Fusion protein platform with improved half-life

Abstract

The present invention relates to a fusion protein platform with improved half-life, which uses an IgG Fc region, an IgM region, a J chain, and / or a linker. The present invention also relates to a fusion protein produced through the platform, and in particular, to a fusion protein comprising an IgG Fc region, an IgM region, a J chain, a linker, and / or a biologically active molecule. The fusion protein platform according to the present invention can be used in combination with various biologically active molecules selected according to the purpose. In addition, the biologically active molecules included in the platform can have increased binding activity. Therefore, the fusion protein produced through the platform has excellent effect of recognizing and regulating the target, and there is little concern of unpredictable side effects due to non-specific binding. In addition, the half-life in the blood of the body is increased, so that the therapeutic effect to be achieved through the fusion protein can be maximized.

Description

[Technical field]

[0001] The present invention relates to a fusion protein platform with improved half-life, which uses an IgG Fc region, an IgM region, a J chain, and / or a linker, and also relates to a fusion protein produced through said platform, specifically, a fusion protein comprising an IgG Fc region, an IgM region, a J chain, a linker, and / or a biologically active molecule. [Background technology]

[0002] Various types of biologically active molecules are used for the purpose of treating diseases, and since the activity of the biologically active molecules is directly related to the therapeutic effect, various approaches have been taken to enhance the activity.

[0003] One method for enhancing the therapeutic effect of biologically active molecules is that biological activity is mainly achieved through the process in which active molecules, including ligands, bind to receptors present in the body and transmit signals. Therefore, by increasing the affinity or avidity of the active molecule with the target receptor, the receptor can be activated even at a lower concentration of the active molecule in the body, thereby improving the therapeutic effect of the active molecule.

[0004] Therefore, in order to increase the binding activity of active molecules, methods have been developed such as fusing the fragment crystallizable region (Fc region), which is the heavy chain invariant region of IgG, one of the immunoglobulin (Ig) isotypes, to proteins or utilizing biotin-streptavidin binding. However, the effect of increasing the binding activity of Fc fusion proteins, which are dimers, is not great, and in the case of biotin-streptavidin-based substances, the process of manufacturing the polymers is complicated, making it difficult to ensure a quality that can be used as a pharmaceutical.

[0005] Therefore, there remains a demand for the development of technologies for enhancing the activity of biologically active molecules, or for protein pharmaceuticals having enhanced therapeutic effects by enhancing the activity of biologically active molecules. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have studied a method for increasing the in vivo blood half-life of a multimeric fusion protein and minimizing side effects, and have developed a platform that (1) imparts FcRn binding ability to a fusion protein containing IgM Fc, and (2) minimizes the binding ability of the fusion protein to an IgM-specific receptor. As a result, it has been experimentally demonstrated that the fusion protein produced through the platform has increased avidity of biological molecules, maximizes target binding ability and blood half-life increase effect, and minimizes the possibility of side effects due to non-specific binding, leading to the completion of the present invention. [Means for solving the problem]

[0007] Each description and embodiment of the present invention is applicable to each other description and embodiment, and the scope of the present invention is not limited to the specific description given below.

[0008] Furthermore, terms not specifically defined herein have the meanings commonly used in the art to which the present invention pertains. Furthermore, unless otherwise defined in the context, the singular includes the plural and the plural includes the singular.

[0009] One aspect of the present invention provides a fusion protein having the following structural formula: [(X) a -(Y) b -(IgG Fc region) c -(IgM Fc region)] m -(Z) n In this case, in the structural formula: X is a biologically active molecule; said Y being a linker; the IgG Fc region is monomeric; the IgM Fc region is monomeric; said Z being a J chain; The a is 1 or 2, the b is 1 or 2, the c is 0 or 1, the m is 5 or 6, and the n is 0 or 1.

[0010] In one embodiment according to the present invention, a may be 2, b may be 2, c may be 1, m may be 5, and n may be 1. For example, when a is 2, b is 2, c is 1, m is 5, and n is 1, the fusion protein may be interchangeably represented as [(X)2-(Y)2-(IgG Fc region)1-(IgM Fc region)]5-(Z)1 or [(X)2-(Y)2-(IgG Fc region)-(IgM Fc region)]5-(Z).

[0011] In another embodiment according to the present invention, a may be 2, b may be 2, c may be 0, m may be 5, and n may be 1. For example, when a is 2, b is 2, c is 0, m is 5, and n is 1, the fusion protein may be interchangeably represented as [(X)2-(Y)2-(IgG Fc region)0-(IgM Fc region)]5-(Z)1 or [(X)2-(Y)2-(IgM Fc region)]5-(Z).

[0012] Furthermore, when m is 5 and n is 1, the fusion protein may be in a form in which a J chain is linked to the first IgM Fc domain monomer and the fifth IgM Fc domain monomer via a disulfide bond, and representative structures thereof are shown in Figures 1A, 1B, 11A, and 11B.

[0013] The fusion protein provided in the present invention has 10-12 binding domains through the IgM Fc region to maximize the avidity of biologically active molecules, and solves the steric hindrance problem of existing IgM antibodies through a linker. In addition, the fusion protein not only minimizes FcRn recycling through the IgG Fc region, but also prevents the possibility of a decrease in blood half-life due to binding with IgM-specific receptors and the occurrence of side effects due to non-specific binding by inducing mutations in the IgM Fc region and / or J chain contained in the fusion protein.

[0014] The term "fusion protein" as used herein refers to a protein in which a part or the whole of two or more different proteins are joined together. The fusion protein includes the amino acid sequence of the proteins that constitute it, and may further include an amino acid sequence such as a signal peptide that allows the protein produced in the cell to be secreted outside the cell, but is not limited thereto.

[0015] The term "IgG Fc region" as used herein refers to the Fc region (fragment crystallizable region) of immunoglobulin G (IgG) and refers to the remaining portion of IgG excluding the Fab region (fragment, antigen binding region). The IgG Fc region may be directly linked to a linker and an IgM Fc region in the fusion protein. The IgG Fc region may also be indirectly linked to a biologically active molecule via a linker in the fusion protein, and the IgG Fc region may serve as a binder, support, carrier, etc. for the biologically active molecule so that one or more biologically active molecules may be included. In the present specification, the "IgG Fc region" may be used interchangeably with the term "IgG Fc fragment".

[0016] Specifically, the IgG Fc domain monomer may be composed of two heavy chains. One heavy chain may include one or more selected from the group consisting of a CH2 domain and a CH3 domain. For example, one heavy chain may include a CH2 domain and a CH3 domain, or may be composed of a CH2 domain and a CH3 domain.

[0017] In the present invention, the IgG Fc region may be derived from IgG1, IgG2, IgG3, or IgG4.

[0018] The IgG Fc region may be derived from a wild-type IgG or a mutant IgG. Specifically, the wild-type IgG may comprise the amino acid sequence of SEQ ID NO: 1 or 3, and the mutant IgG may comprise the amino acid sequence of SEQ ID NO: 2.

[0019] In this case, the mutant IgG may have a mutation that reduces antibody-dependent cell-mediated cytotoxicity (ADCC) and / or a mutation that reduces complement-defendent cytotoxicity (CDC). Specifically, the mutant IgG may have an L14A mutation at the 14th amino acid position in the amino acid sequence of SEQ ID NO: 1, an L15A mutation at the 15th amino acid position in the amino acid sequence of SEQ ID NO: 1, or an L14A and L15A mutation at the 14th and 15th amino acids in the amino acid sequence of SEQ ID NO: 1, respectively.

[0020] The term "IgM Fc region" as used herein refers to the Fc region (fragment crystallizable region) of immunoglobulin M (IgM), and refers to the remaining portion of IgM excluding the Fab region (fragment, antigen binding region). The IgM Fc region may be directly linked to the IgG Fc region in the fusion protein, or may be directly linked to a linker when the IgG Fc region is not present in the fusion protein. The IgM Fc region may be indirectly linked to a biologically active molecule in the fusion protein via the IgG Fc region and a linker, or may be indirectly linked to a biologically active molecule in the fusion protein via only a linker. Here, the IgM Fc region may serve as a binder, support, carrier, etc. for biologically active molecules so that it may contain one or more biologically active molecules. As used herein, the term "IgM Fc region" may be used interchangeably with the term "IgM Fc fragment."

[0021] In the present invention, the IgM Fc domain monomer may form a pentamer, in which each monomer may be linked through a disulfide bond. The pentamer has a structure including five IgM Fc domain monomers, as illustrated in FIGS. 1A, 1B, 11A, and 11B. Meanwhile, when the IgM Fc domain monomer forms a hexamer, the production yield of the fusion protein including the hexamer may be lower than that of the pentamer, and problems related to productivity such as the generation of a large amount of impurities during the purification process may occur. The hexamer has a structure including six IgM Fc domain monomers, as illustrated in FIGS. 1C and 1D.

[0022] Specifically, the IgM Fc domain monomer may be composed of two heavy chains, each of which may include one or more domains selected from the group consisting of cμ2, cμ3, and cμ4 domains.

[0023] For example, one heavy chain may comprise or consist of the cμ2, cμ3 and cμ4 domains, and may comprise or consist of the amino acid sequence of SEQ ID NO: 4 or the nucleic acid sequence of SEQ ID NO: 5, but is not limited thereto.

[0024] As another example, one heavy chain may comprise or consist of the cμ3 domain and the cμ4 domain, and may comprise or consist of the amino acid sequence of SEQ ID NO: 6 or the nucleic acid sequence of SEQ ID NO: 7, but is not limited thereto.

[0025] As another example, one heavy chain may comprise or consist of a cμ2 domain, and in this case, the one heavy chain may comprise or consist of the amino acid sequence of SEQ ID NO: 8 or the nucleic acid sequence of SEQ ID NO: 9, but is not limited thereto.

[0026] As another example, one heavy chain may include or consist of a cμ3 domain, and in this case, the one heavy chain may include or consist of the amino acid sequence of SEQ ID NO: 10 or the nucleic acid sequence of SEQ ID NO: 11, but is not limited thereto.

[0027] As another example, one heavy chain may include or consist of a cμ4 domain, and in this case, the one heavy chain may include or consist of the amino acid sequence of SEQ ID NO: 12 or the nucleic acid sequence of SEQ ID NO: 13, but is not limited thereto.

[0028] Additionally, the IgM Fc region may be derived from a wild-type IgM or a mutant IgM.

[0029] Specifically, the wild-type IgM may comprise or consist of the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, or 12, or the nucleic acid sequence of SEQ ID NO: 5, 7, 9, 11, or 13, but is not limited thereto.

[0030] In this case, the mutant IgM may have one or more of the amino acids at positions 237 and 238 of the amino acid sequence of SEQ ID NO: 4 deleted or substituted. Specifically, the mutant IgM may have any one mutation selected from the group consisting of N237A, N237F, and N237K at the amino acid at position 237 of the amino acid sequence of SEQ ID NO: 4. In addition, the mutant IgM may have any one mutation selected from the group consisting of L238A, L238D, L238E, L238F, L238G, L238H, L238I, L238K, L238M, L238N, L238P, L238Q, L238R, L238S, L238T, L238V, L238W, and L238Y at the amino acid at position 238 of the amino acid sequence of SEQ ID NO: 4.

[0031] In this case, the mutant IgM may have one or more of the amino acids at positions 124 and 125 in the amino acid sequence of SEQ ID NO: 6 deleted or substituted. Specifically, the mutant IgM may have any one mutation selected from the group consisting of N124A, N124F, and N124K at the amino acid at position 124 in the amino acid sequence of SEQ ID NO: 6. In addition, the mutant IgM may have any one mutation selected from the group consisting of L125A, L125D, L125E, L125F, L125G, L125H, L125I, L125K, L125M, L125N, L125P, L125Q, L125R, L125S, L125T, L125V, L125W, and L125Y at the amino acid at position 125 in the amino acid sequence of SEQ ID NO: 6.

[0032] In this case, the mutant IgM may have one or more of the amino acids at positions 19 and 20 in the amino acid sequence of SEQ ID NO: 12 deleted or substituted. Specifically, the mutant IgM may have any one mutation selected from the group consisting of N19A, N19F, and N19K at the amino acid at position 19 in the amino acid sequence of SEQ ID NO: 12. In addition, the mutant IgM may have any one mutation selected from the group consisting of L20A, L20D, L20E, L20F, L20G, L20H, L20I, L20K, L20M, L20N, L20P, L20Q, L20R, L20S, L20T, L20V, L20W, and L20Y at the amino acid at position 20 in the amino acid sequence of SEQ ID NO: 12.

[0033] The term "J chain" as used herein refers to a polypeptide that is a component of an IgM or IgA antibody and binds IgM or IgA monomers to form a polymer. The J chain may be present between IgM Fc domain monomers in a fusion protein and may play a role in linking them. Specifically, the J chain may be included when the IgM Fc domain monomers form a pentamer, and may not be included when the IgM Fc domain monomers form a hexamer. More specifically, when the IgM Fc domain monomers form a pentamer, the J chain may be linked to the first and fifth IgM Fc domain monomers through a disulfide bond, and such a structure is illustrated in FIG. 1A, FIG. 1B, FIG. 11A, and FIG. 11B.

[0034] Specifically, the J chain may be derived from a wild-type J chain or a mutant J chain, and specifically, the wild-type J chain may comprise or consist of the amino acid sequence of SEQ ID NO: 14 or the nucleic acid sequence of SEQ ID NO: 15, but is not limited thereto.

[0035] In this case, the mutant J chain may have one or more of the amino acids at positions 105, 106, and 107 in the amino acid sequence of SEQ ID NO: 14 deleted or substituted. Specifically, the mutant J chain may have a mutation at the amino acid at position 105 in the amino acid sequence of SEQ ID NO: 14 selected from the group consisting of D105A and D105K. The mutant J chain may also have a mutation at the amino acid at position 106 in the amino acid sequence of SEQ ID NO: 14 selected from the group consisting of R106A, R106D, R106E, R106F, R106G, R106H, R106I, R106K, R106L, R106M, R106N, R106P, R106Q, R106S, R106T, R106V, R106W, and R106Y. The mutant J chain may also have any one mutation at the 107th amino acid position in the amino acid sequence of SEQ ID NO:14 selected from the group consisting of N107A, N107E, N107F, and N107K.

