the fusion protein of this embodiment. FIG. 2 shows the results of SDS-PAGE (12.5%) performed to confirm the expression of the fusion protein of this embodiment. FIG. 3 shows the results of separation and purification of the fusion protein (SN-132) using an affinity column displaying the MUC1 glycopeptide KSAPDT (Galβ1→3GalNAcα1)RPAPG. FIG. 4 shows the results of evaluating the affinity of the purified fusion protein (SN-132) for its epitope using the Biacore interaction analysis system. FIG. 5 is a schematic diagram illustrating the structure of the fusion protein carrying nanosomes obtained by adding 10 mM SN-132 solution (D-PBS) to the fusion protein (SN-132) at a molar ratio of 1:5 and allowing the mixture to stand at 4°C for 2 hours. FIG. 6 shows the results of measuring particle size and its distribution using a dynamic light scattering (DLS) system. Figure 7 shows the results of fluorescence microscopy imaging demonstrating that nanoparticles (PCSAM-coated QD655) are taken up into breast cancer cells (MCF7) (A-C) and that endocytosis due to the specific interaction between the MUC1 glycopeptide epitope on the breast cancer cell membrane surface and FMAM (SN-132) is important (C-E). Figure 8 shows the results of flow cytometry demonstrating that the uptake of nanosomes carrying FMAM (SN-132) into cancer cells is specific to MCF-7 cells expressing MUC1 on their cell surface, which contains the glycopeptide epitope recognized by SN-132. Figure 9 shows the results of separation and purification of Herceptin FMAM using a His-trap affinity column. Figure 10 shows the results of comparing the binding affinity of purified Herceptin FMAM and Herceptin Fab to HER2 in a competitive ELISA assay with Herceptin. Figure 11 shows the results of SDS-PAGE (15%) to confirm the expression of the fusion protein (SN-132'). Figure 12 shows the results of separation and purification of the fusion protein (SN-132') using an affinity column displaying the MUC1 glycopeptide KSAPDT(Galβ1→3GalNAcα1)RPAPG.

[0015] The present invention will be described in detail below. The following description may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0016] (Fusion Protein) This embodiment relates to a fusion protein comprising an antibody domain (A) having at least one antigen-binding site, a stabilizing domain (B) having an α-helical structure linked to the antibody domain (A) directly or via a linker, and a membrane-binding domain (C) linked to the stabilizing domain (B) directly or via a linker. The antibody domain (A) has at least one antigen-binding site, and may have two or three identical or different antigen-binding sites. The antigen-binding site is a site capable of recognizing and binding to an antigen site on a target to which the fusion protein is to bind. The stabilizing domain (B) functions to stabilize the structure of the antibody domain (A), thereby ensuring that the antibody domain (A) can function as an antibody and that the membrane-binding domain (C) can function as an adhesive. The membrane-binding domain (C) is a domain capable of binding to an organic membrane containing a lipid having a zwitterion (e.g., a phospholipid membrane) and the like, and functions to link the fusion protein to other elements.

[0017] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein 100 according to this embodiment. As shown in FIG. 1, the fusion protein 100 comprises an antibody domain (A), a stabilizing domain (B) having an α-helical structure, and a membrane-binding domain (C). In the fusion protein 100, the N-terminus of the stabilizing domain (B) is preferably linked to the C-terminus of the antibody domain (A), and the N-terminus of the membrane-binding domain (C) is preferably linked to the C-terminus of the stabilizing domain (B). In the fusion protein 100, the membrane-binding domain (C) may consist of a single membrane-binding peptide chain or multiple membrane-binding peptide chains. For example, the membrane-binding domain (C) preferably comprises 2 to 10 peptide chains, more preferably 2 to 5 chains, even more preferably 2 to 4 chains, and particularly preferably 2 or 3 chains.

[0018] The antibody domain (A) is preferably a domain formed from the variable region of an antibody. The molecular weight of the antibody domain (A) is preferably 500 kDa or less, more preferably 300 kDa or less, and even more preferably 100 kDa or less. The molecular weight of the antibody domain (A) is preferably 5 kDa or more.

[0019] The stabilization domain (B) is preferably a domain formed by, for example, multimerization (e.g., dimerization or trimerization) of two or more peptide chains. It is particularly preferred that the stabilization domain (B) is a domain formed by dimerization of two peptide chains. In a preferred embodiment, the stabilization domain (B) is sometimes referred to as a dimerization domain.

[0020] In this embodiment, the antibody domain (A) preferably comprises a peptide chain (a1) comprising an immunoglobulin heavy chain variable region (VH) domain (hereinafter also referred to as VH peptide chain (a1)) and a peptide chain comprising an immunoglobulin light chain variable region (VL) domain (hereinafter also referred to as VL peptide chain (a2)), and as shown in Figure 1, the antibody domain (A) preferably consists of a VH peptide chain (a1) and a VL peptide chain (a2). When the antibody domain (A) has two or more types of antigen-binding sites, it may comprise two or more types of VH peptide chains (a1) and / or VL peptide chains (a2).

[0021] The stabilization domain (B) preferably comprises a first peptide chain (b1) and a second peptide chain (b2), and the first peptide chain (b1) and the second peptide chain (b2) preferably have a coiled-coil structure, and the N-terminus of the first peptide chain (b1) is linked to the C-terminus of the VH peptide chain (a1), and the N-terminus of the second peptide chain (b2) is linked to the C-terminus of the VL peptide chain (a2).

[0022] The membrane-binding domain (C) may consist of a single membrane-binding peptide chain, but preferably comprises a first membrane-binding peptide chain (c1) and a second membrane-binding peptide chain (c2). When the membrane-binding domain (C) consists of a single membrane-binding peptide chain, the membrane-binding peptide chain is linked to either the first peptide chain (b1) or the second peptide chain (b2). On the other hand, when the membrane-binding domain (C) comprises a first membrane-binding peptide chain (c1) and a second membrane-binding peptide chain (c2), it is preferred that the N-terminus of the first membrane-binding peptide chain (c1) is linked to the C-terminus of the first peptide chain (b1), and the N-terminus of the second membrane-binding peptide chain (c2) is linked to the C-terminus of the second peptide chain (b2).

[0023] In a preferred embodiment of this embodiment, the N-terminus of the first peptide chain (b1) is linked directly or via a linker to the C-terminus of the VH peptide chain (a1), the N-terminus of the second peptide chain (b2) is linked directly or via a linker to the C-terminus of the VL peptide chain (a2), the N-terminus of the first membrane-binding peptide chain (c1) is linked directly or via a linker to the C-terminus of the first peptide chain (b1), and the N-terminus of the second membrane-binding peptide chain (c2) is linked directly or via a linker to the C-terminus of the second peptide chain (b2). The fusion protein of this embodiment is preferably a protein formed by folding a first fusion protein having a VH peptide chain (a1)-first peptide chain (b1)-first membrane-binding peptide chain (c1) configuration and a second fusion protein having a VL peptide chain (a2)-second peptide chain (b2)-second membrane-binding peptide chain (c2) configuration ( FIG. 1 ). The folding of the first fusion protein and the second fusion protein is promoted by the formation of a coiled-coil structure between the first peptide chain (b1) and the second peptide chain (b2).

[0024] The first peptide chain (b1) and the second peptide chain (b2) are not particularly limited as long as they can form a coiled-coil structure, and may be peptide chains having the same amino acid sequence or different amino acid sequences. Similarly, the first membrane-binding peptide chain (c1) and the second membrane-binding peptide chain (c2) may be peptide chains having the same amino acid sequence or different amino acid sequences.

[0025] The first fusion protein and the second fusion protein may each be expressed in a prokaryotic or eukaryotic cell system. That is, the fusion protein of this embodiment may be a fusion protein obtained by folding (refolding) the first fusion protein and the second fusion protein expressed in a prokaryotic or eukaryotic cell system. The first fusion protein and the second fusion protein may each be expressed in E. coli, bacteria, or insect cells, allowing for inexpensive and simple mass purification of the fusion proteins. When the fusion protein of this embodiment is obtained by refolding the first fusion protein and the second fusion protein expressed in E. coli, bacteria, or insect cells, for example, it becomes easy to modify each protein using known methods, making it possible to purify a fusion protein with high applicability and versatility.

[0026] In the fusion protein of this embodiment, the antibody domain (A) has an antigen-binding site and functions as an antibody. In the stabilizing domain (B), a pair of peptide chains having an α-helical structure are arranged in antiparallel, forming a coiled-coil structure. Linking such a stabilizing domain (B) to the peptide chains constituting the antibody domain (A) can enhance the structural stability of the antibody domain (A). The membrane-binding domain (C) contains a peptide chain that has the property of adhering to an organic membrane containing a lipid having a zwitterion, and has the property of adhering to, for example, liposomes composed of phospholipid membranes containing phosphatidylcholine, cells, extracellular microparticles, particles coated with amphiphilic compounds similar to phospholipid membranes containing phosphatidylcholine, and the like. In this embodiment, for example, liposomes, particles, immune cells, etc. encapsulating drugs such as nucleic acid medicines or low-molecular-weight drugs can be supported by the membrane-binding domain (C) of the fusion protein, and the antibody domain (A) functions as an antibody, thereby directing the fusion protein carrying the particles, etc., to a target site (e.g., tumor cells where an antigen is present). This allows drugs, immune cells, and the like to be efficiently delivered to target sites. Furthermore, because the antibody domain (A) of this embodiment has excellent affinity for antigens, side effects (systemic drug exposure) can be more effectively suppressed. Thus, this embodiment provides an innovative technology that enables the creation of a next-generation DDS (drug delivery system) that is original, highly applicable, and versatile, and solves the problems of both ADCs and DDSs. Furthermore, nucleic acid drugs and the like, which have traditionally been difficult to transport to target sites, can be protected from digestion by RNases by encapsulating them in liposomes or particles, enabling their delivery to the target site. This is expected to lead to groundbreaking therapies using nucleic acid drugs. Furthermore, potent anticancer drugs that exhibit cytotoxicity can be encapsulated in liposomes or particles and efficiently delivered to the target site, enabling more effective treatment while appropriately controlling the amount of anticancer drug used.

[0027] <Antibody domain (A)> The antibody domain (A) preferably comprises a peptide chain (a1) comprising an immunoglobulin heavy chain variable region (VH) domain and a peptide chain (a2) comprising an immunoglobulin light chain variable region (VL) domain, and the VH peptide chain (a1) and the VL peptide chain (a2) form an antigen-binding site. The antibody domain (A) has the antigen-binding site and functions as an antibody.

