WO2022080912A1 - Fusion protein platform using igm region - Google Patents

Abstract

The present invention relates to a fusion protein platform using an IgM region. Also, the present invention relates to a fusion protein comprising biologically active molecules and an IgM region. The fusion protein platform according to the present invention enables 10 to 12 target binding domains to be formed by means of an IgM Fc region. Also, the problem of steric hindrance occurring in the existing IgM antibody can be solved by means of a linker region. Therefore, the platform can include 10 to 12 biologically active molecules in one fusion protein, thereby maximizing the avidity of the biologically active molecules and the therapeutic effects and disease diagnosis due to same. Also, since various biologically active molecules can be selected in accordance with the purpose, and various types of biologically active molecules can also be combined and used, the fusion protein prepared by means of the platform exhibits notably enhanced effects of target recognition and control.

Description

Fusion protein platform using IgM region

The present invention relates to a fusion protein platform using an IgM region. In addition, the present invention relates to a fusion protein comprising a biologically active molecule and an IgM region.

Biologically active molecules are of great therapeutic importance. However, biologically active molecules, including soluble proteins, membrane-bound proteins, ligands, and receptors, have weak in vivo stability and have to maintain high concentrations in the body to obtain desired activity. In order to increase the effect, it is required to increase the activity of the biologically active molecule.

Among biologically active molecules, soluble proteins, membrane-bound proteins, ligands, and receptors are produced by recombination of all or part of natural proteins that transmit signals in vivo. However, most of these proteins have low binding strength, so a high in vivo concentration must be secured for a sufficient therapeutic effect. In particular, in the case of recombinant ligands and receptors, there are many cases where they do not exhibit sufficient therapeutic effect even at high concentrations compared to their natural forms on the surface of cell membranes. This is because in the case of natural ligands and receptors, a large number of each exists on the cell surface, and a large number of binding sites are generated by intercellular access, resulting in high binding strength. This is because even if the bonding strength of each bonding portion is not different from the natural state, it will have a low bonding strength compared to the naturally occurring bonding (Mammen et al., Angew. Chem., Int. Ed., Polyvalent Interactions in Biological Systems: Implications) for Design and Use of Multivalent Ligands and Inhibitors, 1998, 37, 2755.)

As a method for enhancing the therapeutic effect of biologically active molecules, human immunoglobulin or fragments thereof that specifically bind to target substances can be used instead of natural ligands and receptors. Human immunoglobulin (Ig) is a major protein constituting blood and is classified into various isotypes such as IgG, IgM, IgA, IgD, and IgE. The Ig monomer consists of four polypeptide chains classified into two heavy chains and two light chains linked by disulfide bonds, and each chain has a variable region and a constant region. ) is composed of The heavy chain constant region can be further divided into three or four regions, depending on the isotype.

Since the variable region of Ig binds to a specific structure (epitope) on the surface of a target material with high binding force, Ig specific for a target can be used as a biologically active molecule instead of a ligand and a receptor. In general, Ig has a much greater binding force than that between ligands or receptors, and thus can effectively inhibit target ligand-receptor binding. This action is called an antagonist. On the other hand, some Igs may activate a target ligand or receptor instead of inhibiting the target ligand-receptor binding, and this action is called an agonist.

The therapeutic effect can be increased by using recombinant Ig as a biologically active molecule in place of the recombinant ligand and receptor, but a panning operation is required to select Igs that specifically bind to the target molecule from a large number of random Igs. It takes a lot of extra time to increase it.

As another method for increasing the therapeutic effect of biologically active molecules, a material having high avidity by binding several biologically active molecules to one molecule may be used. For example, in the case of IgG, one of the isotypes of Ig, since it has two binding sites per molecule and has a higher binding activity than that of a monomer, the Fc region (Fragment crystallizable region), a heavy chain constant region of IgG, is fused to a protein Thus, it is possible to create a substance with increased protein stability and binding activity. In addition, using the high binding force of four biotin molecules to one streptavidin molecule, a biologically active molecule can be labeled with biotin to make a material with high binding activity having four binding sites. However, the effect of enhancing the binding activity of the Fc fusion protein is not large, and biotin is chemically labeled with a biologically active molecule for biotin-streptavidin binding, and a material having four binding sites is prepared and purified for use as a drug. The process is also difficult.

The present inventors have studied a method for enhancing the therapeutic effect of a biologically active molecule. As a result, when the biologically active molecule is bound to the constant region of IgM, that is, the fragment crystallizable region of IgM, the The present invention was completed by experimentally demonstrating that the binding activity (avidity) is improved and thus the therapeutic effect is also increased.

Each description and embodiment disclosed in the present invention is also applicable to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

In addition, it will be understood that terms not specifically defined herein have meanings commonly used in the technical field to which the present invention pertains. Also, unless otherwise defined by context, the singular includes the plural and the plural includes the singular.

One aspect of the present invention provides a fusion protein represented by the following structural formula:

[(X) _a -(Y) _b -(IgM Fc region)] _m -(Z) _n

At this time, in the structural formula,

wherein X is a biologically active molecule,

Y is a linker,

The IgM Fc region is a monomer,

wherein Z is a J chain,

wherein a is 1 or 2, b is 0, 1 or 2, m is 5 or 6, and n is 0 or 1.

In one embodiment according to the present invention, when a is 1, b may be 0 or 1. For example, when a is 1 and b is 0, the fusion protein is [(X)-(Y) ₀ -(IgM Fc region)] _m -(Z) _n , [(X)-(IgM) Fc region)] _m -(Z) _n or [(X) ₁ -(Y) ₀ -(IgM Fc region)] _m -(Z) _n .

In another embodiment, when a is 2, b may be 0 or 2.

Also, in another embodiment, when m is 5, n may be 1. In another embodiment, when m is 6, n may be 0. For example, when m is 5 and n is 1, the fusion protein may be in a form in which the J chain is bonded to the first IgM Fc region monomer and the fifth IgM Fc region monomer by a disulfide bond. and a representative structure thereof is shown in A and B of FIG. 1 .

The fusion protein provided in the present invention has 10 to 12 binding domains through the IgM Fc region to maximize the avidity of biologically active molecules, and to solve the problem of steric hindrance of existing IgM antibodies through a linker. .

As used herein, the term "fusion protein" refers to a protein in which some or all of two or more different proteins are bound. The fusion protein includes the amino acid sequence of the protein constituting it, and in addition to the amino acid sequence that allows the protein to be secreted out of the cell, for example, an amino acid sequence such as a signal peptide, but is not limited thereto .

In addition, the fusion protein may be in a form in which some amino acid sequences constituting the fusion protein are mutated. In proteins and polypeptides, amino acid substitutions that do not entirely alter the activity of the molecule are known in the art. The most common substitutions are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is a substitution between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and a fusion protein of a sequence mutated from the amino acid sequence of the fusion protein provided in the present invention by this substitution are included within the scope of the present invention. In addition, mutated fusion proteins with increased structural stability against heat, pH, etc. or increased antibody activity due to mutations or modifications in the amino acid sequence are also included in the scope of the present invention.

As used herein, the term "IgM Fc region" is an Fc region (fragment crystallizable region) of immunoglobulin M (Immunoglobulin M; IgM), and refers to the remaining portion except for the Fab region (fragment, antigen binding region) in IgM. . The IgM Fc region is directly or indirectly linked to a biologically active molecule in the fusion protein, wherein the fusion protein serves as a linker, support, carrier, etc. of the biologically active molecule so as to include one or two or more of the biologically active molecule. can be performed. In the present specification, the 'IgM Fc region (IgM Fc region)' may be used interchangeably with the same meaning as 'IgM Fc fragment (IgM Fc fragment)'.

Specifically, the IgM Fc region monomer may be composed of two heavy chains. One heavy chain may include one or more selected from the group consisting of a Cμ2 domain, a Cμ3 domain and a Cμ4 domain. For example, one heavy chain may comprise or consist of a Cμ2 domain, a Cμ3 domain and a Cμ4 domain, or a Cμ2 domain, a Cμ3 domain and a Cμ4 domain. As another example, one heavy chain may comprise or consist of a Cμ3 domain and a Cμ4 domain. As another example, the IgM Fc region may include or consist of the nucleic acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2.

In addition, the IgM Fc region monomer may form a pentamer or a hexamer, wherein each monomer may be linked through a disulfide bond. The pentamer represents a structure including 5 monomers, and the hexamer represents a structure including 6 monomers. Examples of the pentamer are shown in FIGS. 1A and B, and examples of the hexamer are shown in FIGS. 1C and D.

As used herein, the term “linker” refers to an amino acid present between each domain of a protein. The linker may exist between the biologically active molecule and the IgM Fc region in the fusion protein, and may serve to connect them. As a specific example, the linker may be at least one selected from the group consisting of an IgD hinge region, an IgA hinge region, an IgG hinge region, and a GS linker.

The "hinge region" refers to a portion located in the central portion of the heavy chain of an antibody and connecting two regions of the heavy chain by a disulfide bond. The hinge region is located between and connected to the CH1 and CH2 domains of the heavy chain. Unlike immunoglobulins having a short hinge region, which have a Y-shape, specifically, IgD has a long hinge region, so it is possible to have great flexibility to form a T-shape with both target binding sites spread apart. This flexibility enables IgD to have a binding activity capable of binding to a multimeric foreign antigen with little binding to its own antigen.