[0036] In the present invention, the J chain may further be bound to a protein molecule. In this case, the protein molecule may exhibit a synergistic effect with respect to the intended effect of the fusion protein of the present invention. Specifically, the protein molecule may be one or more selected from the group consisting of antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, soluble proteins, membrane-bound proteins, ligands, receptors, virus-like particles, protein toxins, and enzymes, but is not limited thereto. More specifically, the protein molecule may be one or more selected from the group consisting of costimulatory receptors, cytokines, and cell engagers. In this case, the cell engager may be an antibody, specifically an antibody that binds to a cancer cell or immune cell specific marker protein, for example, a T-cell engager antibody. The immune cells may include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (NK cells), and lymphocytes such as B cells and T cells.

[0037] The term "biologically active molecule" as used herein means a substance capable of exhibiting a specific action or effect in a living body, for example, a substance capable of promoting or suppressing the expression of a specific gene, enhancing or suppressing the function of a specific protein, or inducing or suppressing cell death by activating or inactivating a specific signaling pathway. Preferably, the biologically active molecule may exhibit immune regulation and / or anticancer activity.

[0038] In the present invention, the biologically active molecule may be indirectly linked to the IgG Fc region in the fusion protein via a linker, or the biologically active molecule may be indirectly linked to the IgM Fc region in the fusion protein via a linker, where the biologically active molecule may act as a functional group or active moiety that allows the fusion protein to exert the desired effect.

[0039] Specifically, the biologically active molecule may be, but is not limited to, one or more selected from the group consisting of antibodies, antigen-binding fragments of antibodies, antibody-drug conjugates, antibody-like molecules, antigen-binding fragments of antibody-like molecules, soluble proteins, membrane-bound proteins, ligands, receptors, virus-like particles, protein toxins, chemokines, cytokines, and enzymes. In this case, the receptor may be, but is not limited to, a cytokine receptor and / or an immune checkpoint receptor. For example, the fusion protein of the present invention may contain two or more biologically active molecules depending on the desired purpose, target, or effect.

[0040] As a specific example, the antigen-binding fragment of the antibody may be one or more selected from the group consisting of Fab, F(ab')2, F(ab)2, Fab', Fab2, Fab3, Fv, scFv, Bis-scFv, minibody, triabody, diabody, tandem diabody (TandAb), nanobody, and tetrabody, but is not limited thereto. In addition, the antigen-binding fragment of the antibody may bind to HLA-G. HLA-G is a type of HLA type 1, which is expressed in the placenta of pregnant women and is involved in fetal immune tolerance. HLA-G binds to ILT2, ILT4, and KIR2DL4, which are inhibitory receptors of NK (natural killer) cells, to inhibit NK cells and to inhibit regulatory T cells (T reg ) differentiation. In addition, when cancer cells overexpress HLA-G on the surface, the activity of cytotoxic T-cells, helper T-cells, and NK cells involved in cytotoxicity is inhibited, thereby reducing the immunotherapeutic effect for cancer treatment by half. Thus, an antigen-binding fragment that selectively binds to HLA-G and inhibits HLA-G from binding to an inhibitory receptor can be used as an anti-cancer therapeutic. For example, the antigen-binding fragment of the antibody may be Fab or scFv. In this case, the light chain (light chain, VL domain and CL domain) constituting the Fab may include or consist of the amino acid sequence of SEQ ID NO: 26 or the nucleic acid sequence of SEQ ID NO: 27; and a part of the heavy chain (VH domain and CH1 domain) may include or consist of the amino acid sequence of SEQ ID NO: 30 or the nucleic acid sequence of SEQ ID NO: 31, but is not limited thereto.

[0041] As another specific example, the receptor may be, but is not limited to, HLA (Human Leukocyte Antigen). The term "HLA" refers to a protein encoded by the human major histocompatibility complex (MHC) gene complex, which is an important immunological molecule that exists mainly in a membrane-bound form and is responsible for regulating the immune system. HLA presents peptide fragments of foreign or self proteins from inside the cell to the cell surface, and the T cell receptor (TCR) of the cytotoxic T cell recognizes and binds to the peptide fragments, thereby activating the cytotoxic T cell. HLA is broadly divided into two types, where type 1 HLA is present on the surface of all cells, while type 2 HLA is present only in specific antigen presenting cells (Antigen Presenting Cells; APCs) such as NK cells, macrophages, and dendritic cells. HLA genes have a high degree of polymorphism and express six different HLA protein α chains (two HLA-A, two HLA-B and two HLA-C). When a specific peptide fragment is loaded onto the HLA type 1, it can selectively activate cytotoxic T-cells expressing TCR, and recombinant HLA type 1 with the membrane-bound domain removed may be used to treat cancer and infectious diseases. In addition, unlike HLA type 2, most HLA type 1 is unstable when the groove is left vacant without a peptide loaded, so recombinant HLA type 1 is generally expressed using animal cells, since there is a concern that refolding may not be sufficient when recombinant HLA type 1 is produced by loading a synthetic peptide of interest or by producing a recombinant HLA protein fused to the α chain and expressing it using a microorganism. In the art, HLA and MHC are used interchangeably with the same meaning, and HLA is used instead of MHC in humans. As used herein, the terms "HLA" and "MHC" may be used interchangeably with the same meaning.

[0042] The HLA may be type 1 HLA such as HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G; or type 2 HLA such as HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. The type 1 HLA is composed of β2m domain, α1 domain, α2 domain, and α3 domain, and the type 2 HLA is composed of α1 domain, α2 domain, β1 domain, and β2 domain. The HLA of the present invention may include β2m domain, α1 domain, α2 domain, or α3 domain, or may include α1 domain, α2 domain, β1 domain, and β2 domain. In this case, the α2 domain may be one in which Gln (glutamine; Q) at position 115 is replaced with Glu (glutamic acid; E). The α1 domain may have a substitution at position 84 from Tyr (tyrosine; Y) to Cys (cysteine; C). When Gln at position 115 of the α2 domain is substituted with Glu, the α1 domain may have a substitution at position 84 from Tyr (tyrosine; Y) to Cys (cysteine; C). + The binding strength with TCR in T-cells is increased by about 1.5 times compared to the case without substitution. When Tyr at position 84 of the α1 domain is substituted with Cys, HLA forms a disulfide bond with the peptide bound to it, thereby increasing the stability of HLA.

[0043] The HLA may also include a substance that specifically binds to the activation site of a target T-cell. The substance may selectively activate a target T-cell expressing a TCR and / or may increase the stability of the HLA. The term "target T-cell" may refer to a T-cell to which the fusion protein of the present invention binds, and may be a cytotoxic T cell (killer T cell; Tc) or a helper T cell (Helper T cell; Th). The cytotoxic T-cell is a cell that can kill cancer cells, cells infected with viruses, or damaged cells, and is, for example, a CD8 + T-cells. CD8+ CD8, a protein receptor of T-cells, is located on the cell surface and binds to HLA type 1. The helper T-cells do not directly kill cells, but they play a role in activating other immune cells, for example, promoting antibody production by B-cells and activation of cytotoxic T-cells. + This includes T-cells. + CD4, the protein receptor for T-cells, is located on the cell surface and binds to HLA type 2.

[0044] A specific example of the substance that specifically binds to the activation site of the target T-cell may be, but is not limited to, a peptide. The peptide may be derived from a virus or an animal cell. In this case, the virus may be, but is not limited to, cytomegalovirus (CMV). CMV is one of the most powerful immunogenic antigens for the human immune system, and when it infects the human body, it induces a very strong CD8 + Stimulates T-cell responses. CD8 + T-cell immune responses were primarily activated by the CMV proteins pp65 and IE-1, and the frequency of CMV-specific T-cells against individual peptides was significantly higher than that of total CD8 + It is known to be highly frequent, at a maximum frequency of about 1% to 2% of the T-cell repertoire. For example, the HLA containing CMV may include, or consist of, the amino acid sequence of SEQ ID NO: 22 or 24, or the nucleic acid sequence of SEQ ID NO: 23 or 25, but is not limited thereto.

[0045] As another specific example, the ligand may be, but is not limited to, EPO (erythropoietin) or an EPO analog. The term "EPO" is a hormone protein essential for the production of red blood cells, which acts on hematopoietic cells in the bone marrow to promote the differentiation and growth of red blood cells, treat anemia, protects nerves in a hypoxic environment such as a stroke, and participates in the wound recovery process. It is also known to be involved in the hippocampal response, synaptic connections, and neuronal networks of the brain, helping to promote memory, and is effective in treating depression. The "EPO analog" is a recombinant protein that performs the same function as EPO but has a different structure, has two additional sugar chains in addition to the three sugar chains that human EPO has, and has a blood half-life that is three times longer than that of existing human EPO. The EPO analog may be a ligand, antibody, or antibody fragment that can bind to and activate the EPO receptor, and as a specific example, may be NESP (Novel erythropoiesis stimulating protein), but is not limited thereto. For example, the EPO analog can comprise or consist of the amino acid sequence of SEQ ID NO:32 or the nucleic acid sequence of SEQ ID NO:33, but is not limited thereto.

[0046] The term "linker" as used herein means an amino acid present between each domain of a protein. The linker may be present between a biologically active molecule and an IgG Fc region in a fusion protein, and may play a role of directly linking the biologically active molecule and the IgG Fc region. In addition, when an IgG Fc region is not present in a fusion protein, the linker may be present between a biologically active molecule and an IgM Fc region, and may play a role of directly linking the biologically active molecule and the IgM Fc region.

[0047] As a specific example, the linker may be one or more selected from the group consisting of an IgD hinge region, an IgA hinge region, an IgG hinge region, and a GS linker.

[0048] The "hinge region" refers to a portion located in the center of the heavy chain of an antibody, connecting two regions of the heavy chain with a disulfide bond. The hinge region is located between and connected to the CH1 and CH2 domains of the heavy chain. Unlike immunoglobulins with short hinge regions that have a Y shape, IgD specifically has a long hinge region and can have a large degree of flexibility to the extent that both target binding sites can open and form a T shape. This flexibility allows IgD to have a binding activity that can bind to multimeric external antigens while hardly binding to self antigens. In this specification, the term "hinge region" can be used interchangeably with the term "hinge domain".

[0049] In the present invention, the hinge region may comprise a part or the whole of the hinge region, a part or the whole of the CH1 domain, a part or the whole of the CH2 domain, or a combination thereof.

[0050] For example, the hinge region may include a part or the whole of an IgD hinge (hereinafter, "a part or the whole" is expressed as "a part / the whole"). Specifically, the hinge region may include a part / the whole of an IgD hinge and a part / the whole of an IgD CH1 domain. Specifically, the hinge region may include a part / the whole of an IgD hinge and a part / the whole of an IgD CH2 domain. Specifically, the hinge region may include a part / the whole of an IgD hinge, a part / the whole of an IgD CH1 domain, and a part / the whole of an IgD CH2 domain.

[0051] As another example, the hinge region may include a part / whole of an IgA hinge. Specifically, the hinge region may include a part / whole of an IgA hinge and a part / whole of an IgA CH1 domain. Specifically, the hinge region may include a part / whole of an IgA hinge and a part / whole of an IgA CH2 domain. Specifically, the hinge region may include a part / whole of an IgA hinge, a part / whole of an IgA CH1 domain, and a part / whole of an IgA CH2 domain.

[0052] As another example, the hinge region may include a part / whole of an IgG hinge. Specifically, the hinge region may include a part / whole of an IgG hinge and a part / whole of an IgG CH1 domain. Specifically, the hinge region may include a part / whole of an IgG hinge and a part / whole of an IgG CH2 domain. Specifically, the hinge region may include a part / whole of an IgG hinge, a part / whole of an IgG CH1 domain, and a part / whole of an IgG CH2 domain.

[0053] Specifically, the IgD hinge region may comprise or consist of the amino acid sequence of SEQ ID NO: 16 or the nucleic acid sequence of SEQ ID NO: 17, but is not limited thereto, and the IgA hinge region may comprise or consist of the amino acid sequence of SEQ ID NO: 18 or the nucleic acid sequence of SEQ ID NO: 19, but is not limited thereto.

[0054] The "GS linker" is (GS) d , (SG) d , (GGGS) d , (GGGGS) d , GCGS(GGGS) d , and GCGGS (GGGGS) dIn this case, d is an integer of 2 to 6. Specifically, it may contain 4 to less than 35 amino acids consisting of glycine (Gly; G) and serine (Ser; S), and may contain 4 to less than 35 amino acids consisting of glycine, serine and cysteine ​​(Cys; C) to enhance stability through disulfide bonds, but is not limited thereto. In addition, the GS linker may include or consist of the amino acid sequence of SEQ ID NO: 20 or the nucleic acid sequence of SEQ ID NO: 21, but is not limited thereto.

[0055] Another aspect of the present invention provides a nucleic acid molecule encoding the fusion protein.

[0056] In the nucleic acid molecules of the present invention, unless otherwise specified, the related terms are understood to have the same meaning as those previously described.

[0057] The term "nucleic acid molecule" as used herein is intended to encompass DNA and RNA molecules, and the nucleotides that are the basic building blocks of the nucleic acid molecule include not only natural nucleotides but also analogues in which the sugar or base moiety is modified. The sequence of the nucleic acid molecule encoding the fusion protein of the present invention may be modified, and the modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0058] In addition, all sequences used in the present invention, including nucleic acid sequences and amino acid sequences, are analyzed to include sequences that show substantial identity with the sequences listed in the sequence listing, taking into account variations with biologically equivalent activity. The term "substantial identity" refers to a sequence that shows at least 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology when the sequence of the present invention is aligned with any other sequence to maximize correspondence and the aligned sequences are analyzed using algorithms commonly used in the art.

[0059] Therefore, sequences having high homology to the sequences shown in SEQ ID NOs: 1 to 33 of the present invention, for example, sequences having a homology of 70% or more, specifically 80% or more, and more specifically 90% or more, must also be considered to be included in the scope of the present invention.

[0060] Another aspect of the present invention provides a vector for expressing a fusion protein, comprising the nucleic acid molecule.

[0061] In the fusion protein expression vector according to the present invention, unless otherwise specified, the relevant terms are understood to have the same meaning as those previously described.

[0062] The term "vector" as used herein refers to a means for expressing the fusion protein of the present invention, such as a plasmid, virus, or other vehicle known in the art, into which a nucleic acid molecule encoding the fusion protein can be inserted or introduced into a host cell. The vector can be constructed as a vector for cloning or a vector for expression. Specific examples thereof include viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors, and more specific examples thereof include plasmids (e.g., pcDNA series such as pcDNA3 or pcDNA3.1, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), phages (e.g., λgt4·λB, λ-Charon, λΔz1, and M13), or viruses (e.g., SV40), but are not limited thereto.

[0063] The vector of the present invention may be one in which the nucleic acid molecule encoding the fusion protein is operably linked to a promoter. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence, such that the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0064] When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and may contain a polyadenylation sequence as a transcription termination sequence. For example, the recombinant vector of the present invention may contain a CMV promoter, but is not limited thereto.