[0028] In this embodiment, the antibody domain (A) preferably consists only of an antibody variable region. That is, the antibody domain (A) preferably consists only of an immunoglobulin heavy chain variable region (VH) domain and an immunoglobulin light chain variable region (VL) domain. This allows the molecular weight of the antibody domain (A) to be reduced, thereby enabling the fusion protein itself to be reduced in molecular weight and facilitating mass purification. Furthermore, if the fusion protein itself can be reduced in molecular weight, its versatility and efficacy as a pharmaceutical can also be effectively improved. As such, the antibody domain (A) consists only of a partial region of an antibody, and therefore can also be referred to as a so-called "mini-antibody." Furthermore, the fusion protein of this embodiment is sometimes referred to as a "functional mini-antibody module (FMAM)" because it is a "mini-antibody" that is provided with the ability to harbor liposomes or particles encapsulating drugs such as nucleic acid medicines or small-molecular-weight drugs, immune cells, or the like.

[0029] The inventors have succeeded not only in reducing the molecular weight of the antibody domain (A) by constructing it from only the variable region of an antibody, but also in increasing the affinity of the antibody domain (A) with an antigen. Generally, miniaturization of a domain constructed from only a partial region of an antibody is a concern, as it may result in a deterioration in antibody function. However, in this embodiment, it has surprisingly been demonstrated that an antibody domain (A) constructed from only the variable region has a higher affinity with the antigen. Thus, the fusion protein of this embodiment is a protein having an antibody domain (A) with extremely high affinity for the antigen, and is therefore efficiently directed to a target site (such as a tumor cell where the antigen is present).

[0030] The antibody domain (A) has a binding site for an epitope. In this specification, an epitope refers to a site in an antigen to which the antibody domain (A), which functions as an antibody, specifically binds. In this embodiment, the epitope is preferably present on the surface of a tumor cell. This allows the antibody domain (A) to specifically bind to the surface of the tumor cell. Then, as described below, particles or the like encapsulating a drug or the like carried by the membrane-binding domain (C) are selectively delivered to tumor cells, making it possible to damage only tumor cells presenting a specific epitope.

[0031] An epitope is a part of an antigen, and is expressed in low amounts in normal cells, but epitopes are known to exist only in tumor-associated antigens. For example, mucin-1 (hereinafter also referred to as MUC1), a type of mucin, is a tumor-associated antigen that is expressed in low amounts in epithelial cells of normal tissues, but is a high-molecular-weight glycoprotein that is expressed in many adenocarcinomas. Known epitopes of MUC1 include the PDTR region present in the tandem repeat domain.

[0032] HER-2, a growth factor receptor-type oncogene product and a transmembrane receptor protein with a tyrosine kinase domain having a molecular weight of 185 kDa, is known to be expressed in low amounts in epithelial cells of normal tissues, but is frequently overexpressed in breast cancer, ovarian cancer, uterine cancer, lung cancer, gastrointestinal cancer, etc. The Thr575-Pro625 epitope is known to be the epitope of the HER-2 antigen (Joan T. Garrett et al., J Immunol (2007) 178 (11): 7120-7131).

[0033] Many antibodies are known that specifically bind to an epitope of an antigen. Representative antibodies include, for example, MUC1-reactive monoclonal antibodies, and recombinant humanized anti-HER2 monoclonal antibodies such as trastuzumab, rituximab, nivolumab, cetuximab, bevacizumab, pertuzumab, necitumumab, pembrolizumab, ustekinumab, atezolizumab, ramucirumab, adalimumab, and emicizumab.

[0034] The amino acid sequence of the antibody domain (A) is preferably designed to include a variable region sequence of an antibody that specifically binds to an epitope of an antigen. For example, each of the immunoglobulin heavy chain variable region (VH) domain and the immunoglobulin light chain variable region (VL) domain of the antibody domain (A) preferably has a CDR sequence listed in the following table. Also, each of the immunoglobulin heavy chain variable region (VH) domain and the immunoglobulin light chain variable region (VL) domain of the antibody domain (A) preferably has a variable region sequence including a CDR sequence listed in Table 1 below.

[0035] Representative antibody variable region sequences are exemplified below, but the amino acid sequence of the antibody domain (A) in this embodiment is not limited to the following.

[0036]

[0037] Examples of antibody domain (A) include the following: (1) The VH peptide chain (a1) of antibody domain (A) has the CDR sequences of SEQ ID NO: 1 (amino acid sequences of NYGLS (SEQ ID NO: 27), ENHPGSGIIYHNEKFRG (SEQ ID NO: 28), and SSGTRGFAY (SEQ ID NO: 29)), and the VL peptide chain (a2) of antibody domain (A) has the CDR sequences of SEQ ID NO: 2 (amino acid sequences of RSSQSIVHSNGNTYLE (SEQ ID NO: 30), KVSNRFS (SEQ ID NO: 31), and FQGSHGPWT (SEQ ID NO: 32)). (2) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 3 (amino acid sequences of GFNIKDTYIH (SEQ ID NO: 33), RIYPTNGYTRYADSVKG (SEQ ID NO: 34), and WGGDGFYAMDY (SEQ ID NO: 35)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 4 (amino acid sequences of RASQDVNTAVA (SEQ ID NO: 36), SASFLYS (SEQ ID NO: 37), and QQHYTTPPT (SEQ ID NO: 38)). (3) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 5 (amino acid sequences of SYNMH (SEQ ID NO: 39), AIYPGNGDTSYNQKFKG (SEQ ID NO: 40), and STYYGGDWYFNV (SEQ ID NO: 41)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 6 (amino acid sequences of RASSSVSYIH (SEQ ID NO: 42), ATSNLAS (SEQ ID NO: 43), and QQWTSNPPT (SEQ ID NO: 44)). (4) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 7 (amino acid sequences of NSGMH (SEQ ID NO: 45), VIWYDGSKRYYADSVKG (SEQ ID NO: 46), and NDDY (SEQ ID NO: 47)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 8 (amino acid sequences of RASQSVSSYLA (SEQ ID NO: 48), DASNRAT (SEQ ID NO: 49), and SSNWPRT (SEQ ID NO: 50)).(5) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 9 (the amino acid sequence of NYGVH (SEQ ID NO: 51), VIWSGGNTDYNTPFTS (SEQ ID NO: 52), and ALTYYDYEFAY (SEQ ID NO: 53)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 10 (the amino acid sequence of RASQSIGTNIH (SEQ ID NO: 54), YASESIS (SEQ ID NO: 55), and QQNNNWPTT (SEQ ID NO: 56)). (6) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 11 (amino acid sequences of GYTFTNYGMN (SEQ ID NO: 57), WINTYTGEPTYAADFKR (SEQ ID NO: 58), and YPHYYGSSHWYFDV (SEQ ID NO: 59)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 12 (amino acid sequences of SASQDISNYLN (SEQ ID NO: 60), FTSSLHS (SEQ ID NO: 61), and QQYSTVPWT (SEQ ID NO: 62)). (7) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 13 (amino acid sequences of GFTFTDYTMD (SEQ ID NO: 63), DVNPNSGGSIYNQRFKG (SEQ ID NO: 64), and NLGPSFYFDY (SEQ ID NO: 65)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 14 (amino acid sequences of KASQDVSIGVA (SEQ ID NO: 66), SASYRYT (SEQ ID NO: 67), and QQYYIYPYT (SEQ ID NO: 68)). (8) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 15 (amino acid sequences of SGDYYWS (SEQ ID NO: 69), YIYYSGSTDYNPSLKS (SEQ ID NO: 70), and VSIFGVGTFDY (SEQ ID NO: 71)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 16 (amino acid sequences of RASQSVSSYLAW (SEQ ID NO: 72), DASNRAT (SEQ ID NO: 73), and HQYGSTPLT (SEQ ID NO: 74)).(9) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 17 (the amino acid sequence of NYYMY (SEQ ID NO: 75), GINPSNGGTNFNEKFKN (SEQ ID NO: 76), and RDYRFDMGFDY (SEQ ID NO: 77)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 18 (the amino acid sequence of RASKGVSTSGYSYLH (SEQ ID NO: 78), LASYLES (SEQ ID NO: 79), and QHSRDLPLT (SEQ ID NO: 80)). (10) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 19 (amino acid sequences of TYWLG (SEQ ID NO: 81), IMSPVDSDIRYSPSFQG (SEQ ID NO: 82), and RRPGQGYFDF (SEQ ID NO: 83)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 20 (amino acid sequences of RASQGISSWLA (SEQ ID NO: 84), AASSLQS (SEQ ID NO: 85), and QQYNIYPYT (SEQ ID NO: 86)). (11) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 21 (amino acid sequences of DSWIH (SEQ ID NO: 87), WISPYGGSTY (SEQ ID NO: 88), and RHWPGG (SEQ ID NO: 89)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequences in SEQ ID NO: 22 (amino acid sequences of DVSTAVA (SEQ ID NO: 90), SASFLY (SEQ ID NO: 91), and QQYLYHPAT (SEQ ID NO: 92)). (12) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence of SEQ ID NO: 23 (amino acid sequences of GFTFSSYSMN (SEQ ID NO: 93), SISSSSSYIYYADSVKG (SEQ ID NO: 94), and VTDAFDI (SEQ ID NO: 95)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence of SEQ ID NO: 24 (amino acid sequences of RASQGIDNWLG (SEQ ID NO: 96), DASNLDT (SEQ ID NO: 97), and QQAKAFPPT (SEQ ID NO: 98)).(13) The VH peptide chain (a1) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 25 (the amino acid sequence of DYAMH (SEQ ID NO: 99), AITWNSGHIDYADSVEG (SEQ ID NO: 100), and VSYLSTASSLDY (SEQ ID NO: 101)), and the VL peptide chain (a2) of the antibody domain (A) has the CDR sequence in SEQ ID NO: 26 (the amino acid sequence of RASQGIRNYLA (SEQ ID NO: 102), AASTLQS (SEQ ID NO: 103), and QRYNRAPYT (SEQ ID NO: 104)).

[0038] Examples of antibody domain (A) include the following: (1) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 1, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 2. (2) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 3, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 4. (3) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 5, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 6. (4) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 7, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 8. (5) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 9, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 10. (6) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 11, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 12. (7) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 13, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 14. (8) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 15, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 16. (9) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 17, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 18. (10) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 19, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 20. (11) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 21, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 22.(12) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 23, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 24. (13) The VH peptide chain (a1) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 25, and the VL peptide chain (a2) of the antibody domain (A) has the amino acid sequence of SEQ ID NO: 26.