In this case, the hinge region may include part or all of the hinge region, part or all of the CH1 domain, part or all of the CH2 domain, or a combination thereof. For example, the hinge region may include a part or all of an IgD hinge (hereinafter, 'part or all' is denoted as 'part/all'). As another example, the hinge region may include a part/all of an IgD hinge and a part/all of an IgD CH1 domain. As another example, the hinge region may include a part/all of an IgD hinge and a part/all of an IgD CH2 domain. As another example, the hinge region may include a part/all of an IgD hinge, a part/all of an IgD CH1 domain, and a part/all of an IgD CH2 domain. As another example, the hinge region may include part/all of an IgA hinge. As another example, the hinge region may include a part/all of an IgA hinge and a part/all of an IgA CH1 domain. As another example, the hinge region may include a part/all of an IgA hinge and a part/all of an IgA CH2 domain. As another example, the hinge region may include a part/all of an IgA hinge, a part/all of an IgA CH1 domain, and a part/all of an IgA CH2 domain. As another example, the hinge region may include a part/all of an IgG hinge. As another example, the hinge region may include a part/all of an IgG hinge and a part/all of an IgG CH1 domain. As another example, the hinge region may include a part/all of an IgG hinge and a part/all of an IgG CH2 domain. As another example, the hinge region may include a part/all of an IgG hinge, a part/all of an IgG CH1 domain, and a part/all of an IgG CH2 domain. In this specification, 'hinge region' may be used interchangeably with the same meaning as 'hinge domain'. For example, the IgD hinge region may include or consist of the nucleic acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO: 6. In addition, the IgA hinge region may include or consist of the nucleic acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 8.

The "GS linker" may be at least one selected from the group consisting of (GS)c, (SG)c, (GGGS)c, (GGGGS)c, GCGS(GGGS)c and GCGGS(GGGGS)c, wherein the c is an integer from 2 to 6. Specifically, it may contain 4 to less than 35 amino acids composed of glycine (Gly; G) and serine (Ser; S), and glycine in order to increase stability through disulfide bonds. It may include 4 to less than 35 amino acids consisting of serine and cysteine (Cys; C), but is not limited thereto. For example, the GS linker may include or consist of the nucleic acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10.

As used herein, the term "biologically active molecule" refers to a substance capable of exhibiting a specific action or effect in an organism, for example, by activating or inactivating a specific signaling pathway, promoting or inhibiting the expression of a specific gene, or It refers to a substance capable of enhancing or inhibiting the function of a specific protein, or inducing or inhibiting cell death. Preferably, the biologically active molecule may exhibit immunoregulation and/or anticancer activity. The biologically active molecule is directly or indirectly linked to the IgM Fc region in the fusion protein, and may serve as a functional group or active moiety capable of exhibiting a desired effect of the fusion protein.

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

Specifically, the antigen-binding fragment of the antibody is Fab, F(ab') ₂ , F(ab) ₂ , Fab', Fab ₂ , Fab ₃ , Fv, scFv, Bis-scFv, minibody, tria It may be one or more selected from the group consisting of body (Triabody), diabody (Diabody), tandem diabody (TandAb), nanobody (Nanobody), tetrabody (Tetrabody), but is not limited thereto. In addition, the antigen-binding fragment of the antibody may bind to HLA-G. HLA-G, a type 1 HLA, is expressed in the placenta of pregnant women and is involved in the immune tolerance of the fetus. HLA-G binds to NK (natural killer) cell inhibitory receptors ILT2, ILT4, and _KIR2DL4 , inhibits NK cells, and promotes the differentiation of regulatory T cells (T regs). In addition, when cancer cells overexpress HLA-G on the surface, the immunotherapeutic effect for cancer treatment is halved by inhibiting the activities of cytotoxic T-cells, helper T-cells and NK cells involved in cytotoxicity. Accordingly, an antigen-binding fragment that selectively binds to HLA-G and inhibits the binding of HLA-G to an inhibitory receptor can be utilized as an anticancer therapeutic substance. For example, the antigen-binding fragment of the antibody may be a Fab or scFv. In addition, the light chain variable region (VL) constituting the Fab or scFv comprises or consists of the nucleic acid sequence of SEQ ID NO: 15 or the amino acid sequence of SEQ ID NO: 16; The heavy chain variable region (VH) and CH1 domains comprise or consist of the nucleic acid sequence of SEQ ID NO: 17 or the amino acid sequence of SEQ ID NO: 18; and/or the heavy chain variable region (VH) may include or consist of the nucleic acid sequence of SEQ ID NO: 19 or the amino acid sequence of SEQ ID NO: 20.

In another specific example, the membrane-binding protein may be Human Leukocyte Antigen (HLA), but is not limited thereto. The term, 'HLA' refers to a protein encoded by a human major histocompatibility complex (MHC) gene complex, and is mainly present in a membrane-bound form and is an important immunological agent responsible for the regulation of the immune system. is a molecule HLA activates cytotoxic T-cells by presenting a peptide fragment of a foreign or autologous protein on the cell surface from the inside of the cell and allowing it to be recognized and bound to the T-cell antigen receptor (TCR) of the cytotoxic T-cell. make it HLA is largely divided into two types. Type 1 HLA is present on all cell surfaces, whereas type 2 HLA presents specific antigens such as NK cells, macrophages, and dendritic cells. It exists only in cells (Antigen Presenting Cell; APC). The HLA gene is highly polymorphic and expresses six different HLA protein α chains (two HLA-A, two HLA-B and two HLA-C). Since the type 1 HLA can selectively activate cytotoxic T-cells expressing TCR when a specific peptide fragment is loaded thereto, the recombinant type 1 HLA from which the membrane-bound domain has been removed can be used in cancer It is also used for the treatment of infectious diseases. In addition, unlike type 2 HLA, most type 1 HLA is unstable in an empty state in which no peptide is loaded in the groove. Recombinant HLA protein is produced in a modified form, and when expressed using a microorganism, there is a risk that refolding may not be performed properly, so it is generally expressed using animal cells. HLA and MHC are used interchangeably in the art, and HLA is used instead of MHC in humans. In this specification, the terms 'HLA' and 'MHC' may be used interchangeably with the same meaning.

The HLA includes 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 consists of a β2m domain, an α1 domain, an α2 domain and an α3 domain, and the type 2 HLA consists of an α1 domain, an α2 domain, a β1 domain and a β2 domain. The HLA of the present invention may include a β2m domain, an α1 domain, an α2 domain or an α3 domain, and may include an α1 domain, an α2 domain, a β1 domain and a β2 domain. In this case, the α2 domain may be one in which position 115 is substituted with Glu (glutamic acid; E) in Gln (glutamine; Q). In the α1 domain, the 84th position may be substituted with Cys (cysteine; C) from Tyr (tyrosine; Y). When Gln at position 115 of the α2 domain is substituted with Glu, the binding force of HLA with TCR present in CD8+ T-cells is increased by about 1.5 times compared to the case of no substitution. In the case of replacing Tyr at position 84 of the α1 domain with Cys, HLA forms a disulfide bond with the peptide bound thereto, thereby increasing the stability of HLA.

In addition, the HLA may include a substance that specifically binds to the active site of the target T-cell. The substance may selectively activate target T-cells expressing TCR, and/or may increase the stability of HLA. The term, 'target T-cell' may be 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 capable of killing cancer cells, cells infected with a virus, or the like, or damaged cells, for example, CD8 ⁺ T-cells. CD8 ⁺ T-cell protein receptor, CD8, is located on the cell surface and binds to type 1 HLA. The helper T-cells do not directly kill cells, but play a role in activating other immune cells, for example, promoting the production of antibodies in B-cells, activation of cytotoxic T-cells, etc., and CD4 ⁺ T - This is the cell. CD4, a protein receptor on CD4 ⁺ T-cells, is located on the cell surface and binds to type 2 HLA.

A specific example of the substance that specifically binds to the active site of the target T-cell may be a peptide, but is not limited thereto. The peptide may be derived from a virus or an animal cell. In this case, the virus may be a cytomegalovirus (CMV), but is not limited thereto. CMV is one of the strongest immunogenic antigens on the human immune system and stimulates a very strong CD8 ⁺ T-cell response upon human infection. The CD8 ⁺ T-cell immune response is primarily activated by the CMV protein pp65, IE-1, and the frequency of CMV-specific T-cells for individual peptides is approximately 1% to 2% of the total CD8 ⁺ T-cell repertoire. It is known that the frequency is very high. For example, the HLA comprising CMV may include or consist of the nucleic acid sequence of SEQ ID NO: 11 and/or 13 or the amino acid sequence of SEQ ID NO: 12 and/or 14.

In another specific example, the ligand may be EPO (erythropoietin) or an EPO analog, but is not limited thereto. The term 'EPO' is a hormone protein essential for the production of red blood cells, which acts on hematopoietic stem cells in the bone marrow to promote the differentiation and growth of red blood cells to treat anemia, protect nerves in hypoxic environments such as stroke, and participate in the wound healing process do. In addition, it is known to be effective in the treatment of depression and improve memory by participating in the hippocampal response of the brain, synaptic connections, and neuronal networks. The 'EPO analog' is a recombinant protein that performs the same function as EPO and has a different structure, has two additional sugar chains in addition to the three sugar chains of human EPO, and has a blood half-life that is three times or more improved compared to the existing human EPO. The EPO analog may be a ligand, antibody, or antibody fragment capable of binding to and activating an EPO receptor, and a specific example may be NESP (Novel erythropoiesis stimulating protein), but is not limited thereto. For example, the EPO analog may comprise or consist of the nucleic acid sequence of SEQ ID NO: 21 or the amino acid sequence of SEQ ID NO: 22.