[0065] When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, it may contain a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. For example, when E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway or the left-facing promoter (pLλ promoter) of phage λ can be used as a regulatory site; when Bacillus bacteria is used as a host cell, the promoter of the toxin protein gene of Bacillus thuringiensis or any promoter that can be expressed in Bacillus bacteria can be used as a regulatory site.

[0066] The recombinant vector system of the present invention can be constructed through various methods known in the art and may contain antibiotic resistance genes commonly used in the art as selection markers (e.g., resistance genes against ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline).

[0067] Another aspect of the present invention provides a host cell into which the fusion protein expression vector is introduced.

[0068] In the host cell of the present invention, unless otherwise specified, the related terms are understood to have the same meaning as those previously described.

[0069] The term "host cell" as used herein refers to a cell that contains the fusion protein expression vector and can stably and continuously clone and express the fusion protein of the present invention. For example, prokaryotic host cells such as Bacillus species such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas, Proteus mirabilis, or Staphylococcus; fungi such as Aspergillus species, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, or Neurospora crassa; higher eukaryotic cells such as insect-derived cells; plant cells; or mammalian-derived cells such as COS7 cells (monkey kidney cells), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, or 293 cells, but are not limited thereto, and host cells commonly used in the art can be used without limitation.

[0070] Yet another aspect of the present invention provides a method for producing the fusion protein, comprising the step of introducing the fusion protein expression vector into a host cell.

[0071] In the method for producing a fusion protein according to the present invention, unless otherwise specified, the relevant terms are understood to have the same meaning as those previously explained.

[0072] Specifically, the method for producing the fusion protein of the present invention comprises the steps of: (a) introducing the vector of the present invention into a host cell; (b) culturing the host cell; and (c) obtaining the fusion protein in said host cell.

[0073] The step (a) of introducing the vector of the present invention into a host cell may be a step of producing a transformant containing the fusion protein expression vector, and the step of producing the transformant may be a step of transforming the host cell.

[0074] The term "transformant" refers to an organism in which DNA has been introduced into a host so that the DNA can replicate as a chromosomal factor or by chromosomal integration, and refers to an organism in which a genetic change has been artificially produced by introducing exogenous DNA into the cell.

[0075] Transformation of the host cells may be performed using any transformation method, and may be easily performed by a method commonly used in the art, including, but not limited to, the CaCl2 precipitation method, the Hanahan method in which the efficiency of the CaCl2 method is increased by using DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fiber, agrobacteria-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation, and any transformation or transfection method commonly used in the art may be used without limitation.

[0076] The step (b) of culturing the host cell of the present invention may be carried out using a medium and culture conditions known in the art. Those skilled in the art can easily adjust and use such a culture process depending on the selected strain. Depending on the cell growth method, suspension culture or adherent culture may be used; depending on the culture method, batch, fed-batch, or continuous culture may be used.

[0077] In animal cell culture, the medium may contain various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, or cellulose; fats such as soybean oil, sunflower oil, castor oil, or coconut oil; fatty acids such as palmitic acid, stearic acid, or linolenic acid; alcohols such as glycerol or ethanol; or organic acids such as acetic acid, and these carbon sources may be used alone or in combination. Examples of nitrogen sources may include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor, or soybean meal; or inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, or ammonium nitrate, and these nitrogen sources may be used alone or in combination. Examples of trace elements may include phosphorus sources such as potassium dihydrogen phosphate or dipotassium hydrogen phosphate; metal salts such as magnesium sulfate or ferrous sulfate. Others may include amino acids, vitamins, or appropriate precursors.

[0078] In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid or sulfuric acid can be added to the culture in an appropriate manner during cultivation to adjust the pH of the culture, antifoaming agents such as fatty acid polyglycol esters can be used during cultivation to suppress foam formation, and oxygen or oxygen-containing gases (e.g., air) can be injected into the culture to maintain an aerated state of the culture.

[0079] The culture temperature can be maintained at 20°C to 45°C, specifically 25°C to 40°C.

[0080] The step (c) of expressing the fusion protein in the host cell of the present invention may be a step of recovering, purifying and concentrating the fusion protein of the present invention from the culture medium obtained through the step (b).

[0081] The fusion protein may be used without purification, or may be recovered, purified, and concentrated. Specifically, methods commonly used in the art (e.g., dialysis, salt precipitation, chromatography, etc.) may be used, through which recovery, purification, and concentration can be performed simultaneously. More specifically, the fusion protein may be recovered, purified, and concentrated using chromatography (e.g., ion exchange chromatography, size exclusion chromatography, or affinity chromatography), and the type and order of columns used therein may be appropriately selected depending on the characteristics of the fusion protein, the culture method, etc.

[0082] Another aspect of the present invention provides a use of the fusion protein for the manufacture of a therapeutic, prophylactic or diagnostic agent.

[0083] Another aspect of the present invention provides a pharmaceutical composition comprising the fusion protein.

[0084] Yet another aspect of the present invention provides a method for treating or preventing a disease in an individual, comprising administering the fusion protein to the individual.

[0085] In the uses, pharmaceutical compositions and methods of the present invention, unless otherwise specified, the associated terminology shall be understood to have the same meaning as previously described.

[0086] When the fusion protein contains a biologically active molecule that exhibits, for example, immunoregulatory or anti-cancer effects, it can be used in individuals in need of treatment, prevention, amelioration, or diagnosis of diseases caused by impaired or lost immune regulatory function, or diseases such as cancer.

[0087] The "individual" may include, without limitation, a human, any non-human animal, fish, or plant. The non-human animal may be a vertebrate, such as a primate, dog, cow, horse, pig, rodent, such as a mouse, rat, hamster, guinea pig, etc. In this specification, the "individual" may be used interchangeably with a "subject" or a "patient."

[0088] In one embodiment of the present invention, the biologically active molecule contained in the fusion protein may be, but is not limited to, one that exhibits immunomodulatory activity, anti-cancer activity, or anti-inflammatory activity. In addition, the biologically active molecule may include, but is not limited to, the amino acid sequence of SEQ ID NO: 22, 24, 26, 28, 30, or 32, or the nucleic acid sequence of SEQ ID NO: 23, 25, 27, 29, 31, or 33, or may consist of the amino acid sequence or nucleic acid sequence. A person of ordinary skill in the art should be able to appropriately select a biologically active molecule depending on the type of disease of an individual and its severity or condition.

[0089] As used herein, "A, B and / or C" means A, or B, or C, or A and B, or A and C, or B and C, or A, B and C. Effect of the Invention

[0090] The fusion protein platform of the present invention can be used in combination with various biologically active molecules selected according to the purpose, and the binding activity of the biologically active molecules included in the platform can be increased.

[0091] Therefore, the fusion protein produced through this platform has excellent target recognition and regulation effects, and there is little risk of unpredictable side effects due to non-specific binding. In addition, the half-life in the bloodstream is long, so the therapeutic effect to be achieved through the fusion protein can be maximized. [Brief description of the drawings]

[0092] [Figure 1A] FIG. 1 is a schematic diagram of a fusion protein according to the present invention, the fusion protein comprising a biologically active molecule, a linker, an IgM Fc region, and a J chain, wherein the IgM Fc and J chain form a pentamer. [Figure 1B] Schematic diagram of a fusion protein of the invention, comprising a biologically active molecule, an IgM Fc region and a J chain, but no linker, wherein the IgM Fc region forms a pentamer. [Figure 1C] FIG. 1 is a schematic diagram of a fusion protein of the invention, comprising a biologically active molecule, a linker, and an IgM Fc region but no J chain, wherein the IgM Fc region forms a hexamer. [Figure 1D] Schematic diagram of a fusion protein of the invention, comprising a biologically active molecule and an IgM Fc domain, but excluding a linker and a J chain, wherein the IgM Fc domain forms a hexamer. [Figure 2A] 1 is an image showing the results of reducing SDS-PAGE analysis of a fusion protein containing HLA (CMVpHLA) as a biologically active molecule. "Ladder" is a protein size marker; "1" is a fusion protein having the structure [(CMVpHLA)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain); "2" is a fusion protein having the structure [(CMVpHLA)2-(linker)0-(IgM Fc region)]5-(J chain); "3" is a fusion protein having the structure [(CMVpHLA)2-((GGGGS)3)2-(IgM Fc region)]5-(J chain); "4" is a fusion protein having the structure [(CMVpHLA)2-(IgA hinge region)2-(IgM Fc region)]5-(J chain); "5" is a fusion protein having the structure [(CMVpHLA)2-(IgD hinge region)2-(IgM Fc region)]6-(J chain)0; and "6" is a control CMVpHLA protein. [Figure 2B]1 is an image showing the results of reducing SDS-PAGE analysis of a fusion protein containing an antibody fragment (IgG Fab; A2014) as a biologically active molecule. "Ladder" is a protein size marker; "1" is a fusion protein having the structure of [(A2014)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain); "2" is a fusion protein having the structure of [(A2014)2-(linker)0-(IgM Fc region)]5-(J chain); "3" is a fusion protein having the structure of [(A2014)2-((GGGGS)3)2-(IgM Fc region)]5-(J chain); "4" is a fusion protein having the structure of [(A2014)2-(IgA hinge region)2-(IgM Fc region)]5-(J chain); "5" is a fusion protein having the structure of [(A2014)2-(IgD hinge region)2-(IgM Fc region)]6-(J chain)0; and "6" is a control group (A2014)-(IgG Fc) refers to fusion proteins. [Figure 2C] This is an image showing the results of reducing SDS-PAGE analysis of fusion proteins containing an EPO analog (NESP) as a biologically active molecule. "Ladder" is a protein size marker; "1" is a fusion protein having the structure of [(NESP)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain); "2" is a fusion protein having the structure of [(NESP)2-(linker)0-(IgM Fc region)]5-(J chain); and "3" is NESP as a control. [Figure 3A] 1 is a graph showing ELISA results analyzing the target binding ability of a fusion protein containing HLA (CMVpHLA) as a biologically active molecule. [Figure 3B] 1 is a graph showing the ELISA results of analyzing the target binding ability of a fusion protein containing an antibody fragment (IgG Fab; A2014) as a biologically active molecule. [Figure 3C] 1 is a graph showing ELISA results analyzing the target binding ability of a fusion protein containing an EPO analog (NESP) as a biologically active molecule. [Figure 4]1 is a graph showing the results of ELISPOT analysis of the IFN-γ content of the fusion protein according to the present invention, which shows that the fusion protein can induce the activation of CD8+ T cells. [Figure 5A] 1 is a graph showing the FACS results of analyzing the degree of CD8+ T cell proliferation by the fusion protein according to the present invention, [(CMVpHLA)2-(linker)0-(IgM Fc region)]5-(J chain), which shows that the fusion protein can induce the proliferation of CD8+ T cells. [Figure 5B] 1 is a graph showing the FACS results of analyzing the degree of CD8+ T cell proliferation by the fusion protein according to the present invention, [(CMVpHLA)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain), which shows that the fusion protein can induce the proliferation of CD8+ T cells. [Figure 5C] 1 is a graph showing the FACS results of analyzing the degree of CD8+ T cell proliferation by the fusion protein according to the present invention, [(CMVpHLA)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain), which shows that the fusion protein can induce the proliferation of CD8+ T cells. [Figure 5D] 1 is a graph showing the FACS result of analyzing the degree of CD8+ T cell proliferation by the fusion protein according to the present invention, [(CMVpHLA)2-((GGGGS)3)2-(IgM Fc region)]5-(J chain), which shows that the fusion protein can induce the proliferation of CD8+ T cells. [Figure 5E] 1 is a graph showing the FACS results of analyzing the degree of CD8+ T cell proliferation by the fusion protein according to the present invention, [(CMVpHLA)2-(IgA hinge region)2-(IgM Fc region)]5-(J chain), which shows that the fusion protein can induce the proliferation of CD8+ T cells. [Figure 5F]1 is a graph showing the FACS results of analyzing the degree of CD8+ T cell proliferation by the fusion protein according to the present invention, [(CMVpHLA)2-(IgD hinge region)2-(IgM Fc region)]6-(J chain)0, which shows that the fusion protein can induce the proliferation of CD8+ T cells. [Figure 5G] 1 is a graph showing the results of FACS analyzing the degree of CD8+ T cell proliferation caused by the fusion protein according to the present invention, with reference to the control group, CMVpHLA. [Figure 6] 1 is a graph showing the FACS result of analyzing the cytotoxicity of the fusion protein according to the present invention, which shows that the fusion protein can activate CD8+ T cells and induce the death of cancer cells. [Figure 7] 1 is a graph showing the assay results of analyzing the binding activity of the fusion protein of the present invention to HLA-G expressing cells, showing that the fusion protein can bind to intracellular HLA-G receptors. [Figure 8] 1 is a graph showing the results of an ELISA assay analyzing the degree of binding between recombinant HLA-G protein and recombinant ILT-2 protein by the fusion protein of the present invention, which shows that the fusion protein can block the binding of HLA-G and ILT-2 proteins. [Figure 9] 1 is a graph showing the FACS results of analyzing the degree of binding between recombinant HLA-G protein and intracellularly expressed ILT-2 protein by the fusion protein according to the present invention, which shows that the fusion protein can block the binding between HLA-G protein and intracellularly expressed ILT-2 receptor protein. [Figure 10] 1 is a graph showing the assay results of analyzing the proliferation ability of EPO-expressing cells by the fusion protein according to the present invention, which shows that the fusion protein can bind to an EPO receptor expressed intracellularly and promote the proliferation of cells expressing the same. [Figure 11A]Schematic diagram of a fusion protein according to the present invention, comprising a biologically active molecule, a linker, an IgM Fc region, and a J chain, the IgM Fc and J chain forming a pentamer, and the IgM Fc region and J chain in a form that includes amino acid substitutions and deletions. [Figure 11B] Schematic diagram of a fusion protein according to the present invention, comprising a biologically active molecule, a linker, an IgG Fc region, an IgM Fc region, and a J chain, the IgM Fc and J chain forming a pentamer, and the IgM Fc region and J chain in a form that includes amino acid substitutions and deletions. [Figure 12A] 1 is an image showing the results of reducing SDS-PAGE analysis of fusion proteins containing CMVpHLA as a biologically active molecule. "Ladder" refers to protein size marker; "1" refers to [(CMVpHLA)2-(linker)2-(IgG Fc region)-(IgM Fc region containing amino acid mutation)]5-(J chain containing amino acid mutation) pentameric fusion protein; "2" refers to [(CMVpHLA)2-(linker)2-(IgG Fc region)-(IgM Fc region)]5-(J chain) pentameric fusion protein; "3" refers to [(CMVpHLA)2-(linker)2-(IgM Fc region)]5-(J chain) pentameric fusion protein; "4" refers to (CMVpHLA)2-(IgG Fc region) fusion protein. [Figure 12B] This is an image showing the results of reducing SDS-PAGE analysis of fusion proteins containing an HLA-G target-like antibody (A2014) as a biologically active molecule. "Ladder" refers to protein size markers; "1" refers to [(A2014)2-(linker)2-(IgG Fc region)-(IgM Fc region with amino acid mutation)]5-(J chain with amino acid mutation) pentameric fusion protein; "2" refers to [(A2014)2-(linker)2-(IgG Fc region)-(IgM Fc region)]5-(J chain) pentameric fusion protein; "3" refers to [(A2014)2-(linker)2-(IgM Fc region)]5-(J chain) pentameric fusion protein; "4" refers to (A2014)2-(IgG Fc region) fusion protein. [Figure 13A]1 is a graph showing the results of FACS analysis of memory CD8+ T cell proliferation in response to a fusion protein containing HLA as a biologically active molecule. [Figure 13B] 1 is a graph showing the ILT-2 blocking ability of a fusion protein containing an HLA-G target-like antibody (A2014) as a biologically active molecule, confirmed by ELISA assay. [Figure 14A] 1 is a graph showing the ELISA analysis results for measuring the affinity of the fusion protein according to the present invention to FcRn, in which IMB-4000, IMB-4001, IMB-4011, and IMB-4012 were analyzed at pH 6.0, showing that the FcRn recycling effect of the fusion protein can be maximized. [Figure 14B] 1 is a graph showing the ELISA analysis results for measuring the affinity of the fusion protein according to the present invention to FcRn, in which IMB-4000, IMB-4001, IMB-4011, and IMB-4012 were analyzed at pH 7.4, showing that the FcRn recycling effect of the fusion protein can be maximized. [Figure 14C] 1 is a graph showing the ELISA analysis results for measuring the affinity of the fusion protein according to the present invention to FcRn, where A2014-1, A2014-2, A2014-3, and A2014-4 were analyzed at pH 6.0, showing that the FcRn recycling effect of the fusion protein can be maximized. [Figure 14D] 1 is a graph showing the ELISA analysis results for measuring the affinity of the fusion protein according to the present invention to FcRn, where A2014-1, A2014-2, A2014-3, and A2014-4 were analyzed at pH 7.4, showing that the FcRn recycling effect of the fusion protein can be maximized. [Figure 15] 1 is a graph showing the results of ELISA analysis measuring the affinity of the fusion protein according to the present invention to pIg receptor (pIgR), which shows that the half-life of the fusion protein in blood can be improved. [Figure 16]1 is a graph showing the ELISA analysis results for measuring the affinity of the fusion protein of the present invention to the Fcα / μ receptor, which shows that the fusion protein can reduce non-specific binding and improve half-life in blood. [Figure 17] 1 is a graph showing the ELISA analysis results for measuring the affinity of the fusion protein according to the present invention to the Fcμ receptor, which shows that the fusion protein can reduce non-specific binding and improve half-life in blood. [Figure 18] 1 shows the results of a measurement test of the half-life in blood of the fusion protein according to the present invention. The fusion protein exhibits the effect of maximizing the half-life in blood through inhibition of FcRn recycling and IgM specific receptor binding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0093] The present invention will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0094] Example 1. Preparation of an IgM Fc region-containing fusion protein platform In this Example 1, a fusion protein platform was produced that includes a biologically active molecule, a linker, an IgM Fc region, and / or a J chain.