[0039] The antibody domain (A) may consist of the variable region sequence described above. In this case, one or several amino acid residues in the variable region sequence may be substituted, added, inserted, or deleted, as long as the epitope-binding function is not lost. As used herein, "several" preferably means 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 amino acid residues. Conservative amino acid substitutions are preferred as amino acid substitutions herein. Conservative amino acid substitutions are substitutions that occur within a group of amino acids related in amino acid side chains. For example, if the amino acid before substitution is a nonpolar amino acid, it can be substituted with another nonpolar amino acid; if the amino acid before substitution is an uncharged amino acid, it can be substituted with another uncharged amino acid; if the amino acid before substitution is an acidic amino acid, it can be substituted with another acidic amino acid; and if the amino acid before substitution is a basic amino acid, it can be substituted with another basic amino acid. The identity between the amino acid sequence of each peptide chain constituting the antibody domain (A) and the amino acid sequences listed in the table above is preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0040] The antibody domain (A) may be chemically or biologically modified. Chemical modifications include attachment of a chemical moiety to the amino acid backbone, chemical modifications of N- or O-linked carbohydrate chains, etc. Biological modifications include post-translational modifications (e.g., N- or O-linked glycosylation, N- or C-terminal processing, deamidation, aspartic acid isomerization, and methionine oxidation), and addition of a methionine residue to the N-terminus by expression in a prokaryotic host cell. Furthermore, the antibody domain (A) may be labeled with an enzyme, a fluorescent label, or an affinity label to facilitate detection and isolation.

[0041] <Stabilization domain (B)> The stabilization domain (B) preferably comprises a first peptide chain (b1) having an α-helical structure and a second peptide chain (b2) having an α-helical structure arranged in antiparallel to form a coiled-coil structure. The stabilization domain (B) having such a structure can enhance the structural stability of the antibody domain (A) linked to the stabilization domain (B). Therefore, the stabilization domain (B) functions as a binding module in the fusion protein and is also called a dimerization domain.

[0042] The length of the stabilization domain (B) forming the coiled-coil structure is, for example, preferably 0.5 nm or more, more preferably 1 nm or more, and even more preferably 2 nm or more, and the length of the stabilization domain (B) forming the coiled-coil structure is preferably 50 nm or less, more preferably 40 nm or less, and even more preferably 30 nm or less.

[0043] The stabilization domain (B) preferably consists of a SARAH domain derived from human Mst1 kinase (hMst1 SARAH domain). The SARAH (Salvador / RASSF1A / Hippo) domain is known as a small helical domain of 50 residues or less found in proteins involved in the Hippo signaling pathway, including MST, RASSF, and WW45. The SARAH domain is also known to form homodimers or heterodimers.

[0044] The amino acid sequence of the peptide chain constituting the stabilization domain (B) preferably used in this embodiment is not particularly limited as long as it can form an antiparallel coiled-coil structure, and examples include the following. In the following example, the amino acid sequences of the first peptide chain (b1) and the second peptide chain (b2) are different, but the amino acid sequences of the first peptide chain (b1) and the second peptide chain (b2) may be the same. (VH side (first peptide chain (b1))) DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAK (SEQ ID NO: 119) (VL side (second peptide chain (b2)) DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAK (SEQ ID NO: 120)

[0045] Each peptide chain constituting the stabilization domain (B) preferably consists of the above-mentioned amino acid sequence, but one or several amino acid residues of the above-mentioned amino acid sequence may be substituted, added, inserted, or deleted, as long as the function of the stabilization domain (B) is not lost. Furthermore, each peptide chain constituting the stabilization domain (B) may have an additional amino acid sequence added to the C-terminus and / or N-terminus of the above-mentioned amino acid sequence. The identity of the amino acid sequence of each peptide chain constituting the stabilization domain (B) with the above-mentioned amino acid sequences is preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0046] In this embodiment, it is preferred that the N-terminus of the first peptide chain (b1) is linked to the C-terminus of the VH peptide chain (a1), and the N-terminus of the second peptide chain (b2) is linked to the C-terminus of the VL peptide chain (a2). The respective peptide chains may be linked directly or via a linker. In this case, an example of the linker is a two-residue linker (Gly-Ser). Linking the respective peptide chains via such a linker can more effectively enhance the structural stability of the antibody domain (A) to which the stabilizing domain (B) is linked.

[0047] <Membrane-binding domain (C)> The membrane-binding domain (C) preferably contains a peptide chain capable of adhering to an organic membrane containing a lipid having a zwitterion, and more preferably may contain a peptide chain capable of adhering to a lipid membrane having a zwitterion. In this specification, "adhesion" generally refers to the act of sticking or adhering, or the contact and inseparability of the surfaces of two objects (Sanseido Daijirin Second Edition). In this embodiment, however, the term "adhesion" is used interchangeably with "fixation" or "immobilization." That is, in addition to cases where the membrane-adhesive portion of a membrane-adhesive peptide chain comes into contact with the surface of an organic membrane and adheres to it, it also includes, for example, cases where the membrane-adhesive portion of a membrane-adhesive peptide chain penetrates or penetrates the organic membrane, making it impossible for the membrane-adhesive portion of the membrane-adhesive peptide chain to separate from the organic membrane.

[0048] The organic membrane containing the zwitterionic lipid may be an organic membrane containing a phospholipid and another polymer. Such an organic membrane may be a biomimetic membrane or an artificial membrane. The organic membrane may contain optional components or drugs (e.g., lipid-soluble drugs, etc.) as needed.

[0049] In particular, the membrane-binding domain (C) preferably contains a peptide chain capable of adhering to lipid membranes containing zwitterions, and more preferably contains a peptide chain capable of adhering to phospholipid membranes containing zwitterions. As used herein, a phospholipid refers to a molecule having two or more hydrophobic groups, each consisting of a phosphate group and an acyl group and / or an alkyl group. Examples of phospholipids containing zwitterions include phosphatidylcholine (lecithin), phosphatidylethanolamine, sphingomyelin, and mixtures of one or more of these derivatives. In this embodiment, phosphatidylcholine is preferably used as the preferred phospholipid, and the membrane-binding domain (C) particularly preferably contains a peptide chain capable of adhering to lipid membranes containing phosphatidylcholine as the primary phospholipid.

[0050] The fatty acids constituting the phospholipids are not particularly limited, and examples thereof include saturated or unsaturated fatty acids such as caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, arachidic acid, palmitoleic acid, oleic acid, linoleic acid, α- and γ-linoleic acid, erucic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, and tetracosatetraenoic acid.

[0051] The membrane-binding domain (C) is preferably amphiphilic and composed of a basic peptide chain. A basic peptide is a peptide containing a basic amino acid such as arginine, histidine, ornithine, or lysine. An amphipathic peptide is a peptide having both a hydrophobic portion and a hydrophilic portion within one peptide molecule. An amphipathic peptide can be obtained, for example, by hydrophilizing a portion of a hydrophobic peptide (e.g., a peptide having a hydrophobic α-helix structure), and it is preferable to introduce a hydrophilic amino acid in a specific direction of the hydrophobic α-helix structure. The membrane-binding domain (C) can be immobilized to the organic membrane by binding between the zwitterion (hydrophilic group) of the organic membrane and the hydrophilic amino acid side chain of the membrane-binding domain (C).

[0052] The membrane-binding domain (C) preferably consists of a peptide chain having an α-helical structure. The membrane-binding domain (C) preferably has the reversible property of adopting a so-called random structure in an aqueous solvent to be soluble, and adopting an α-helical structure in a neutral environment in the presence of phospholipids. That is, under neutral conditions, the membrane-binding domain (C) adheres to an organic membrane while adopting an α-helical structure, but when placed under alkaline conditions of about pH 10, it adopts a random structure and can be detached from the organic membrane.

[0053] The membrane-binding domain (C) preferably consists of a C-terminal polypeptide of α1,3 / α1,4-fucosyltransferase derived from Helicobacter pylori. Helicobacter pylori-derived α1,3 / α1,4-fucosyltransferase naturally has a membrane-binding peptide chain at the C-terminus, and the membrane-binding domain (C) of this embodiment preferably comprises the C-terminal membrane-binding site. It is known that there are several types of amino acid sequences for the C-terminal polypeptide of Helicobacter pylori-derived α1,3 / α1,4-fucosyltransferase depending on the strain of H. pylori. The C-terminal polypeptide of Helicobacter pylori-derived α1,3 / α1,4-fucosyltransferase preferably comprises, for example, the following amino acid sequence: More specifically, the membrane-binding domain (C) comprises a first membrane-binding peptide chain (c1) and a second membrane-binding peptide chain (c2), and each of the first membrane-binding peptide chain (c1) and the second membrane-binding peptide chain (c2) preferably comprises an amino acid sequence selected from the following amino acid sequences (i) to (vi): (i) SKIYRKAYQKSLPLLRAIRRWVKK (SEQ ID NO: 105) (ii) FKIYRKAYQKSLPLLRAIRRWVRK (SEQ ID NO: 106) (iii) FKIYRKIYQKSLPLLRVIRRWVKK (SEQ ID NO: 107) (iv) FKIYRKAYQKSLPLLRAVRKLVKK (SEQ ID NO: 108) (v) FKIYRKAYQKSLPLLRTIRRWVKK (SEQ ID NO: 109) (vi) FKIYRKAYQKSLPLLRAIRRWVKK (SEQ ID NO: 110)

[0054] Each membrane-binding peptide chain constituting the membrane-binding domain (C) preferably consists of the above-mentioned amino acid sequence, but one or several amino acid residues of the above amino acid sequence may be substituted, added, inserted, or deleted, as long as the function of the membrane-binding domain (C) is not lost. Furthermore, each membrane-binding peptide chain constituting the membrane-binding domain (C) may have an additional amino acid sequence added to the C-terminus and / or N-terminus of the above-mentioned amino acid sequence. The identity of the amino acid sequence of each membrane-binding peptide chain constituting the membrane-binding domain (C) with the above-mentioned amino acid sequences is preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0055] Specific examples of targets to which the membrane-binding domain (C) can adhere include liposomes composed of phospholipid membranes containing phosphatidylcholine, cells (immune cells, cancer cells, etc.), and extracellular particles such as exosomes. Furthermore, particles (nanoparticles, etc.) coated with amphipathic compounds similar to phospholipid membranes containing phosphatidylcholine can also be targets to which the membrane-binding domain (C) can adhere. Furthermore, targets to which the membrane-binding domain (C) can adhere are not limited to particles, but may also be, for example, sections (strips) of phospholipid membranes containing phosphatidylcholine. Thus, the membrane-binding domain (C) has excellent adhesive properties for liposomes composed of phospholipid membranes containing phosphatidylcholine, cells, extracellular particles, and particles or strips coated with amphipathic compounds or peptides similar to phospholipid membranes containing phosphatidylcholine.