As used herein, the term “J chain (joining chain)” refers to a polypeptide that is a component of an IgM or IgA antibody and forms a multimer by combining IgM or IgA monomers. The J chain may exist between the IgM Fc region monomers in the fusion protein, and may serve to connect them. Specifically, the J chain may be included when the IgM Fc region monomer forms a pentamer, and may not be included when the IgM Fc region monomer forms a hexamer. More specifically, when the IgM Fc region monomer forms a pentamer, the J chain may be bonded to the first IgM Fc region monomer and the fifth IgM Fc region monomer via a disulfide bond, and this structure is 1A and B are shown. For example, the J chain may include or consist of the nucleic acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4.

In addition, the J chain may be a protein molecule is additionally bound. In this case, the protein molecule may exhibit a synergistic effect with respect to the desired effect of the fusion protein of the present invention. Specifically, an antibody, antigen-binding fragments of antibody, antibody-drug conjugate, antibody-like molecule, antigen-binding of antibody-like molecule selected from the group consisting of antigen-binding fragments of antibody-like molecule, soluble protein, membrane-bound protein, ligand, receptor, virus-like particle, protein toxin and enzyme It may be one or more, but is not limited thereto. More specifically, the protein molecule may be one or more selected from the group consisting of a co-stimulatory receptor, a cytokine, and a cell engager. In this case, the cell tube receptor may be an antibody, specifically, an antibody that binds to a cancer cell or immune cell-specific labeling protein, for example, a T-cell engager. In addition, the immune cells are neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, NK cells (natural killer cells), and lymphocytes such as B cells and T cells.

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

In the nucleic acid molecule according to the present invention, each term is the same as described for the fusion protein unless otherwise specified.

As used herein, the term "nucleic acid molecule" has a meaning comprehensively including DNA and RNA molecules, and the nucleotide, which is the basic structural unit in the nucleic acid molecule, includes not only natural nucleotides, but also analogs ( analogues) are also included. The sequence of the nucleic acid molecule encoding the fusion protein of the present invention may be modified, and the modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

In addition, all sequences used in the present invention, including nucleic acid sequences and amino acid sequences, are construed to include sequences exhibiting substantial identity to the sequences described in the sequence listing, provided that mutations with biologically equivalent activity are considered. . The term, 'substantial identity' refers to aligning the sequence of the present invention and any other sequences as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art. 60% homology, more specifically 70% homology, even more specifically 80% homology, most specifically 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% homology. , means a sequence showing 98% or 99% homology.

Accordingly, a sequence having high homology with the sequence represented by SEQ ID NOs: 1 to 22 of the present invention, for example, has a high homology of 70% or more, specifically 80% or more, more specifically 90% or more Sequences should also be construed to be included within the scope of the present invention.

Another aspect of the present invention provides a vector for expression of a fusion protein comprising the nucleic acid molecule.

In the vector for expression of a fusion protein according to the present invention, each term is the same as described for the fusion protein and nucleic acid molecule, unless otherwise specified.

As used herein, the term "vector" is a means for expressing the fusion protein of the present invention, and a plasmid known in the art that can insert or introduce a nucleic acid molecule encoding the fusion protein into a host cell. ), viruses or other vectors. The vector may be constructed as a vector for cloning or a vector for expression. Specific examples may be viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors, and more specifically, plasmids (eg, pcDNA3 or pcDNA3.1, such as pcDNA3.1). series, pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19, etc.), phage (such as λgt4) λB, λ-Charon, λΔz1 and M13, etc.) or a virus (eg, SV40, etc.), but is not limited thereto.

The vector of the present invention may be one in which a nucleic acid molecule encoding the fusion protein is operably linked to a promoter. As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcription regulator binding sites) and another nucleic acid sequence, whereby the control sequence is to regulate the transcription and/or translation of said other nucleic acid sequences.

When the vector of the present invention is an expression vector and a eukaryotic cell is a host, a promoter derived from the genome of a mammalian cell (eg, metallotionine promoter, β-actin promoter, human hegglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, promoter of Moloney virus, promoter of Epstein Barr virus (EBV) and promoter of Loose sarcoma virus (RSV)) may be used, and may include a polyadenylation sequence as a transcription termination sequence. For example, the recombinant vector of the present invention may include a CMV promoter, but is not limited thereto.

When the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of propagating transcription (eg, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. For example, when Escherichia coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthesis pathway, or the left-handed promoter of phage λ (pLλ promoter) can be used as a regulatory region. there is; 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 may be used as a regulatory region.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and antibiotic resistance genes commonly used in the art as selection markers (eg, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin) , kanamycin, geneticin, neomycin and tetracycline resistance genes).

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

In the host cell according to the present invention, each term is the same as described in the fusion protein, nucleic acid molecule, and vector for expression of the fusion protein, unless otherwise specified.

As used herein, the term "host cell" refers to a cell that includes the vector for expression of the fusion protein and can stably and continuously clone and express the fusion protein of the present invention. For example, E. coli ( Escherichia coli ), Bacillus subtilis ( Bacillus subtilis ) and Bacillus thuringiensis ( Bacillus thuringiensis ), such as Bacillus genus strains, Streptomyces , Pseudomonas , Proteus mirabilis ( Proteus mirabilis ) or prokaryotic host cells such as Staphylococcus ; Fungi such as Aspergillus species , Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces or Neurospora crassa ), such as eukaryotic host cells; lower eukaryotic cells; higher eukaryotic cells such as insect-derived cells; plant cells; or COS7 cells (monkey kidney cells), NSO cells, SP2/0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell line, HuT 78 cells Alternatively, it may be a cell derived from a mammal such as 293 cells, but is not limited thereto, and host cells commonly used in the art may be used without limitation.

Another aspect of the present invention provides a method for producing the fusion protein, comprising introducing the vector for expression of the fusion protein into a host cell.

In the method for producing a fusion protein according to the present invention, each term is the same as described for the fusion protein, nucleic acid molecule, vector and host cell for expression of the fusion protein, unless otherwise specified.

Specifically, the method for preparing the fusion protein of the present invention comprises:

(a) introducing the vector of the present invention into a host cell;

(b) culturing the host cell; and

(c) obtaining a fusion protein from the host cell.

The step (a) of introducing the vector of the present invention into a host cell may be a step of preparing a transformant comprising the vector for expression of the fusion protein, and the step of preparing the transformant comprises transforming the host cell. It could be a conversion.

As used herein, the term "transformant" refers to an organism in which DNA is introduced into a host and DNA can be replicated as a factor of chromosomes or by the completion of chromosome integration. do.

Transformation of the above-described host cells can be performed using any transformation method, and can be easily performed according to conventional methods in the art. For example, CaCl ₂ precipitation method, CaCl ₂ method using DMSO (dimethyl sulfoxide) to increase the efficiency, Hanahan method, electroporation (electroporation), calcium phosphate precipitation method, protoplast fusion method, silicon carbide fiber It may include, but is not limited to, a stirring method using Transformation or transfection methods commonly used in the present invention may be used without limitation.

The step (b) of culturing the host cell of the present invention may be performed according to a medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Suspension culture or adherent culture depending on the cell growth method; Depending on the culture method, batch, fed-batch, or continuous culture methods can be used.

In animal cell culture, the medium may contain various carbon sources, nitrogen sources and trace elements. Examples of the carbon source 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 linoleic acid; alcohols such as glycerol or ethanol; or an organic acid such as acetic acid, and these carbon sources may be used alone or in combination. Examples of the nitrogen source include, for example, organic nitrogen sources such as peptone, yeast extract, broth, malt extract, corn steep liquor or soybean wheat; or an inorganic nitrogen source such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, these nitrogen sources may be used alone or in combination. Examples of the trace component include phosphorus such as potassium dihydrogen phosphate or dipotassium hydrogen phosphate; metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, or suitable precursors may be included.

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 culture to adjust the pH of the culture, and fatty acid polyglycol esters and The same anti-foaming agent may be used, and oxygen or oxygen-containing gas (eg, air) may be injected into the culture to maintain the aerobic state of the culture.

In addition, it can be cultured by maintaining the temperature of the culture at 20 °C to 45 °C, specifically 25 °C to 40 °C.

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 solution obtained through the step (b).

The fusion protein may be used in an unpurified state, or may be used after further recovery, purification and concentration. Specifically, methods commonly used in the art (eg, dialysis, salt precipitation, chromatography, etc.) may be used, through which recovery, purification and concentration may be simultaneously performed. More specifically, it can be recovered, purified, and concentrated using chromatography (eg, ion exchange chromatography, size exclusion chromatography, or affinity chromatography), and the type and sequence of columns used for this depend on the characteristics of the fusion protein. , can be appropriately selected according to the culture method, etc.

The fusion protein platform according to the present invention can form 10 to 12 target binding sites through the IgM Fc region. In addition, it is possible to solve the problem of steric hindrance occurring in the existing IgM antibody through the linker region.

Accordingly, the platform can include 10 to 12 biologically active molecules in one fusion protein, thereby maximizing the avidity of the biologically active molecules and thus the therapeutic effect and disease diagnosis. In addition, since it is possible to select various biologically active molecules according to the purpose and to use various types of biologically active molecules in combination, the fusion protein prepared through the platform can significantly improve the effect of recognizing and controlling the target. .