[0095] Example 1-1. Preparation of a fusion protein containing an IgM Fc region, a J chain, and a linker In this Example 1-1, recombinant plasmids of the IgM Fc region, J chain, and / or linker were produced. The IgM Fc region used was the cμ2 domain, cμ3 domain, and cμ4 domain of wild-type IgM.

[0096] Specifically, a recombinant plasmid was constructed to express the IgM Fc region (SEQ ID NO: 5) containing the cμ2, cμ3, and cμ4 domains, and the J chain (SEQ ID NO: 15) required for pentamer formation. In addition, to solve the steric hindrance problem of existing IgM antibodies, a fusion protein recombinant plasmid was constructed in which the human IgD hinge region (SEQ ID NO: 17), human IgA hinge region (SEQ ID NO: 19), and (GGGGS)3 linker (SEQ ID NO: 21) were linked to the IgM Fc region.

[0097] For gene cloning, each of the above substances was codon optimized, and then codon-optimized nucleotide molecules were synthesized by Cosmogenetek (Seoul, Korea).

[0098] Using the optimized nucleotides and primers, PCR was performed to prepare insert DNA so that overlap PCR could be performed on the MCS (multiple cloning site) of pcDNA 3.1 (manufacturer: Thermo Scientific) vector. After overlap PCR was performed on each of the obtained insert DNAs and the pcDNA 3.1 vector, the template was removed by treating with DpnI restriction enzyme, and vectors into which each substance was inserted were prepared.

[0099] The vector was inserted into Stella® competent cells (manufacturer: Clontech), and then smeared on an agar plate containing ampicillin antibiotic that matches the antibiotic resistance gene in the vector, and colonies were selected. The base sequence was confirmed through DNA sequencing using an Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Scientific).

[0100] Examples 1-2. Preparation of a fusion protein containing an IgM Fc region, a J chain, a linker, and a biologically active molecule In this Example 1-2, a recombinant plasmid for a biologically active molecule was prepared and linked to the recombinant plasmid for the IgM Fc region, J chain, and / or linker prepared in Example 1-1, and a fusion protein containing these was expressed and purified.

[0101] As biologically active molecules, HLA-A, IgG antibody fragments (IgG Fab) or EPO analogues were used.

[0102] Example 1-2-1. HLA-A containing fusion protein Fusion proteins containing HLA-A as the biologically active molecule and containing the IgM Fc region, J chain, and / or a linker were produced.

[0103] Specifically, to increase the stability of the HLA-A protein, a peptide derived from CMV (cytomegalovirus) was further loaded, and β2m (Beta-2 microglobulin) was subsequently loaded onto the peptide derived from CMV. Through this, an HLA-A protein with improved stability (hereinafter, referred to as "CMVpHLA"; SEQ ID NO: 23) was constructed. In addition, the α3 region of the CMVpHLA was found to be similar to that of mouse K b CMVpHLAK in the form substituted with b (SEQ ID NO: 25) was prepared. Then, in order to facilitate protein purification, a FLAG tag was attached to the C-terminus, and the detailed cloning method was performed according to Example 1-1.

[0104] The CMVpHLA protein sequence, with the FLAG tag removed from the constructed CMVpHLA recombinant plasmid, was linked to an IgM Fc region sequence, and the final recombinant plasmid vector thus constructed was introduced into Expi293 (Thermo Scientific) animal cells to establish a transient expression system for obtaining the fusion protein within a short period of time.

[0105] Specifically, one day before transformation, 3.0 x 10 6After dispensing cells at a concentration of 1.0 × 10 cells / mL, the cells were cultured for 24 hours. On the day of transduction, Expi293® expression medium was added to the cells, and 3.0 × 10 6 The cells were diluted to a concentration of 10000 cells / mL. Then, 1 μg / mL of each recombinant plasmid vector was mixed with Opti-MEM® medium, and ExpiFectamine® reagent and Opti-MEM® medium were mixed to the appropriate volume, and then left at room temperature for 5 minutes. Then, the DNA and ExpiFectamine® reagent were reacted for 15 minutes and added to the flask, and enhancers 1 and 2 were treated 16 to 22 hours later. The cell viability was confirmed to be 75% or higher on the 4th to 5th day, and the fusion protein was collected.

[0106] The expressed fusion protein was then purified using IgM affinity chromatography. Specifically, the culture medium obtained from the transient expression system was filtered through a PES filter membrane with a pore size of 0.2 μm to remove impurities. The collected filtrate was then purified using the IgM affinity matrix POROS. TM CaptureSelect TM The fusion protein was purified by loading onto IgM Affinity Matrix® (Thermo Scientific). IgM affinity chromatography was performed under the following conditions:

[0107] <Chromatography conditions> -Resin: POROS TM CaptureSelect TM IgM Affinity Matrix® -Flow velocity: 480cm / h -Equilibrium: PBS (Phosphate-buffered saline) buffer - Loading: up to 6g protein / L resin volume -Regeneration and sterilization: 0.01M NaOH solution -Elution: 0.1M glycine-HCl, pH 2.7 buffer -Storage: 20% EtOH

[0108] Then, 1 / 10 of the eluate volume was neutralized with 1.0 M Tris-HCl, pH 8.5 buffer. The recovered fusion protein was concentrated using ultrafiltration, dialyzed against PBS, and stored at -20°C.

[0109] Meanwhile, the purification and concentration of the CMVpHLA control protein not containing the IgM Fc region was carried out as follows: The culture medium obtained from the transient expression system was filtered through a PES membrane filter with a pore size of 0.2 μm to remove impurities. The collected filtrate was loaded onto a DYKDDDDK (FLAG-tag) affinity matrix, Pierce Anti-DYKDDDDK Affinity Resin (registered trademark) (Thermo Scientific), and purified.

[0110] FLAG-tag affinity chromatography was carried out under the following conditions.

[0111] <Chromatography conditions> -Resin: Pierce Anti-DYKDDDDK Affinity Resin (registered trademark) -Flow velocity: 300cm / h -Equilibrium: PBS (Phosphate-buffered saline) buffer - Loading: Maximum 3g protein / L resin volume -Regeneration and sterilization: 0.1M glycine-HCl, pH 2.7 buffer -Elution: 0.1M glycine-HCl, pH2.7 buffer -Storage: 20% EtOH

[0112] After FLAG-tag affinity chromatography, 1 / 10 of the eluate volume was neutralized with 1.0 M Tris-HCl, pH 8.5 buffer, and the recovered fusion protein was concentrated using ultrafiltration, dialyzed against PBS, and stored at -20°C.

[0113] The fusion protein prepared by the above method was represented by the following structural formula: [(biologically active molecule)-(linker)-(IgM Fc region)]-(J chain). The number of biologically active molecules, linkers, IgM Fc regions, and / or J chains contained in one fusion protein is indicated by the following subscripts, and is not indicated if there is only one.

[0114] [Table 1]

[0115] These are listed in Table 1. For example, "[(CMVpHLA)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain)" means a fusion protein in which two CMVpHLA molecules are bound to an IgM Fc region monomer via two IgD hinge regions as a biologically active molecule, and five of the IgM Fc region monomers are bound to form a pentamer, and one J chain is included to form the pentamer. The pentamer structure of such a fusion protein can be seen in the form illustrated in Figures 1A and 11A.

[0116] In addition, "[(CMVpHLA)2-(linker)0-(IgM Fc region)]5-(J chain)" means a fusion protein in which two CMVpHLA molecules are directly linked to an IgM Fc region monomer without a linker as a biologically active molecule, and the IgM Fc region monomer is a pentamer in which five molecules are linked, and one J chain is included to form the pentamer. The pentamer structure of such a fusion protein can be seen in the form illustrated in FIG. 1B.

[0117] In addition, "[(CMVpHLA)2-(IgD hinge region)2-(IgM Fc region)]6-(J chain)0" refers to a fusion protein in which two CMVpHLA molecules are bound to an IgM Fc region monomer via two IgD hinge regions as a biologically active molecule, and the IgM Fc region monomer is a hexamer of six molecules bound together, and does not include a J chain to form the hexamer. The hexamer structure of such a fusion protein can be seen in the form illustrated in FIG 1C.

[0118] Example 1-2-2. Fusion protein containing IgG antibody fragment Fusion proteins containing an IgG antibody fragment as the biologically active molecule, and containing an IgM Fc region, a J chain, and / or a linker, have been produced.

[0119] Specifically, an antibody portion that binds to HLA-G protein (IgG Fab; hereinafter, referred to as "A2014") was used. The light chain (VL domain and CL domain; SEQ ID NO: 27) and a portion of the heavy chain (VH domain and CH1 domain; SEQ ID NO: 31) of the IgG Fab portion of A2014 were constructed using the cloning method described in Example 1-1 above.

[0120] In addition, a fusion protein containing an IgG Fc region instead of an IgM Fc region was prepared as a control group. The IgG Fc region was prepared by the cloning method described in Example 2-1 above using the CH2 and CH3 domains of IgG, specifically, the light chain (VL domain and CL domain; SEQ ID NO: 27) and the heavy chain (VH domain, CH1 domain, CH2 domain, and CH3 domain; SEQ ID NO: 29).

[0121] The constructed A2014 recombinant plasmid sequence was linked to an IgM Fc region or IgG Fc region sequence, and the final recombinant plasmid vector thus constructed was introduced into cells by the method described in Example 1-2-1 to express the fusion protein.

[0122] Thereafter, the culture medium was purified and concentrated in the same manner as in 1-2-1 above, dialyzed against PBS, and stored at -20°C.

[0123] Meanwhile, the fusion protein containing the IgG Fc region was purified and concentrated as follows: The culture medium obtained from the transient expression system was loaded onto the IgG affinity matrix MabSelect SuRe (registered trademark) (Cytiva) and purified according to the method described in 1-2-1 above.

[0124] IgG affinity chromatography was performed under the following conditions.

[0125] <Chromatography conditions> -Resin: MabSelect SuRe (registered trademark) -Flow velocity: 300cm / h -Equilibrium: PBS (Phosphate-buffered saline) buffer - Loading: Maximum 35g protein / L resin volume -Regeneration and sterilization: 0.01M NaOH solution -Elution: 0.1M glycine-HCl, pH2.7 buffer -Storage: 20% EtOH

[0126] After IgG affinity chromatography, 1 / 10 of the eluate volume was neutralized with 1.0 M Tris-HCl, pH 8.5 buffer, and the recovered fusion protein was concentrated using ultrafiltration, dialyzed against PBS, and stored at -20°C.

[0127] [Table 2]

[0128] The fusion proteins prepared by the above-mentioned method are listed in Table 2. For example, "(A2014)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain)" means a fusion protein in which two A2014 molecules are bound to an IgM Fc region monomer via two IgD hinge regions as a biologically active molecule, and five IgM Fc region monomers are bound to form a pentamer, and one J chain is included to form the pentamer. The pentamer structure of such a fusion protein can be seen in the form illustrated in Figures 1A and 11A.

