Novel modified immunoglobulin FC fusion protein and its uses

Abstract

To provide a novel modified Fc domain protein in which the therapeutic protein fused without effector functions such as ADCC and CDC exhibits a longer half-life while not affecting the production and activity of bioactive proteins.SOLUTION: Provided are novel immunoglobulin Fc domain variant proteins and uses thereof. When expressed in the form of a fusion protein with a different biologically active protein, the immunoglobulin Fc domain variant protein according to an embodiment of the present invention has the advantages of not only increasing the half-life of the biologically active protein in vivo, but also effectively inhibiting effector functions such as ADCC or CDC, thereby allowing unexpected side effects to be minimized.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under the Paris Convention for the Protection of Industrial Property to U.S. Patent Application No. 62 / 847,470, filed May 14, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The present invention relates to novel modified immunoglobulin Fc fusion proteins and uses thereof, more particularly to novel modified immunoglobulin Fc fusion proteins that have increased half-life and can minimize side effects caused by antibody Fc fragments, such as ADCC and CDC, and uses thereof.

[0003] The immunoglobulin (antibody) Fc domain is widely used as a carrier protein for various therapeutic and diagnostic proteins. Antibodies contain two functionally independent portions: a variable domain known as "Fab" that binds to antigens, and a constant domain known as "Fc" that is linked to effector functions such as complement activation and attack by phagocytes. Fc has a long serum half-life, while Fab has a short lifespan (Capon et al., Nature 337:525-531, 1989). When combined with a therapeutic protein or peptide, the Fc domain can provide a longer half-life or integrate functions such as Fc receptor binding, protein A binding, complement fixation, and even placental delivery. Notably, the longer half-life of such Fc domains is due to their binding affinity to the neonatal Fc receptor (FcRn).

[0004] Many techniques have been developed to fuse the Fc domain and biologically active proteins to increase the half-life of the biologically active proteins. A representative example is etanercept (trade name: Enbrel), a fusion protein in which TNFα receptor 2 is linked to the Fc domain of an IgG1 antibody. (登録商標)) (U.S. Patent No. 5,447,851). In addition, there are also some cytokines and growth hormones that are fused to the Fc domain of IgG antibodies.

[0005] However, unlike fusion of the extracellular domain of a cell surface receptor, fusion of a water-soluble protein to IgG results in reduced biological activity compared to the unfused cytokine or growth factor. The chimeric protein exists as a dimer, and the presence of two active proteins in close proximity to each other induces steric hindrance in binding to target molecules such as receptors. Therefore, these issues must be overcome to generate efficient Fc fusion proteins.

[0006] Another limitation of Fc fusion technology is unintended immune responses. The Fc domain of immunoglobulins also has effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In particular, the four human IgG isotypes bind with different affinities to activating Fcγ receptors (FcγRs, including FcγRI, FcγRIIa, and FcγRIIIa), inhibitory FcγRIIb receptors, and the first factor of complement (C1q), inducing very different effector functions (Bruhns, P. et al., Blood 113(16):3716-3725, 2009). IgG binding to FcγRs or C1q depends on residues located in the hinge region and CH2 domain. However, the effector functions of the Fc domain can cause cell death, cytokine secretion, or inflammation in an individual when a therapeutic protein fused to the Fc domain is administered to the individual in vivo; therefore, if these are not absolutely necessary, they should be suppressed to reduce undesired reactions.

[0007] To overcome these identified problems, modified Fc domain proteins have been developed, including those described in US2006 / 0074225A1, Armour, KLet et al. (Eur. J. Immunol., 29(8):2613-2624, 1999), Shields, RLet et al. (J. Biol. Chem. 276(9):6591-6604, 2001), and Idusogie, E. E. et al. (J. Immunol. 164(8):4178-4184, 2000).

[0008] Additionally, despite improvements in half-life, there remains a need to further improve the half-life of therapeutic proteins fused to an Fc domain. Summary of the Invention [Problem to be solved by the invention]

[0009] However, the above-mentioned conventional techniques have drawbacks such as not being able to achieve a satisfactory level of half-life extension or having other side effects.

[0010] Therefore, the present invention aims to solve the various problems described above, and an object of the present invention is to provide a novel modified Fc domain protein that does not affect the production and activity of biologically active proteins while allowing fused therapeutic proteins to exhibit a longer half-life without effector functions such as ADCC and CDC. However, these problems are merely exemplary and do not limit the scope of the present invention. [Means for solving the problem]

[0011] According to one aspect of the present invention, there is provided an immunoglobulin Fc domain variant protein in which the amino acids at positions 18 and 196 of a modified IgG4 Fc domain protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 have been mutated to other amino acids.

[0012] Another aspect of the present invention provides a fusion protein in which one or more biologically active proteins (APIs, active pharmaceutical ingredients) are linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein.

[0013] In accordance with another aspect of the present invention, there is provided a polynucleotide encoding the immunoglobulin Fc domain variant protein or the fusion protein.

[0014] In yet another aspect of the present invention, there is provided a homo- or hetero-dimer comprising the above fusion protein.

[0015] In yet another aspect, the present invention provides a composition comprising the fusion protein or a polynucleotide encoding the same, or the homo- or heterodimer as an active ingredient.

[0016] According to yet another aspect of the present invention, there is provided a method for extending the in vivo half-life of a biologically active protein, comprising the step of administering to an individual a fusion protein in which a biologically active protein is fused to the immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the fusion protein. [Effects of the Invention]

[0017] The immunoglobulin Fc domain variant protein according to one embodiment of the present invention can be fused to another biologically active protein to increase the half-life of the biologically active protein while inactivating antibody Fc domain-dependent effector functions, such as ADCC and CDC, which are side effects associated with the use of antibody-based protein therapeutics. However, the effects of the present invention are not limited to the above. Further aspects of the present invention are described below: [Section 1] An immunoglobulin Fc domain mutant protein, in which the amino acids at positions 18 and 196 of a modified IgG4 Fc domain protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 have been mutated to other amino acids. [Section 2] Item 2. The immunoglobulin Fc domain variant protein according to Item 1, wherein the mutation at the 18th amino acid is a mutation selected from the group consisting of T18N, T18K, and T18Q. [Section 3] Item 2. The immunoglobulin Fc domain variant protein according to Item 1, wherein the mutation at amino acid position 196 is selected from the group consisting of M196A, M196F, M196I, M196L, M196V, M196P, and M196W. [Section 4] Item 1. The immunoglobulin Fc domain variant protein according to Item 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 9. [Section 5] A fusion protein in which at least one biologically active protein (API, active pharmaceutical ingredient) is linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein according to item 1. [Section 6] Item 6. The fusion protein according to Item 5, wherein the biologically active protein is linked to the N-terminus or C-terminus of the immunoglobulin Fc domain variant protein by a linker peptide or an antibody hinge region. [Section 7] Item 6. The fusion protein of item 5, wherein the biologically active protein is a cytokine, an enzyme, a blood clotting factor, an extracellular domain of a membrane receptor, a growth factor, a peptide hormone, or an antibody mimetic. [Section 8] A polynucleotide encoding the immunoglobulin Fc domain variant protein of Item 1 or the fusion protein of Item 5. [Section 9] A recombinant vector comprising the polynucleotide according to Item 8. [Section 10] A homodimer comprising the fusion protein according to item 5. [Section 11] A heterodimer comprising the fusion protein of item 5. [Section 12] The heterodimer of item 11, wherein the immunoglobulin Fc domain variant protein has a knobs-into-holes structure. [Section 13] A composition comprising at least one selected from the group consisting of the fusion protein of Item 5, the homodimer of Item 10, and the heterodimer of Item 11 as an active ingredient. [Section 14] A method for extending the in vivo half-life of a biologically active protein, comprising the step of administering to an individual a fusion protein in which a biologically active protein is fused to the immunoglobulin Fc domain variant protein of Item 1, or a homodimer or heterodimer containing the fusion protein. [Section 15] A method for treating a disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of a fusion protein in which a biologically active protein is fused to the immunoglobulin Fc domain variant protein of Item 1, or a homodimer or heterodimer containing the fusion protein. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a monospecific fusion protein according to one embodiment of the present invention. [Figure 2] FIG. 1 is a schematic diagram showing the structure of a monospecific fusion protein according to one embodiment of the present invention. [Figure 3] FIG. 1 is a schematic diagram showing the structure of a monospecific fusion protein according to one embodiment of the present invention. [Figure 4]FIG. 1 is a schematic diagram showing the structure of a bispecific fusion protein according to one embodiment of the present invention. [Figure 5] FIG. 1 is a schematic diagram showing the structure of a homodimer formed by a bispecific fusion protein according to one embodiment of the present invention. [Figure 6] FIG. 1 is a schematic diagram showing the structure of a homodimer of a monospecific fusion protein according to one embodiment of the present invention. [Figure 7] FIG. 1 is a schematic diagram showing the structure of a heterodimer of two different monospecific fusion proteins according to one embodiment of the present invention. [Figure 8] FIG. 1 is a schematic diagram showing the structure of a heterodimer composed of two different bispecific fusion proteins according to one embodiment of the present invention. [Figure 9] FIG. 1 is a schematic diagram showing the structure of a homodimer of a monospecific fusion protein having an IgG1 hinge region and a linker peptide according to one embodiment of the present invention. [Figure 10] FIG. 1 is a schematic diagram showing the structure of a heterodimer composed of two different monospecific fusion proteins according to one embodiment of the present invention. [Figure 11] FIG. 1 is a schematic diagram showing the structure of a homodimer of a monospecific fusion protein with a biologically active protein at the C-terminus according to one embodiment of the present invention. [Figure 12] FIG. 1 is a schematic diagram showing the structure of a heterodimer composed of two different bispecific fusion proteins with biologically active proteins at their C-termini according to one embodiment of the present invention.

[0019] [Figure 13] FIG. 1 is a schematic diagram showing the process by which antibodies are recycled by FcRn. [Figure 14a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-11) according to an embodiment of the present invention. [Figure 14b] 1 is a chromatogram showing the results of analyzing the produced PG-11 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 15a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-22) according to an embodiment of the present invention. [Figure 15b] 1 is a chromatogram showing the results of analyzing the produced PG-22 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 16a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-088) according to an embodiment of the present invention. [Figure 16b] 1 is a chromatogram showing the results of analyzing the produced PG-088 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 17a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-088m) according to an embodiment of the present invention. [Figure 17b] 1 is a chromatogram showing the results of analyzing the produced PG-088m by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 18a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-075) according to an embodiment of the present invention. [Figure 18b] 1 is a photograph showing the results of Western blot analysis of the produced PG-075 using an anti-IgG antibody. [Figure 19a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-110) according to an embodiment of the present invention. [Figure 19b] 1 is a chromatogram showing the results of analyzing the produced PG-110 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 20a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-129) according to an embodiment of the present invention. [Figure 20b] 1 is a chromatogram showing the results of analyzing the produced PG-129 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 21a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-400) according to an embodiment of the present invention. [Figure 21b] 1 is a chromatogram showing the results of analyzing the produced PG-400 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 22a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-410) according to an embodiment of the present invention.