1 is a schematic diagram of a fusion protein according to the present invention. A relates to a fusion protein comprising a biologically active molecule, a linker, an IgM Fc region and a J chain, wherein the IgM Fc region forms a pentamer, and B is a biologically active molecule, an IgM Fc region and a J chain It includes, but does not include a linker, and relates to a fusion protein in which the IgM Fc region forms a pentamer, C includes a biologically active molecule, a linker and an IgM Fc region, but does not include a J chain, and the IgM It relates to a fusion protein in which an Fc region forms a hexamer, D includes a biologically active molecule and an IgM Fc region, but does not include a linker and a J chain, wherein the IgM Fc region forms a hexamer It relates to a fusion protein that

Figure 2a is an image showing the result 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 of [(CMVpHLA) ₂ -(IgD hinge region) ₂ -(IgM Fc region)] ₅ - (J chain); '2' is [(CMVpHLA) ₂ - (linker) ₀ - (IgM Fc region)] ₅ - (J chain) fusion protein having a structure; '3' is a fusion protein having the structure of [(CMVpHLA) ₂ -((GGGGS) ₃ ) ₂ -(IgM Fc region)] ₅ - (J chain); '4' is [(CMVpHLA) ₂ - (IgA hinge region) ₂ - (IgM Fc region)] ₅ - (J chain) fusion protein having the structure; '5' is a fusion protein having a structure of [(CMVpHLA) ₂ -(IgD hinge region) ₂ -(IgM Fc region)] ₆ -(J chain) ₀ ; '6' indicates the control CMVpHLA protein.

Figure 2b is an image showing the result 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 [(A2014) ₂ - (IgD hinge region) ₂ - (IgM Fc region)] ₅ - (J chain) fusion protein having the structure; '2' is [(A2014) ₂ - (linker) ₀ - (IgM Fc region)] ₅ - (J chain) fusion protein having the structure; '3' is a fusion protein having the structure of [(A2014) ₂ -((GGGGS) ₃ ) ₂ -(IgM Fc region)] ₅ - (J chain); '4' is [(A2014) ₂ - (IgA hinge region) ₂ - (IgM Fc region)] ₅ - (J chain) fusion protein having the structure; '5' is [(A2014) ₂ - (IgD hinge region) ₂ - (IgM Fc region)] ₆ - (J chain) fusion protein having a structure of ₀ ; '6' indicates a control (A2014)-(IgG Fc) fusion protein.

Figure 2c is an image showing the result of reducing SDS-PAGE analysis of a fusion protein containing an EPO analog (NESP) as a biologically active molecule. 'Ladder' is a protein size marker; '1' is [(NESP) ₂ - (IgD hinge region) ₂ - (IgM Fc region)] ₅ - (J chain) fusion protein having the structure; '2' is [(NESP) ₂ - (linker) ₀ - (IgM Fc region)] ₅ - (J chain) fusion protein having the structure; '3' indicates NESP as a control.

Figure 3a is a graph showing the results of ELISA analysis of the target binding ability of the fusion protein, including HLA (CMVpHLA) as a biologically active molecule.

Figure 3b is a graph showing the results of ELISA analysis of the target binding ability of the fusion protein, including the antibody fragment (IgG Fab; A2014) as a biologically active molecule.

Figure 3c is a graph of the results of ELISA analysis of the target binding ability of the fusion protein, including the EPO analog (NESP) as a biologically active molecule.

4 is a graph showing the result of ELISPOT analysis of IFN-γ secretion by the fusion protein according to the present invention, showing that the fusion protein can induce the activation of CD8 ⁺ T cells.

FIG. 5a is a graph of FACS results analyzing the degree of CD8 ⁺ T cell proliferation by the fusion protein according to the present invention, showing that the fusion protein can induce CD8 ⁺ T cell proliferation. A relates to [(CMVpHLA) ₂ -(linker) ₀ -(IgM Fc region)] ₅ -(J chain), B is [(CMVpHLA) ₂ -(IgD hinge region) ₂ -(IgM Fc region)] ₅ - (J chain).

FIG. 5b is a graph of FACS results analyzing the degree of CD8 ⁺ T cell proliferation by the fusion protein according to the present invention, showing that the fusion protein can induce CD8 ⁺ T cell proliferation. A relates to [(CMVpHLA) ₂ -(IgD hinge region) ₂ -(IgM Fc region)] ₅ - (J chain), B relates to [(CMVpHLA) ₂ -((GGGGS) ₃ ) ₂ -(IgM Fc region)] ₅ - (J chain), C is [(CMVpHLA) ₂ - (IgA hinge region) ₂ - (IgM Fc region)] ₅ - (J chain), D is [(CMVpHLA) ₂ -(IgD hinge region) ₂ -(IgM Fc region)] ₆ -(J chain) ₀ , and E relates to the control CMVpHLA.

6 is a graph showing the FACS result of analyzing the cytotoxicity caused by the fusion protein according to the present invention, and shows that the fusion protein can induce cancer cell death by activating CD8 ⁺ T cells.

7 is a graph showing the assay result of analyzing the binding activity of the fusion protein according to the present invention to HLA-G-expressing cells, showing that the fusion protein can bind to the HLA-G receptor in the cell.

8 is a graph showing the results of an ELISA assay analyzing the degree of binding between the recombinant HLA-G protein and the recombinant ILT-2 protein by the fusion protein according to the present invention, wherein the fusion protein binds the HLA-G and ILT-2 proteins. shows that it can be blocked.

9 is a graph showing the results of FACS analysis of the degree of binding between the recombinant HLA-G protein and the intracellularly expressed ILT-2 protein by the fusion protein according to the present invention, wherein the fusion protein is the HLA-G protein and the intracellularly expressed graph. It shows that it can block the binding of ILT-2 receptor protein.

10 is a graph of the assay result analyzing the proliferative capacity of EPO-expressing cells by the fusion protein according to the present invention. The fusion protein binds to the intracellularly expressed EPO receptor, and can promote the proliferation of cells expressing it. shows

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

Example 1. Preparation of fusion protein

In Example 1, a fusion protein comprising an IgM Fc region, a biologically active molecule, a J chain and/or a linker was prepared.

Example 1-1. Recombinant plasmid preparation of IgM Fc region, J chain and linker

In Example 1-1, a recombinant plasmid of IgM Fc region, J chain and/or linker was prepared. The cμ2 domain, cμ3 domain and cμ4 domain of wild-type IgM were used as the IgM Fc region.

Specifically, a recombinant plasmid for expressing the IgM Fc region (SEQ ID NO: 1 or 2) including the cμ2 domain, the cμ3 domain and the cμ4 domain and the J chain (SEQ ID NO: 3 or 4) required for pentamer formation was constructed. In addition, in order to solve the steric hindrance problem of the existing IgM antibody, human IgD hinge region (SEQ ID NO: 5 or 6), human IgA hinge region (SEQ ID NO: 7 or 8) and (GGGGS) ₃ linkers (SEQ ID NO: 9 or 10) A fusion protein recombinant plasmid linked to the IgM Fc region was constructed.

For gene cloning, after codon optimization of each of the above materials, codon-optimized nucleotide molecules were synthesized by requesting Cosmogene Tech (Seoul, Korea).

Insert DNA was prepared by PCR so that overlap PCR could be performed on the MCS (multiple cloning site) of pcDNA 3.1 (manufacturer: Thermo Scientific) vector using the optimized nucleotides and primers. After overlap PCR, each of the obtained insert DNA and pcDNA 3.1 vectors was treated with DpnI restriction enzyme to remove the template, and each material inserted vector was prepared.

After the vector was inserted into Stella ^® competent cell (manufacturer: Clontech), colonies were selected by plating on an agar plate to which ampicillin antibiotic suitable for the antibiotic resistance gene in the vector was added, and Applied biosystems 3730xl DNA analyzer (manufacturer: The nucleotide sequence was confirmed through DNA sequencing using Thermo Scientific).

Example 1-2. Preparation of fusion protein containing biologically active molecules

In this Example 1-2, a recombinant plasmid of a biologically active molecule was prepared, and it was linked with the recombinant plasmid of the IgM Fc region, J chain and/or linker prepared in Example 1-1, and a fusion protein containing them was prepared. Expression and purification.

As biologically active molecules, HLA-A, an IgG antibody fragment (IgG Fab), or an EPO analog was used.

Example 1-2-1. Preparation of fusion protein containing HLA-A

A fusion protein including HLA-A as a biologically active molecule, IgM Fc region, J chain and/or linker was prepared.

Specifically, in order to increase the stability of the HLA-A protein, CMV (cytomegalovirus)-derived peptide was additionally bound (loaded), and additionally β2m (Beta-2 microglobulin) was subsequently bound to the CMV-derived peptide. Through this, an HLA-A protein with improved stability (hereinafter referred to as 'CMVpHLA') was constructed, and the specific sequence thereof is described in SEQ ID NO: 11 or 12. And in order to facilitate protein purification later, a FLAG tag was attached to the C-terminus, and the cloning method according to Example 1-1 was used for the detailed execution method.

In the CMVpHLA recombinant plasmid constructed above, the HLA-A protein sequence excluding the FLAG tag was linked to the IgM Fc region sequence, and the final recombinant plasmid vector thus constructed was introduced into Expi293 (Thermo Scientific) animal cells, and the fusion protein was quickly introduced A transient expression system was established to secure it.