[0129] In addition, "[(A2014)2-(linker)0-(IgM Fc region)]5-(J chain)" means a fusion protein in which two A2014 molecules as biologically active molecules are directly linked to an IgM Fc region monomer without a linker, the IgM Fc region monomer is a pentamer in which five molecules are linked, and one J chain is included to form the pentamer. The pentamer structure of such a fusion protein can be seen in the form illustrated in FIG 1B.

[0130] In addition, "[(A2014)2-(IgD hinge region)2-(IgM Fc region)]6-(J chain)0" refers to a fusion protein in which two A2014 molecules are bound to an IgM Fc region monomer via two IgD hinge regions as a biologically active molecule, and the IgM Fc region monomer is a hexamer of six molecules bound together, and does not include a J chain to form the hexamer. The hexamer structure of such a fusion protein can be seen in the form illustrated in FIG 1C.

[0131] Example 1-2-3. Fusion protein containing EPO analogue Fusion proteins containing an EPO analog as the biologically active molecule and including an IgM Fc region, a J chain, and / or a linker have been prepared.

[0132] Specifically, NESP (Novel erythropoiesis stimulating protein) protein was used as the EPO analog. The NESP (SEQ ID NO: 33) was linked to an IgM Fc region sequence, and a plasmid vector was constructed using the cloning method described in Example 1. The constructed recombinant plasmid vector was introduced into cells using the method described in Example 1-2-1 to express the fusion protein, which was then purified and concentrated, then dialyzed against PBS, and stored at -20°C.

[0133] [Table 3]

[0134] The fusion proteins prepared by the above-mentioned method are listed in Table 3. For example, "[(NESP)2-(IgD hinge region)2-(IgM Fc region)]5-(J chain)" means a fusion protein in which two NESPs as biologically active molecules are bound to an IgM Fc region monomer via two IgD hinge regions, and five IgM Fc region monomers are bound to form a pentamer, and one J chain is included to form the pentamer. The pentamer structure of such a fusion protein can be seen in the forms illustrated in Figures 1A and 11A.

[0135] In addition, "[(NESP)2-(linker)0-(IgM Fc region)]5-(J chain)" refers to a fusion protein in which two NESPs as biologically active molecules are directly linked to an IgM Fc region monomer without a linker, the IgM Fc region monomer is a pentamer in which five IgM Fc region monomers are linked, and one J chain is included to form the pentamer. The pentamer structure of such a fusion protein can be seen in the form illustrated in Figure 1B.

[0136] Examples 1-3. Fusion Protein Construction It was confirmed whether the fusion protein prepared in Example 1-2 had the desired structure. Meanwhile, schematic diagrams of a fusion protein including a biologically active molecule, a linker, an IgM Fc domain monomer, and a J chain, in which the IgM Fc domain forms a pentamer, are shown in FIG. 1A and FIG. 11A. Also, a schematic diagram of a fusion protein including a biologically active molecule, an IgM Fc domain monomer, and a J chain, but not including a linker, in which the IgM Fc domain forms a pentamer, is shown in FIG. 1B. Also, a schematic diagram of a fusion protein including a biologically active molecule, a linker, and an IgM Fc domain monomer, but not including a J chain, in which the IgM Fc domain forms a hexamer, is shown in FIG. 1C. Also, a schematic diagram of a fusion protein including a biologically active molecule and an IgM Fc domain monomer, but not including a linker and a J chain, in which the IgM Fc domain forms a hexamer, is shown in FIG. 1D.

[0137] Specifically, the structure of the fusion protein was confirmed using reducing SDS-PAGE. 2 μg of each fusion protein was mixed with 5× reducing-PAGE sample buffer, heated at 100°C for 5 minutes, and then left at room temperature for 20 minutes. After inserting a polyamide gel (12-20%, Invitrogen) into the gel kit, the sample was loaded and electrophoresed at 200V for 30 minutes, and the gel was then separated from the kit and stained with Coomassie Brilliant Blue staining solution and left for 1 hour. The left gel was transferred to a destaining buffer (70% triple distilled water, 20% methanol, 10% acetic acid) and the results were confirmed overnight.

[0138] As a result, as shown in Figures 2A, 2B and 2C, each fusion protein containing an IgM Fc region, a biologically active molecule, a J chain and / or a linker includes all the sizes of the corresponding substances that compose it.

[0139] The above results suggest that the fusion protein prepared in Example 1-2 has the desired structure and is expressed sufficiently.

[0140] Examples 1-4. Fusion protein target binding ability In this Example 1-4, the binding ability of the fusion proteins prepared in Example 1-2 to the targets was analyzed. At this time, the targets were set differently depending on the biologically active molecules (CMVpHLA, IgG antibody fragment, or EPO analog) contained in each fusion protein.

[0141] Example 1-4-1. CMVpHLA-containing fusion protein The biologically active molecule was the target of the fusion protein containing CMVpHLA, and its binding ability to anti-CMVpHLA antibody was confirmed.

[0142] Specifically, the results were analyzed through ELISA assay and SPR (Surface Plasmon Resonance).

[0143] The ELISA assay was performed as follows. Anti-CMVpHLA antibody (Ajou University) was coated on a 96-well plate at 10 ng / well for 16 hours at 4°C. The plate was then washed with 0.05% PBS-tween and blocked with 3% nonfat dry milk. After blocking, a dilution solution containing each fusion protein or control protein was added to the wells and incubated at 37°C for 2 hours. The plate was then washed and HRP-linked anti-IgM (F5μ) antibody (MERCK) or HRP-linked anti-FLAG antibody (Abcam) was diluted 1:5000 and incubated at 37°C for 1 hour. The plate was then washed 9 times with 0.05% PBS-tween and developed with TMB solution (Sigma), and the reaction was terminated with 1N sulfuric acid (Oi Chemical Industry Co., Ltd.) and analyzed with VARIOSKAN LUX (Thermo Scientific).

[0144] SPR analysis was performed as follows. The SPR analysis device used was a Biacore T200 from Cytiva. To label the anti-CMVpHLA mouse Fc antibody (Ajou University) onto the CM5 chip (Cytiva), recombinant protein G (Abcam) was attached using an EDC / NHS reaction and blocked with 1M ethanolamine. Next, the CMVpHLA mouse Fc antibody was poured to coat the chip in a directionally specific manner. Next, a dilution solution containing each fusion protein or a control protein was poured, and a dissociation reaction was carried out with 10 mM glycine-HCl, pH 1.5.

[0145] As a result, as shown in Figure 3A, the CMVpHLA control protein not containing the IgM Fc region had significantly lower binding activity to the target (anti-CMVpHLA antibody). In contrast, the fusion protein containing the IgM Fc region had very high binding activity to the target (anti-CMVpHLA antibody), and EC 50 Values ​​were several dozen fold improved compared to the CMVpHLA control protein.

[0146] [Table 4]

[0147] (In Table 4, ka is the rate at which two substances adhere to each other (association rate constant), kd is the rate at which two substances dissociate (dissociation rate constant), KD is the binding strength, Rmax is the maximum RU value at which the angle in SPR changes when two substances adhere to each other, and avidity score is the Rmax / kd value, which indicates the degree of avidity in SPR.)

[0148] Furthermore, as shown in Table 4, the CMVpHLA control protein not containing the IgM Fc region had a significantly low KD value and avidity score (Rmax / Kd) for the target (CMVpHLA mouse Fc antibody). In contrast, the fusion protein containing the IgM Fc region had a significantly high KD value and avidity score (Rmax / Kd) for the target (CMVpHLA mouse Fc antibody), and showed a 100- to 1000-fold improved binding ability compared to the CMVpHLA control protein. The results suggest that the IgM Fc region can improve the binding ability of a biologically active molecule to a target, and therefore the fusion protein containing the IgM Fc region and a biologically active molecule according to the present invention can exhibit a high level of the desired biological activity in cells.

[0149] Example 1-4-2. Fusion protein containing IgG antibody fragment The fusion protein containing an antibody fragment (IgG Fab; A2014) as a biologically active molecule was targeted, and its binding ability to the HLA-G protein antigen was confirmed.

[0150] Specifically, the analysis was carried out through the ELISA assay and SPR according to Example 1-4-1.

[0151] As a result, as shown in Figure 3B, the (A2014)-(IgGFc) control protein containing the IgG Fc region had significantly lower binding activity to the HLA-G antigen. In contrast, the fusion protein containing the IgM Fc region had very high binding activity to the HLA-G antigen and was significantly higher in EC 50 This value was improved by approximately 1000-fold or more compared to the (A2014)-(IgGFc) control protein.

[0152] [Table 5]

[0153] (In Table 5, ka is the rate at which two substances adhere to each other (association rate constant), kd is the rate at which two substances dissociate (dissociation rate constant), KD is the binding force, Rmax is the maximum RU value at which the angle in SPR changes when two substances adhere to each other, and avidity score is the Rmax / kd value, which indicates the degree of avidity in SPR.)

[0154] Furthermore, as shown in Table 5, the (A2014)-(IgGFc) control protein containing an IgG Fc region had a significantly low KD value and avidity score (Rmax / Kd) for the HLA-G antigen. In contrast, the fusion protein containing an IgM Fc region had a significantly high KD value and avidity score (Rmax / Kd) for the HLA-G antigen, and showed a 10- to 500-fold improved binding ability compared to the (A2014)-(IgGFc) control protein. The results suggest that the IgM Fc region can improve the binding ability of a biologically active molecule to its target, and therefore the fusion protein containing the IgM Fc region and a biologically active molecule according to the present invention can exhibit a high level of the desired biological activity in cells.

[0155] Example 1-4-3. Fusion protein containing EPO analogue The binding ability of the fusion protein containing an EPO analog (NESP) as a biologically active molecule to the EPO receptor was confirmed.

[0156] Specifically, the analysis was carried out through the ELISA assay and SPR according to Example 1-4-1.

[0157] As a result, as shown in FIG. 3C, the fusion protein containing the IgM Fc region and NESP had extremely high binding activity to the EPO receptor.

[0158] [Table 6]

[0159] (In Table 6, ka is the rate at which two substances adhere to each other (association rate constant), kd is the rate at which two substances dissociate (dissociation rate constant), KD is the binding force, Rmax is the maximum RU value at which the angle in SPR changes when two substances adhere to each other, and avidity score is the Rmax / kd value, which indicates the degree of avidity in SPR.)

[0160] Furthermore, as shown in Table 6, the NESP control protein not containing the IgM Fc region had a significantly low KD value and avidity score (Rmax / Kd) for the EPO receptor. In contrast, the fusion protein containing the IgM Fc region and NESP had a significantly high KD value and avidity score (Rmax / Kd) for the EPO receptor, and showed a binding ability that was 100- to 400-fold improved compared to the NESP control protein. The results suggest that the IgM Fc region can improve the binding ability of a biologically active molecule to its target, and therefore the fusion protein containing the IgM Fc region and a biologically active molecule according to the present invention can exhibit a high level of the desired biological activity in cells.

[0161] In particular, the results of the above-mentioned Examples suggest that the effect of the fusion protein is exhibited regardless of the type or MoA (Mode of Action) of the biologically active molecule, as long as the biologically active molecule binds to the IgM Fc region.

[0162] Examples 1-5. Fusion protein biological activity

[0163] Example 1-5-1.CD8 + T cell activation, proliferation and cytotoxicity To confirm the biological activity of the fusion protein prepared in Example 1-2, CD8 + The ability to induce activation and proliferation of T cells was analyzed.

[0164] First, CD8+ T cell activation was confirmed by IFN-γ ELISPOT analysis. PBMCs were purchased from Immunospot and used as CD8 + T cells are CD8 + T cells were obtained using a T cell isolation kit (Miltenyi Biotec), and ELISPOT analysis was performed using an ELISPOT kit (Immunospot). After that, the plate coated with IFN-γ antibody was washed with PBS, and RPMI-1640 (10% FBS) was added to stabilize the antibody. After removing RPMI-1640, dilutions containing each fusion protein or control protein were added to the plate. Then, PBMCs (6 × 10 5 cells / well) and CD8 + T cells (2×10 5 The plates were then washed with PBS to remove the cells and other substances, and then incubated with biotinylated IFN-γ antibody for 2 hours at room temperature. After washing with PBS, the plates were incubated with Strep-AP for 1 hour at room temperature, and then washed with PBS and incubated with the substrate for 10 minutes to induce color development. The results were then analyzed using an ELISPOT reader (AID) and statistical processing was performed using the GraphPad Prism Version 5.0 (GraphPad software) analysis program.

[0165] On the other hand, CD8 + The ability to induce proliferation of T cells was confirmed by FACS. PBMC 1 × 10 6 Suspend the cells / mL in RPMI-1640 (10% FBS) and plate them in 12 wells at 1 × 10 6The cells were seeded at a density of 100 cells / well. Then, each fusion protein or control protein was treated at a concentration of 2 nM per well, and IL-2 (Peprotech) was added at 20 U / mL each and cultured. After 3-4 days, IL-2 20 U / mL, IL-7 (Peprotech) 25 ng / mL, and IL-15 (Peprotech) 25 ng / mL were added, and on the 7th-8th day of culture, the three cytokines were mixed and the initial volume of culture medium was added. On the 10th-11th or 19th-20th days, each cell was obtained and washed with FACS buffer (5% FBS in PBS). Then, the cell suspension was treated with isotype antibody (Biolegend) to carry out Fc blocking, and then washed with FACS buffer. Then, PerCP cy5.5 anti-human CD8 antibody (BD bioscience) was treated and washed, and HLA-A was detected. * 02:01 CMV pp65 tetramer (MBL) was treated and then washed. After the process was completed, flow cytometry (BD bioscience, FACSLyric TM ) using CMV-specific CD8 + The degree of T cell proliferation was confirmed.

[0166] In addition, CD8 + Cytotoxicity analysis of T cells was confirmed by FACS. 6 × 10 PBMC purchased from Immunospot 5 Suspend the cells / mL in RPMI-1640 (10% FBS) and plate 1.8 × 10 6After seeding, each fusion protein or control protein was treated at a concentration of 100 nM per well, and IL-2 (Peprotech) at 20 U / mL and anti-human CD28 antibody (Ebioscience) at 2 μg / mL were added and cultured. After 3 to 4 days, IL-2 20 U / mL, IL-7 (Peprotech) at 25 ng / mL, IL-15 (Peprotech) at 25 ng / mL, and anti-human CD28 antibody at 2 μg / mL were added, and on the 7th to 8th day of culture, the three cytokines and one antibody were mixed, and the same volume of culture medium as the initial volume was added, and each fusion protein or control protein was added at the same time. After that, the three cytokines and one antibody were mixed and added every 3 to 4 days, and on the 19th to 20th days, each cell was washed with FACS buffer and CD8 + T cells are CD8 + The T cells were isolated using a T cell isolation kit (Miltenyi Biotec). Then, the target cells, T2 cells (ATCC), were reacted with 50 μg / mL of CMVpp65 peptide and 3 μg / mL of B2M (Sino) for 1 hour at 37°C in a 5% CO2 incubator. After washing with FACS buffer, 1 × 10 6 After treatment with 4 μM DiO (Invitrogen), the cells were incubated at 37°C in a 5% CO2 incubator and then washed. + T cells were mixed in different ratios and incubated with each fusion protein or control protein for 4 hours. After 10 minutes of incubation with 7AAD (Invitrogen), the death of T2 cells was analyzed by flow cytometry.