[0020] [Figure 22b] 1 is a chromatogram showing the results of analyzing the produced PG-410 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 23] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of heterodimeric proteins MG12-4 (left) and MG12-5 (right) according to one embodiment of the present invention. [Figure 24a] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-50-1) according to an embodiment of the present invention. [Figure 24b] 1 is a photograph showing the results of SDS-PAGE analysis showing the results of sample production of a fusion protein (PG-50-2) according to an embodiment of the present invention. [Figure 24c] 1 is a photograph (left) showing the results of SDS-PAGE analysis of a sample of a fusion protein (PG-073) according to an embodiment of the present invention, and a schematic diagram (right) showing the structures of various cleavage forms of the fusion protein predicted based on the SDS-PAGE analysis results. [Figure 24d] 1 is a photograph (top) showing the results of SDS-PAGE analysis of a sample of a fusion protein (PG-210-5) according to an embodiment of the present invention, and a chromatogram (bottom) showing the results of analyzing the produced PG-210-5 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 24e]1 is a photograph (top) showing the results of SDS-PAGE analysis of a sample of a fusion protein (PG-210-6) according to an embodiment of the present invention, and a chromatogram (bottom) showing the results of analyzing the produced PG-210-6 by size exclusion high performance liquid chromatography (SEC-HPLC). [Figure 25] 1 is a series of graphs showing the results of analyzing the binding affinity between a fusion protein (PG-11) prepared according to one embodiment of the present invention and recombinant human fetal Fc receptor (rhFcRn) using surface plasmon resonance (SPR) analysis. Each of the three graphs shows the results of three independent experiments. [Figure 26] 1 is a series of graphs showing the results of surface plasmon resonance (SPR) analysis of the binding affinity between a fusion protein (PG-11, top) prepared according to one embodiment of the present invention and recombinant human fetal Fc receptor (rhFcRn), compared with Trulicity (bottom), which uses an IgG4 Fc domain. The graphs on the left, middle, and right each show the results of three independent experiments. [Figure 27] 1 is a series of graphs showing the results of surface plasmon resonance (SPR) analysis of the binding affinity between a fusion protein (PG-11, top panel) prepared according to an embodiment of the present invention and recombinant human fetal Fc receptor (rhFcRn), compared with that of rituximab (bottom panel), a human recombinant antibody containing an IgG1 Fc domain. The graphs on the left, middle, and right of the bottom panel each show the results of three independent experiments. [Figure 28] Figure 10 is a series of graphs showing the results of SPR analysis comparing the binding affinities to rhFcRn of a bispecific fusion protein in which anti-PD-L1 scFγ and IL-2 proteins are linked to the N- and C-termini, respectively, of NTIG protein according to an embodiment of the invention (left), and a control bispecific fusion protein in which anti-PD-L1 scFγ and IL-2 proteins are linked to the N- and C-termini, respectively, of a modified IgG4 Fc protein of SEQ ID NO: 2 (right), using rhFcRn as the ligand and the two substances as the analyte. [Figure 29] Figure 27 is a series of graphs showing the results of SPR analysis comparing the binding affinities of a bispecific fusion protein in which anti-PD-L1 scFγ and IL-2 proteins are linked to the N- and C-termini, respectively, of an NTIG protein according to an embodiment of the present invention (left), and a control bispecific fusion protein in which anti-PD-L1 scFγ and IL-2 proteins are linked to the N- and C-termini, respectively, of a modified IgG4 Fc protein of SEQ ID NO: 2 (right), using the two substances as ligands and rhFcRn as the analyte, in the opposite manner to Figure 28 above.

[0021] [Figure 30] 1 is a series of graphs showing the results of BLI analysis of the binding affinity of a fusion protein (PG-11) according to one embodiment of the present invention to Fc gamma receptor I (FcgR1, left) and Fc gamma receptor IIIa (FcgR3a, right) compared to that of rituximab, an IgG1-series humanized antibody. The top, middle, and bottom columns show the results of three independent experiments. [Figure 31] 1 is a series of graphs showing the results of BLI analysis of the binding affinity of a fusion protein (PG-11) according to one embodiment of the present invention to various Fc gamma receptors (FcgR2a, FcgR2b, FcgR3b) in comparison with rituximab, an IgG1 series humanized antibody, with PBS used as a control. [Figure 32] 1 is a graph showing the results of an analysis of the binding affinity of a fusion protein (PG-11) according to an embodiment of the present invention to complement C1q in comparison with rituximab. [Figure 33] 1 is a series of graphs showing the results of a comparative analysis of the GLP-2 activity of fusion proteins containing GLP-2 analogs according to various embodiments of the present invention (PG-22, PG-210-5, PG-210-6, MG12-4, and MG12-5). The two graphs at the top and bottom show the results of two independent tests, respectively. [Figure 34]1 is a series of graphs showing the results of BLI analysis of the affinity of the dimeric IL-10 mutant fusion protein of Example 2-3 (left) and the monomeric IL-10 mutant fusion protein of Example 2-4 (right) for IL-10R1 depending on the treatment concentration. [Figure 35a] 1 is a graph showing the results of quantitative analysis by ELISA of the release of TNF-α from macrophages depending on the treatment concentration of the fusion protein PG-210-6 according to an embodiment of the present invention. [Figure 35b] 1 is a graph showing the results of quantitative analysis by ELISA of the release of TNF-α from macrophages depending on the treatment concentration of the fusion protein PG-410 according to an embodiment of the present invention. [Figure 35c] 1 is a graph showing the results of quantitative analysis of the release of TNF-α in macrophages according to the treatment concentrations of fusion proteins PG-110 and PG-088m, according to an embodiment of the present invention, by ELISA. [Figure 36a] FIG. 10 is a series of graphs showing the results of BLI analysis of the affinity of FcεRIα-modified IgG4 Fc (top) and the fusion protein FcεRIα-NTIG-IL-10Vm (bottom) of Example 3-3 of the present invention for mouse IgE, as a comparative example. [Figure 36b] 1 is a graph showing the results of BLI analysis of the affinity of the fusion protein FcεRIα-NTIG-IL-10Vm of Example 3-3 of the present invention for human IgE. [Figure 37] 1 is a graph showing the results of analyzing the IgE-suppressing activity of the fusion protein PG-110 and FcεRIα-modified IgG4 Fc according to an embodiment of the present invention by β-hexosaminidase release assay. [Figure 38] 1 is a graph showing the results of BLI analysis of the binding affinity of fusion proteins PG-400 (left) and PG-410 (right) to CD40L according to an embodiment of the present invention. [Figure 39]1 is a graph showing the results of analyzing the pharmacodynamic profile of the fusion protein PG-110 according to an embodiment of the present invention via various administration routes (SC, IV, IP, and IM). DETAILED DESCRIPTION OF THE INVENTION

[0022] According to one aspect of the present invention, there is provided an immunoglobulin Fc domain variant protein in which the amino acids at positions 18 and 196 of a modified IgG4 Fc domain protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4 are mutated to other amino acids.

[0023] The term "modified IgG4 immunoglobulin Fc domain protein" or "modified IgG4 Fc domain protein" as used herein refers to a recombinant antibody Fc domain protein prepared by combining all or part of the hinge, CH2, and CH3 components of the immunoglobulin Fc domain derived from different types of antibody molecules, i.e., IgG, IgD, IgE, IgM, etc. A representative modified IgG4 immunoglobulin Fc domain protein is disclosed in Korean Patent No. 897938.

[0024] As used herein, the term "immunoglobulin Fc domain variant protein" or "Fc domain variant protein" refers to a variant protein in which at least one amino acid in the modified IgG4 Fc domain protein has been substituted, deleted, or added.

[0025] In the immunoglobulin Fc domain mutant protein, the mutation at the 18th amino acid is a mutation selected from the group consisting of T18N, T18K, and T18Q, and the mutation at the 196th amino acid is a mutation selected from the group consisting of M196A, M196F, M196I, M196L, M196V, M196P, and M196W, more preferably, the mutation at the 18th amino acid is T18Q, and more preferably, the mutation at the 196th amino acid is M196L.

[0026] The immunoglobulin Fc domain variant protein may or may not have a lysine (K) removed from its C-terminus. Whether or not lysine is removed depends on whether or not another API is linked to the C-terminus. For example, if an API is linked to the C-terminus of the immunoglobulin Fc domain variant protein of the present invention, removing lysine is advantageous in terms of protein production, whereas if an API is not linked to the C-terminus, there is no need to remove lysine.

[0027] The immunoglobulin Fc domain variant protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 5-9.

[0028] Another aspect of the present invention provides a fusion protein in which one or more biologically active proteins (APIs, active pharmaceutical ingredients) are linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein (FIGS. 1 to 4).

[0029] The term "biologically active protein" as used in this document is a concept distinct from structural proteins, which are proteins that constitute the tissues and organs of the human body. It refers to proteins that perform specific functions in the human body, such as metabolic processes and intercellular or intracellular signal transduction, and are used in the diagnosis and / or treatment of diseases caused by a deficiency or excess of the protein in question.

[0030] As shown in Figures 1 to 4, in the fusion proteins 10 to 40 of the present invention, a biologically active protein or active pharmaceutical ingredient 11 (API) is linked to the N-terminus or C-terminus of an immunoglobulin Fc domain variant protein 12 via a linker peptide 14, such as an IgG1 hinge region 13 and / or a G4S linker. However, the hinge region 13 is essential for dimerization. When the biologically active protein 11 is directly linked to the IgG1 hinge region 13, the hinge region (i.e., SEQ ID NOS: 20 and 21) is considered a linker, as shown in Figure 3. The hinge region provides flexibility and a dimerization moiety capable of forming intermolecular disulfide bonds via cysteine ​​residues.

[0031] A fusion protein according to one embodiment of the present invention contains one or two biologically active proteins. When the fusion protein contains two biologically active proteins, the fusion protein itself exhibits bispecificity (FIG. 4). As shown in FIG. 4a, a first biologically active protein (11a) is linked to the N-terminus of an immunoglobulin Fc domain variant protein (12) via an IgG1 hinge region (13), and a second biologically active protein (11b) is linked to the C-terminus of the immunoglobulin Fc domain variant protein (12) via a linker peptide (14). Additionally, as shown in FIG. 4b, a linker peptide (14), such as a G4S linker, is further linked to the N-terminus of the IgG1 hinge region (13), and the first biologically active protein (11a) is linked to the N-terminus of the linker peptide (14), and the second biologically active protein (11b) is linked to the C-terminus of the immunoglobulin Fc domain variant protein (12) via the linker peptide (14). In this case, the linker peptide (14) linked to the N-terminus and the linker peptide (14) linked to the C-terminus have the same sequence and have properties similar to those of a G4S linker, but the specific sequence may differ in part.

[0032] In the fusion protein, the biologically active protein is a cytokine, an enzyme, a blood clotting factor, an extracellular domain of a membrane receptor, a growth factor, a peptide hormone, or an antibody mimetic.