[0056] In this embodiment, it is preferred that the N-terminus of the first membrane-binding peptide chain (c1) is linked to the C-terminus of the first peptide chain (b1), and the N-terminus of the second membrane-binding peptide chain (c2) is linked to the C-terminus of the second peptide chain (b2). The respective peptide chains may be linked directly or via a linker. In this case, examples of linkers include a repeat sequence consisting of seven residues of (Asp-Asp-Leu-Arg-Val-Asn-Tyr) called Heptad Repeat (SEQ ID NO: 111) and a sequence consisting of five residues of (Leu-Pro-Glu-Thr-Gly) (SEQ ID NO: 112).

[0057] (Method for producing fusion protein) In this embodiment, it is preferable to use genetic recombination techniques to prepare an expression vector (plasmid vector) having polynucleotides encoding the antibody domain (A), the stabilizing domain (B), and the membrane-binding domain (C), and then introduce the expression vector into host cells or the like to obtain a transformant, and then culture the transformant to produce the fusion protein.

[0058] More specifically, the method for producing the fusion protein preferably comprises the steps of: preparing a first expression vector having polynucleotides encoding the VH peptide chain (a1), the first peptide chain (b1), and the membrane-binding peptide chain (c1); introducing the first expression vector into a host cell or the like to obtain a transformant and culturing the transformant to obtain the first fusion protein; preparing a second expression vector having polynucleotides encoding the VL peptide chain (a2), the second peptide chain (b2), and the membrane-binding peptide chain (c2); introducing the second expression vector into a host cell or the like to obtain a transformant to obtain the second fusion protein; and folding the first fusion protein and the second fusion protein.

[0059] The step of preparing the first expression vector may include a step of linking a polynucleotide encoding a first peptide chain (b1) to a polynucleotide encoding a VH peptide chain (a1), and a step of linking a polynucleotide encoding a first membrane-binding peptide chain (c1) to the polynucleotide encoding the first peptide chain (b1), or a first expression vector having polynucleotides encoding the VH peptide chain (a1), the first peptide chain (b1), and the membrane-binding peptide chain (c1) may be prepared without the linking step. Similarly, the step of preparing the second expression vector may include a step of linking a polynucleotide encoding a second peptide chain (b2) to a polynucleotide encoding a VL peptide chain (a2), and a step of linking a polynucleotide encoding the second membrane-binding peptide chain (c2) to the polynucleotide encoding the second peptide chain (b2), or a first expression vector having polynucleotides encoding the VL peptide chain (a2), the second peptide chain (b2), and the membrane-binding peptide chain (c2) may be prepared without the linking step. When linking the respective polynucleotides, the order of linking is not limited.

[0060] Polynucleotides encoding the antibody domain (A), stabilizing domain (B), and membrane-binding domain (C) can be artificially produced using known genetic engineering techniques. Furthermore, if necessary, the polynucleotides may be modified. Modifications can be performed using commercially available kits or known methods. Examples of modifications include digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, and insertion of start codons (ATG) and / or stop codons (TAA, TGA, or TAG). Furthermore, in the production of proteins by genetic engineering, if the codons used in the original gene encoding the target protein are used infrequently in the host, high expression of the target protein may be achieved by optimizing the codon usage frequency for the host without changing the encoded amino acid sequence.

[0061] In addition to a promoter and a terminator, the expression vector may contain, as control elements, transcription elements such as an enhancer, a CCAAT box, a TATA box, or an SPI site, as necessary. The plasmid vector into which the DNA encoding each peptide chain is introduced can be used in an appropriate combination with the host cell. The vector used in the present invention can be appropriately selected by those skilled in the art and is not particularly limited. Examples of vectors that can be used include pET-26b, pCOS1, pME18S, pEF-BOS, pCDM8, pRSVneo, pSV2-neo, pcDNAI / Amp, pcDNAI, pAMoERC3Sc, pAGE107, pREP4, pAGE103, pAMoA, pAS3-3, pCAGGS, pBK-CMV, pcDNA3.1, and pZeoSV.

[0062] The DNA sequence can be confirmed by known methods, such as the dideoxynucleotide chain termination method (Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)). Alternatively, an automated base sequencer (e.g., DNA Sequencer PRISM 377 or DNA Sequencer PRISM 310; both manufactured by Perkin-Elmer) can be used.

[0063] When introducing an expression vector into a host cell, known methods can be used, such as the calcium phosphate method (Chen, C. et al. (1987) Mol. Cell. Biol. 7, 2745-272), lipofection (Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417), or electroporation (Potter, H. (1988) Anal. Biochem. 174, 361-373). For electroporation, a gene transfer device (GenePulser; Bio-Rad) can be used.

[0064] After culturing the transformant into which the expression vector has been introduced, the first fusion protein and the second fusion protein are each solubilized under alkaline conditions and recovered. The expressing cell line can be pulverized by any known method, but it is preferable to employ a pulverization method using an ultrasonic pulverizer.

[0065] The step of folding the first fusion protein and the second fusion protein may be a step of refolding each fusion protein that was denatured during expression. Refolding conditions can be determined, for example, by employing the method described in Takagi et al., Structure 2017, 25, 1611-1622. Alternatively, optimal protein refolding conditions can be easily determined using a protein refolding kit. Commercially available protein refolding kits can be used, such as the ProteoStat Protein Refolding and Aggregation Detection Kit manufactured by Cosmo Bio Co., Ltd. Appropriate control of the refolding step can also effectively increase the yield of the resulting fusion protein.

[0066] The fusion protein obtained by refolding is preferably purified by a known method. As a purification method, separation and purification methods commonly used for separating and purifying antibodies may be used. For example, the fusion protein can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reversed-phase chromatography, and adsorption chromatography. In this embodiment, purification can be performed by gel filtration column chromatography or an affinity column, and it is preferable that the antigen (epitope) to which the antibody domain (A) binds is presented on the affinity column.

[0067] (Polynucleotide, Expression Vector, Transformant) This embodiment may relate to a polynucleotide encoding the above-described fusion protein. That is, this embodiment may relate to a polynucleotide encoding the antibody domain (A), the stabilizing domain (B), and the membrane-binding domain (C). The polynucleotide encoding the antibody domain (A) is linked to a polynucleotide encoding the stabilizing domain (B), and the polynucleotide encoding the stabilizing domain (B) is linked to a polynucleotide encoding the membrane-binding domain (C). When the antibody domain (A) has a VH peptide chain (a1) and a VL peptide chain (a2), the stabilization domain (B) has a first peptide chain (b1) and a second peptide chain (b2), and the membrane-binding domain (C) has a first membrane-binding peptide chain (c1) and a second membrane-binding peptide chain (c2), this embodiment may relate to polynucleotides encoding the VH peptide chain (a1), the first peptide chain (b1), and the first membrane-binding peptide chain (c1), or may relate to polynucleotides encoding the VL peptide chain (a2), the second peptide chain (b2), and the second membrane-binding peptide chain (c2), or may relate to combinations thereof.

[0068] This embodiment may also relate to an expression vector containing the polynucleotide, or an expression kit including the expression vector. The expression vector is preferably one that can replicate in a prokaryotic or eukaryotic cell system. When a prokaryotic cell system is used as the host, for example, Escherichia coli or Bacillus subtilis can be used. When a eukaryotic cell system is used as the host, animal cells, plant cells, or eukaryotic microorganisms can be used.

[0069] This embodiment may relate to a transformant transformed with the expression vector. The host for the transformant may be, for example, a prokaryotic cell or a eukaryotic cell, but is not particularly limited thereto. In this embodiment, Escherichia coli, bacteria, insect cells, or the like can be used as the host, so that the transformant can be obtained inexpensively and easily.

[0070] (Complex) This embodiment relates to a complex comprising the above-described fusion protein and a particle having an organic membrane containing a lipid having a zwitterion. This embodiment may also relate to a complex comprising the above-described fusion protein and a cell (e.g., an immune cell, etc.). In this specification, the particle also includes extracellular particles such as liposomes and exosomes composed of an organic membrane containing a lipid having a zwitterion. The particle also includes particles (nanoparticles, etc.) coated with an amphiphilic compound similar to the organic membrane containing the lipid having a zwitterion. Among these, the particle is preferably a liposome.

[0071] In particular, the complex of this embodiment is preferably a complex containing the above-mentioned fusion protein and a liposome composed of a phospholipid membrane containing phosphatidylcholine. Furthermore, the complex of this embodiment is also preferably a complex containing the above-mentioned fusion protein and a particle (nanoparticle, etc.) coated with an amphipathic compound similar to the phospholipid membrane containing phosphatidylcholine. Furthermore, the complex of this embodiment is also preferably a complex containing the above-mentioned fusion protein and an immune cell. In particular, the complex of this embodiment is preferably a complex containing the above-mentioned fusion protein and a liposome composed of a phospholipid membrane containing phosphatidylcholine.

[0072] In the complex of this embodiment, the particles preferably contain a drug. The drug is not particularly limited as long as it is acceptable as a pharmaceutical preparation. The pharmaceutical preparation is for use in animals and humans. For example, the drug may be a nucleic acid drug or a low-molecular-weight drug.

[0073] Examples of low-molecular-weight drugs include anticancer drugs. The anticancer drug is preferably an anticancer drug for use in lung cancer, urothelial cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, vulva cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

[0074] Examples of anticancer agents include chemotherapeutic agents, toxins, radioisotopes, and substances containing the same. Examples of chemotherapeutic agents include DNA damaging agents, antimetabolites, enzyme inhibitors, DNA intercalating agents, DNA cleaving agents, topoisomerase inhibitors, DNA binding inhibitors, tubulin binding inhibitors, cytotoxic nucleosides, platinum compounds, antiangiogenic agents, signal transduction inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, and cell cycle inhibitors. Examples of toxins include bacterial toxins (e.g., diphtheria toxin) and plant toxins (e.g., ricin). Examples of radioisotopes include radioisotopes of hydrogen atoms (e.g., ３ H), radioactive isotopes of carbon atoms (e.g., １４ C), radioactive isotopes of phosphorus atoms (e.g., ３２ P), radioactive isotopes of sulfur atoms (e.g., 35 Ｓ ), radioactive isotopes of yttrium (e.g., ９０ Y), radioactive isotopes of technetium (e.g., ９９ｍ Tc), radioactive isotopes of indium (e.g., １１１ In), radioactive isotopes of iodine atoms (e.g., １２３ I, １２５ I, １２９ I, １３１ I), radioactive isotopes of samarium (e.g., １５３ Sm), radioactive isotopes of rhenium (e.g., １８６ Re), radioactive isotopes of astatine (e.g., ２１１ At), radioactive isotopes of bismuth (e.g., ２１２ Bi).