[0167] As a result, as shown in Figure 4, the fusion protein containing the IgM Fc region and CMVpHLA produced about 2-fold more IFN-γ than the control CMVpHLA protein not containing the IgM Fc region. In addition, the fusion protein containing both the IgM Fc region and the linker produced higher IFN-γ than the fusion protein containing the IgM Fc region but not the linker. The above results suggest that the IgM Fc enhances the target binding ability of the fusion protein, and that the addition of a linker will result in even better target binding ability.

[0168] In addition, as shown in FIG. 5A, the fusion protein containing both the IgM Fc region and the linker was significantly more effective in inhibiting CD8 + The effect of inducing T cell proliferation was more than three times greater.

[0169] As shown in FIG. 5B, the fusion protein containing the IgM Fc region and the linker and CMVpHLA inhibited CD8 + The effect of inducing T cell proliferation was more than 30 times greater.

[0170] Furthermore, as shown in FIG. 6, the fusion protein containing the IgM Fc region and CMVpHLA was more toxic to cancer cells than the CMVpHLA control protein not containing the IgM Fc region.

[0171] The results showed that fusion proteins containing the IgM Fc region were able to bind to CD8 at higher levels than proteins that did not contain the IgM Fc region. + These results suggest that the IgM Fc region can induce T cell activation, proliferation, and cancer cell death, and that it can exhibit even superior biological activity when the IgM Fc region and the biologically active molecule are linked via a linker.

[0172] Example 1-5-2. Binding activity to HLA-G expressing cells To confirm the biological activity of the fusion protein prepared in Example 1-2, its binding activity to JEG-3 cells expressing HLA-G was analyzed.

[0173] Specifically, JEG-3 cells (ATCC, 3.5 × 10 5 Dilutions containing each fusion protein or control protein were added to the cells (100 μL / cell) and incubated at 4°C for 1 hour. After washing with FACS buffer (5% FBS in PBS), the cells were treated with anti-IgM-PE (ebioscience) or anti-IgG Fc-FITC (Biolegend) and incubated at 4°C for 30 minutes. Finally, the cells were washed with FACS buffer and analyzed by flow cytometry (BD bioscience, FACSLyric TM ) was used to confirm the extent of fusion protein and control binding to JEG-3 cells.

[0174] As a result, the (A2014)-(IgGFc) control protein containing the IgG Fc region had significantly lower binding activity to HLA-G expressing cells, as shown in Figure 7. In contrast, the fusion protein containing the IgM Fc region had extremely high binding activity to the cells, with the maximum MFI (median fluorescence intensity) value being approximately 2.5-fold higher than that of the (A2014)-(IgGFc) control protein.

[0175] The above results suggest that the fusion protein containing the IgM Fc region has higher intracellular receptor binding activity than a protein not containing the IgM Fc region, i.e., the fusion protein of the present invention containing the IgM Fc region and a biologically active molecule can exhibit a high level of the desired biological activity in cells.

[0176] Example 1-5-3. ILT-2 protein binding blocking activity To confirm the biological activity of the fusion protein prepared in Example 1-2, the binding blocking activity of HLA-G protein and ILT-2 (LILRB1) protein was analyzed. The ILT-2 protein is a receptor protein expressed by immune cells, and when HLA-G protein binds to it, the activity of cytotoxic T cells, NK cells, and B cells is suppressed, preventing normal immune responses.

[0177] First, the binding blocking activity of recombinant HLA-G protein and recombinant ILT-2 protein was analyzed by ELISA assay. ILT-2 antigen (R&D systems) was added to a 96-well plate at 150 ng / well and then coated at 4°C for 16 hours. The plate was then washed with 0.05% PBS-Tween and blocked with 3% skim milk powder. After blocking, dilutions containing each fusion protein or control protein were added to the wells, and each well was treated with HLA-G-6x his tag (IM Biologics) at a certain concentration and incubated at 37°C for 2 hours. The plate was then washed and HRP-linked anti-6x his antibody (abcam) was diluted 1:10,000 and incubated at 37°C for 1 hour. Thereafter, the plate was washed nine times with 0.05% PBS-Tween, and the color was developed with TMB solution (Sigma), and the reaction was terminated with 1N sulfuric acid (Oi Chemical Industry) and analyzed using VARIOSKAN LUX (Thermo Scientific).

[0178] On the other hand, the binding blocking activity between HLA-G protein and ILT-2 protein naturally expressed from cells was analyzed using JEG-3 cells. JEG-3 cells (3 × 10 5The cells (cells / tube) were released into MEM containing 10% FBS. Dilutions containing each fusion protein or control protein were then added to the tubes and incubated at 4°C for 30 minutes. After washing with FACS buffer (5% FBS in PBS), 1.5 μg of ILT-2 Fc chimera (R&D systems) was added and incubated at 4°C for 30 minutes. After washing with FACS buffer, PE-anti-human IgG Fc (Biolegnd) was added and incubated at 4°C for 20 minutes. Finally, after washing with FACS buffer, the cells were analyzed by flow cytometry (BD bioscience, FACSLyric TM ) was used to confirm the degree of blocking of the binding between ILT-2 protein and intracellularly expressed HLA-G protein.

[0179] As a result, as shown in Figure 8, the (A2014)-(IgGFc) control protein containing the IgG Fc region had significantly low binding blocking activity for recombinant HLA-G protein and ILT-2 protein. In contrast, the fusion protein containing the IgM Fc region had very high binding blocking activity for the above proteins, and showed high IC 50 The values ​​were improved by about 5 to 7 times or more compared to the (A2014)-(IgGFc) control protein.

[0180] In addition, as shown in Figure 9, the fusion protein containing the IgM Fc region had a very high binding blocking activity against the HLA-G protein expressed from cells, suggesting that it can effectively inhibit the binding of HLA-G expressed on the surface of cancer cells in vivo to ILT-2, an inhibitory receptor of immune cells, thereby increasing the death of cancer cells.

[0181] The above results suggest that the fusion protein containing the IgM Fc region has a higher activity of blocking the binding of an intracellularly expressed receptor protein to an antigen than a protein not containing the IgM Fc region. In other words, the fusion protein containing the IgM Fc region and a biologically active molecule according to the present invention can exhibit a high level of the desired biological activity in cells.

[0182] Example 1-5-4. Binding activity to intracellular EPO receptor To confirm the biological activity of the fusion protein prepared in Example 1-2, the binding activity to cells expressing EPO receptor was analyzed.

[0183] Specifically, the binding activity was confirmed using the EPOR reporter assay purchased from Indigobioscience. Cells were seeded into a 96-well cell culture plate (Corning) at a density of 150uL / well and then incubated in a 5% CO2 incubator at 37℃ for 4-6 hours. Then, dilutions containing each fusion protein or control protein were added to the wells and incubated in a 5% CO2 incubator at 37℃ for one day. Then, 100uL of reaction solution was added according to the manufacturer's protocol, and after color development at room temperature for 5 minutes, analysis was performed using VARIOSKAN LUX (Thermo Scientific).

[0184] As a result, the fusion protein containing the IgM Fc region and linker induced a higher level of proliferation of cells overexpressing EPOR compared to the NESP control protein not containing the IgM Fc region, as shown in Figure 10. In other words, the fusion protein containing the IgM Fc region showed excellent binding activity to the intracellular EPO receptor, activating the signaling pathway stimulated by the EPO receptor, ultimately improving the proliferation ability of cells.

[0185] The above results suggest that the fusion protein containing the IgM Fc region has a higher ability to bind to intracellular receptors and thus induce biological activity than a protein that does not contain the IgM Fc region. In other words, the fusion protein containing the IgM Fc region and a biologically active molecule according to the present invention can exhibit a high level of the desired biological activity in cells.

[0186] In particular, the results of the above examples suggest that the effect of the IgM Fc region in inducing the biological activity of a fusion protein is exhibited regardless of the type or MoA of the biologically active molecule, so long as the biologically active molecule binds to the IgM Fc region.

[0187] Example 2. Preparation of IgG Fc region and IgM Fc region containing fusion protein platform In this Example 2, a fusion protein platform was prepared that includes a biologically active molecule, a linker, an IgG Fc region, an IgM Fc region, and / or a J chain, and a fusion protein platform was prepared using mutations that deleted or substituted amino acids in the wild-type IgG Fc region, the wild-type IgM Fc region, and / or the wild-type J chain.

[0188] Example 2-1. Preparation of wild-type fusion protein platform In this Example 2-1, recombinant plasmids of a linker, an IgG Fc region, an IgM Fc region, and a J chain were produced. The linker was produced using an IgD hinge region (a part of the IgD CH1 domain, a part of the hinge and CH2 domain), the IgG Fc region was produced using the CH2 domain and CH3 domain of a wild-type IgG, the IgM Fc region was produced using the cμ3 domain and cμ4 domain of a wild-type IgM, and the J chain was produced using a wild-type J chain.

[0189] Specifically, recombinant plasmids were constructed to express the IgG Fc region (SEQ ID NO: 1, 2, or 3), the IgM Fc region (SEQ ID NO: 6), a linker (SEQ ID NO: 16) linked to the N-terminal portion of the IgG Fc region, and the J chain (SEQ ID NO: 14) required for pentamer formation.

[0190] To proceed with gene cloning, each of the above substances was codon optimized, and codon-optimized nucleotides were synthesized by Cosmogenetek (Seoul, Korea). Using the optimized nucleotides and primers, insert DNA was amplified by PCR so that overlap PCR could be performed on the MCS (multiple cloning site) of pcDNA3.1 (manufacturer: Thermo Scientific) vector. After overlap PCR of each of the obtained insert DNAs and the pcDNA3.1 vector, the template was digested using DpnI restriction enzyme to prepare vectors into which each substance was inserted.

[0191] The vector was inserted into Stella® competent cells (manufacturer: Clontech), and then smeared on an agar plate containing ampicillin antibiotic that matches the antibiotic resistance gene in the vector, and colonies were selected. The base sequence was confirmed through DNA sequencing using an Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Scientific).

[0192] Example 2-2. Preparation of mutant fusion protein platform In this Example 2-2, recombinant plasmids of the linker, IgG Fc region, IgM Fc region, and J chain were prepared, and a fusion protein in which the IgM Fc region and J chain were mutated was prepared. Based on the plasmid vector prepared in Example 2-1, some amino acids in the wild-type IgM domain were deleted or substituted, and the J chain was also used with some amino acids deleted or substituted.

[0193] Specifically, random point mutations were made in the cμ3 and cμ4 domains (SEQ ID NO: 6) of the IgM Fc region, and random point mutations were made in the wild-type J chain (SEQ ID NO: 14), and then recombinant plasmids were constructed.

[0194] To proceed with gene cloning, we asked Cosmogenetek (Seoul, Korea) to synthesize primers. Using these primers, overlap PCR was performed on the MCS (multiple cloning site) of pcDNA3.1 (manufacturer: Thermo Scientific) vector, and the template was removed by treating with DpnI restriction enzyme to prepare vectors into which each substance was inserted.

[0195] The vector was inserted into Stella® competent cells (manufacturer: Clontech), and then smeared on an agar plate containing ampicillin antibiotic that matches the antibiotic resistance gene in the vector, and colonies were selected. The base sequence was confirmed through DNA sequencing using an Applied biosystems 3730xl DNA analyzer (manufacturer: Thermo Scientific).

[0196] Examples 2-3. Preparation of fusion proteins containing biologically active molecules In this Example 2-3, a fusion protein containing a biologically active molecule was produced.

[0197] Specifically, a recombinant plasmid of a biologically active molecule was prepared, and then linked to the N-terminus of the fusion protein platform recombinant plasmid prepared in Example 2-1 or 2-2. The fusion protein was expressed and purified using the plasmid prepared in this manner.

[0198] In this case, HLA-A or IgG antibody fragment (IgG Fab) was used as an example of a biologically active molecule.

[0199] Example 2-3-1. Preparation of HLA-A-containing fusion protein A fusion protein was prepared that contained HLA-A as the biologically active molecule, a linker, an IgG Fc region, an IgM Fc region, and a J chain.

[0200] Specifically, to increase the stability of the HLA-A protein, a peptide derived from CMV (cytomegalovirus) was further loaded, and then β2m (Beta-2 microglobulin) was subsequently loaded onto the peptide derived from CMV, thereby constructing a CMV pHLA protein with increased stability, the specific amino acid sequence of which is set forth in SEQ ID NO:22.

[0201] The final recombinant plasmid vector of the fusion protein (hereinafter referred to as "CMVPHLA fusion protein") containing the constructed biologically active molecule CMVpHLA and including a linker, an IgG Fc region, an IgM Fc region, and a J chain was introduced into Expi293 (Thermo Scientific) animal cells to establish a transient expression system for the CMVpHLA fusion protein.

[0202] CMVpHLA fusion protein plasmid DNA and J chain plasmid DNA were transfected at a ratio of 10:1. The combined recombinant plasmid vectors were mixed at a total amount of 1μg / mL with Opti-MEM® medium, and ExpiFectamine® reagent and Opti-MEM® medium were mixed to the appropriate volume and left at room temperature for 5 minutes. After that, the DNA and ExpiFectamine® reagent were reacted for 15 minutes and added to the flask, and enhancer 1 and 2 were treated 16 to 22 hours later. The cell viability was confirmed to be 75% or higher on the 4th to 5th day, and the fusion protein was collected.

[0203] The expressed CMVpHLA fusion protein was then purified using IgM affinity chromatography. TM CaptureSelect TMThe fusion protein was purified by loading onto IgM Affinity Matrix® (Thermo Scientific). IgM affinity chromatography was performed under the following conditions.

[0204] <Chromatography conditions> -Resin: POROS TM CaptureSelect TM IgM Affinity Matrix® -Flow velocity: 480cm / h -Equilibrium: PBS (Phosphate-buffered saline) buffer - Loading: up to 6g protein / L resin volume -Regeneration and sterilization: 0.01M NaOH solution -Elution: 0.1M glycine-HCl, pH2.7 buffer -Storage: 20% EtOH

[0205] Then, 1 / 10 of the eluate volume was neutralized with 1.0 M Tris-HCl, pH 8.5 buffer. The recovered CMVpHLA fusion protein was concentrated using ultrafiltration, dialyzed against PBS, and stored at -20°C.