[0033] In the fusion protein, the linker peptide has a length of 2 to 60 amino acids (aa), 4 to 55 aa, 5 to 50 aa, 5 to 46 aa, 5 to 45 aa, or 5 to 30 aa. The linker peptide that connects the API to the N-terminus of the immunoglobulin Fc domain variant protein contains part or all of the hinge region of an IgG1 antibody heavy chain. When a part of the hinge region is included, an artificial linker peptide is added to the N-terminus or C-terminus of the hinge region. The hinge region may also be a hybrid hinge region that combines parts of hinge regions from two or more antibody heavy chains. Specifically, the linker peptides are GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 14), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 15), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 16), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 17), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 18), EPKSSDKTHTCPPCP (SEQ ID NO: 19), EPKSCDKTHTCPPCP (SEQ ID NO: 20), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 21), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 22), (G4S) n (n is an integer from 1 to 10, unit sequence number 23), (GSSGGS) n(Unit: SEQ ID NO: 24, n is an integer from 1 to 10), SGGGSGGGGSGGGGSGGEEQEEGGS (SEQ ID NO: 25), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 26), KESGSVSSEQLAQFRSLD (SEQ ID NO: 27), EGKSSGSGSESKST (SEQ ID NO: 28), GSAGSAAGSGEF (SEQ ID NO: 29), (EAAAK) n (Unit: SEQ ID NO: 30, n is an integer from 1 to 10), CRRRRREAEAC (SEQ ID NO: 31), GGGGGGGG (SEQ ID NO: 32), GGGGGG (SEQ ID NO: 33), PAPAP (SEQ ID NO: 34), (Ala-Pro) n (n is an integer of 1 to 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 35), PLGLWA (SEQ ID NO: 36), TRHRQPRGWE (SEQ ID NO: 37), AGNRVRRSVG (SEQ ID NO: 38), RRRRRRRR (SEQ ID NO: 39), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 40), GGGGSGGGSGGGGS (SEQ ID NO: 41), AEAAAKEAAAAKA (SEQ ID NO: 42), GFLG (SEQ ID NO: 43), AAKATTAPATATRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 44), GGSGG (SEQ ID NO: 45), GGSGGSGGS (SEQ ID NO: 46), GGGSGG (SEQ ID NO: 47), and GSTSGSGKPGSGEGS (SEQ ID NO: 48).

[0034] In this case, the cytokine is a chemokine, an interferon, an interleukin, a colony-stimulating factor, or a tumor necrosis factor, and the chemokine is CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, XCL1, XCL2, or CX3CL1, wherein the interferon is interferon-α, interferon-β, interferon-ε, interferon-κ, interferon-δ, interferon-γ, interferon-τ, interferon-ω, interferon-ν, or interferon-ζ, the interleukin is IL-2, IL-4, IL-7, IL-10, IL-12α, IL-12β, IL-13, IL-15, or IL-21, and the colony-stimulating factor is macrophage colony-stimulating factor (MCSF). The tumor necrosis factor is selected from the group consisting of tumor necrosis factor (TNF-α), tumor necrosis factor (TNF-β), CD40 ligand (CD40L), Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL), and promegapoietin.

[0035] In the fusion protein, the enzyme is selected from the group consisting of tissue-type plasminogen activator (tPA), urokinase, alteplase, reteplase, tenecteplase, streptokinase, anlsteplase, staphylokinase, nattokinase, lumbrokinase, collagenase, glutenase, and alglucerase. Examples of enzymes that may be used include, but are not limited to, alglucerase, velaglucerase, imiglucerase, taliglucerase-α, β-glucocerebrosidase, α-galactosidase A, α-glucosidase, α-L-iduronidase, arylsulphatase B, agalsidase, adenosine deaminase, phenylalanine ammonia lyase, dornase, rasburicase, pegloticase, glucarpidase, or ocriplasmin.

[0036] In the fusion protein, the blood coagulation factor is factor VIII or factor IV.

[0037] In the fusion protein, the membrane receptor is a receptor tyrosine kinase (RTK) superfamily, a T cell receptor, an Fc receptor, a chemokine state, a Toll-like receptor family, a guanylyl cyclase-coupled receptor (GCCR), or a receptor serine / threonine kinase, and the receptor tyrosine kinase is an EGFR family, an insulin receptor family, a platelet-derived growth factor receptor (PDGFR) family, a vascular endothelial growth factor receptor (VEGFR) family, a fibroblast growth factor receptor (FGFR) family, a colon cancer kinase (CRK), or a leukocyte growth factor receptor (LGF). kinase (CCK) family, nerve growth factor receptor (NGFR) family, hepatocyte growth factor receptor (HGFR) family, erythropoietin-producing human hepatocyte receptor (EphR) family, tyrosine protein kinase receptor (AXL) family, angiopoietin receptor (TIER) family, receptor tyrosine kinase-coupled receptor (RYKR) family, discoidin domain receptor (DDR) family, RET (rearranged during transfection) family, leukocyte receptor tyrosine kinase (LTK) family, ROR (receptor tyrosine kinase-like orphan receptors) family, muscle specific kinase (MuSK) family, CD80 (B7.1) CD83, the Fc receptor is an Fcγ receptor, an Fcα receptor, or an Fcε receptor, the cytokine receptor is a type 1 cytokine receptor, a type 2 cytokine receptor, a tumor growth factor receptor family, a chemokine receptor, or a TGF-β receptor family, the Toll-like receptor family is TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, or TLR13, the guanylate cyclase-linked receptor is natriuretic factor receptor 1 (NPR1), NPR2, or NPR3, and the receptor serine / threonine kinase is a TGF-β superfamily receptor, a bone morphogenetic protein (BMP) receptor, or an activin receptor-like kinase (ALK).

[0038] In the fusion protein, the growth factor may be adrenomedullin, angiopoietin, bone morphogenetic protein, ciliary neurotrophic factor, leukemia inhibitory factor, epidermal growth factor (EGF), ephrin, erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF) family, growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, insulin-like growth factor (IGF), keratinocyte growth factor (KGF), migration-stimulating factor (MSF), or the like. These include macrophage-stimulating factors (MSFs), macrophage-stimulating factors, neuregulins, neurotrophins, placental growth factors (PGFs), platelet-derived growth factors (PDGFs), T-cell growth factors (TCGFs), thrombopoietin (TPO), the transforming growth factor (TGF) family, and vascular endothelial growth factors (VEGFs).

[0039] In the fusion protein, the peptide hormone may be human growth hormone (hGF), GLP-1 analog, GLP-2 analog, insulin, adrenocorticotropic hormone, amylin, angiotensin, neuropeptide Y, enkephalin, neurotensin, atrial natriuretic peptide, calcitonin, cholecystokinin, gastrin, ghrelin, glucagon, follicle-stimulating hormone, leptin, melanocyte-stimulating hormone (MSH), oxytocin, parathyroid hormone, or the like. These are thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), vasopressin, or vasoactive intestinal peptide.

[0040] In the fusion protein, the antibody mimetic is an scFv, V NAR , V H H, affibody, affilin, affimer, affitin, alphabody, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, repebody, VLR, or nanoCLAMP.

[0041] In the fusion protein, the IL-10 protein is an IL-10 mutant protein in which the 87th amino acid, isoleucine, is replaced with alanine, and a linker peptide having a length of 6 to 10 aa is inserted between the 134th amino acid, asparagine, and the 135th amino acid, lysine, and is a monomeric IL-10 mutant protein having the amino acid sequence set forth in SEQ ID NO: 54 or 56.

[0042] The term "fusion protein" as used herein refers to a recombinant protein in which two or more proteins or domains responsible for specific functions within a protein are linked together so that each protein or domain retains its original function. A linker peptide with a flexible structure is usually inserted between the two or more proteins or domains. The linker peptides are GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 14), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 15), GGGGSGGGGSGGGGSEPKSCDKTHTCPPCP (SEQ ID NO: 16), GSGGGSGTLVTVSSESKYGPPCPPCP (SEQ ID NO: 17), GGGGSGGGGSGGGGSEPKSSDKTHTCPPCP (SEQ ID NO: 18), EPKSSDKTHTCPPCP (SEQ ID NO: 19), EPKSCDKTHTCPPCP (SEQ ID NO: 20), GGGGSGGGGSGGGGSAKNTTAPATTRNTTRGGEEKKKEKEKEEQEERTHTCPPCP (SEQ ID NO: 21), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 22), (G4S) n (n is an integer from 1 to 10, unit sequence number 23), (GSSGGS) n(Unit: SEQ ID NO: 24, n is an integer from 1 to 10), SGGGSGGGGSGGGGSGGEEQEEGGS (SEQ ID NO: 25), AAGSGGGGGSGGGGSGGGGS (SEQ ID NO: 26), KESGSVSSEQLAQFRSLD (SEQ ID NO: 27), EGKSSGSGSESKST (SEQ ID NO: 28), GSAGSAAGSGEF (SEQ ID NO: 29), (EAAAK) n (Unit: SEQ ID NO: 30, n is an integer from 1 to 10), CRRRRREAEAC (SEQ ID NO: 31), GGGGGGGG (SEQ ID NO: 32), GGGGGG (SEQ ID NO: 33), PAPAP (SEQ ID NO: 34), (Ala-Pro) n (n is an integer between 1 and 10), VSQTSKLTRAETVFPDV (SEQ ID NO: 35), PLGLWA (SEQ ID NO: 36), TRHRQPRGWE (SEQ ID NO: 37), AGNRVRRSVG (SEQ ID NO: 38), RRRRRRRR (SEQ ID NO: 39), GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 40), GGGGSGGGSGGGGS (SEQ ID NO: 41), AEAAAKEAAAAKA (SEQ ID NO: 42), GFLG (SEQ ID NO: 43), AAKATTAPATATRNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 44), GGSGG (SEQ ID NO: 45), GGSGGSGGS (SEQ ID NO: 46), GGGSGG (SEQ ID NO: 47), and GSTSGSGKPGSGEGS (SEQ ID NO: 48).

[0043] As used herein, the term "antibody" refers to an immunoglobulin molecule, a double tetrameric protein formed by the association of two identical heavy chains and two identical light chains, the variable region (V L ) and the variable region of the heavy chain (V H Antibodies bind specifically to antigens through antigen-binding sites composed of IgA, IgB, IgD, IgE, IgM, IgY, etc., depending on their origin.

[0044] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment derived from an antibody that has antigen-binding ability, and includes both fragments produced by cleaving an antibody with a protease as well as single-chain fragments produced by recombinant methods, including Fab, F(ab')2, scFv, diabody, triabody, sdAb, and V. H Contains H.

[0045] The term "Fab" as used in this document refers to an antigen-binding antibody fragment, a fragment produced by cleaving an antibody molecule with the protease papain. H -CH1 and V L -C L The other fragment, which is a dimer of these two peptides and is produced by papain, is called Fc (fragment crystallizable).

[0046] As used herein, the term "F(ab')2" refers to a tetramer of two Fab fragments linked by disulfide bonds that contain an antigen-binding site and are produced by cleaving an antibody with the protease pepsin. The other fragment produced by pepsin is called pFc'.

[0047] The term "Fab" as used herein refers to a molecule structurally similar to Fab, which is generated by separating the F(ab')2 under mildly reducing conditions.

[0048] The term "scFv" used in this document is an abbreviation for "single chain variable fragment" and is not a fragment of an actual antibody, but rather a fragment of the heavy chain variable region (V H ) and the light chain variable region (V L) with a linker peptide of approximately 25 a.a. in size, and is known to have antigen-binding ability despite not being an inherent antibody fragment (Glockshuber et al., Biochem. 29(6):1362-1367, 1990).

[0049] As used herein, the terms "diabody" and "triabody" refer to antibody fragments in which two and three scFvs are linked by linkers, respectively.

[0050] The term "single domain antibody (sdAb)" used in this document refers to an antibody fragment consisting of a single variable region fragment of an antibody, also known as a nanobody. While sdAbs derived from heavy chains are primarily used, single variable region fragments derived from light chains have also been reported to specifically bind to antigens. Unlike conventional antibodies consisting of heavy and light chains, V consisting of the variable region fragment of a shark antibody consists of only a single chain dimer. NAR , and V consisting of variable region fragments of camelid antibodies H H is also included in sdAb.