[0075] The anticancer agent may be an antitumor drug, and examples of the antitumor drug include doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin or its derivatives), taxol or its derivatives, other camptothecin or its derivatives (antineoplastic agents described in Japanese Patent Laid-Open No. 6-87746), and exatecan, a camptothecin derivative (antineoplastic compound described in Japanese Patent No. 5,998,289).

[0076] The drug may be a nucleic acid drug. Examples of nucleic acid drugs include siRNA, antisense, miRNA, decoy, aptamer, ribozyme, CpG oligo, and plasmid DNA. These nucleic acid drugs may be those whose in vivo stability has been improved by a method known per se, such as phosphorothioation (changing the oxygen atom double-bonded to phosphorus in the phosphate ester moiety to a sulfur atom) or PEGylation (linking a polyethylene glycol chain to one end).

[0077] In addition, in order to facilitate the uptake of the drug into the cells, a suitable ligand may be bound to the drug or the organic membrane. Examples of the ligand include the ligands disclosed in WO2019 / 208820.

[0078] When a particle encapsulates a drug, the amount of drug encapsulated can be adjusted appropriately, taking into account the particle size, the type of drug, etc. For example, in conventional ADCs, the number of payloads (amount of small molecule drugs) that can be bound is limited by the linker structure, but by using the fusion protein of this embodiment, it is possible to significantly increase the amount of drug that can be carried. This is expected to result in better cancer treatment effects.

[0079] In the conjugate of the embodiment, the fusion protein has a membrane-binding domain (C), and thus can adhere to particles (e.g., particles encapsulating a drug) or immune cells having an organic membrane containing a zwitterionic lipid, thereby encapsulating these. Furthermore, the antibody domain (A) constituting the fusion protein has an antigen-binding site and functions as an antibody, thereby exhibiting high affinity for the antigen and efficiently directing the conjugate encapsulating the particles or cells to a target site (e.g., tumor cells containing the antigen). Upon reaching the target site, the drug is released from the conjugate, thereby damaging the tumor cells and inhibiting their proliferation. Furthermore, the antibody domain (A) has excellent affinity for the antigen, resulting in high target site delivery efficiency, thereby preventing the drug encapsulated in the particles from being released outside the target site. As a result, the side effects (systemic drug exposure) that are a problem with anticancer drugs can be more effectively suppressed.

[0080] (Pharmaceutical Composition) This embodiment relates to a pharmaceutical composition containing the above-described complex. This embodiment may also relate to an anticancer agent containing the above-described complex. The complex of this embodiment can be administered alone as it is, but may also be administered generally as various pharmaceutical preparations (pharmaceutical compositions). Furthermore, pharmaceutical preparations (pharmaceutical compositions) are used in animals and humans.

[0081] The route of administration of the pharmaceutical composition is preferably the most effective route for treatment, and examples thereof include oral or parenteral routes such as rectal, buccal, subcutaneous, intramuscular, and intravenous. Dosage forms include capsules, tablets, granules, powders, syrups, emulsions, suppositories, and injections. Liquid preparations suitable for oral administration, such as emulsions and syrups, can be prepared using water, sugars such as sucrose, sorbitol, and fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, and soybean oil, preservatives such as p-hydroxybenzoic acid esters, and flavors such as strawberry flavor and peppermint. Furthermore, capsules, tablets, powders, granules, etc. can be produced using excipients such as lactose, glucose, sucrose, mannitol, etc., disintegrants such as starch, sodium alginate, etc., lubricants such as magnesium stearate, talc, etc., binders such as polyvinyl alcohol, hydroxypropyl cellulose, gelatin, etc., surfactants such as fatty acid esters, plasticizers such as glycerin, etc.

[0082] Preparations suitable for parenteral administration preferably consist of a sterile aqueous preparation containing an active compound that is isotonic with the recipient's blood. For example, in the case of an injection, a solution for injection is prepared using a carrier such as a saline solution, a glucose solution, or a mixture of saline and glucose solution. In addition, parenteral preparations may also contain one or more auxiliary ingredients selected from glycols, oils, flavors, preservatives (including antioxidants), excipients, disintegrants, lubricants, binders, surfactants, plasticizers, etc., as exemplified for oral preparations.

[0083] The effective dose and frequency of administration of a pharmaceutical composition vary depending on the type and amount of drug encapsulated in the particles, the administration form, the age and body weight of the patient, the nature or severity of the symptoms to be treated, etc., and are determined by comprehensively considering these factors. Furthermore, the higher the affinity for the antigen (e.g., the lower the dissociation constant Kd value), the more likely it is that a smaller dose will produce a medicinal effect.

[0084] The features of the present invention will be explained in more detail below with reference to examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0085] Example 1 (Preparation of SN-131 Fv-Clasp-VH and SN-131 Fv-Clasp-VL) The nucleotide sequence of SEQ ID NO: 117 (SARAH domain nucleotide sequence) was introduced at the C-terminus of the nucleotide sequence of SEQ ID NO: 113 (nucleotide sequence of the heavy chain variable region (VH) of an antibody having a mucin-1-binding site), to prepare DNA encoding SN-131 Fv-Clasp-VH (SEQ ID NO: 121). This was cloned into the NdeI-XhoI site of the expression vector pET-26b. Furthermore, the nucleotide sequence of SEQ ID NO: 118 (SARAH domain nucleotide sequence) was introduced at the C-terminus of the nucleotide sequence of SEQ ID NO: 115 (nucleotide sequence of the light chain variable region (VL) of an antibody having a mucin-1-binding site), to prepare DNA encoding SN-131 Fv-Clasp-VL (SEQ ID NO: 123). This was cloned into the NdeI-XhoI site of the expression vector pET-26b. The amino acid sequences of SN-131 Fv-Clasp-VH and SN-131 Fv-Clasp-VL are shown as SEQ ID NOs: 122 and 124, respectively.

[0086] (Preparation of FMAM (SN-132-VH and SN-132-VL)) Next, the nucleotide sequence (MAP sequence: SEQ ID NO: 125) constituting the membrane-binding domain (C) was introduced into the C-terminus of each of SN-131 Fv-Clasp-VH and SN-131 Fv-Clasp-VL to construct DNAs (SEQ ID NOs: 130 and 132) encoding FMAM (SN-132-VH and SN-132-VL). Specifically, PCR was performed using primers having the nucleotide sequences of SEQ ID NOs: 127 to 129 below and pET-26b containing DNA of SN-131 Fv-Clasp-VH or SN-131 Fv-Clasp-VL as a template to construct DNAs (SEQ ID NOs: 130 and 132) encoding FMAM (SN-132-VH and SN-132-VL). The amino acid sequences of SN-132-VH and SN-132-VL are as shown in SEQ ID NOs: 131 and 133, respectively. Primer (Forward) (SEQ ID NO: 127) ccagaaaagtctgccgttactgcgtgcgattcgccgttgggtgcgcaaataagaattcgagctccgtcgacaagcttg Primer (VH Revers) (SEQ ID NO: 128) taggctttgcgatagatcttaaacgaggtgttttggctcccaccgccgcctttggcttcaatcgcatcgagaatcggc Primer (VL Revers) (SEQ ID NO: 129) taggctttgcgatagatcttaaacgaggtgttttggctcccaccgccgcctttggcttcgattgcgtcgagaatcgg

[0087] The PCR product obtained above was treated with DnpI, and the resulting DNA was ligated and introduced into XL10Gold. The transformed E. coli was cultured overnight on agar medium. The plasmid was purified from the cultured E. coli. Next, PCR was performed using Big Dye 3.1, followed by sequencing to confirm that the plasmid contained the FMAM sequence, including the MAP sequence.

[0088] (Expression of fusion protein) Each of the prepared plasmids was introduced into BL21star (DE3) Escherichia coli strain, inoculated into 5 mL of LB medium (containing 25 μg / mL Kanamycin), and cultured at 37° C. with shaking at 150 rpm. ６００ｎｍ Expression was induced by adding IPTG to a final concentration of 0.5 mM at pH 7.0 or 0.8, and the cells were cultured overnight at 37°C with shaking at 150 rpm. SDS-PAGE confirmed that the cultured E. coli expressed the target protein. As shown in Figure 2, the result of SDS-PAGE (12.5%) confirmed the target protein band at around 20 kDa.

[0089] (Large-scale culture, refolding, and purification of fusion proteins) Each of the prepared plasmids was introduced into BL21star (DE3) Escherichia coli strain, inoculated into 5 mL of LB medium (containing 25 μg / mL Kanamycin), and cultured with shaking at 37° C. and 150 rpm. Next, the culture was inoculated into 2 L of LB medium (containing 25 μg / mL Kanamycin) and cultured at OD of 1000. ６００ｎｍExpression was induced by adding IPTG to a final concentration of 0.5 mM at pH 7.0 = 0.8, and the mixture was cultured at 37 ° C. with shaking at 150 rpm for 5 hours. The E. coli suspension was centrifuged at 5,000 g for 20 minutes to recover the E. coli. The E. coli was disrupted using ultrasound, then centrifuged at 16,000 g to remove the supernatant, and each was dissolved in 6 mL of solubilization buffer (6 M guanidine hydrochloride, Tris-HCl, 150 mM NaCl, pH 8.0, 375 μM 2-mercaptoethanol). SN-132 (SN-132-VH + SN-132-VL) was refolded, precipitated with ammonium sulfate, and then dialyzed according to the method described in Takagi et al., Structure 2017, 25, 1611-1622. The resulting fusion protein (SN-132) was purified by gel filtration chromatography and an affinity column (Nishimura S.-I. et al., ACS Omega 2017, 2, 7493-7505; RSC Chem. Biol. 2023) displaying the MUC1 glycopeptide KSAPDT(Galβ1→3GalNAcα1)RPAPG (SEQ ID NO: 134) on Hi-trap NHS activated (1 mL) (Figure 3). The yield of purified SN-132 was 0.1 mg.