[0206] The CMVpHLA fusion protein prepared by the above method will be represented as follows. First, the substance starting from the N-terminus of the fusion protein is represented by a structural formula: [(biologically active molecule)-(linker)-(IgG Fc region)-(IgM Fc region)]-(J chain). The mutants are represented by the amino acids substituted for the existing amino acids in the corresponding regions based on the numbering of the reference IgM sequence number 6 and the J chain sequence number 14. Hereinafter, the CMVpHLA fusion protein will be named with the substance numbering as shown in Table 7 below. In the case of fusion proteins containing random point mutations, if there are problems with expression or they do not form pentamers, they are not named separately.

[0207] - Wild-type IgM Fc region sequence (SEQ ID NO: 6, where amino acids 124 and / or 125 underlined and bolded may contain mutations): TIFF2025513907000008.tif55168

[0208] - Wild type J chain sequence (SEQ ID NO: 14, where amino acids 105, 106, and / or 107 may contain mutations as underlined and bolded): TIFF2025513907000009.tif31168

[0209] [Table 7] TIFF2025513907000011.tif232168TIFF2025513907000012.tif232168TIFF2025513907 000013.tif232168TIFF2025513907000014.tif231168TIFF2025513907000015.tif95168

[0210] On the other hand, a fusion protein not containing the IgM Fc region (hereinafter referred to as "CMVpHLA-IgG Fc") was prepared as a control. The IgG Fc region contained in the fusion protein used was the CH2 domain and CH3 domain of IgG. Purification and concentration of the control were performed as follows. The culture medium obtained from the transient expression system was loaded onto an IgG affinity matrix MabSelect SuRe (registered trademark) (Cytiva) and purified.

[0211] IgG affinity chromatography was performed under the following conditions.

[0212] <Chromatography conditions> -Resin: MabSelect SuRe (registered trademark) -Flow velocity: 300cm / h -Equilibrium: PBS (Phosphate-buffered saline) buffer - Loading: up to 35g protein / L resin volume -Regeneration and sterilization: 0.01M NaOH solution -Elution: 0.1M glycine-HCl, pH2.7 buffer -Storage: 20% EtOH

[0213] After IgG affinity chromatography, 1 / 10 of the eluate volume was neutralized with 1.0 M Tris-HCl, pH 8.5 buffer, and the recovered fusion protein was concentrated using ultrafiltration, dialyzed against PBS, and stored at -20°C.

[0214] This resulted in a decreased binding ability to pIgR, which in turn increased the half-life in blood. Furthermore, the fusion proteins containing the mutations showed decreased binding ability to pIgR compared to the fusion proteins without the mutations (IMB-4001 and IMB-4011).

[0215] Example 2-3-2. Preparation of fusion protein containing IgG antibody fragment A fusion protein (hereinafter referred to as "A2014 fusion protein") was produced that contained an IgG antibody fragment (IgG Fab; hereinafter referred to as "A2014") as a biologically active molecule, and also contained a linker, an IgG Fc region, an IgM Fc region, and a J chain.

[0216] The A2014 was prepared by the cloning method according to Example 2-1 using a light chain (VL domain and CL domain; SEQ ID NO: 27) and a part of a heavy chain (VH domain and CH1 domain; SEQ ID NO: 31) from the IgG Fab portion.

[0217] Meanwhile, a fusion protein not containing the IgM Fc region (hereinafter referred to as "A2014-IgG") was prepared as a control. The IgG Fc region contained in the A2014-IgG was prepared by the cloning method described in Example 2-1 above, using the CH2 and CH3 domains of IgG, specifically, the light chain (VL and CL domains; SEQ ID NO: 27) and the heavy chain (VH, CH1, CH2, and CH3 domains; SEQ ID NO: 29).

[0218] The constructed A2014 recombinant plasmid sequence was ligated to the fusion protein platform plasmid sequence by the method according to Example 2-3-1, and the final recombinant plasmid vector thus constructed was introduced into cells to express each protein.

[0219] Specifically, the A2014 heavy chain part, A2014 light chain and J chain plasmid DNA were transfected at a ratio of 4.5:4.5:1. The combined recombinant plasmid vectors were mixed at a total amount of 1 μg / mL with Opti-MEM (registered trademark) medium, and the ExpiFectamine (registered trademark) reagent and Opti-MEM (registered trademark) medium were mixed to the appropriate volume and then left at room temperature for 5 minutes. After that, the DNA and ExpiFectamine (registered trademark) reagent were reacted for 15 minutes and added to the flask, and enhancer 1 and 2 were treated 16 to 22 hours later. The cell viability was confirmed to be 75% or more on the 4th to 5th day, and the fusion protein was collected.

[0220] Thereafter, the culture medium was purified and concentrated in the same manner as in Example 2-3-1, dialyzed against PBS, and stored at -20°C.

[0221] The A2014 fusion protein prepared by the above method is represented by the structural formula as shown in Table 7, and the substance numbering is the same as that in Table 8.

[0222] [Table 8]

[0223] Example 2-4. Structure of the fusion protein produced It was confirmed whether the substances prepared in Examples 2-3-1 and 2-3-2 had the desired structures.

[0224] Meanwhile, a schematic diagram of a pentameric fusion protein containing a biologically active molecule, a linker, an IgG Fc region, an IgM Fc variant, and a variant of the J chain is shown in FIG. 11B.

[0225] Specifically, the structure of the fusion protein was confirmed using reducing SDS-PAGE. 1 μg of each fusion protein was mixed with 5x reducing-PAGE sample buffer, heated at 100°C for 5 minutes, and then left at room temperature for 20 minutes. After connecting a polyamide gel (12-20%, Invitrogen) to the gel kit, the sample was loaded and electrophoresed at 200V for 30 minutes, and the gel was removed from the kit and stained with Coomassie Brilliant Blue staining solution for 1 hour. The gel was then transferred to a destaining buffer (70% triple distilled water, 20% methanol, 10% acetic acid) and the results were confirmed overnight.

[0226] As a result, as shown in Figures 12A and 12B, it was confirmed that each fusion protein containing a biologically active molecule, linker, IgG Fc region, IgM Fc, and J chain contains all the sizes of the corresponding substances that constitute it.

[0227] The above results suggest that the fusion protein prepared in Example 2-3 has the desired structure and is expressed sufficiently.

[0228] Examples 2-5. Biological activity of fusion proteins In this Example 2-5, the biological activity of the fusion proteins prepared in Example 2-3 was analyzed. The purpose of this test was to confirm whether the effect of increasing the binding activity to the target shown by the fusion protein without mutations was maintained by the fusion protein with mutations. In this case, the target was set differently depending on the biologically active molecule (CMVpHLA or IgG antibody fragment) contained in each fusion protein.

[0229] Example 2-5-1. Memory CD8 of CMVpHLA fusion protein + T cell proliferation To confirm the biological activity of the fusion protein prepared in Example 2-3-1, CD8 + The ability of T cells to induce memory cell proliferation was analyzed.

[0230] CD8 + The ability to induce memory cell proliferation in T cells was confirmed by FACS. After approval by the Bioethics Committee, PBMCs were isolated from human donor blood obtained through the Gyeonggi Red Cross using Ficoll gradient centrifugation. The PBMCs were 1×10 6 Suspend the cells / mL in RPMI-1640 (10% FBS) and plate them in 6 wells at 1 × 10 6 The cells were seeded at a density of 100 cells / well. Thereafter, the CMVpHLA fusion protein or control protein was treated at a concentration of 100 nM per well, and IL-2 (Peprotech) was added at 20 U / mL each and cultured. After 3-4 days, IL-2 20 U / mL was added, and on the 7th-8th day of culture, half the initial volume of culture medium with each protein and IL-2 was added. On the 13th-14th day, each cell was obtained and washed with FACS buffer (5% FBS in PBS).

[0231] The cell suspension was then treated with isotype antibody (Biolegend) for Fc blocking, and washed with FACS buffer. Then, PerCP cy5.5 anti-human CD8 antibody (BD bioscience), APC anti-human CD45RA antibody (BD bioscience), and FITC anti-human CCR7 antibody (Biolegen) were treated and washed. After completing this process, the cells were analyzed by flow cytometry (BD bioscience, FACSLyric TM ) using CD8 + The degree of T cell proliferation was confirmed.

[0232] As a result, as shown in FIG. 13A, naive CD8 + The proportion of T cells analyzed was 36%, where CMVpHLA fusion proteins IMB-4001, IMB-4011, and IMB-4012 all expressed naive CD8 + The proportion of T cells is measured at about 3-4%, and the remaining proportion exceeding 30% is memory CD8 + However, the control IMB-4000 (CMVpHLA-IgG) had low avidity and inhibited only 13% of naive CD8 T cells. + The remaining T cells are memory CD8 + It was analyzed that the conversion rate to T cells was low.

[0233] The above results suggest that the effect of increasing the binding activity to the target shown by the fusion proteins IMB-4001 and IMB-4011, which do not contain a mutation, is maintained by the fusion protein IMB-4012, which contains a mutation.

[0234] Example 2-5-2. ILT-2 protein binding blocking activity To confirm the biological activity of the A2014 fusion protein prepared in Example 2-3-2 and the control group, the binding blocking activity of HLA-G protein and ILT-2 (LILRB1) protein was analyzed. The ILT-2 protein is a receptor protein expressed by immune cells, and when HLA-G protein binds to it, the activity of cytotoxic T cells, NK cells, and B cells is suppressed, preventing normal immune responses.

[0235] To confirm the biological activity of the fusion protein, the binding blocking activity between recombinant HLA-G protein and recombinant ILT-2 protein was analyzed by ELISA assay. ILT-2 antigen (R&D systems) was added to a 96-well plate at 150 ng / well and then coated at 4°C for 16 hours. The plate was then washed with 0.05% PBS-Tween and blocked with 3% skim milk powder. After blocking, a dilution solution containing each A2014 fusion protein, A2014 mutant fusion protein, or control protein was added to the wells, and each well was treated with HLA-G-6x his tag (IM Biologics) at a certain concentration and incubated at 37°C for 2 hours. The plate was then washed, and HRP-linked anti-6x his antibody (abcam) was diluted 1:10,000 and incubated at 37°C for 1 hour. Thereafter, the plate was washed with 0.05% PBS-Tween, and the color was developed with TMB solution (Sigma), and the reaction was terminated with 1N sulfuric acid (Oi Chemical Industry) and analyzed with VARIOSKAN LUX (Thermo Scientific).

[0236] As a result, as shown in Figure 13B, the control group A2014-1 (A2014-IgG) had significantly low binding blocking activity for recombinant HLA-G protein and ILT-2 protein. In contrast, the A2014 fusion proteins (A2014-2, A2014-3, and A2014-4) had very high binding blocking activity for the above proteins, and showed IC 50 The value was improved by about 5-fold or more compared to the control protein.

[0237] The above results suggest that the binding activity enhancing effect on the targets shown by A2014-2 and A2014-3, which are fusion proteins without mutations, is also maintained in A2014-4, which is a fusion protein with mutations.

[0238] Example 2-6. Receptor affinity of fusion proteins

[0239] Example 2-6-1. Affinity for FcRn interaction In order to confirm whether the fusion proteins produced in Example 2-3 increase the in vivo half-life through binding to FcRn, an ELISA analysis was performed to confirm the interaction with FcRn. The binding to FcRn must occur near weakly acidic (<pH 6.5), and in order to achieve recycling, it is expected that the half-life will increase only when there is no binding at neutral (pH 7.4).

[0240] Specifically, to confirm the binding force with FcRn, a conjugate of FcRn and β2m antigen (ACRO biosystems) was added to a 96-well plate at 1 μg / well, and then coated at 4°C for 16 hours. All solutions for confirming binding under weakly acidic conditions were prepared at pH 6.0, and all solutions for confirming binding under neutral conditions were prepared at pH 7.4. Subsequently, the plate was washed with 0.05% PBS-Tween and then blocked with 3% skim milk powder. After blocking, a dilution containing each fusion protein, mutant fusion protein, or control group protein was added to the wells and incubated at 37°C for 2 hours. Subsequently, the plate was washed, and an anti-IgM (F5μ) antibody (MERCK) conjugated with HRP or an anti-IgG Fc antibody (Invitrogen) was diluted 1:5,000 and incubated at 37°C for 1 hour. Thereafter, the plate was washed with 0.05% PBS-Tween, developed with a TMB solution (Sigma), and then the reaction was terminated with 1N sulfuric acid (Ohi Kinka) and analyzed with a VARIOSKAN LUX (Thermo Scientific).

[0241] As a result, as shown in Figures 14A to 14d, the FcRn-binding ability of the fusion proteins not containing IgG Fc (IMB-4001 or A2014-1) was significantly lower than that of the fusion proteins containing IgG Fc (IMB-4011 or A2014-3; IMB-4012 or A2014-4).

[0242] In contrast, the fusion proteins containing IgG Fc (IMB-4011 or A2014-3; IMB-4012 or A2014-4) had much higher binding strength to FcRn than the control group, and in particular, the fusion proteins containing mutations (IMB-4012 or A2014-4) were analyzed to have better binding efficacy than the fusion proteins without mutations (IMB-4011 or A2014-3).

[0243] These results suggest that the fusion protein is endowed with an advantage in FcRn-binding efficacy due to the introduction of the IgG Fc region and the mutations induced in the IgM Fc region and J chain, thereby improving the strength of interaction and enhancing the efficiency of FcRN recycling.

[0244] Example 2-6-2. Affinity for pIgR (polymeric Ig Receptor) interaction In order to confirm whether the fusion protein prepared in Example 2-3 has a decreased binding ability to pIgR and therefore has an increased half-life in blood, an ELISA analysis was performed to confirm the interaction with pIgR.

[0245] Specifically, to confirm the binding strength with pIgR, pIgR (R&D systems) was added to a 96-well plate at 100 ng / well and coated at 4°C for 16 hours. The plate was then washed with 0.05% PBS-Tween and blocked with 3% skim milk powder. After blocking, dilutions containing each fusion protein and mutant fusion protein were added to the wells and incubated at 37°C for 2 hours. The plate was then washed, and anti-IgM (F5μ) antibody (MERCK) linked to HRP was diluted 1:5,000 and incubated at 37°C for 1 hour. The plate was then washed with 0.05% PBS-Tween, developed with TMB solution (Sigma), and the reaction was terminated with 1N sulfuric acid (Oi Chemical Industry Co., Ltd.) and analyzed with VARIOSKAN LUX (Thermo Scientific).