[0051] The term "antibody mimetic" as used in this document refers to a concept that includes proteins that have similar functions to antibodies, i.e., antigen-binding ability, but are manufactured from non-antibody protein frameworks such as monobodies and variable lymphocyte receptors (VLRs), unlike conventional full-length antibodies that function by forming a quaternary structure of two heavy chains and two light chains. Such antibody mimetics include Affibodies derived from the Z domain of protein A (Nygren, PA, FEBS J. 275(11):2668-2676, 2008), Affilins derived from Gamma-B crystallin or Ubiquitin (Ebersbach et al., J. Mol. Biol. 372(1):172-185, 2007), Affimers derived from Cystatin (Johnson et al., Anal. Chem. 84(15):6553-6560, 2012), Affitins derived from Sac7d (Krehenbrink et al., J. Mol. Biol. 383 (5):1058-1068, 2008), and Alphabodies derived from triple helix-coiled coil proteins (Desmet et al., J. Mol. Biol. 383(5):1058-1068, 2008). al., Nat. Commun. 5:5237, 2014), Anticalin derived from lipocalin (Skerra et al., FEBS J. 275(11):2677-2683, 2008), Avimers derived from domains of various membrane receptors (Silverman et al., Nat. Biotechnol. 23(12):1556-1561, 2005), DARPins derived from the Ankyrin repeat motif (Stumpp et al., Drug Discov. Today. 13(15-16):695-701, 2008), Fynomers derived from the SH3 domain of the Fyn protein (Grabulovski et al., J. Biol. Chem. 282(5):3196-3204, 2007), and Kunitz domain peptides derived from the Kunitz domains of various protein inhibitors (Nixon and Wood, Curr. Opin. Drug. Discov.Dev.9(2):261-268, 2006), a monobody derived from the tenth type 3 domain of fibronectin (Koide and Koide, Methods Mol. Biol. 352:95-109, 2007), nanoCLAMP derived from carbohydrate-binding module 32-2 (Suderman et al., Protein Exp. Purif. 134:114-124, 2017), a variable lymphocyte receptor (VLR) derived from hagfish (Boehm et al., Ann. Rev. Immunol. 30:203-220, 2012), and a repebody engineered to improve antigen affinity based on the VLR (Lee et al., Proc. Natl. Acad. Sci. USA, 109:3299-3304, 2012).

[0052] In accordance with another aspect of the present invention, there is provided a polynucleotide encoding the immunoglobulin Fc domain variant protein or the fusion protein.

[0053] The polynucleotide may be any polynucleotide that encodes a protein containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 9, and is codon-optimized for the type of host cell used to produce the protein. Such codon optimization techniques using host cells are well known in the art (Fuglsang et al., Protein Expr. Purif. F31(2):247-249, 2003). More preferably, the polynucleotide contains a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10 to 13.

[0054] In yet another aspect, the present invention provides a recombinant vector comprising the polynucleotide.

[0055] In the recombinant vector, the polynucleotide is contained in the form of a gene construct operably linked to a regulatory sequence.

[0056] As used herein, the term "operably linked to" means that a target sequence (e.g., in an in vitro transcription / translation system or in a host cell) is linked to said regulatory sequence in a manner that allows its expression.

[0057] The term "regulatory sequence" is intended to include promoters, enhancers, and other regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a target gene in many host cells, those that direct expression of a target gene only in specific tissues (e.g., tissue-specific regulatory sequences), and those that direct expression in response to a specific signal (e.g., inducible regulatory sequences). Those skilled in the art will appreciate that the design of an expression vector can vary depending on factors such as the choice of host cell to be transformed and the desired expression level of the protein. The expression vector of the present invention is introduced into a host cell to express the fusion protein. Regulatory sequences enabling expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As described above, these typically include a regulatory sequence responsible for transcription initiation and, optionally, a poly-A signal responsible for transcription termination and stabilization of the transcript. In addition to transcriptional regulatory elements, additional regulatory sequences include translational enhancers and / or naturally occurring or heterologous promoter regions. For example, possible regulatory sequences enabling expression in mammalian host cells include the CMV-HSV thymidine kinase promoter, SV40, RSV promoter (Rous sarcoma virus), human kidney element 1α promoter, glucocorticoid-inducible MMTV promoter (Moloney murine tumor virus), metallothionein-inducible or tetracycline-inducible promoter, or amplifiers such as the CMV amplifier or SV-40 amplifier. For expression in neuronal cells, it is contemplated that the neurofilament promoter, PGDF promoter, NSE promoter, PrP promoter, or thy-1 promoter may be used. Such promoters are known in the art and are described in the literature (Charron J. Biol. Chem. 270:25739-25745, 1995). For expression in prokaryotic cells, numerous promoters have been disclosed, including the lac promoter, tac promoter, or trp promoter.In addition to factors that initiate transcription, the regulatory sequence may also include a transcription termination signal, such as an SV40 poly-A site or a TK poly-A site, downstream of the polynucleotide according to one embodiment of the present invention. In this document, suitable expression vectors are known in the art, including, for example, the Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pRc / CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1 (GIBCO BRL), pGX-27 (Japanese Patent No. 1442254), pX (Pagano et al., Science 255:1144-1147, 1992), yeast two-hybrid vectors such as pEG202 and dpJG4-5 (Gyuris et al., Cell 75:791-803, 1995), or prokaryotic expression vectors such as λgt11 or pGEX (Amersham Pharmacia). In addition to the nucleic acid molecule of the present invention, the vector may further comprise a polynucleotide encoding a secretion signal. Such secretion signals are well known to those skilled in the art. Depending on the expression system used, a leader sequence that directs the fusion protein according to one embodiment of the present invention to a cellular compartment is combined with the coding sequence of the polynucleotide according to one embodiment of the present invention, preferably a leader sequence that directs the encoded protein or the protein to the periplasm or extracellular medium.

[0058] The vectors of the present invention can be constructed using standard recombinant DNA techniques, including, for example, blunt-end and sticky-end ligation, restriction enzyme treatment to provide suitable termini, removal of phosphate groups by alkaline phosphatase treatment to prevent incompatible ligations, and enzymatic ligation using T4 DNA ligase. The vectors of the present invention can be constructed by recombining DNA encoding a signal peptide obtained by chemical synthesis or genetic recombination technology, DNA encoding an immunoglobulin Fc domain variant protein according to one embodiment of the present invention, or a fusion protein containing the same, into a vector containing appropriate regulatory sequences. Vectors containing the regulatory sequences can be commercially purchased or manufactured. In one embodiment of the present invention, the pBispecific backbone vector (Genexine, Inc., Korea), pAD15 vector, pGP30 (Genexine, Inc., Korea), or pN293F vector (Y-Biologics, Inc., Korea) is used as the backbone vector.

[0059] The expression vector further comprises a polynucleotide encoding a secretory signal sequence, which induces extracellular secretion of the recombinant protein expressed in the cell, and is a tissue plasminogen activator (tPA) signal sequence, a herpes simplex virus (HSV) gDs (glycoprotein D) signal sequence, or a growth hormone signal sequence.

[0060] According to one embodiment of the present invention, the expression vector is an expression vector that allows the protein to be expressed in a host cell, and the expression vector may take any form, such as a plasmid vector, a virus vector, a cosmid vector, a phagemid vector, or a human artificial chromosome.

[0061] In yet another aspect, the present invention provides a homodimer or heterodimer comprising the fusion protein.

[0062] In the case of the homodimer, as shown in FIG. 5, when different biologically active proteins 11a and 11b are linked to the N-terminus and C-terminus, respectively, of the immunoglobulin Fc domain variant protein 12, the resulting homodimer 100 exhibits bispecificity. However, as shown in FIG. 6, when only one monospecific fusion protein containing biologically active protein 11 is dimerized, the resulting homodimer 200 still exhibits monospecificity (FIG. 6). In the case of heterodimers, as shown in FIG. 7, when two different monospecific fusion proteins containing biologically active proteins 11a and 11b, respectively, are dimerized, the resulting heterodimer 300 exhibits bispecificity. As shown in FIG. 8, when two different bispecific fusion proteins containing two different biologically active proteins 11a, 11b, 11c, and 11d, respectively, are dimerized, the resulting heterodimer 400 exhibits tetraspecificity.

[0063] Optionally, the homodimers or heterodimers 500, 600, 700, and 800 according to one embodiment of the present invention contain one or more linker peptides 14 between the biologically active proteins 11, 11a, 11b, 11c, and 11d and the hinge region 13, or between the immunoglobulin Fc domain variant protein 12 and the biologically active proteins 11, 11a, 11b, 11c, and 11d. Such fusion partners are linked to the N-terminus or C-terminus of the immunoglobulin Fc domain variant protein by the linker peptides 14 (FIGS. 9 to 12).

[0064] In particular, the biologically active protein 11a linked to the N-terminus of the fusion protein is a ligand that acts as a targeting moiety, and another biologically active protein 11b linked to the C-terminus of the same fusion protein is a functional protein possessing the desired biological activity (Figures 5 and 8). Conversely, the functional protein may be linked to the N-terminus and the ligand to the C-terminus. These bispecific fusion proteins form homodimers via intermolecular disulfide bonds formed by cysteine ​​residues in the hinge region 13, and selective hydrophobic interactions between the immunoglobulin Fc domain variant proteins aid in dimerization. Two different bispecific fusion proteins form heterodimers. In this manner, a homodimeric or heterodimeric protein according to one embodiment of the present invention may contain one to four biologically active proteins, and linker peptides may be added to the biologically active proteins at each end to allow for the addition of more proteins.

[0065] When preparing heterodimers according to one embodiment of the present invention, a knobs-into-holes (KIH) technique, which is well known in the art, is used to minimize the formation of homodimers.

[0066] The term "Knobs-into-Holes (KIH)" as used herein refers to a design strategy in antibody engineering used for heavy chain heterodimerization in the production of bispecific IgG antibodies. When two fusion proteins that form heterodimers are classified into a first fusion protein and a second fusion protein, the KIH technique involves substituting the 10th amino acid, serine, with cysteine ​​(C) and the 22nd amino acid, threonine (T) with tryptophan (W) in the CH3 domain of the modified IgG Fc region of the first fusion protein (Knobs structure). Furthermore, the 5th amino acid, tyrosine (Y), with cysteine ​​(C), the 22nd amino acid, threonine with serine (S), the 24th amino acid, leucine (L) with alanine (A), and the 63rd amino acid, threonine with tryptophan (W) in the CH3 domain of the Fc region of the second fusion protein (Knobs structure). In the first fusion protein, the fifth amino acid, tyrosine (Y), is substituted with valine (V) (hole structure), and conversely, in the first fusion protein, the fifth amino acid, tyrosine (Y), in the CH3 domain of the Fc region is substituted with cysteine ​​(C), the 22nd amino acid, threonine (T), is substituted with serine (S), the 24th amino acid, leucine (L), is substituted with alanine (A), and the 63rd amino acid, tyrosine (Y), is substituted with valine (V) (hole structure). In the second fusion protein, the tenth amino acid, serine, in the CH3 domain of the modified IgG4 Fc region is substituted with cysteine ​​(C), and the 22nd amino acid, threonine (T), is substituted with tryptophan (W) (knobs structure).