Specifically, one day before transformation, 3.0 × 10 ⁶ cells/mL of cells were dispensed into the flask and cultured for 24 hours. On the day of transduction, Expi293 ^® expression medium was added and diluted to a concentration of 3.0 × 10 ⁶ cells/mL. Thereafter, 1 μg/mL of each recombinant plasmid vector was mixed with Opti-MEM ^® medium, and the ExpiFectamine ^® reagent and Opti-MEM ^® medium were mixed according to volume and left at room temperature for 5 minutes. After that, DNA and ExpiFectamine ^® reagent were reacted for 15 minutes and added to the flask, treated with enhancer1 and 2 after 16 to 22 hours, and confirmed when the cell viability (viability) is 75% or more on days 4 to 5 and fusion protein was recovered.

Then, the expressed fusion protein was purified using IgM affinity chromatography. Specifically, the culture solution obtained from the temporary expression system of Example 5 was filtered through a PES filtration membrane having a pore size of 0.2 μm to remove impurities. The recovered filtrate was loaded onto an IgM affinity matrix POROS™ CaptureSelect™ IgM Affinity Matrix ^® (Thermo Scientific) to purify the fusion protein. IgM affinity chromatography was performed under the following conditions.

<Chromatography Conditions>

- Resin: POROS™ CaptureSelect™ IgM Affinity Matrix ^®

- Flow rate: 480 cm/h

- Equilibration: PBS (Phosphate-buffered saline) buffer

- Loading: up to 6 g protein/L resin volume

- Regeneration and sterilization: 0.01 M NaOH solution

- Elution: 0.1 M glycine-HCl, pH 2.7 buffer

- Storage: 20% EtOH

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

On the other hand, purification and concentration of the CMVpHLA control protein not including the IgM Fc region was performed as follows. The culture solution obtained from the temporary expression system was filtered through a PES filtration membrane having a pore size of 0.2 μm to remove impurities. The recovered filtrate was purified by loading on DYKDDDDK (FLAG-tag) affinity matrix Pierce Anti-DYKDDDDK Affinity Resin ^® (Thermo Scientific).

FLAG-tag affinity chromatography was performed under the following conditions.

<Chromatography Conditions>

- Resin: Pierce Anti-DYKDDDDK Affinity Resin ^®

- Flow rate: 300 cm/h

- Equilibration: PBS (Phosphate-buffered saline) buffer

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

- Regeneration and sterilization: 0.1 M glycine-HCl, pH 2.7 buffer

- Elution: 0.1 M glycine-HCl, pH 2.7 buffer

- Storage: 20% EtOH

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

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

fusion protein	note
[(CMVpHLA) 2 - (IgD hinge region) 2 - (IgM Fc region)] 5 - (J chain)	Linker is IgD hinge region, IgM Fc region forms pentamer
[(CMVpHLA) 2 - (IgA hinge region) 2 - (IgM Fc region)] 5 - (J chain)	Linker is IgA hinge region, IgM Fc region forms pentamer
[(CMVpHLA) 2 -((GGGGS) 3 ) 2 -(IgM Fc region)] 5 - (J chain)	The linker is (GGGGS) 3 and the IgM Fc region forms a pentamer.
[(CMVpHLA) 2 - (Linker) 0 - (IgM Fc region)] 5 - (J chain)	Does not contain a linker, the IgM Fc region forms a pentamer
[(CMVpHLA) 2 -(IgD hinge region) 2 -(IgM Fc region)] 6 -(J chain) 0	Linker is IgD hinge region, IgM Fc region forms a hexamer
CMVpHLA	Does not contain linker, IgM Fc region and J chain
CMVpHLAK b	Does not contain linker, IgM Fc region and J chain, α3 region is substituted with mouse K f

These are listed in Table 1 above. For example, "[(CMVpHLA) ₂ -(IgD hinge region) ₂ -(IgM Fc region)] ₅ -(J chain)" is a biologically active molecule, in which two CMVpHLA are transferred through two IgD hinge regions to an IgM Fc region monomer. are bound to each, and the IgM Fc region monomer is a pentamer bound to five, and refers to a fusion protein including one J chain to form the pentamer. The pentameric structure of such a fusion protein may refer to the form shown in FIG. 1A .

In addition, "[(CMVpHLA) ₂ - (linker) ₀ - (IgM Fc region)] ₅ - (J chain)" is a biologically active molecule in which each of two CMVpHLA is directly bonded to the IgM Fc region monomer without a linker, and , The IgM Fc region monomer is a pentamer bonded to five, and refers to a fusion protein including one J chain to form the pentamer. The pentameric structure of such a fusion protein may refer to the form shown in FIG. 1B.

In addition, "[(CMVpHLA) ₂ -(IgD hinge region) ₂ - (IgM Fc region)] ₆ - (J chain) ₀ " is a biologically active molecule, in which two CMVpHLA are linked to the IgM Fc region monomer through two IgD hinge regions. Each of them is bound, and the IgM Fc region monomer is a hexamer to which six are bound, and refers to a fusion protein that does not include a J chain to form the hexamer. The hexameric structure of such a fusion protein may refer to the form shown in FIG. 1C.

Example 1-2-2. Preparation of fusion protein including IgG antibody fragment

A fusion protein including an IgG antibody fragment as a biologically active molecule and an IgM Fc region, a J chain and/or a linker was prepared.

Specifically, a portion of the antibody that binds to the HLA-G protein (IgG Fab; hereinafter referred to as 'A2014') was used. The A2014 was constructed using the cloning method according to Example 1-1 in which the light chain (SEQ ID NO: 15 or 16), and parts of the heavy chain, VH and CH1 (SEQ ID NO: 17 or 18), among the moieties of the IgG Fab were used.

In addition, a fusion protein containing an IgG Fc region rather than an IgM Fc region was prepared as a control. As the IgG Fc region (SEQ ID NO: 19 or 20), the CH2 and CH3 domains of IgG were used.

The A2014 recombinant plasmid sequence constructed above was linked to the IgM Fc region or IgG Fc region sequence, and the final recombinant plasmid vector constructed in this way was introduced into the cell by the method performed in Example 1-2-1, and the fusion protein was expressed. made it

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

On the other hand, purification and concentration of the fusion protein containing the IgG Fc region was performed as follows. According to the method of 1-2-1 above, the culture solution obtained from the transient expression system was loaded onto an IgG affinity matrix MabSelect SuRe ^® (Cytiva) and purified.

IgG affinity chromatography was performed under the following conditions.

<Chromatography Conditions>

- Resin: MabSelect SuRe ^®

- Flow rate: 300 cm/h

- Equilibration: PBS (Phosphate-buffered saline) buffer

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

- Regeneration and sterilization: 0.01 M NaOH solution

- Elution: 0.1 M glycine-HCl, pH 2.7 buffer

- Storage: 20% EtOH

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

fusion protein	note
[(A2014) 2 - (IgD hinge region) 2 - (IgM Fc region)] 5 - (J chain)	Linker is IgD hinge region, IgM Fc region forms pentamer
[(A2014) 2 - (IgA hinge region) 2 - (IgM Fc region)] 5 - (J chain)	Linker is IgA hinge region, IgM Fc region forms pentamer
[(A2014) 2 -((GGGGS) 3 ) 2 -(IgM Fc region)] 5 - (J chain)	The linker is (GGGGS) 3 and the IgM Fc region forms a pentamer.
[(A2014) 2 - (Linker) 0 - (IgM Fc region)] 5 - (J chain)	Does not contain a linker, the IgM Fc region forms a pentamer
[(A2014) 2 - (IgD hinge region) 2 - (IgM Fc region)] 6 - (J chain) 0	Linker is IgD hinge region, IgM Fc region forms a hexamer
(A2014)-(IgG Fc region)	Does not contain linker, IgM Fc region and J chain, but contains IgG Fc region

The fusion proteins prepared by the above method are listed in Table 2 above.

For example, "[(A2014) ₂ - (IgD hinge region) ₂ - (IgM Fc region)] ₅ - (J chain)" is a biologically active molecule, in which two A2014 are IgM Fc region monomers through two IgD hinge regions. are bound to each, and the IgM Fc region monomer is a pentamer bound to five, and refers to a fusion protein including one J chain to form the pentamer. The pentameric structure of such a fusion protein may refer to the form shown in FIG. 1A .

In addition, "[(A2014) ₂ - (linker) ₀ - (IgM Fc region)] ₅ - (J chain)" is a biologically active molecule, wherein each of two A2014s is directly bound to the IgM Fc region monomer without a linker, and , The IgM Fc region monomer is a pentamer bonded to five, and refers to a fusion protein including one J chain to form the pentamer. The pentameric structure of such a fusion protein may refer to the form shown in FIG. 1B.

In addition, "[(A2014) ₂ - (IgD hinge region) ₂ - (IgM Fc region)] ₆ - (J chain) ₀ " is a biologically active molecule, in which two A2014 Each of them is bound, and the IgM Fc region monomer is a hexamer to which six are bound, and refers to a fusion protein that does not include a J chain to form the hexamer. The hexameric structure of such a fusion protein may refer to the form shown in FIG. 1C.

Example 1-2-3. Preparation of fusion protein containing EPO analogues

A fusion protein including an EPO analog as a biologically active molecule and an IgM Fc region, a J chain and/or a linker was prepared.

Specifically, NESP (Novel erythropoiesis stimulating protein) protein was used as an EPO analog. The NESP (SEQ ID NO: 21 or 22) was linked to the IgM Fc region sequence, and a plasmid vector was constructed using the cloning method according to Example 1. The constructed recombinant plasmid vector was introduced into the cells in the same manner as in Example 1-2-1, the fusion protein was expressed, purified and concentrated, and then dialyzed against PBS and stored at -20°C.

fusion protein	note
[(NESP) 2 - (IgD hinge region) 2 - (IgM Fc region)] 5 - (J chain)	Linker is IgD hinge region, IgM Fc region forms pentamer
[(NESP) 2 - (Linker) 0 - (IgM Fc region)] 5 - (J chain)	Does not contain a linker, the IgM Fc region forms a pentamer
NESP	Does not contain linker, IgM Fc region and J chain

The fusion proteins prepared by the above method are listed in Table 3 above.