[0246] As a result, as shown in FIG. 15, when mutations were introduced into N124 of the IgM Fc region, L125 of the IgM Fc region, D105 of the J chain, R106 of the J chain, and / or N107 of the J chain, the binding strength to pIgR was reduced compared to the wild-type fusion protein (IMB-4001, IMB-4011) in which no mutations were introduced. In detail, the relative binding strength values ​​when the binding strength between IMB-4011 and pIgR was set to 100% are shown in Table 9. When the amino acids at the corresponding five sites were substituted, differences in binding strength to pIgR occurred depending on the converted amino acids. In addition, since the difference in binding strength did not have a certain tendency according to the groups classified according to the charge, polarity, and functional group of the amino acids, the change in binding strength due to a specific amino acid was confirmed only experimentally.

[0247] [Table 9] TIFF2025513907000018.tif166168

[0248] The results showed that the fusion proteins with mutations in the IgM Fc region and / or J chain had reduced binding to pIgR through the mutations, thereby increasing their half-life in serum. Furthermore, the fusion proteins with the mutations had reduced binding to pIgR compared to the fusion proteins without the mutations (IMB-4001 and IMB-4011).

[0249] Example 2-6-3. Affinity measurement test for Fcα / μ receptor interaction ELISA analysis was performed to determine whether the fusion proteins prepared in Example 2-3 that had effectively reduced binding ability to pIgR had non-specific binding to Fcα / μ receptors and increased half-life in the blood.

[0250] Specifically, to confirm the binding strength with the Fcα / μ receptor, Fcα / μ receptor (R&D systems) was added to a 96-well plate at 100 ng / well and coated at 4°C for 16 hours. The plate was then washed with 0.05% PBS-Tween and blocked with 3% skim milk powder. After blocking, dilutions containing each fusion protein and mutant fusion protein were added to the wells and incubated at 37°C for 2 hours. The plate was then washed, and anti-IgM (F5μ) antibody (MERCK) linked to HRP was diluted 1:5,000 and incubated at 37°C for 1 hour. The plate was then washed with 0.05% PBS-Tween, developed with TMB solution (Sigma), and the reaction was terminated with 1N sulfuric acid (Oi Chemical Industry Co., Ltd.) and analyzed with VARIOSKAN LUX (Thermo Scientific).

[0251] As a result, as shown in FIG. 16, when mutations were introduced into N124 of the IgM Fc region, L125 of the IgM Fc region, D105 of the J chain, R106 of the J chain, and / or N107 of the J chain, the binding strength to the Fcα / μ receptor was reduced compared to the wild-type fusion protein (IMB-4001, IMB-4011) in which no mutations were introduced. In detail, the relative binding strength values ​​when the binding strength of IMB-4011 to the Fcα / μ receptor was set to 100% are shown in Table 10. When the amino acids at the corresponding 5 sites were replaced, differences in binding strength to the Fcα / μ receptor occurred depending on the converted amino acid. In addition, since the difference in binding strength did not have a certain tendency according to the groups classified according to the charge, polarity, and functional group of the amino acid, the change in binding strength due to a specific amino acid was confirmed only experimentally.

[0252] [Table 10]

[0253] The results showed that the fusion proteins with mutations in the IgM Fc region and / or J chain had decreased binding to the Fcα / μ receptor through the mutations, which increased their half-life in serum. In addition, the fusion proteins with the mutations had decreased binding to the Fcα / μ receptor compared to the fusion proteins without the mutations (IMB-4001 and IMB-4011).

[0254] Example 2-6-4. Affinity measurement test for Fcμ receptor interaction ELISA analysis was performed to determine whether the mutant fusion proteins prepared in Example 2-3 that have reduced binding ability to pIgR would have non-specific binding to Fcμ receptors and increased half-life in the blood.

[0255] Specifically, to confirm the binding strength with the Fcμ receptor, Fcμ receptor (R&D systems) was added to a 96-well plate at 100ng / well and coated at 4℃ for 16 hours. The plate was then washed with 0.05% PBS-tween and blocked with 3% skim milk powder. After blocking, dilutions containing each fusion protein and mutant fusion protein were added to the wells and incubated at 37℃ for 2 hours. The plate was then washed, and anti-IgM (F5μ) antibody (MERCK) linked to HRP was diluted 1:5,000 and incubated at 37℃ for 1 hour. The plate was then washed with 0.05% PBS-tween, developed with TMB solution (Sigma), and the reaction was terminated with 1N sulfuric acid (Oi Kakin) and analyzed with VARIOSKAN LUX (Thermo Scientific).

[0256] As a result, as shown in FIG. 17, when mutations were introduced into N124 of the IgM Fc region, L125 of the IgM Fc region, D105 of the J chain, R106 of the J chain, and / or N107 of the J chain, the binding strength to the Fcμ receptor was reduced compared to the wild-type fusion protein (IMB-4001, IMB-4011) in which no mutations were introduced. In detail, the relative binding strength values ​​when the binding strength between IMB-4011 and the Fcμ receptor was set to 100% are shown in Table 11. When the amino acids at the corresponding 5 sites were replaced, differences in binding strength to the Fcμ receptor occurred depending on the converted amino acids. In addition, since the difference in binding strength did not have a certain tendency according to the groups classified according to the charge, polarity, and functional group of the amino acid, the change in binding strength due to a specific amino acid was confirmed only experimentally.

[0257] [Table 11]

[0258] The above results show that fusion proteins with mutations in the IgM Fc region and / or J chain, except for one mutation (IMB-4012-43), mostly exhibit reduced binding to the Fcμ receptor, thereby reducing non-specific binding and increasing serum half-life of the fusion protein. The results of the above examples suggest that an appropriate combination of IgM Fc region mutations and J chain mutations can reduce non-specific binding of the fusion protein to the target and further increase serum half-life.

[0259] Example 2-7. Affinity for IgM receptor interaction In this Example 2-7, it was attempted to confirm whether a fusion protein in which a mutation in the IgM Fc region and a mutation in the J chain were simultaneously induced would have reduced binding ability to all three types of IgM receptors.

[0260] Specifically, the ELISA analysis for the three types of IgM receptors was performed in the same manner as in Examples 4, 5, and 6, and the relative binding strength values ​​when the binding strength of IMB-4011 to the three types of IgM receptors was set at 100% were entered in Table 12. In addition, since the difference in binding strength does not show a certain tendency according to the groups classified according to the charge, polarity, and functional group of the amino acid, the change in binding strength due to a specific amino acid could only be confirmed experimentally.

[0261] [Table 12]

[0262] As a result, the fusion protein containing all of the IgM Fc region mutations and J chain mutations had reduced binding to all three types of IgM receptors, as shown in Table 12. The results suggest that the IgM Fc region mutations and J chain mutations can mutually complement each other to reduce binding to IgM receptors, which can reduce in vivo nonspecific binding and maximize the increase in serum half-life.

[0263] Example 2-8. Half-life of fusion protein in blood In this Example 2-8, it was attempted to confirm whether the half-life in blood of a fusion protein in which a mutation in the IgM Fc region and a mutation in the J chain were simultaneously induced was increased.

[0264] Specifically, the amount of protein present in plasma was measured after administration of the fusion protein to rats. The half-life in blood refers to the time period during which the protein remains at 50% of the initial concentration while circulating in the body. Pharmacokinetic parameters were calculated using PKsolver. The experimental animals were 6-week-old rats, and after a one-week acclimation period, the individual doses were slowly administered intravenously at 100 nM / kg based on the body weight measured on the day of administration of the test substance, taking into account the different molecular weights of each fusion protein. After administration, blood was collected from the animals by orbital blood sampling at 0.08, 2, 4, 8, 24, 48, 72, 96, 120, and 144 hours. The collected blood was centrifuged to separate plasma, and analyzed using an IgG ELISA kit (Abcam) and an IgM ELISA kit (Abcam) according to the enclosed test method.

[0265] As a result, as shown in Figure 18, the half-life in blood of the fusion protein not containing the IgG Fc region (IMB-4001) was measured to be 4 hours, while the half-life in blood of the fusion protein containing the IgG Fc region (IMB-4011) was increased to 29 hours, despite binding to the IgM receptor, due to the ability of FcRn recycling.

[0266] In particular, the fusion protein (IMB-4012) containing the IgG Fc region and a mutation that prevents IgM receptor binding was measured to have the longest half-life in blood of 59 hours, suggesting that this is the result of FcRn recycling and the inhibitory effect on IgM receptor binding, and that the half-life in blood was maximized.

[0267] That is, the results of the above examples suggest that the fusion protein platform according to the present invention, which comprises an IgG Fc region, an IgM Fc region, a J chain, and a linker, can increase the avidity of a biologically active molecule contained in the fusion protein, and can increase the serum half-life of the fusion protein through inhibition of FcRn recycling and inhibition of IgM-specific receptor binding. It also suggests that the above effects can be further enhanced when mutations are introduced into N124 of the IgM Fc region, L125 of the IgM Fc region, D105 of the J chain, R106 of the J chain, and / or N107 of the J chain contained in the fusion protein.

[0268] From the above description, a person skilled in the art to which the present invention pertains can understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limited thereto. The scope of the present invention should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A fusion protein represented by the following structural formula: [(X) ａ - (Y) ｂ -(IgG Fc domain)c-(IgM Fc domain)] ｍ - (Z) ｎ In this case, in the structural formula: X is a biologically active molecule; said Y being a linker; the IgG Fc region is monomeric; the IgM Fc region is monomeric; said Z being a J chain; The a is 1 or 2, the b is 1 or 2, the c is 0 or 1, the m is 5 or 6, and the n is 0 or 1.

2. The fusion protein of claim 1 , wherein the IgG Fc region comprises one or more domains selected from the group consisting of a CH2 domain and a CH3 domain.

3. The fusion protein of claim 1 , wherein the IgG Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

4. The fusion protein of claim 1 , wherein the IgG Fc region is derived from a wild-type IgG or a mutant IgG.

5. The fusion protein of claim 4 , wherein the wild-type IgG comprises the amino acid sequence of SEQ ID NO: 1 or 3.

6. The fusion protein of claim 4 , wherein the mutant IgG comprises the amino acid sequence of SEQ ID NO:

2.

7. The fusion protein of claim 1 , wherein the IgM Fc region comprises one or more selected from the group consisting of a cμ2 domain, a cμ3 domain, and a cμ4 domain.

8. The fusion protein of claim 1 , wherein the IgM Fc region is derived from a wild-type IgM or a mutant IgM.

9. The fusion protein of claim 8 , wherein the wild-type IgM comprises the amino acid sequence of SEQ ID NO:

6.

10. The fusion protein according to claim 8 , wherein the mutant IgM has one or more amino acids at positions 124 and 125 of the amino acid sequence of SEQ ID NO:6 deleted or substituted.

11. The fusion protein of claim 8, wherein the mutant IgM has a mutation at the 124th amino acid position in the amino acid sequence of SEQ ID NO:6 selected from the group consisting of N124A, N124F, and N124K.

12. The fusion protein of claim 8, wherein the mutant IgM has any one of the mutations at the 125th amino acid position in the amino acid sequence of SEQ ID NO:6 selected from the group consisting of L125A, L125D, L125E, L125F, L125G, L125H, L125I, L125K, L125M, L125N, L125P, L125Q, L125R, L125S, L125T, L125V, L125W, and L125Y.

13. The fusion protein of claim 1 , wherein the J chain is derived from a wild-type J chain or a mutant J chain.

14. The fusion protein of claim 13 , wherein the wild-type J chain comprises the amino acid sequence of SEQ ID NO:

14.

15. The fusion protein of claim 13, wherein the mutant J chain has one or more amino acids at positions 105, 106, and 107 of the amino acid sequence of SEQ ID NO:14 deleted or substituted.

16. The fusion protein of claim 13, wherein the mutant J chain has a mutation at the 105th amino acid position in the amino acid sequence of SEQ ID NO: 14 selected from the group consisting of D105A and D105K.

17. The fusion protein of claim 13, wherein the mutant J chain has any one of the mutations at the 106th amino acid position in the amino acid sequence of SEQ ID NO: 14 selected from the group consisting of R106A, R106D, R106E, R106F, R106G, R106H, R106I, R106K, R106L, R106M, R106N, R106P, R106Q, R106S, R106T, R106V, R106W, and R106Y.

18. The fusion protein of claim 13, wherein the mutant J chain has any one of the mutations at the 107th amino acid position in the amino acid sequence of SEQ ID NO: 14 selected from the group consisting of N107A, N107E, N107F, and N107K.

19. The fusion protein of claim 1 , wherein the J chain further comprises a protein molecule bound thereto.

20. 20. The fusion protein of claim 19, wherein the protein molecule is one or more selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, an antibody-drug conjugate, an antibody-like molecule, an antigen-binding fragment of an antibody-like molecule, a lytic protein, a membrane-bound protein, a ligand, a receptor, a virus-like particle, a protein toxin, an enzyme, a co-stimulatory receptor, a cytokine, and a cell engager.

21. The fusion protein of claim 1, wherein the biologically active molecule is one or more selected from the group consisting of an antibody, an antigen-binding fragment of an antibody, an antibody-drug conjugate, an antibody-like molecule, an antigen-binding fragment of an antibody-like molecule, a lytic protein, a membrane-bound protein, a ligand, a receptor, a virus-like particle, a protein toxin, a chemokine, a cytokine, and an enzyme.

22. The fusion protein of claim 1 , wherein the linker is one or more selected from the group consisting of an IgD hinge region, an IgA hinge region, an IgG hinge region, and a GS linker.

23. 23. The fusion protein of claim 22, wherein the hinge region comprises a portion or all of a hinge region, a portion or all of a CH1 domain, a portion or all of a CH2 domain, or a combination thereof.

24. The GS linker is (GS) ｄ , (S.G.) ｄ , (GGGS) ｄ , (GGGGS) ｄ , GCGS (GGGS) ｄ , and GCGGS (GGGGS) ｄ wherein d is an integer from 2 to 6.

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

26. A vector for expressing a fusion protein, comprising the nucleic acid molecule of claim 25.

27. A host cell into which the fusion protein expression vector described in claim 26 has been introduced.

28. A method for producing the fusion protein described in claim 1, comprising the step of introducing the fusion protein expression vector described in claim 26 into a host cell.

29. 13. Use of the fusion protein according to claim 1 for the manufacture of a diagnostic or therapeutic drug.

30. A pharmaceutical composition comprising the fusion protein of claim 1.

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