[0067] In this case, the position of the mutated amino acid is determined based on the reference sequence (the amino acid sequence of the human IgG1 CH3 domain of SEQ ID NO: 77). Even if additional mutations such as addition, deletion, or substitution of amino acids occur at sites in the CH3 domain that are not related to the knob-into-holes structure, the amino acid corresponding to the mutated position based on the reference sequence can be used. Alternatively, the knob-into-holes structure may be introduced by other amino acid mutations well known in the art. Such mutations have been well described in previous literature (Wei et al., Oncotarget 8(31):51037-51049, 2017; Ridgway et al., Protein Eng. 7):617-621, 1996; Carter, P., J. Immunol. Methods 48(1-2):7-15, 2001; Merchant et al., Nat. Biotechnol. 16(7):677-681, 1998). For example, a bispecific dimeric fusion protein can be generated by combining a Knobs structure in which the 22nd amino acid, threonine, in the CH3 domain of the first fusion protein is replaced with tyrosine, and a Hole structure in which the 63rd amino acid, tyrosine, in the CH3 domain of the second fusion protein is replaced with threonine. Conversely, the Knobs-into-Holes structure may be formed by introducing a Hole structure into the first fusion protein and a Knobs structure into the second fusion protein.

[0068] In yet another aspect, the present invention provides a composition comprising the fusion protein or a polynucleotide encoding the same, or the homo- or heterodimer as an active ingredient.

[0069] According to yet another aspect of the present invention, there is provided a method for extending the in vivo half-life of a biologically active protein, comprising the step of administering to an individual a fusion protein in which a biologically active protein is fused to the immunoglobulin Fc domain variant protein, or a homodimer or heterodimer containing the fusion protein.

[0070] In yet another aspect, the present invention provides a composition comprising, as an active ingredient, a fusion protein in which a biologically active protein is fused to the immunoglobulin Fc domain variant protein, or a homodimer or heterodimer containing the fusion protein.

[0071] The composition contains a pharmaceutically acceptable carrier, and may further contain pharmaceutically acceptable adjuvants, excipients, or diluents in addition to the carrier.

[0072] The term "pharmaceutically acceptable" as used herein refers to a composition that is physiologically tolerable and does not typically cause gastrointestinal upset, dizziness, or other allergic or similar reactions when administered to humans. Examples of carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may also contain fillers, anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, and preservatives.

[0073] The pharmaceutical composition according to one embodiment of the present invention may be formulated using methods known in the art to enable rapid, sustained, or delayed release of the active ingredient when administered to a mammal, including powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, and sterile powders.

[0074] The composition according to one embodiment of the present invention may be administered by various routes, including oral, parenteral, e.g., suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, and intraspinal administration, and may be administered using an implantable device for sustained, continuous, or repeated release. The number of doses may be once a day or several times a day, as desired, but may also be once a week, twice a week, or once a month, and the administration period is not particularly limited.

[0075] The composition according to one embodiment of the present invention may be formulated in a suitable form with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, parenteral carriers such as water, suitable oils, saline, aqueous glucose, and glycols, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl or propyl paraben, and chlorobutanol. Furthermore, the composition according to the present invention may appropriately contain suspending agents, solubilizers, stabilizers, isotonicity agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, antioxidants, and the like, as needed depending on the administration method and dosage form. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, latest edition.

[0076] The dosage of the composition administered to a patient may vary depending on many factors, including the patient's height, body surface area, age, the particular compound administered, sex, time and route of administration, general health, and other drugs administered concomitantly. The pharmaceutically active protein is administered in an amount of 100 ng / kg body weight to 10 mg / kg body weight, more preferably 1 to 500 μg / kg body weight, and most preferably 5 to 50 μg / kg body weight, although dosages may be adjusted to take these factors into consideration.

[0077] According to one aspect of the present invention, there is provided a method for treating a disease in an individual, comprising the step of administering to the individual a therapeutically effective amount of a fusion protein in which a biologically active protein is fused to the immunoglobulin Fc domain variant protein, or a homo- or hetero-dimer containing the same.

[0078] The term "therapeutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level is determined by factors including the type and severity of the subject, age, sex, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The therapeutically effective amount of the composition of the present invention is 0.1 mg / kg to 1 g / kg, more preferably 1 mg / kg to 500 mg / kg, but the effective dose is appropriately adjusted depending on the age, sex, and condition of the patient.

[0079] The disease is one that requires administration of the biologically active protein. For example, if the biologically active protein is a GLP-1 analog, the disease to be treated is diabetes or metabolic syndrome; if the biologically active protein is a GLP-2 analog, the disease to be treated is short bowel syndrome or inflammatory bowel disease; if the biologically active protein is IL-2, the disease is malignant tumor; if the biologically active protein is FceRIa, the disease is IgE-mediated allergic disease such as asthma or atopic dermatitis; or if the biologically active protein is an anti-CD40L antibody analog, the disease is autoimmune disease or organ transplant rejection. [Example]

[0080] The present invention will be described in more detail below through examples and experimental examples. However, the present invention is not limited to the examples and experimental examples disclosed below, and may be embodied in various different forms. The following examples and experimental examples are provided to fully disclose the present invention and to fully convey the scope of the invention to those skilled in the art.

[0081] Example 1: Design of immunoglobulin Fc domain variant proteins Linking the Fc domain of an antibody to a biologically active protein or active pharmaceutical ingredient (API) to increase the half-life of the API is a widely used technique. However, the Fc domain of an antibody is formed by the addition of an N-linked glycan to the asparagine at amino acid position 297. Depending on the characteristics of the host cells used to produce the antibody, the glycan pattern may differ from that of human antibodies, or the glycan pattern may be heterogeneous. This can result in inconsistent activity of the fusion protein linked to the API, or it may exhibit unwanted effects such as ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity), potentially inducing side effects. Furthermore, the reason for the increased half-life of antibodies after administration to the body is known to be that after antibodies are absorbed by vascular endothelial cells in the body, they bind to neonatal Fc receptors (hereinafter referred to as "FcRn") at low pH in the endosome, thereby avoiding degradation in the lysosome, and then are separated from FcRn under extracellular physiological pH conditions during the process of recycling of endosomes to the plasma membrane (Figure 13). Such antibody recycling by FcRn is known to have the effect of extending the half-life of antibodies to approximately 21 days (Raghavan, M. et al., Biochem, 34(45):14649-14657, 1995).

[0082] However, conventional antibody Fc domains have different affinities for FcRn depending on the type of IgG, which limits the improvement in the half-life of APIs.

[0083] Therefore, the present inventors designed an Fc domain protein mutant that has an increased affinity for FcRn compared to conventionally used Fc domain proteins, but lacks ADCC and CDC activity.

[0084] To this end, the present inventors designed mutants (SEQ ID NOS: 6 to 9) in which the 18th amino acid, threonine (T), of a modified IgG4 Fc protein consisting of the amino acid sequences set forth in SEQ ID NOS: 1 to 4, was substituted with glutamine (Q) and the 196th amino acid, methionine (M), was substituted with leucine (L), and designated these mutants as "NTIG (SEQ ID NOS: 5)."

[0085] Example 2: Preparation of monospecific fusion protein constructs Next, the present inventors prepared various fusion proteins in which various biologically active proteins were linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein (NTIG) prepared in Example 1.

[0086] 2-1: Design and production of GLP-1E-NTIG The present inventors designed a fusion protein (GLP-1E-NTIG, SEQ ID NO: 50) by linking the NTIG protein (SEQ ID NO: 7) prepared in Example 1 to the C-terminus of a GLP-1 / Exendin-4 hybrid peptide (SEQ ID NO: 49), a hybrid peptide of GLP-1 and Exendin-4, via a linker peptide containing a hybrid hinge region ((G4S)3-IgD / IgG1 hybrid hinge, SEQ ID NO: 14). The fusion protein was named "PG-11." The GLP-1E-NTIG was designed to include a tPA signal sequence (SEQ ID NO: 51) for secretion in host cells. A polynucleotide encoding PG-11 was synthesized using oligonucleotide synthesis, PCR amplification, and site-directed mutagenesis techniques and then inserted into the pGP30 expression vector (Genexine, Inc., Korea). The resulting vector was transiently expressed using Thermo Fisher's ExpiCHO kit. Specifically, the vector prepared as described above and the ExpiFectamine reagent included in the kit were mixed with ExpiCHO-S cells, and the cells were cultured in an incubator equipped with 8% CO2 and 37°C for 1 day, and then the temperature was lowered to 32°C and the cells were cultured for 7 days.

[0087] Next, Protein A capture purification was performed, and the candidate substance was confirmed to be purified by SDS-PAGE analysis under non-reducing and reducing conditions. The candidate substance was then formulated in a formulation buffer that matched its pI value. The formulated substance was quantified using Nanodrop, and the final purity was confirmed using SEC-HPLC (Figures 14a and 14b).

[0088] 2-2: Design and production of GLP-2-2G-NTIG The present inventors designed a fusion protein (GLP-2-2G-NTIG, SEQ ID NO: 53) by linking NTIG (SEQ ID NO: 7) to the C-terminus of a GLP-2 analog (GLP-2-2G, SEQ ID NO: 52), a variant in which the second amino acid of the human GLP-2 peptide, alanine (A), is replaced with glycine (G), via the linker peptide (SEQ ID NO: 14). The fusion protein was named "PG-22." The PG-22 was also engineered to contain a tPA signal sequence (SEQ ID NO: 51) for secretion. As in Example 2-1, a polynucleotide encoding PG-22 was prepared and inserted into the pGP30 expression vector (Genexine, Inc., Korea). The polynucleotide was then produced using the ExpiCHO system, and the final purity was confirmed (Figures 15a and 15b).

[0089] 2-3: Design and production of NTIG-IL-10V The present inventors designed a fusion protein (NTIG-IL-10V, SEQ ID NO: 55) by linking an IL-10 variant (SEQ ID NO: 54) to the C-terminus of NTIG via a linker peptide (SEQ ID NO: 26), and named it "PG-088." As in Example 2-1, a polynucleotide encoding PG-088 was prepared and inserted into a pBispec expression vector (Genexine, Inc., Korea). The polynucleotide was then produced using the ExpiCHO system, and the final purity was confirmed (Figures 16a and 16b).

[0090] 2-4: Design and production of NTIG-IL-10V monomer The present inventors designed a fusion protein (NTIG-IL-10Vm, SEQ ID NO: 57) by linking a monomeric IL-10 variant (SEQ ID NO: 56) to the C-terminus of NTIG via a linker peptide (SEQ ID NO: 26), and named it "PG-088m." As in Example 2-1, a polynucleotide encoding PG-088m was prepared and inserted into a pBispec expression vector. The fusion protein was then produced using the ExpiCHO system, and the final purity was confirmed (Figures 17a and 17b). The monomeric IL-10 variant (IL-10Vm) incorporates mutations to selectively suppress the immunostimulatory activity of the dual-activity IL-10 protein, immunostimulatory and immunosuppressive. To address the problem of self-dimerization leading to aggregate formation during protein production, a linker of GGSGGSGGS (SEQ ID NO: 46) was inserted into the middle of the protein, allowing it to be produced in a monomeric form.

[0091] Example 3: Production of bispecific fusion protein gene constructs 3-1: Production of anti-PD-L1-NTIG-IL-2 Polynucleotides encoding a fusion protein in which a PD-L1-targeting scFv was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-2 was linked to the C-terminus (anti-PD-L1-NTIG-IL-2) and a fusion protein in which the modified IgG4 Fc domain protein of SEQ ID NO: 2 was used in place of NTIG (anti-PD-L1-modified IgG4 Fc-IL-2) were inserted into the pBispec expression vector, and the proteins were produced using the ExpiCHO system.