[0090] The affinity of purified SN-132 for the epitope was evaluated using the interaction analysis system Biacore. As a result, SN-132 showed high affinity (K Ｄ = 0.6 nM). This value is the same as that of the SN131 antibody Fab (K ＤThis value exceeded the 1.58 nM (Figure 4) (Nishimura, S.-I., Structural and molecular insight into antibody recognition of dynamic neoepitopes in membrane-tethered MUC1 of pancreatic cancer cells and secreted exosomes. RSC Chemical Biol. 2023).

[0091] (Immobilization on lipid membrane-coated nanoparticles (nanosomes)) MeOH (150 μL) and i-PrOH (300 μL) were added to 150 μL of TOPO-QD655 solution (1 μM, Invitrogen), and the nanoparticles were precipitated by centrifugation, after which the solvent was removed. Hexane was added to resuspend the nanoparticles, and the nanoparticles were immobilized with PC linker (100 mM, 20 mL / MeOH) and NaBH as a phospholipid model compound in which phosphocholine is ester-linked. ４ The nanosomes were purified by ultrafiltration (Amicon Ultra 0.5, 100K) and the solvent was replaced with D-PBS. The final solution volume was concentrated to 50 μL. A 10 mM SN-132 solution (D-PBS) was added to the nanosomes at a molar ratio of 1:5 to the fusion protein (SN-132), and the mixture was left to stand at 4°C for 2 hours (Figure 5). Excess SN-132 was removed using an ultrafiltration device (Amicon Ultra 0.5, 100K). Before and after mixing with the fusion protein, the particle size and its distribution were measured using a dynamic light scattering (DLS) device. As a result, it was revealed that the particle size distribution of the SN-132-loaded nanoparticles was shifted to the larger side, and that the fusion protein had bound to form SN-132-loaded nanosomes (complex) (Figure 6).

[0092] (Cancer cell-specific active DDS function of nanosome-carrying SN-132) Human breast cancer cells (MCF-7) were seeded at a split ratio of 1 / 5, 200 μL / well, and incubated in D-MEM High glucose, 10% fetal bovine serum (FBS) at 37°C, 5% CO ２ The cells were incubated for 48 hours under a 50°C ambient atmosphere. Furthermore, 10 nM nanoparticles (PCSAM-coated-QD655) or 10 nM nanosome-carrying FMAM (SN-132) were added and incubated for 24 hours. A system was also prepared in which 10 μM of MUC1 glycopeptide (antigen), which can be a competitive inhibitor of SN-132, and SN-131 mAb (monoclonal antibody) (0.01 mg / mL) were co-cultured, and the cells were co-cultured for 24 hours. As a negative control, a mixture of nanoparticles (PCSAM-coated-QD655) / SN-131Fab (a mixture of nanosomes and 3 mg / mL of SN-131Fab) was also prepared and co-cultured with cancer cells under similar conditions. SN-131Fab is the antigen-binding site that is part of SN-131mAb.

[0093] After removing the medium and washing once with Opti-MEM, the medium was replaced with Opti-MEM containing 100 nM LysoTracker Green DND-26 and 2.5 μg / mL Heochst 33342, and the cells were incubated at 37°C, 5% CO ２ The nanoparticles were incubated under ambient conditions for 30 minutes. After washing twice with Opti-MEM, they were imaged using a fluorescence microscope (Figure 7). The results shown in Figure 7 clearly demonstrate that the nanoparticles (PCSAM-coated QD655) were only taken up by breast cancer cells (MCF7) once FMAM (SN-132) was encapsulated in the surface phospholipid membrane (A-C), and further demonstrate the importance of endocytosis due to the specific interaction between the MUC1 glycopeptide epitope on the breast cancer cell membrane and FMAM (SN-132) (C-E).

[0094] Furthermore, the MUC1 glycopeptide epitope-specific uptake of nanosome-carrying FMAM (SN-132) into human breast cancer cells (MCF-7) was confirmed by flow cytometry. Human breast cancer cells (MCF-7) and human lung epithelial adenocarcinoma cells (A549) were cultured in D-MEM (10% FBS) containing 5 nM nanoparticles (PCSAM-coated QD655), 5 nM nanosome-carrying FMAM (SN-132), and 5 nM nanosome-carrying FMAM (SN-132) + 1 μM MUC1 glycopeptide. ２ The cells were incubated for 24 hours under ambient conditions. The culture medium was removed, and the cells were washed with D-PBS. A 0.25 w / v% trypsin-1 mM EDTA 4Na solution was added and incubated at 37°C for 5 minutes. This detached the cells from the dish, and the FMAM (SN-132) carrying the nanosomes bound to the cancer cell surface was also removed. D-MEM (10% FBS) was added to the dish to stop the action of trypsin, and the cell suspension was collected in a centrifuge tube and the supernatant was removed by centrifugation. The cells were washed twice with D-PBS and then suspended in a D-PBS solution containing 2 mM EDTA 2Na. The fluorescence intensity of the cancer cells was measured using a FACSCanto (BD biosciences). The excitation wavelength was set to 633 nm, and the detection wavelength was set to 650-670 nm. Analysis was performed using an FCS analyzer.

[0095] Figure 8 shows the results of flow cytometry. The results also clearly demonstrate that the uptake of nanosome-carrying FMAM (SN-132) into cancer cells is specific to MCF-7 cells, which express MUC1 on their cell surface, containing the glycopeptide epitope recognized by SN-132, as evidenced by the inhibitory effect of 1 mM MUC1 glycopeptide (Figure 8A). In contrast, lung cancer cells A549 do not express the MUC1 epitope recognized by SN-132 (Nishimura S.-I. et al., ACS Omega 2017, 2, 7493-7505), and therefore, no uptake into these cancer cells (Figure 8B).

[0096] As described above, it has been demonstrated that FMAM (SN-132) with antibody functionality can be easily immobilized by controlling its orientation on the surface of phospholipids with phosphorylcholine groups at their head groups, such as phospholipid-coated quantum dots (PCSAM-coated nanosomes). FMAM (SN-132) can be easily immobilized by controlling its orientation on the surface of various nanoparticles, such as liposomes whose main phospholipid is phosphatidylcholine containing phosphorylcholine groups, extracellular particles such as immune cells and exosomes whose main components are phosphatidylcholine and sphingomyelin, and nanoparticles coated with amphiphilic compounds similar to phospholipids with phosphorylcholine groups at their head groups, such as the phospholipid-coated quantum dots (PCSAM-coated nanosomes) of this example. Therefore, for example, as in this example, a complex (FMAM (SN-132) carrying nanosomes) can be prepared. Then, FMAM (SN-132) with antibody functionality binds to a specific antigen and is directed to cells that present that specific antigen. By encapsulating nucleic acid medicines or small molecule drugs in nanosomes, the drugs can be efficiently delivered to cells that present specific antigens.

[0097] SEQ ID NO: 113 (Fv-VH base sequence) caagttcagctgcaacagtctggtgcggaattagcgcgtcctggagcaagcgtcaaactctcctgcaaagcgagtggctataccttcaccaactatggcctgagctgggtgaaacaacgcaccggtcaaggcttggagtggattggcgaaaatcatccagggagtggtatcatctac cacaacgagaagtttcgcggaaaagcaacgctgactgctgacaagtcgtcgtctaccgcctatgtgcagctgagctcgttaacgtccg aagattccgcggtgtacttttgcgcccgttcaagtggtactcgtggctttgcctattggggtcaagggacccttgttacggtatcggct

[0098] SEQ ID NO: 114 (Fv-VH amino acid sequence) QVQLQQSGAELARPGASVKLSCKASGYTFTNYGLSWVKQRTGQGLEWIGENHPGSGIIYHNEKFRGKATLTADKSSSTAYVQLSSLTSEDSAVYFCARSSGTRGFAYWGQGTLVTVSA

[0099] SEQ ID NO: 115 (Fv-VL base sequence) gacgtcttgatgacccaaaccccgctttctttacccgtttcgttgggcgatcaagcgagcattagctgtcgttcgtcgcagagcattgtgcactccaatggcaacacgtatctggaatggtatctgcagaaaccgggtcagtcaccgaaactgctgatctacaaagtcagc aaccgcttttcaggcgtaccagatcggttttctgggagtgggagtggcactgatttcaccctgaagatcagtcgcgttgaagcggaagatctgggtgtgtactactgcttccaaggctcccatggtccttggacgtttggtggaggaaccaaactggaaatcaagcgtgcg

[0100] SEQ ID NO: 116 (Fv-VL amino acid sequence) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHGPWTFGGGTKLEIKRA

[0101] SEQ ID NO: 117 (SARAH base sequence) (VH) gactacgagttcctgaaaagctggacagtcgaagatctgcagaaacgcttgctggcactggatccgatgatggaacaggaaatcgaggaaattcgccagaagtatcagagcaaacggcagccgattctcgatgcgattgaagccaaa SEQ ID NO: 118 (SARAH base sequence) (VL) gactatgagttcctgaaatcctggacagtggaagatctccagaaacgcttacttgctctggatccaatgatggaacaggaaattgaggagattcgccagaagtatcagtgcaaacgtcaaccgattctcgacgcaatcgaagccaaa

[0102] SEQ ID NO: 119 (SARAH amino acid sequence) (VH) DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAK SEQ ID NO: 120 (SARAH amino acid sequence) (VL) DYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAK

[0103] SEQ ID NO: 121 (SN-131 Fv-Clasp-VH base sequence) caagttcagctgcaacagtctggtgcggaattagcgcgtcctggagcaagcgtcaaactctcctgcaaagcgagtggctataccttcaccaactatggcctgagctgggtgaaacaacgcaccggtc aaggcttggagtggattggcgaaaatcatccagggagtggtatcatctaccacaacgagaagtttcgcggaaaagcaacgctgactgctgacaagtcgtcgtctaccgcctatgtgcagctgagctcg ttaacgtccgaagattccgcggtgtacttttgcgcccgttcaagtggtactcgtggctttgcctattggggtcaagggacccttgttacggtatgtgctggctcagactacgagttcctgaaaagct ggacagtcgaagatctgcagaaacgcttgctggcactggatccgatgatggaacaggaaatcgaggaaattcgccagaagtatcagagcaaacggcagccgattctcgatgcgattgaagccaaataa

[0104] SEQ ID NO: 122 (amino acid sequence of SN-131 Fv-Clasp-VH) QVQLQQSGAELARPGASVKLSCKASGYTFTNYGLSWVKQRTGQGLEWIGENHPGSGIIYHNEKFRGKATLTADKSSSTAYVQLSSLTSEDSAVYFCARSSGTRGFAYWGQGTLVTVCAGSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAK

[0105] SEQ ID NO: 123 (SN-131 Fv-Clasp-VL base sequence) gacgtcttgatgacccaaaccccgctttctttacccgtttcgttgggcgatcaagcgagcattagctgtcgttcgtcgcagagcattgtgcactccaatggcaacacgtatctggaatggtat ctgcagaaaccgggtcagtcaccgaaactgctgatctacaaagtcagcaaccgcttttcaggcgtaccagatcggttttctgggagtgggagtggcactgatttcaccctgaagatcagtcgcg ttgaagcggaagatctgggtgtgtactactgcttccaaggctcccatggtccttggacgtttggtggaggaaccaaactggaaatcaagcgtggtagcgactatgagttcctgaaatcctggac agtggaagatctccagaaacgcttacttgctctggatccaatgatggaacaggaaattgaggagattcgccagaagtatcagtgcaaacgtcaaccgattctcgacgcaatcgaagccaaataa

[0106] SEQ ID NO: 124 (amino acid sequence of SN-131 Fv-Clasp-VL) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHGPWTFGGGTKLEIKRGSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAK

[0107] SEQ ID NO: 125 (nucleotide sequence of MAP sequence) agccaaaacacctcgtttaagatctatcgcaaagcctaccagaaaagtctgccgttactgcgtgcgattcgccgttgggtgcgcaaa

[0108] SEQ ID NO: 126 (amino acid sequence of MAP sequence) SQNTSFKIYRKAYQKSLPLLRAIRRWVRK

[0109] SEQ ID NO: 127 (Primer (Forward)) ccagaaaagtctgccgttactgcgtgcgattcgccgttgggtgcgcaaataagaattcgagctccgtcgacaagcttg

[0110] SEQ ID NO: 128 (Primer (VH Revers)) taggctttgcgatagatcttaaacgaggtgttttggctcccaccgccgcctttggcttcaatcgcatcgagaatcggc

[0111] SEQ ID NO: 129 (Primer (VL Revers)) taggctttgcgatagatcttaaacgaggtgttttggctcccaccgccgcctttggcttcgattgcgtcgagaatcgg

[0112] SEQ ID NO: 130 (SN-132 VH base sequence) tctaaaattaaacaagttcagctgcaacagtctggtgcggaattagcgcgtcctggagcaagcgtcaaactctcctgcaaagcgagtggctataccttcaccaactatggcctgagctgggtgaaacaacgcaccggtcaaggcttggagtggat tggcgaaaatcatccagggagtggtatcatctaccacaacgagaagtttcgcggaaaagcaacgctgactgctgacaagtcgtcgtctaccgcctatgtgcagctgagctcgttaacgtccgaagattccgcggtgtacttttgcgcccgttcaa gtggtactcgtggctttgcctattggggtcaagggacccttgttacggtatgtgctggctcagactacgagttcctgaaaagctggacagtcgaagatctgcagaaacgcttgctggcactggatccgatgatggaacaggaaatcgaggaaatt cgccagaagtatcagagcaaacggcagccgattctcgatgcgattgaagccaaaggcggcggtgggagccaaaacacctcgtttaagatctatcgcaaagcctaccagaaaagtctgccgttactgcgtgcgattcgccgttgggtgcgcaaataa

[0113] SEQ ID NO: 131 (amino acid sequence of SN132 VH) SKIKQVQLQQSGAELARPGASVKLSCKASGYTFTNYGLSWVKQRTGQGLEWIGENHPGSGIIYHNEKFRGKATLTADKSSSTAYVQLSSLTSEDSAVYFCARSSGTRGFAYWGQGTLVTVCAGSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAKGGGGSQNTSFKIYRKAYQKSLPLLRAIRRWVRK

[0114] SEQ ID NO: 132 (SN-132 VL base sequence) gacgtcttgatgacccaaaccccgctttctttacccgtttcgttgggcgatcaagcgagcattagctgtcgttcgtcgcagagcattgtgcactccaatggcaacacgtatctggaatggtatctgcagaaaccgggtcagtcaccga aactgctgatctacaaagtcagcaaccgcttttcaggcgtaccagatcggttttctgggagtgggagtggcactgatttcaccctgaagatcagtcgcgttgaagcggaagatctgggtgtgtactactgcttccaaggctcccatggt ccttggacgtttggtggaggaaccaaactggaaatcaagcgtggtagcgactatgagttcctgaaatcctggacagtggaagatctccagaaacgcttacttgctctggatccaatgatggaacaggaaattgaggagattcgccaga agtatcagtgcaaacgtcaaccgattctcgacgcaatcgaagccaaaggcggcggtgggagccaaaacacctcgtttaagatctatcgcaaagcctaccagaaaagtctgccgttactgcgtgcgattcgccgttgggtgcgcaaataa

[0115] SEQ ID NO: 133 (amino acid sequence of SN132 VL) DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHGPWTFGGGTKLEIKRGSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAKGGGGSQNTSFKIYRKAYQKSLPLLRAIRRWVRK

[0116] Example 2 (Preparation of Herceptin FMAM VH and Herceptin FMAM VL) DNAs (SEQ ID NOs: 136 and 138) encoding FMAM (Herceptin FMAM VH and Herceptin FMAM VL) containing all of the sequences of Herceptin Fv VH and Herceptin Fv VL, as well as the SARAH domain and MAP sequence, were prepared. These were cloned into the NdeI-XhoI site of the expression vector pET-26b to prepare plasmids. The prepared plasmids were each introduced into XL10Gold. The transformed E. coli was cultured overnight on agar medium. The plasmid was purified from the cultured E. coli. Next, PCR was performed using Big Dye 3.1, followed by sequencing to confirm that the plasmid had an FMAM sequence including a MAP sequence. The amino acid sequences of Herceptin FMAM VH and Herceptin FMAM VL are set forth in SEQ ID NOs: 137 and 139, respectively.

[0117] (Expression of fusion protein) Each of the prepared plasmids was introduced into BL21star (DE3) Escherichia coli strain, inoculated into 5 mL of LB medium (containing 25 μg / mL Kanamycin), and cultured at 37° C. with shaking at 150 rpm. ６００ｎｍ IPTG was added to a final concentration of 1.0 mM at a pH of 1.0 to induce expression, and the mixture was cultured overnight with shaking at 37°C and 150 rpm. SDS-PAGE confirmed that the cultured E. coli expressed the target protein.

[0118] (Large-scale culture, refolding, and purification of fusion proteins) Each of the prepared plasmids was introduced into BL21star (DE3) Escherichia coli strain, inoculated into 5 mL of LB medium (containing 25 μg / mL Kanamycin), and cultured with shaking at 37° C. and 150 rpm. Next, the culture was inoculated into 2 L of LB medium (containing 25 μg / mL Kanamycin) and cultured at OD of 1000. ６００ｎｍExpression was induced by adding IPTG to a final concentration of 1.0 mM at a pH of 1.0, and the cells were cultured at 37°C and 150 rpm for 5 hours with shaking. The E. coli suspension was centrifuged at 5,000 g for 20 minutes to recover the E. coli. The E. coli cells were disrupted using ultrasound, then centrifuged at 16,000 g to remove the supernatant, and each cell was dissolved in 6 mL of solubilization buffer (6 M guanidine hydrochloride, Tris-HCl, 150 mM NaCl, pH 8.0, 375 μM 2-mercaptoethanol). Herceptin FMAM (Herceptin FMAM VH and Herceptin FMAM VL) was refolded, precipitated with ammonium sulfate, and then dialyzed according to the method described in Takagi et al., Structure 2017, 25, 1611-1622. The resulting fusion protein (Herceptin FMAM) was purified by gel filtration chromatography and a His-trap affinity column (FIG. 9).

[0119] The affinity of purified Herceptin FMAM for HER2 was evaluated by competitive ELISA assay with Herceptin. The results showed that Herceptin FMAM binds to HER2 with higher affinity than Herceptin Fab (Figure 10). The IC calculated from Figure 10 ５０ was 0.36 μg / mL for Herceptin Fab and 0.04 μg / mL for Herceptin FMAM.

[0120] Sequence number 136 (base sequence of Herceptin FMAM VH) catcatcaccatcatcacgaagtgcaactggtggaatccggtggcggactggtccaaccaggtggttcgttacgcctgtcatgtgcggcttccgggtttaacatcaaagacacgtacattcactgggtacgtcaagctcctgggaaaggccttgaatgggttgcacggatttatccgactaatggctatacacgctatgcagattccgttaagggacgctttacgatctcagccgataccagcaagaataccgcgtatcttcagatgaacagcttgcgtgcggaagataccgcagtctactactgctctcgttggggtggcgatggcttttacgccatggactattggggccaaggcactctcgtaacggtgtgcagtgggtcggactatgagttcctgaaaagttggacagttgaggatctgcagaaacgtctgctggccttggaccccatgatggaacaggaaatcgaggaaattcgccagaaataccagagcaaacgccagccgattctggatgcgattgaagccaaaggcggtggtgggagtggcggtggcggttctggtggtggcggaagccagaacaccagcttcaaaatctaccgcaaagcgtatcagaaatcgctgccgctcttacggaccattcgtcgctgggtgaagaaataa

[0121] Sequence number 137 (amino acid sequence of Herceptin FMAM VH) HHHHHHEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVCSGSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQSKRQPILDAIEAKGGGGSGGGGSGGGGSQNTSFKIYRKAYQKSLPLLRTIRRWVKK

[0122] SEQ ID NO: 138 (base sequence of Herceptin FMAM VL) gatatccagatgacccagtctccgtcctcactttctgctagcgtaggtgatcgtgtgacaatcacgtgtcgtgcaagccaagacgtcaatacggctgttgcgtggtatcaacagaaaccggggaaagcgcccaaactgctgatttactcggc gtcattcctgtatagcggcgttccttcccgctttagcggcagtcgttcgggcacagactttaccttgaccattagcagcttgcagccagaagattttgccacgtattattgccaacagcattacaccactccgcctacctttgggcaaggca cgaaagtggagatcaaacgcggcagtgactacgagttcctgaaatcgtggactgtggaagatctgcagaaacggctgttagcactggatccgatgatggaacaggaaatcgaagagattcgccagaaataccaatgcaaacgccaaccgatt ctcgatgcgattgaagccaaaggtggtggtggctccggtggaggtgggtcaggtggcggaggcagtcagaacacctcgttcaagatctatcgcaaggcctatcagaaatctctgccactcttacgcaccattcgtcgttgggtcaagaagtaa

[0123] SEQ ID NO: 139 (amino acid sequence of Herceptin FMAM VL) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAKGGGGSGGGGSGGGGSQNTSFKIYRKAYQKSLPLLRTIRRWVKK

[0124] Example 3 (Preparation of FMAM (SN-132-VL')) A DNA (SEQ ID NO: 140) encoding FMAM (SN-132-VL') containing the SARAH domain and MAP sequence in addition to the sequence of SN-131 Fv VL was prepared. This was cloned into the NedI-XhoI site of the expression vector pET-26b. The plasmid obtained above was introduced into a DH5α Escherichia coli strain and cultured overnight on an agar medium. The plasmid was purified from the cultured E. coli. Next, PCR was performed using Big Dye 3.1, followed by sequencing to confirm that the plasmid contained the sequence of FMAM (SN-132-VL'). The amino acid sequence of SN-132-VL' is set forth in SEQ ID NO: 141.