For example, "[(NESP) ₂ -(IgD hinge region) ₂ -(IgM Fc region)] ₅ -(J chain)" is a biologically active molecule in which two NESPs are IgM Fc region monomers through two IgD hinge regions. are bound to each, and the IgM Fc region monomer is a pentamer bound to five, and refers to a fusion protein including one J chain to form the pentamer. The pentameric structure of such a fusion protein may refer to the form shown in FIG. 1A .

In addition, "[(NESP) ₂ - (linker) ₀ - (IgM Fc region)] ₅ - (J chain)" is a biologically active molecule, wherein each of the two NESPs is directly bound to the IgM Fc region monomer without a linker, and , The IgM Fc region monomer is a pentamer bonded to five, and refers to a fusion protein including one J chain to form the pentamer. The pentameric structure of such a fusion protein may refer to the form shown in FIG. 1B.

Examples 1-3. Confirmation of the structure of the prepared fusion protein

It was confirmed whether the fusion proteins prepared in Examples 1-1 and 1-2 had the desired structure. On the other hand, a schematic diagram of a fusion protein comprising a biologically active molecule, a linker, an IgM Fc region monomer and a J chain, wherein the IgM Fc region forms a pentamer is shown in FIG. 1A . In addition, a schematic diagram of a fusion protein comprising a biologically active molecule, an IgM Fc region monomer, and a J chain, but not a linker, in which the IgM Fc region forms a pentamer is shown in FIG. 1B . In addition, a schematic diagram of a fusion protein comprising a biologically active molecule, a linker, and an IgM Fc region monomer, but not including a J chain, in which the IgM Fc region forms a hexamer is shown in FIG. 1C . In addition, a schematic diagram of a fusion protein including a biologically active molecule and an IgM Fc region monomer, but not a linker and a J chain, in which the IgM Fc region forms a hexamer is shown in FIG. 1D .

Specifically, the structure of the fusion protein was confirmed using reducing SDS-PAGE. After mixing 5x reducing-PAGE sample buffer with 2 μg of each fusion protein, it was heated at 100° C. for 5 minutes, and then left at room temperature for 20 minutes. After inserting polyamide gel (12-20%, Invitrogen) into the gel kit, loading the sample and electrophoresing it at 200V for 30 minutes, separating the gel from the kit and adding a staining solution (Coomassie Brilliant Blue staining solution) for 1 hour left for a while. The left gel was transferred to a buffer (destaining buffer; 70% tertiary distilled water, 20% methanol, 10% acetic acid), and the result was confirmed after overnight.

As a result, as shown in FIGS. 2a, 2b and 2c, each fusion protein comprising an IgM Fc region, a biologically active molecule, a J chain and/or a linker includes all the sizes of the corresponding substances constituting it. did

From the above results, it can be seen that the fusion protein prepared in Example 1 has the desired structure and is properly expressed.

Example 2. Target binding ability of fusion protein

In this Example 2, the binding ability of the fusion protein prepared in Example 1 to the target was analyzed. At this time, the target was set differently depending on the biologically active molecule (HLA-A, IgG antibody fragment or EPO analog) included in each fusion protein.

Example 2-1. HLA-A-containing fusion protein

As a target of a fusion protein containing HLA (CMVpHLA) as a biologically active molecule, binding ability to an anti-CMVpHLA antibody was confirmed.

Specifically, it was analyzed through ELISA assay and SPR (Surface Plasmon Resonance).

The ELISA assay was performed as follows. A 96-well plate was coated with anti-CMVpHLA antibody (Ajou University) at 10 ng/well at 4°C for 16 hours. Plates were then washed with 0.05% PBS-Tween and blocked with 3% dry skim 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. Then, the plate was washed, and HRP-conjugated anti-IgM (F5μ) antibody (MERCK) or HRP-conjugated anti-FLAG antibody (Abcam) was diluted 1:5000 and incubated at 37° C. for 1 hour. Thereafter, the plate was washed 9 times with 0.05% PBS-Tween, and after color development with TMB solution (Sigma), the reaction was terminated with 1N sulfuric acid (Daejeong Hwageum) and analyzed by VARIOSKAN LUX (Thermo Scientific).

SPR analysis was performed as follows. The SPR analyzer used Cytiva's Biacore T200. To label Anti-CMVpHLA mouse Fc antibody (Ajou University) on CM5 chip (Cytiva), recombinant protein G (Abcam) was attached using EDC/NHS reaction and blocked with 1M ethanolamine. Then, CMVpHLA mouse Fc antibody was flowed and coated on the chip in a directional manner. Then, a dilution solution containing each fusion protein or control protein was flowed, and the dissociation reaction was performed with 10 mM glycine-HCl, pH 1.5.

As a result, as shown in FIG. 3a , it was confirmed that the CMVpHLA control protein not including the IgM Fc region had significantly lower binding activity to the target (anti-CMVpHLA antibody). On the other hand, it was confirmed that the fusion protein including the IgM Fc region had very high binding activity to the target (anti-CMVpHLA antibody), and the EC ₅₀ value was improved several tens of times compared to the CMVpHLA control protein.

fusion protein	ka (1/Ms)	kd (1/s)	KD (M)	Rmax	Avidity score
[(CMVpHLA) 2 - (IgD hinge region) 2 - (IgM Fc region)] 5 - (J chain)	5.56×10 6	3.78×10 -5	6.81×10 -12	33.72	8.92×10 5
[(CMVpHLA) 2 - (Linker) 0 - (IgM Fc region)] 5 - (J chain)	5.44×10 6	5.56×10 -5	1.02×10 -11	33.6	6.05×10 5
[(CMVpHLA) 2 -((GGGGS) 3 ) 2 -(IgM Fc region)] 5 - (J chain)	4.74×10 6	2.94×10 -5	6.22×10 -12	32.06	1.09×10 6
[(CMVpHLA) 2 - (IgA hinge region) 2 - (IgM Fc region)] 5 - (J chain)	5.43×10 6	3.18×10 -5	5.86×10 -12	30.2	9.50×10 5
[(CMVpHLA) 2 -(IgD hinge region) 2 -(IgM Fc region)] 6 -(J chain) 0	8.44×10 6	1.97×10 -4	2.33×10 -11	29.88	1.52×10 5
CMVpHLA	2.52×10 6	0.004645	1.84×10 -9	10.33	2.22×10 3

(In Table 4, ka is the rate at which two substances are attached (association rate constant), kd is the rate at which two substances are dissociated (dissociation rate constant), KD is the binding force, and Rmax is the angle in SPR when two substances are attached. The maximum RU value and the Avidity score are Rmax/kd values, which indicate the high degree of avidity in SPR.)

In addition, as shown in Table 4, it was confirmed that the CMVpHLA control protein not including the IgM Fc region had significantly lower KD value and avidity score (Rmax/Kd) for the target (CMVpHLA mouse Fc antibody). On the other hand, the fusion protein containing the IgM Fc region has significantly higher KD value and avidity score (Rmax / Kd) for the target (CMVpHLA mouse Fc antibody), and shows 100- to 1000-fold improved binding capacity compared to the CMVpHLA control protein. was confirmed.

Through the above results, since the IgM Fc region can improve the binding ability of the biologically active molecule to the target, the fusion protein comprising the IgM Fc region and the biologically active molecule according to the present invention has a high level of desired biological activity in cells. It was found that it can be expressed as

Example 2-2. Fusion protein including IgG antibody fragment

As a target of a fusion protein including an antibody fragment (IgG Fab; A2014) as a biologically active molecule, binding ability to HLA-G protein antigen was confirmed.

Specifically, it was analyzed through the ELISA assay and SPR according to Example 2-1.

As a result, as shown in FIG. 3b , it was confirmed that the (A2014)-(IgGFc) control protein including the IgG Fc region had significantly lower binding activity to the HLA-G antigen. On the other hand, it was confirmed that the fusion protein including the IgM Fc region had very high binding activity to the HLA-G antigen, and the EC ₅₀ value was improved by about 1000 times or more compared to the (A2014)-(IgGFc) control protein.

fusion protein	ka (1/Ms)	kd (1/s)	KD (M)	Rmax	Avidity score
[(A2014) 2 - (IgD hinge region) 2 - (IgM Fc region)] 5 - (J chain)	9.58×10 -6	4.25×10 -5	4.44×10 -12	93.38	2.20×10 -6
[(A2014) 2 - (Linker) 0 - (IgM Fc region)] 5 - (J chain)	2.63×10 -7	1.12×10 -4	4.25×10 -12	59.87	5.36×10 -5
[(A2014) 2 -((GGGGS) 3 ) 2 -(IgM Fc region)] 5 - (J chain)	1.96×10 -7	9.66×10 -5	4.94×10 -12	56.68	5.87×10 -5
[(A2014) 2 - (IgA hinge region) 2 - (IgM Fc region)] 5 - (J chain)	1.39×10 -7	5.05×10 -5	3.63×10 -12	73.38	1.45×10 -6
[(A2014) 2 - (IgD hinge region) 2 - (IgM Fc region)] 6 - (J chain) 0	1.48×10 -7	3.51×10 -5	2.32×10 -12	86.71	2.47×10 -6
(A2014)-(IgG Fc)	1.18×10 -7	3.05×10 -4	2.59×10 -11	17.44	5.71×10 -4

(In Table 5, ka is the rate of association of two substances (association rate constant), kd is the rate of dissociation of two substances (dissociation rate constant), KD is binding force, and Rmax is the angle in SPR when two substances are attached. The maximum RU value and the Avidity score are Rmax/kd values, indicating the high degree of avidity in SPR.)