[0092] 3-2: Design and production of FcεRIα-NTIG-IL-10V The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-10V, SEQ ID NO: 59) by linking the extracellular domain of FcεRIα (SEQ ID NO: 58), an IgE-specific receptor, to the N-terminus of the NTIG-IL-10V designed in Examples 2-3 via a linker peptide (SEQ ID NO: 18). This protein was designated "PG-075." A polynucleotide encoding this fusion protein was prepared and inserted into the pBispec expression vector. The PG-075 construct was also designed to have a tPA signal sequence (SEQ ID NO: 51) at the N-terminus for secretion. The PG-075 construct was produced using the ExpiCHO system and analyzed by SDS-PAGE and Western blotting (Figures 18a and 18b).

[0093] 3-3: Design and production of FcεRIα-NTIG-IL-10Vm The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-10Vm, SEQ ID NO: 60) by linking the extracellular domain of FcεRIα (SEQ ID NO: 58), an IgE-specific receptor, to the N-terminus of NTIG-IL-10Vm (designated in Examples 2-4) via a linker peptide (SEQ ID NO: 18). This protein was designated "PG-110." A polynucleotide encoding this fusion protein was prepared and inserted into the pAD15 expression vector. The PG-110 construct was also designed to contain a tPA signal sequence (SEQ ID NO: 51) for secretion at its N-terminus. The gene inserted into the pAD expression vector was transfected into CHO DG44 cells, followed by methotrexate (MTX) amplification. High-efficiency expressing cell lines were then generated through single clone selection, and PG-110 protein was then produced and purified from the high-efficiency expressing cell lines (Figures 19a and 19b).

[0094] Example 4: Production of anti-CD40L hybrid antibodies 4-1: Preparation of chimeric anti-CD40L antibodies The present inventors have produced an anti-CD40L antibody (C10) in which the Fc region of the mouse-derived anti-CD40L (anti-CD154) antibody described in WO2016 / 182335A1 was replaced with the human IgG1 Fc region, and have introduced FcγRIIα-binding inhibitory mutants into the heavy chain CH2 and CH3 domains of this antibody, which they named "C10M."

[0095] 4-2: Preparation of chimeric anti-CD40L-NTIG hybrid antibody The present inventors designed a hybrid heavy chain protein in which the heavy chain portion of the Fab fragment of the C10M antibody was linked to the N-terminus of the NTIG protein produced in Example 1, which does not bind to FcγR, and named it "PG-129."

[0096] The hybrid heavy chain protein comprises the heavy chain portion (V) of the Fab of the C10M antibody. H A polynucleotide encoding a hybrid heavy chain was prepared by linking the Fab portion of the C10M antibody (V-CH1, SEQ ID NO: 61) with NTIG (SEQ ID NO: 7), and inserted into the pAD15 expression vector (WO 2015 / 009052A). L -C L A polynucleotide encoding the nucleotide sequence ...

[0097] The expression vector thus prepared was also transiently expressed using the ExpiCHO kit from Thermo Fisher Scientific. Specifically, the vector construct thus prepared and the ExpiFectamine reagent included in the kit were mixed in ExpiCHO-S cells and cultured in an incubator at 8% CO2 and 37°C for 1 day, after which the temperature was lowered to 32°C and cultured for 7 days.

[0098] Next, Protein A capture purification was performed, and the candidate substance was confirmed to be purified by PAGE analysis and Western blotting analysis under non-reducing and reducing conditions, and then formulated into a formulation buffer according to the pI value of the candidate substance. The formulated substance was quantified using Nanodrop, and the final purity was confirmed using SEC-HPLC.

[0099] As shown in Figures 20a and 20b, the light and heavy chains were not only expressed normally but also formed normal heterotetramers as single bands under non-reducing conditions. SEC-HPLC analysis also showed a very high purity of 97.9%.

[0100] 4-3: Preparation of humanized anti-CD40L-NTIG hybrid antibody Although the chimeric antibody sufficiently overcomes the shortcomings of conventional human antibodies by incorporating the NTIG technology (immunoglobulin Fc domain mutant protein) of the present invention, the inventors determined that the antigen-binding site is still derived from a mouse antibody, which may induce unnecessary immune responses in the human body. Therefore, in order to produce a safer hybrid antibody, they designed a humanized antibody (anti-CD40L Fab-NTIG) in which the antigenic determinants of the Fab, the antigen-binding fragment of the antibody that forms the basis of the present invention, were removed and the remaining framework region was replaced with a human antibody sequence (Tables 1 and 2), and named this antibody "PG-400."

[0101] In addition, the present inventors designed a bispecific hybrid antibody (anti-CD40L Fab-NTIG-IL-10Vm) in which a monomeric mutant protein of IL-10 (SEQ ID NO: 56), a cytokine with immunosuppressive activity, was linked to the C-terminus of the heavy chain of the hybrid antibody (Table 3), and named it "PG-410." The structure of the light chain of the bispecific hybrid antibody is as shown in Table 2.

[0102] [Table 1]

[0103] [Table 2]

[0104] [Table 3]

[0105] To produce the humanized anti-CD40L hybrid antibodies (PG-400 and PG-410), polynucleotides encoding the heavy and light chains of the antibodies were prepared, and then HEK293F cells were transfected with plasmid DNA using the N293F vector system (Y-Biologics Inc.) for transient expression in animal cells. To prepare the nucleic acid, 25 μg of plasmid DNA was added to 3 ml of medium and mixed, followed by the addition of 25 μl of 2 mg / ml PEI (Polyethylenimine, PolyPlus, USA). The reaction mixture was left at room temperature for 15 minutes and then diluted with 1x10 6 The cells were placed in 40 ml of culture medium containing 100 cells / ml of lysate and cultured for 24 hours at 120 rpm, 37°C, and 8% CO2. 24 hours after DNA transfection, nutrient supplements (Soytone, BD, USA) were added to a final concentration of 10 g / L. After 7 days of culture, the cell culture medium was centrifuged at 500 rpm for 10 minutes, and the supernatant was collected.

[0106] Next, Protein A resin was packed into a column and washed with 1X DPBS. The collected supernatant was bound to the resin at 0.5 ml / min at 4°C, and the protein was eluted with 0.1 M glycine. To exchange the buffer, the eluted solution was placed in a dialysis tube (GeBAflex tube, Geba, Israel) and dialyzed against 1X DPBS at 4°C. The obtained material was subjected to gel filtration using Superdex 200 resin and then formulated in PBS (pH 7.4) buffer.

[0107] The yield and purity of the hybrid antibody purified above were assayed by SDS-PAGE analysis, size-exclusion FPLC, and size-exclusion HPLC analysis.

[0108] As a result, as shown in Figures 21a, 21b, 22a, and 22b, it was found that PG-400 and PG-410, to which the humanized anti-CD40L hybrid antibody of the present invention was applied, were produced with high purity.

[0109] 4-4: Design of anti-CD40L scFv-NTIG-IL-10 fusion protein The present inventors selected the variable region of the Fab of the humanized anti-CD40L antibody and used it to design an scFv. This was used as the V of the heavy chain construct of the anti-CD40L antibody used in Example 4-3. H A single-chain-based antibody-binding fragment fusion protein was designed by inserting the IL-10 monomeric variant (SEQ ID NO: 56) used in Example 4-3 into the C-terminus of the anti-CD40L scFv-NTIG using a linker peptide (SEQ ID NO: 26) to design a bispecific fusion protein (Table 4). In this example, the anti-CD40L scFv contained a light chain variable region (V L )-linker-heavy chain variable region (V H ), but conversely, V H -Linker-V L Although the CD40L binding ability is maintained even when the linker is configured in the above order, various types of linkers may also be used.

[0110] [Table 4]

[0111] Example 5: Preparation of heterodimers The present inventors designed bispecific fusion proteins containing both a GLP-1 analog and a GLP-2 analog, as shown in Table 5. These bispecific fusion proteins incorporate a knobs-into-holes (KIH) structure to form heterodimers via intermolecular disulfide bonds and hydrophobic interactions between the first and second fusion proteins.

[0112] The difference between Examples 5-1 and 5-2 below is whether or not the linker connecting the GLP-1 analog and NTIG in the first fusion protein contains an N-linked glycan attachment site for N-glycan attachment. This is because, in addition to GLP-1, Exendin 4 (Ex4), oxyntomodulin (OXM), and GLP-2 all bind to the GLP-1 receptor GLP-1R, whereas only GLP-2 binds to GLP-2R. GLP-1R is expressed in various organs, including the brain, heart, liver, muscle, and pancreas in addition to the gastrointestinal tract (GI tract), and therefore, in order to allow GLP-1 to act specifically in the gastrointestinal tract, it is necessary to somewhat reduce its affinity for GLP-1R. To this end, the present inventors used a GLP-1 analogue with a glycan attachment domain added to the linker portion (including the IgD / IgG1 hybrid hinge) of the analogue, so that glycans could be attached to the linker portion (Example 5-1) in order to somewhat reduce the binding strength of the analogue to GLP-1R.

[0113] [Table 5]

[0114] The vector construct prepared as described above was transiently expressed using the ExpiCHO kit from Thermo Fisher Scientific. Specifically, the vector construct prepared as described above and the ExpiFectamine reagent included in the kit were mixed into ExpiCHO-S cells and cultured in an incubator equipped with 8% CO2 and 37°C for 1 day, then the temperature was lowered to 32°C and cultured for 7 days.

[0115] The supernatant obtained from the incubation was purified through a Protein A column and a second column to prepare the heterodimeric proteins of Examples 5-1 and 5-2 (designated "MG12-4" and "MG12-5," respectively). The supernatant was then diluted appropriately with 4X LSD sample buffer and water for injection to a final concentration of 3-10 μg / 20 μL. For samples under reducing conditions, each substance to be analyzed was diluted appropriately with 4X LSD sample buffer, 10X reducing agent, and water for injection to a final concentration of 3-10 μg / 20 μL and heated in a 70°C heating block for 10 minutes. 30 μL of the prepared sample was added to each well of the gel installed in the electrophoresis equipment. For size markers, 3-5 μL per well was added. Electrophoresis was performed after setting the power supply to 120 V and 90 minutes. After electrophoresis, the gel was separated and stained using staining solution and gastric juice for de-staining, and the results were analyzed. As a result, as can be seen in FIG. 23, it was found that the heterodimer according to one embodiment of the present invention was produced normally.

[0116] Example 6: Analysis of the effect of hinge on fusion protein production The present inventors linked various APIs to the N-terminus and C-terminus of the NTIG protein of the present invention, and investigated how the protein production efficiency differs depending on the type of hinge when an API is linked to the N-terminus of the NTIG protein.

[0117] 6-1: Production of FcεRIα-NTIG-IL-13αR2 The present inventors designed a bispecific fusion protein (FcεRIα-NTIG-IL-13Rα2) consisting of NTIG (SEQ ID NO: 7) linked to FcεRIα (SEQ ID NO: 58) at its N-terminus and IL-13Rα2 (SEQ ID NO: 74) at its C-terminus, designated "PG-50." While producing PG-50, we investigated the protein productivity when the hinge region linking FcεRIα and NTIG was replaced with an IgD hinge (SEQ ID NO: 15) or a combination of a (G4S)3 linker and an IgG1 hinge (SEQ ID NO: 16). The fusion protein using the IgD hinge was designated "PG-50-1," and the fusion protein using the (G4S)3 linker / IgG1 hinge was designated "PG-50-2." In the case of the IgD hinge, one intermolecular disulfide bond is formed, and in the case of the (G4S)3 linker / IgG1 hinge, two intermolecular disulfide bonds are formed.

[0118] As a result, as shown in Figures 24a and 24b, the production yield of the target substance of the fusion protein (PG-50-2) containing the IgG1 hinge was found to be approximately 15% higher than that of PG-50-1 containing the IgD hinge.