[0125] (Expression and Mass Culturing of Fusion Proteins) The plasmid containing the SN-132-VH sequence prepared in Example 1 and the plasmid containing the SN-132-VL' sequence prepared in Example 3 were each introduced into the BL21star (DE3) Escherichia coli strain, inoculated into 5 mL of LB medium (containing 25 μg / mL Kanamycin), and cultured overnight with shaking at 37 ° C. and 150 rpm. Next, the strains were inoculated into 1 L of LB medium (containing 25 μg / mL Kanamycin), and cultured until SN-132-VH reached an O.D. 600 nm of 1.0 and SN-132-VL' reached an O.D. 600 nm of 0.6. Thereafter, expression was induced by adding IPTG to a final concentration of 0.5 mM, and the strains were cultured with shaking at 37 ° C. and 150 rpm for 5 hours. The E. coli suspension was centrifuged at 5,000 g for 20 minutes to recover the E. coli. The E. coli was disrupted using ultrasound, and then centrifuged at 15,000 g for 15 minutes to remove the supernatant. The precipitate was washed with Triton wash buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5% Triton). ＴＭ The cells were washed with PBS (X-100, pH 8.0). SDS-PAGE confirmed that the E. coli expressed the target protein and that the target protein was present in the insoluble fraction after disruption. As shown in Figure 11, the result of SDS-PAGE (15%) confirmed the target protein band at around 20 kDa.

[0126] (Refolding and purification of fusion protein) Each disrupted sample was dissolved in 6 mL of solubilization buffer (6 M guanidine hydrochloride, 50 mM Tris-HCl, 150 mM NaCl, pH 8.0, 375 μM 2-mercaptoethanol), and then centrifuged at 15,000 g for 20 minutes to collect the supernatant. SN-132-VH and SN-132-VL' were mixed in equimolar amounts, and 1 M DTT was added with stirring to a final concentration of 1 mM. Stirring was stopped, and the mixture was incubated at room temperature for 1 hour. Using a dropping funnel, refolding buffer (100 mM Tris-HCl, 0.4 mM L-Arg Hydrochloride, 2 mM EDTA (3Na), 3.73 mM Cystamine, 3.73 mM Cystamine) was added to the sample, and the sample was diluted 4-fold to a concentration of 1.5 M guanidine hydrochloride. The sample was added dropwise to the refolding buffer while stirring on ice. Refolding was carried out by leaving the solution at 4°C for 2 days with stirring. The resulting fusion protein (SN-132') was purified by gel filtration chromatography and an affinity column (Nishimura S.-I. et al., ACS Omega 2017, 2, 7493-7505; RSC Chem. Biol. 2023) displaying the MUC1 glycopeptide KSAPDT(Galβ1→3GalNAcα1)RPAPG (SEQ ID NO: 134) on Hi-trap NHS activated (1 mL) (Figure 12). The yield of purified SN-132' was 2.7 mg, a significant improvement achieved by improving the fusion protein sequence and refolding process.

[0127] SEQ ID NO: 140 (base sequence of SN-132-VL') gacgtcttgatgacccaaaccccgctttctttacccgtttcgttgggcgatcaagcgagcattagctgtcgttcgtcgcagagcattgtgcactccaatggcaacacgtatctggaatggtatctgcagaaaccgggtcagtca ccgaaactgctgatctacaaagtcagcaaccgcttttcaggcgtaccagatcggttttctgggagtgggagtggcactgatttcaccctgaagatcagtcgcgttgaagcggaagatctgggtgtgtactactgcttccaaggct cccatggtccttggacgtttggtggaggaaccaaactggaaatcaagcgtggtagcgactatgagttcctgaaatcctggacagtggaagatctccagaaacgcttacttgctctggatccaatgatggaacaggaaattgagga gattcgccagaagtatcagtgcaaacgtcaaccgattctcgacgcaatcgaagccaaaggcggtggcggtagctttaaaatctatcgcaaagcctaccagaaaagtctgccgttactgcgtaccattcgccgttgggtgaaaaaa

[0128] SEQ ID NO: 141 (amino acid sequence of SN-132-VL') DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHGPWTFGGGTKLEIKRGSDYEFLKSWTVEDLQKRLLALDPMMEQEIEEIRQKYQCKRQPILDAIEAKGGGGSFKIYRKAYQKSLPLLRTIRRWVKK

[0129] 100 Fusion protein A: antibody domain (A) B: stabilizing domain (B) C: membrane-binding domain (C) a1: peptide chain comprising an immunoglobulin heavy chain variable region (VH) domain (VH peptide chain (a1)) a2: peptide chain comprising an immunoglobulin light chain variable region (VL) domain (VL peptide chain (a2)) b1: first peptide chain constituting the stabilizing domain (B) (b1) b2: second peptide chain constituting the stabilizing domain (B) (b2) c1: first membrane-binding peptide chain (c1) c2: second membrane-binding peptide chain (c2)

Claims

1. A fusion protein comprising an antibody domain (A) having at least one antigen-binding site, a stabilizing domain (B) having an α-helix structure and linked to the antibody domain (A) directly or via a linker, and a membrane adhesion domain (C) linked to the stabilizing domain (B) directly or via a linker.

2. The antibody domain (A) comprises a peptide chain (a1) containing an immunoglobulin heavy chain variable region (VH) domain and a peptide chain (a2) containing an immunoglobulin light chain variable region (VL) domain. The stabilizing domain (B) comprises a first peptide chain (b1) and a second peptide chain (b2), and the first peptide chain (b1) and the second peptide chain (b2) have a coiled-coil structure. The membrane adhesion domain (C) comprises a first membrane adhesion peptide chain (c1) and a second membrane adhesion peptide chain (c2). The C-terminus of the peptide chain (a1) is linked to the N-terminus of the first peptide chain (b1) directly or via a linker, and the C-terminus of the peptide chain (a2) is linked to the N-terminus of the second peptide chain (b2) directly or via a linker. The C-terminus of the first peptide chain (b1) is linked to the N-terminus of the first membrane adhesion peptide chain (c1) directly or via a linker, and the C-terminus of the second peptide chain (b2) is linked to the N-terminus of the second membrane adhesion peptide chain (c2) directly or via a linker. The fusion protein according to claim 1.

3. The fusion protein according to claim 1, wherein the antigen-binding site is a binding site to an epitope.

4. The fusion protein according to claim 1, wherein the antigen-binding site is a binding site to an epitope present on the surface of tumor cells.

5. The fusion protein according to claim 1, wherein the stabilizing domain (B) consists of a SARAH domain derived from human Mst1 kinase.

6. The fusion protein according to claim 1, wherein the membrane adhesion domain (C) has the property of adhering to an organic membrane containing a zwitterionic lipid.

7. The fusion protein according to claim 1, wherein the membrane adhesion domain (C) is amphiphilic and consists of a basic peptide chain.

8. The fusion protein according to claim 1, wherein the membrane adhesion domain (C) has the property of adhering to a phospholipid membrane.

9. The fusion protein according to claim 1, wherein the membrane adhesion domain (C) consists of the C-terminal polypeptide of Helicobacter pylori-derived α1,3 / α1,4-fucosyltransferase.

10. The fusion protein according to claim 1, wherein the membrane adhesion domain (C) has an amino acid sequence selected from the following (i) to (vi). (i) SKIYRYKAYQKSLPLLRAIRRWVRKK (SEQ ID NO: 105) (ii) FKIYRYKAYQKSLPLLRAIRRWVRK (SEQ ID NO: 106) (iii) FKIYRKITYQKSLPLLRVIRRWVRKK (SEQ ID NO: 107) (iv) FKIYRYKAYQKSLPLLRAVRKLVRKK (SEQ ID NO: 108) (v) FKIYRYKAYQKSLPLLRTIRRWVRKK (SEQ ID NO: 109) (vi) FKIYRYKAYQKSLPLLRAIRRWVRKK (SEQ ID NO: 110) 11. A complex comprising the fusion protein according to any one of claims 1 to 10 and particles having an organic membrane containing a lipid having an amphoteric ion.

12. The complex according to claim 11, wherein the particles encapsulate a drug.

13. The complex according to claim 11, wherein the particles are liposomes.

14. A pharmaceutical composition comprising the complex according to claim 11.

15. An anticancer agent comprising the complex according to claim 11.

16. A polynucleotide encoding the fusion protein according to any one of claims 1 to 10.

17. An expression vector containing the polynucleotide according to claim 16.

18. A transformant transformed with the expression vector according to claim 17.

19. A method for producing a fusion protein, comprising preparing an expression vector containing a polynucleotide encoding the fusion protein according to any one of claims 1 to 10, and culturing a transformant obtained by introducing the expression vector into a host cell.

20. The antibody domain (A) includes a peptide chain (a1) containing an immunoglobulin heavy chain variable region (VH) domain and a peptide chain (a2) containing an immunoglobulin light chain variable region (VL) domain. The stabilization domain (B) includes a first peptide chain (b1) having an α-helix structure and a second peptide chain (b2) having an α-helix structure, and the first peptide chain (b1) and the second peptide chain (b2) have a coiled-coil structure. The membrane adhesion domain (C) includes a first membrane adhesion peptide chain (c1) and a second membrane adhesion peptide chain (c2). A step of obtaining a first fusion protein by preparing a first expression vector having a polynucleotide encoding the peptide chain (a1), the first peptide chain (b1), and the first membrane adhesion peptide chain (c1), introducing the first expression vector into a host cell, and culturing the obtained transformant; a step of preparing a second expression vector having a polynucleotide encoding the peptide chain (a2), the second peptide chain (b2), and the second membrane adhesion peptide chain (c2); a step of obtaining a second fusion protein by introducing the second expression vector into a host cell and culturing the obtained transformant; and a step of folding the first fusion protein and the second fusion protein. The method for producing a fusion protein according to claim 19.

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