In addition, as shown in Table 5, it was confirmed that the (A2014)-(IgGFc) control protein including the IgG Fc region had significantly lower KD value and avidity score (Rmax/Kd) for the HLA-G antigen. On the other hand, the fusion protein including the IgM Fc region has significantly higher KD value and avidity score (Rmax / Kd) for the HLA-G antigen, and improved 10-fold to 500-fold compared to the (A2014)-(IgGFc) control protein. It was confirmed that the binding ability was shown.

Through the above results, since the IgM Fc region can improve the binding ability of the biologically active molecule to the target, the fusion protein comprising the IgM Fc region and the biologically active molecule according to the present invention has a high level of desired biological activity in cells. It was found that it can be expressed as

Example 2-3. Fusion protein containing EPO analogues

Binding ability to the EPO receptor as a target of a fusion protein containing an EPO analog (NESP) as a biologically active molecule was confirmed.

Specifically, it was analyzed through the ELISA assay and SPR according to Example 2-1.

As a result, as shown in FIG. 3c , it was confirmed that the fusion protein including the IgM Fc region and NESP had very high binding activity to the EPO receptor.

fusion protein	ka (1/Ms)	kd (1/s)	KD (M)	Rmax	Avidity score
[(NESP) 2 - (IgD hinge region) 2 - (IgM Fc region)] 5 - (J chain)	1.383×10 -6	1.240×10 -5	8.968×10 -12	402.0	3.24×10 7
[(NESP) 2 - (Linker) 0 - (IgM Fc region)] 5 - (J chain)	1.612×10 -6	4.051×10 -5	2.513×10 -11	354.8	8.76×10 6
NESP	1.241×10 -6	1.456×10 -3	1.173×10 -9	111.2	7.64×10 4

(In Table 6, ka is the rate at which two substances are attached (association rate constant), kd is the rate at which two substances are dissociated (dissociation rate constant), KD is the binding force, and Rmax is the angle in SPR when two substances are attached. The maximum RU value and the Avidity score are Rmax/kd values, which indicate the high degree of avidity in SPR.)

In addition, as shown in Table 6, it was confirmed that the NESP control protein not including the IgM Fc region had significantly lower KD value and avidity score (Rmax/Kd) for the EPO receptor. On the other hand, the fusion protein comprising the IgM Fc region and NESP has significantly higher KD value and avidity score (Rmax / Kd) for the EPO receptor, and it was confirmed that the NESP control protein exhibits a 100-fold to 400-fold improved binding ability compared to the control protein. .

Through the above results, since the IgM Fc region can improve the binding ability of the biologically active molecule to the target, the fusion protein comprising the IgM Fc region and the biologically active molecule according to the present invention has a high level of desired biological activity in cells. It was found that it can be expressed as

In particular, as a result of synthesizing the results of Examples 2-1 to 2-3, the above-described effects are obtained regardless of the type of biologically active molecule or MoA (Mode of Action), provided that the biologically active molecule binds to the IgM Fc region. could be seen to appear.

Example 3. Biological activity of fusion protein

Example 3-1. Activation, proliferation induction and cytotoxicity analysis of CD8+ T cells

In order to confirm the biological activity of the fusion protein prepared in Example 1, the activation and proliferation inducing ability of CD8 ⁺ T cells was analyzed.

First, activation of CD8 ⁺ T cells was confirmed through IFN-γ ELISPOT assay. PBMC were purchased from Immunospot and used, CD8 ⁺ T cells were obtained using a CD8 ⁺ T cell isolation kit (Miltenyi Biotec), and ELISPOT analysis was performed using an ELISPOT kit (Immunospot). Then, after washing the plate coated with IFN-γ antibody with PBS, RPMI-1640 (10% FBS) was added to stabilize the antibody, and after removing RPMI-1640, a dilution solution containing each fusion protein or control protein was applied to the plate. were placed in each Then, PBMCs (6 × 10 ⁵ cells/well) and CD8 ⁺ T cells (2 × 10 ⁵ cells/well) were put into the wells containing each fusion protein, respectively, and reacted in a 37°C 5% CO ₂ incubator for 48 hours. did The reacted plate was washed with PBS to remove cells and materials, and then reacted with Biotinylated IFN-γ antibody at room temperature for 2 hours. After washing with PBS, Strep-AP was added and reacted at room temperature for 1 hour, washed with PBS, and reacted with the substrate for 10 minutes to induce color development. After that, it was analyzed with an ELISPOT reader (AID) and statistical processing was performed using the GraphPad Prism Version 5.0 (GraphPad software) analysis program.

On the other hand, proliferation-inducing ability to CD8 ⁺ T cells was confirmed through FACS. PBMCs purchased from Immunospot 1×10 ⁶ cells/mL were suspended in RPMI-1640 (10% FBS), which were seeded in 12 wells at a density of 1×10 ⁶ cells/well. Thereafter, each fusion protein or control protein was treated at a concentration of 2 nM per well, and 20 U/mL of IL-2 (Peprotech) was added and cultured. After 3 to 4 days, IL-2 20 U/mL, IL-7 (Peprotech) 25 ng/mL, and IL-15 (Peprotech) 25 ng/mL were added, and the above three Cytokines were mixed, and a culture medium equal to the initial volume was added. Each cell was harvested on days 10-11 or 19-20 and washed with FACS buffer (5% FBS in PBS). Thereafter, the cell suspension was treated with an isotype antibody (Biolegend) to perform Fc blocking, followed by washing with FACS buffer. Thereafter, it was washed with PerCP cy5.5 anti-human CD8 antibody (BD bioscience), and washed after treatment with HLA-A*02:01 CMV pp65 tetramer (MBL). After completion of the process, the proliferation of CMV-specific CD8 ⁺ T cells was checked using a flow cytometer (BD bioscience, FACSLyric™).

In addition, cytotoxicity analysis of CD8 ⁺ T cells was confirmed by FACS. PBMCs purchased from Immunospot 6 × 10 ⁵ cells/mL were suspended in RPMI-1640 (10% FBS), which were seeded into 6 wells at a density of 1.8 × 10 ⁶ cells/well. After seeding, each fusion protein or control protein was treated at a concentration of 100 nM per well, and 20 U/mL of IL-2 (Peprotech) and 2 μg/mL of Anti-human CD28 antibody (Ebioscience) were added and cultured. After 3 to 4 days, IL-2 20 U/mL, IL-7 (Peprotech) 25 ng/mL, IL-15 (Peprotech) 25 ng/mL, and anti-human CD28 antibody 2 μg/mL were added and cultured. On the 7th to 8th days, the above three cytokines and one antibody were mixed, and each fusion protein or control protein was added at the same time as the culture medium was added as much as the initial volume. Then, after every 3 to 4 days, the above three cytokines and one antibody were mixed and added, and on days 19 to 20, each cell was washed with FACS buffer, and then CD8 ⁺ T cells were converted to CD8 ⁺ It was obtained using a T cell isolation kit (Miltenyi Biotec). Thereafter, the target cells, T2 cells (ATCC), were reacted with CMVpp65 peptide 50 μg/mL and 3 μg/mL B2M (Sino) at 37° C. in a 5% CO ₂ incubator for 1 hour. After washing with FACS buffer, 1 × 10 ⁶ cells/4 μM DiO (Invitrogen) was treated and washed after reaction in an incubator at 37° C. 5% CO ₂ . Then, the DiO-labeled T2 cells and the CD8 ⁺ T cells were mixed in each ratio, and after each fusion protein or control protein was added, the reaction was performed for 4 hours. And 7AAD (Invitrogen) was treated and after 10 minutes of reaction, the degree of apoptosis of T2 cells was analyzed by flow cytometry.

As a result, as shown in FIG. 4, compared to the CMVpHLA control protein not containing the IgM Fc region, the fusion protein containing the IgM Fc region and CMVpHLA increased the amount of IFN-γ produced by about 2 times or more. It was confirmed once again that the binding capacity was high due to IgM Fc. In addition, since the amount of IFN-γ produced in the fusion protein including both the IgM Fc region and the linker is higher than that of the fusion protein including the IgM Fc region but not including the linker, better biological activity is shown when the linker is linked. could see that

In addition, as shown in Figure 5a, compared to the fusion protein containing the IgM Fc region but not including the linker, the fusion protein containing both the IgM Fc region and the linker has a 3-fold effect of inducing proliferation of CD8 ⁺ T cells. It was confirmed that the above was excellent.

In addition, as shown in FIG. 5b , compared to the CMVpHLA control protein not containing the IgM Fc region, the fusion protein containing the IgM Fc region and the linker and CMVpHLA was 30 times more effective in inducing proliferation of CD8 ⁺ T cells. Confirmed. Furthermore, as shown in FIG. 6 , it was confirmed that the fusion protein including the IgM Fc region and CMVpHLA had higher toxicity to cancer cells, compared to the CMVpHLA control protein not including the IgM Fc region.

According to the above results, the fusion protein including the IgM Fc region can induce activation, proliferation ^and death of cancer cells at a higher level than the protein without the IgM Fc region, and in particular, the IgM Fc region and biological It was found that better biological activity can be exhibited when the active molecule is linked by a linker.