[0119] 6-2: Preparation of GLP-2-2G-NTIG-IL-10wt The present inventors analyzed the effect of various hinge types on protein production yield using other APIs. Specifically, a construct was prepared in which GLP-2-2G (SEQ ID NO: 52) was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-10wt (SEQ ID NO: 75) was linked to the C-terminus of NTIG. GLP-2-2G and NTIG were linked using a (G4S)3 linker + IgD / IgG1 hybrid hinge (SEQ ID NO: 14), and IL-10wt was linked to the C-terminus of NTIG using a linker peptide consisting of the amino acid sequence set forth in SEQ ID NO: 26. This resulted in the design of a bispecific fusion protein (GLP-2-2G-NTIG-IL-10wt), which was designated "PG-073." The polynucleotide encoding the designed fusion protein was prepared and inserted into the pBispec expression vector. The fusion protein was expressed using the ExpiCHO system as described in Example 2-1, and the expression level of the culture supernatant was confirmed by SDS-PAGE. The IgD / IgG1 hybrid hinge forms two intermolecular disulfide bonds.

[0120] As shown in Figure 24c, the main band of PG-073 under non-reducing conditions was 110 kDa, indicating that it was a bispecific fusion protein in which both GLP-2-2G and IL-10 were linked. However, bands representing one or two cleaved GLP-2 fragments were observed below the main band. This indicated that the (G4S)3 linker + IgD / IgG1 hybrid hinge linker used between GLP-2 and NTIG was somewhat unstable. Furthermore, the amount of fusion protein produced per 100 ml of culture medium was only 0.5 mg (PG-073), indicating a low protein production and a low purity of approximately 66%.

[0121] 6-3 and 6-4: Preparation of GLP-2-2G (3 points)-NTIG-IL-10Vm Therefore, the present inventors modified the GLP-2-2G in the GLP-2-2G-NTIG-IL-10wt fusion protein to a mutant containing three mutations (SEQ ID NO: 76), replaced the hinge portion with another type of linker or hinge (SEQ ID NO: 17 or 18, respectively), and replaced IL-10wt with IL-10Vm prepared in Example 2-4 to design a fusion protein (GLP-2V(3 point)-NTIG-IL-10Vm), which was named "PG210-5 (Example 6-3)" and "PG210-6 (Example 6-4)," respectively.

[0122] The polynucleotide encoding the fusion protein was inserted into a pBispec expression vector, and the fusion protein was expressed using the ExpiCHO system according to the method used in Example 2-1. The expression level of the protein in the culture supernatant was then confirmed by SDS-PAGE.

[0123] As a result, as shown in Figures 24d and 24e, in the case of PG-210-5, 3.48 mg of protein was confirmed based on 200 ml of culture medium, and the purity was confirmed to be approximately 88.3%, while in the case of PG-210-6, 5 mg of protein was confirmed, and the purity was confirmed to be approximately 90.7%.

[0124] Experimental Example 1: Analysis of FcRn binding affinity The present inventors compared the binding affinity of the NTIG protein of the present invention to FcRn with that of IgG4 Fc and IgG1 antibodies.

[0125] Specifically, the ligand, recombinant FcRn (rhFcRn, R&D Systems, USA), was dissolved in acetate buffer at a concentration of 5 μg / ml and then immobilized on a biosensor chip (Series S CM5 sensor chip, GE Healthcare, USA) by amine coupling.

[0126] Next, to compare the FcRn-binding affinity of the NTIG of the present invention and IgG4 Fc, the inventors analyzed the FcRn-binding affinity of PG-11 prepared in Example 2-1 and dulaglutide (trade name Trulicity), a commercially available fusion protein in which GLP-1 is linked to IgG4 Fc, by surface plasmon resonance (SPR) analysis. A Biocore 8K (GE Healthcare, USA) was used as the analytical instrument, and all reagents used in the analysis were purchased from GE Healthcare. PG-11 and Trulicity were dissolved in running buffer (PBST, pH 6.0) and diluted in half increments starting from 1,000 nM, followed by multi-cycle kinetic analysis. The experiment was performed at 25°C and a flow rate of 30 μl / min, with the analyte association reaction running for 60 seconds, followed by the dissociation reaction running for 60 seconds. Based on the experimental results, optimal antibody concentrations were selected (Trulicity: 8,000 nM and PG-11: 2,000 nM) and multi-cycle kinetic analysis was performed to determine the equilibrium dissociation constant (K) using the Biacore Insight program (GE Healthcare, USA). D During regeneration, the buffer solution was replaced with PBS buffer at pH 7.4.

[0127] As a result, as confirmed in Table 6 and Figures 25 and 26, the steady state affinity K D Comparative analysis revealed that Trulicity's KD value was 2,090 nM, while PG-11's KD value was 199 nM, indicating that the affinity of PG-11 for FcRn according to one embodiment of the present invention is approximately 5.3 times higher than that of Trulicity. The binding affinity for FcRn is known to be related to the pharmacokinetics of the corresponding Fc fusion protein, and therefore, PG-11 is predicted to have a longer in vivo half-life than Trulicity (Mackness et al., MAbs. 11(7):1276-1288, 2019).

[0128] [Table 6]

[0129] Additionally, to compare the FcRn-binding affinity of the NTIG of the present invention with that of IgG1 Fc, the inventors analyzed the FcRn-binding affinity of PG-11 and rituximab, a commercially available recombinant chimeric anti-CD20 antibody of the IgG1 series, by surface plasmon resonance (SPR) analysis. Various concentrations (2,000 nM to 1.96 nM for PG-11, 4,000 nM to 3.9 nM for rituximab) were dissolved in running PBST buffer (pH 6.0) and then subjected to binding, dissociation, and regeneration under the same conditions as in the comparative analysis of PG-11 and Trulicity described above: binding for 60 seconds, dissociation for 60 seconds, and regeneration for 30 seconds. Regeneration was performed using PBS buffer at pH 7.4.

[0130] As a result, as confirmed in Table 7 and Figure 27, the steady state affinity K D Comparative analysis showed that rituximab D The KD value of PG-11 was measured to be 256 nM, which means that the affinity of PG-11 according to one embodiment of the present invention for FcRn is approximately 9.3 times higher than that of rituximab.

[0131] [Table 7]

[0132] As described above, NTIG according to one embodiment of the present invention has a higher affinity for FcRn than the Fc regions of conventional IgG1 or IgG4, and is expected to have a significantly improved half-life when administered in vivo.

[0133] Next, the present inventors compared the affinity of the modified IgG4 Fc domain protein of SEQ ID NO: 2, which is the basis for NTIG (SEQ ID NO: 7) according to one embodiment of the present invention, with FcRn.

[0134] To this end, we use a simple K D Instead of calculating the static affinity (K D ) was calculated (Piche-Nicholas et al., MAbs. 10(1):81-94, 2018). Specifically, the binding affinity of the anti-PD-L1-NTIG-IL-2 and anti-PD-L1-modified IgG4 Fc-IL-2 prepared in Example 3-1 above to rhFcRn was analyzed by SPR analysis under different pH conditions for binding and dissociation, as described above.

[0135] As a result, as can be seen in Table 8 and Figure 28, the affinity of anti-PD-L1-NTIG-IL-2, which uses NTIG, for FcRn tended to be higher than that of anti-PD-L1-modified IgG4 Fc-IL-2, which uses a modified IgG4 Fc. However, during the binding buffer exchange process, a large amount of protein aggregates were formed in the anti-PD-L1-modified IgG4 Fc-IL-2 protein sample, which reduced the reliability of the results. Therefore, the same experiment was performed again using the ligand as the test substance and FcRn as the sample.

[0136] These results suggest that the application of NTIG not only enhances the in vivo half-life of the produced protein by increasing its FcRn binding ability, but also has superior effects on the physical properties and stability of the protein, compared to the application of the conventionally known modified IgG4 Fc of SEQ ID NO: 2.

[0137] [Table 8]

[0138] The buffers used in the analysis were running buffer (1X PBST, pH 7.3), sample dilution buffer (1X PBST, pH 6.0), dissociation buffer (1X PBST, pH 7.3), and renaturation buffer (1X PBST, pH 8.0).

[0139] As shown in Table 9 and Figure 29, both proteins exhibited rapid binding and rapid dissociation. Both proteins exhibited rapid dissociation from FcRn under neutral conditions (pH 7.3). However, at pH 6.0, the affinity of the anti-PD-L1-NTIG-IL-2 protein was 5.06-fold higher than that of the anti-PD-L1-modified IgG4 Fc-IL-2 protein. In conclusion, the anti-PD-L1-NTIG-IL-2 protein showed a tendency to bind more rapidly to FcRn at lower concentrations than the anti-PD-L1-modified IgG4 Fc-IL-2 protein at pH 6.0. This strongly suggests that NTIG-containing fusion proteins may be more efficiently recycled by FcRn, given that Fc-containing proteins internalized into cells bind to FcRn at the low pH of endosomes.

[0140] [Table 9]

[0141] Experimental Example 2: Analysis of binding affinity to various Fcγ receptors The Fc region of IgG1 is known to have binding affinity to FcγRI and FcγRIIIa. These Fc receptors are involved in effector functions such as ADCC and CDC, and are useful in anti-cancer therapy when antibodies or Fc fusion proteins are used for anti-cancer therapy. However, they have the disadvantage of potentially inducing side effects when used with APIs intended to treat diseases other than anti-cancer therapy.

[0142] Therefore, the present inventors analyzed the affinity of an NTIG fusion protein according to one embodiment of the present invention for FcγRI and FcγRIIIa by comparing it with an IgG1 antibody using biolayer interferometry (BLI).

[0143] As a result, as shown in Figure 30, it was found that rituximab, an IgG1 series antibody, specifically binds to FcγRI and FcγRIIIa, whereas PG-11, a fusion protein containing NTIG according to one embodiment of the present invention, does not specifically bind to these antibody receptors.

[0144] Furthermore, the present inventors conducted a comparative analysis of the binding affinity of rituximab and PG-11 according to one embodiment of the present invention to FcγRIIa, FcγRIIb, and FcγRIIIb.

[0145] As a result, as can be seen in Figure 31, it was found that rituximab specifically binds to the three Fc receptors. On the other hand, PG-11 according to one embodiment of the present invention showed weak binding, but judging from the sensogram pattern, it is judged to be non-specific binding.

[0146] Experimental Example 3: Complement C1q binding analysis The antibody Fc region induces complement-dependent cytotoxicity (CDC) by binding to the complement C1q, which can pose a serious problem when Fc fusion proteins for general therapeutic purposes other than cancer treatment are clinically applied. Therefore, the affinity of the fusion protein containing NTIG according to one embodiment of the present invention for complement C1q was analyzed in comparison with that of rituximab, an IgG1 antibody.

[0147] Specifically, PG-11 and rituximab were diluted to 2 μg / ml each using coating buffer (0.1 M NaHPO, pH 9.6). 100 μl of the diluted samples were then added to each well and incubated overnight at 4°C. The plate was then washed three times with 300 μl of wash buffer. 200 μl of blocking buffer (2% BSA / PBS) was added to each well and incubated for 2 hours at room temperature. The plate was then washed three times with 300 μl of wash buffer. Anti-C1q antibody was diluted 1 / 3 in assay buffer to 10, 3, 1, 0.3, and 0.1 μg / ml, and 50 μl of each was added to each well (duplication). The plate was then washed eight times with 300 μl of wash buffer. C1q-HRP conjugate was diluted 400-fold and added in 50 μl portions to each well. The plate was washed eight times with 300 μl of wash buffer, and then 50 μl of TMB substrate was added and incubated at room temperature. Upon the development of a blue color, 50 μl of stop solution was added to each well to terminate the reaction. Optical density was measured at 450 nm using a microplate reader.