Example 3-2. Binding activity to HLA-G expressing cells

In order to confirm the biological activity of the fusion protein prepared in Example 1, binding activity to JEG-3 cells expressing HLA-G was analyzed.

Specifically, a dilution solution containing each fusion protein or control protein was added to JEG-3 cells (ATCC, 3.5 × 10 ⁵ cells/100 μl) that naturally express HLA-G, and reacted at 4° C. for 1 hour. Then, after washing with FACS buffer (5% FBS in PBS), anti-IgM-PE (ebioscience) or anti-IgG Fc-FITC (Biolegend) was treated and reacted at 4°C for 30 minutes. Finally, after washing with FACS buffer, the degree of the fusion protein binding to JEG-3 cells and the control was confirmed using a flow cytometer (BD bioscience, FACSLyric™).

As a result, as shown in FIG. 7 , it was confirmed that the (A2014)-(IgGFc) control protein including an IgG Fc region had significantly lower binding activity to HLA-G expressing cells. On the other hand, the fusion protein containing the IgM Fc region has very high binding activity to the cells, and the maximum MFI (median fluorescence intensity) value is improved by about 2.5 times or more compared to the (A2014)-(IgGFc) control protein. .

From the above results, it was found that the fusion protein including the IgM Fc region had a higher activity of binding to the intracellular receptor than the protein not including the IgM Fc region. Therefore, it was found that the fusion protein comprising the IgM Fc region and the biologically active molecule according to the present invention can exhibit a desired biological activity in a cell at a high level.

Example 3-3. Binding blocking activity of ILT-2 protein

In order to confirm the biological activity of the fusion protein prepared in Example 1, the binding blocking activity of the HLA-G protein and the ILT-2 (LILRB1) protein was analyzed. The ILT-2 protein is a receptor protein expressed in immune cells, and when the HLA-G protein binds, the activities of cytotoxic T cells, NK cells, and B cells are suppressed, so that a normal immune response is not caused.

First, the binding blocking activity between the recombinant HLA-G protein and the recombinant ILT-2 protein was analyzed by ELISA assay. ILT-2 antigen (R&D systems) was put in a 96-well plate at 150 ng/well and then coated at 4°C for 16 hours. Plates were then washed with 0.05% PBS-Tween and blocked with 3% dry skim milk. After blocking, a dilution containing each fusion protein or control protein was added to the wells, and HLA-G-6x his tag (IM Biologics) was treated at a constant concentration in each well and incubated at 37° C. for 2 hours. Then, the plate was washed, and HRP-conjugated anti-6x his antibody (abcam) was diluted 1:10,000 and incubated at 37°C for 1 hour. Thereafter, the plate was washed 9 times with 0.05% PBS-Tween, developed with TMB solution (Sigma), and then the reaction was terminated with 1N sulfuric acid (Daejeong Hwageum) and analyzed by VARIOSKAN LUX (Thermo Scientific).

On the other hand, the binding blocking activity between HLA-G protein and ILT-2 protein naturally expressed in cells was Analysis was performed using JEG-3 cells. JEG-3 cells (3 × 10 ⁵ cells/tube) purchased from ATCC were lysed with MEM containing 10% FBS. Then, a dilution solution containing each fusion protein or control protein was added to the tube and reacted 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 treated and reacted at 4° C. for 30 minutes. Washed with FACS buffer, treated with PE-anti-human IgG Fc (Biolegnd), and reacted at 4° C. for 20 minutes. Finally, after washing with FACS buffer, the degree of blocking of the binding between the ILT-2 protein and the intracellularly expressed HLA-G protein was confirmed using a flow cytometer (BD bioscience, FACSLyric™).

As a result, as shown in FIG. 8 , it was confirmed that the (A2014)-(IgGFc) control protein including the IgG Fc region had significantly lower binding blocking activity of the recombinant HLA-G protein and the ILT-2 protein. On the other hand, it was confirmed that the fusion protein including the IgM Fc region had a very high binding blocking activity of the proteins, and the IC ₅₀ value was improved by about 5 to 7 times or more compared to the (A2014)-(IgGFc) control protein.

In addition, as shown in FIG. 9 , it was confirmed that the fusion protein including the IgM Fc region had a very high binding blocking activity to the HLA-G protein expressed in the cells. This suggests that HLA-G expressed on the surface of cancer cells in the body can effectively inhibit binding to ILT-2, an inhibitory receptor of immune cells, thereby increasing the death of cancer cells.

From the above results, it was found that the fusion protein including the IgM Fc region had a higher activity of blocking the binding of the antigen to the receptor protein expressed in the cell, compared to the protein not including the IgM Fc region. Therefore, it was found that the fusion protein comprising the IgM Fc region and the biologically active molecule according to the present invention can exhibit a desired biological activity in a cell at a high level.

Example 3-4. Binding activity with intracellular EPO receptors

In order to confirm the biological activity of the fusion protein prepared in Example 1, binding activity to cells expressing the EPO receptor was analyzed.

Specifically, binding activity was confirmed using the EPOR reporter assay purchased from Indigobioscience. Cells were seeded at a density of 150 uL/well in a 96-well cell culture plate (Corning), and then cultured in a 5% CO ₂ incubator at 37° C. for 4 to 6 hours. Thereafter, a dilution solution containing each fusion protein or control protein was added to the wells, and cultured in a 5% CO ₂ incubator at 37° C. for one day. Then, according to the manufacturer's protocol, 100 uL of the reaction solution was added, and the color was developed at room temperature for 5 minutes, followed by analysis with VARIOSKAN LUX (Thermo Scientific).

As a result, as shown in FIG. 10 , compared to the NESP control protein not including the IgM Fc region, the fusion protein including the IgM Fc region and the linker induced the proliferation of EPOR overexpressed cells to a higher level. . That is, it was confirmed that the fusion protein comprising the IgM Fc region exhibits excellent binding activity with the intracellular EPO receptor, and the signaling pathway is activated by EPO receptor stimulation, thereby resulting in an effect of improving the proliferative capacity of cells.

From the above results, it was found that the fusion protein including the IgM Fc region had a higher ability to bind to an intracellular receptor and thus to induce biological activity, compared to a protein not including the IgM Fc region. Therefore, it was found that the fusion protein comprising the IgM Fc region and the biologically active molecule according to the present invention can exhibit a desired biological activity in a cell at a high level.

In particular, as a result of synthesizing the results of Examples 3-1 to 3-4, the biological activity-inducing effect of the fusion protein by the above-described IgM Fc region is that of the biologically active molecule if the biologically active molecule binds to the IgM Fc region. It was found that it appeared regardless of the type or MoA.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limited thereto. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (16)

1. Represented by the following structural formula, the fusion protein:

   [(X) _a -(Y) _b -(IgM Fc region)] _m -(Z) _n

   At this time, in the structural formula,

   wherein X is a biologically active molecule,

   Y is a linker,

   The IgM Fc region is a monomer,

   wherein Z is a J chain,

   wherein a is 1 or 2, b is 0, 1 or 2, m is 5 or 6, and n is 0 or 1.
2. According to claim 1,

   The IgM Fc region is a fusion protein comprising at least one selected from the group consisting of a Cμ2 domain, a Cμ3 domain and a Cμ4 domain.
3. According to claim 1,

   The linker is at least one selected from the group consisting of an IgD hinge region, an IgA hinge region, an IgG hinge region, and a GS linker, a fusion protein.
4. 4. The method of claim 3,

   The hinge region is a part or all of the hinge region, a part or all of the CH1 domain, a part or all of the CH2 domain, or a combination thereof, a fusion protein.
5. 4. The method of claim 3,

   The GS linker is at least one selected from the group consisting of (GS) _c , (SG) _c , (GGGS) _c , (GGGGS) _c , GCGS(GGGS) _c and GCGGS(GGGGS) _c , wherein c is 2 An integer from 6 to 6.
6. According to claim 1,

   The biologically active molecule is 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 soluble protein, a membrane-bound protein, a ligand, a receptor, a virus-like particle. , At least one selected from the group consisting of protein toxins, chemokines, cytokines and enzymes, fusion proteins.
7. 7. The method of claim 6,

   The antigen-binding fragment of the antibody is Fab, F(ab') ₂ , F(ab) ₂ , Fab', Fab ₂ , Fab ₃ , Fv, scFv, Bis-scFv, minibody, triabody, diabody, tandem At least one selected from the group consisting of a diabody, a nanobody, and a tetrabody, a fusion protein.
8. 7. The method of claim 6,

   The membrane-bound protein is HLA.
9. 7. The method of claim 6,

   The ligand is EPO or an EPO analog, a fusion protein.
10. According to claim 1,

    The J chain is a fusion protein that is further bound to a protein molecule.
11. 11. The method of claim 10,

    The protein molecule may be 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 soluble protein, a membrane-bound protein, a ligand, a receptor, a virus-like particle, At least one selected from the group consisting of protein toxins and enzymes, a fusion protein.
12. 11. The method of claim 10,

    The protein molecule is at least one selected from the group consisting of a co-stimulatory receptor (co-stimulatory receptor), cytokine and cell engager (cell engager), a fusion protein.
13. A nucleic acid molecule encoding the fusion protein of claim 1.
14. A vector for expression of a fusion protein, comprising the nucleic acid molecule of claim 13 .
15. A host cell into which the vector for expression of the fusion protein of claim 14 is introduced.
16. A method for producing the fusion protein of claim 1, comprising the step of introducing the vector for expression of the fusion protein of claim 15 into a host cell.

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