[0148] As a result of the analysis, as can be seen in FIG. 32, it was found that rituximab binds to C1q in a concentration-dependent manner, whereas PG-11 according to an embodiment of the present invention does not bind to C1q.

[0149] Therefore, NTIG according to one embodiment of the present invention was found to be a safe protein carrier that can improve the half-life of API while minimizing side effects caused by the Fc region, such as ADCC and CDC.

[0150] Experimental Example 4: GLP-2 activity analysis In the above examples, cAMP Hunter was used to determine whether or not GLP-2 activity was fully exerted for a number of fusion proteins containing GLP-2. (商標) eXpress GPCR analysis (Table 10).

[0151] [Table 10]

[0152] As shown in Figure 33 and Table 11, the GLP-2 activity of PG-210-6, one of the fusion proteins prepared according to an embodiment of the present invention, was similar to that of PG-22 (Comparative Example), while PG-210-5, another embodiment of the present invention, exhibited approximately half the activity of PG-210-6. The heterodimers MG12-4 and MG12-5 also exhibited approximately half the GLP-2 activity of PG-22. However, since MG12-4 and MG12-5 contain only one GLP-2 molecule compared to PG-22, which contains two GLP-2 molecules, the 50% decrease in activity was expected. PG-210-5 utilizes the linker peptide used in the previous patent document (US7812121B2), while PG-210-6, which utilizes the mixed linker peptide (G4S)3 + IgG1 hinge) used in the present invention, exhibited superior activity.

[0153] [Table 11]

[0154] Experimental Example 5: Analysis of immunosuppressive activity of IL-10Vm fusion protein 5-1: Analysis of affinity with IL-10 receptor The present inventors analyzed the affinity of the fusion proteins of Examples 2-3 and 2-4 to the IL-10 receptor (IL-10R1) by biolayer interferometry (BLI).

[0155] To achieve this, the inventors first performed Dip and Read (商標) Amine Reactive 2 ndIL-10R His-tagged protein was attached to a 96-well plate using the Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, 200 μl of DW was dispensed into a 96-well plate, and the amine biosensor included in the kit was inserted and allowed to hydrate for 10 minutes. Next, 200 μl of DW was dispensed into a new 96-well plate. EDC:NHS was mixed at a 1:1 ratio in 1 / 20 of the required sample volume, diluted with DW, and dispensed into the 96-well plate in 200 μl aliquots. IL-10R His-tagged protein was then diluted to 10 μg / ml in 10 mM acetic acid, pH 5.0, and added in 200 μl aliquots to the 96-well plate. Next, 200 μl of 1 M ethanolamine was added to the 96-well plate, and the Biosensor plate and sample plate were placed in an Octet tube. (登録商標) The plate was then placed in an Octet-K2 instrument and measured. After the measurement was completed, 200 μl of 1x Kinetics buffer was added to the sample plate, and the baseline was determined. Next, the NTIG-IL-10 fusion protein prepared in the above example was diluted in 1x Kinetics buffer to various concentrations (0, 62.5, 125, 250, 500, and 1,000 nM), and then dispensed in 200 μl portions onto the sample plate. (登録商標) Bio-layer interferometry (BLI) was measured using the -K2 instrument.

[0156] [Table 12]

[0157] As a result, as shown in FIG. 34 and Table 12, the fusion protein (NTIG-IL-10Vm) according to Example 2-4 of the present invention exhibited a K D The K value of the dimeric IL-10 fusion protein of Example 2-3 was 29.2 nM. DThe observed affinity was approximately three times that of the dimeric IL-10 mutant protein (11.1 nM). This is interpreted as meaning that the monomeric IL-10 mutant protein has a slightly lower affinity for the receptor than the dimeric IL-10 mutant protein, but this is within the expected range of the possibility that the affinity may be reduced by the formation of the monomer.

[0158] 5-2: Analysis of immunosuppressive activity of IL-10Vm fusion protein The present inventors analyzed the in vitro immunosuppressive activity of various fusion proteins (PG-088m, PG-110, PG-410, and PG-210-6) containing a monomeric IL-10 mutant protein (IL-10Vm) according to one embodiment of the present invention.

[0159] To do this, in detail, the supernatant was removed from a T75 flask containing cultured macrophages, Raw264.7, and then washed once with PBS. (商標) Three ml of Select Enzyme was added and incubated at 37°C for 3 to 5 minutes. Next, 10 ml of fresh medium was added to the T75 flask, and the cell suspension that had separated from the bottom of the flask was transferred to a 50 ml conical tube and centrifuged at 1,500 rpm for 5 minutes. The supernatant was then removed, and the tube was gently tapped to loosen the cells. Next, 5 ml of fresh medium was added and the cells were counted. When the cell concentration was 1 x 10 5 After adjusting the concentration to cells / ml, 100 μl of the solution was dispensed into a 96-well flat-bottom plate.

[0160] Next, PG-088m (Example 2-4), PG-110 (Example 3-3), PG-410 (Example 4-3), and PG-210-6 (Example 6-4) were serially diluted to various concentrations (0-1,000 nM for PG-210-6 and PG-410, 0-1,507 nM for PG-110 and PG-088m), and 50 μl of each was added to the 96-well plate containing the Raw264.7 macrophages. After 20 minutes, 50 μl of each solution was added to a final concentration of 400 ng / ml of lipopolysaccharide (LPS) and incubated at 36°C for 16 hours. The supernatants were collected and analyzed by TNF-α ELISA (Biolegend, USA) according to the manufacturer's protocol.

[0161] As a result, as shown in Figures 35a to 35c, it was found that all fusion proteins containing IL-10Vm according to an embodiment of the present invention inhibited TNF-α secretion in macrophages in a concentration-dependent manner. No significant difference was found depending on the type of fusion protein. This indicates that the monomeric IL-10 mutant protein (IL-10Vm) according to an embodiment of the present invention functions properly even when linked to the C-terminus of NTIG protein.

[0162] 5-3: Analysis of IgE binding capacity The present inventors analyzed the affinity of the fusion protein (FcεRIα-NTIG-IL-10Vm) prepared in Example 2-3 to mouse IgE and human IgE by biolayer interferometry (BLI).

[0163] To achieve this, the inventors first performed Dip and Read (商標) Amine Reactive 2 ndUsing the Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092), the FcεRIα-NTIG protein without IL-10Vm as a control and the fusion protein prepared in Example 3-3 were added to a 96-well plate. Specifically, 200 μl of DW was dispensed into the 96-well plate, and the amine biosensor included in the kit was inserted and allowed to hydrate for 10 minutes. Next, 200 μl of DW was dispensed, and EDC:NHS was mixed at a ratio of 1:1 in 1 / 20 of the required sample volume, diluted with DW, and dispensed into 96-well plates in 200 μl aliquots. Next, the FcεRIα-modified IgG4 Fc protein and FcεRIα-NTIG-IL-10Vm fusion protein were diluted to 10 μg / ml in 10 mM acetic acid, pH 5.0, and then added to the 96-well plate in 200 μl aliquots. Next, 200 μl of 1 M ethanolamine was added to each 96-well plate, and the Biosensor plate and sample plate were then placed in the Octet (登録商標) After the measurement was completed, 200 μl of 1x Kinetics buffer was added to the sample plate, and the baseline was determined. Next, anti-DNP mouse IgE (Sigma) was diluted in 1x Kinetics buffer to various concentrations (50 pM to 3.125 nM) and dispensed in 200 μl portions into the sample plate. (登録商標) Bio-layer interferometry (BLI) was measured using the -K2 instrument.

[0164] [Table 13]

[0165] As a result, as shown in FIG. 36a and Table 13, the K DThe value was 0.284 nM, which was slightly lower than that of the control FcεRIα-modified IgG4 Fc, but this difference was not significant, indicating that the addition of monomeric IL-10 protein did not reduce the IgE-binding ability.

[0166] In addition to the above results, to measure the binding strength of the human FcεRIα used in the present invention to human IgE, the inventors analyzed the affinity of the fusion protein (PG-110) of Example 3-3 to human IgE using the BLI method described above.

[0167] [Table 14]

[0168] As a result, as shown in FIG. 36b and Table 14, the fusion protein according to one embodiment of the present invention exhibited affinity for both human IgE and mouse IgE to a similar degree.

[0169] 5-4: Analysis of inhibitory activity against IgE Next, the present inventors conducted an in vitro experiment to examine whether the fusion protein (FcεRIα-NTIG-IL-10Vm) prepared in Example 3-3 exhibits inhibitory activity against IgE.

[0170] Specifically, FcεRIα-modified IgG4 Fc was used as a control, and the fusion protein (PG-110) of Example 3-3 was used as an experimental substance, and the diluted solutions were prepared at various concentrations. These solutions were mixed with 1 μl / ml of anti-DNP mouse IgE and incubated at room temperature for 30 minutes. Next, bone marrow-derived mast cells were harvested from mice and washed with HBSS buffer, and 5×10 5The cells / 60 μl concentration was adjusted and dispensed into a 96-well plate. 20 μl of the sample reacted with IgE and test drug was added to the pre-prepared mast cells and incubated for 25 minutes in a 37°C, 5% CO2 incubator. Next, 200 μl of DNP BSA (500 ng / ml) was added, followed by an additional 25 minutes of incubation in a 37°C, 5% CO2 incubator. The cells were then centrifuged at 1,500 rpm at 4°C to separate 300 μl of supernatant. This 300 μl of supernatant was mixed with 30 μl of substrate (4-nitrophenyl N-acetyl-β-glucosaminide, 5.84 mM) and incubated for 25 minutes in a 37°C, 5% CO2 incubator. The reaction was terminated by adding 400 μl of 0.1 M sodium carbonate buffer (pH 10). The absorbance at 405 nm was then measured to compare the relative amounts of β-hexosaminidase secreted from the mast cells, and the inhibitory effect of each drug concentration on mast cell proliferation was confirmed. The analysis was performed three times independently and the average was calculated.

[0171] As a result, as can be seen from FIG. 37 and Table 15, the fusion protein PG-110 according to an embodiment of the present invention inhibited IgE activity in a concentration-dependent manner, and the degree of inhibition was similar to that of the control FcεRIα-modified IgG4 Fc. However, the IC 50 was slightly lower than that of FcεRIα-modified IgG4 Fc.

[0172] [Table 15]

[0173] Experimental Example 6: CD40L binding analysis The present inventors investigated the binding affinity of PG-400 and PG-410, which were prepared in Examples 4-2 and 4-3 of the present invention, respectively, to human CD40L by BLI analysis.

[0174] Specifically, 200 μl of distilled water was dispensed onto a flat-bottom 96-well black plate, and then a biosensor tray was placed on the hydrated black plate. The amine biosensor was inserted and allowed to hydrate for 10 minutes. Next, 200 μl of distilled water was dispensed into a new 96-well black sample plate. EDC:NHS was mixed 1:1 at 1 / 20 of the required volume, diluted with distilled water, and dispensed into the 96-well black sample plate in 200 μl portions. Next, human CD40L (Peptrotech, Korea) was diluted to 5 μg / ml in 10 mM nitric acid solution (pH 6.0) and dispensed into the 96-well black sample plate in 200 μl portions. Next, 200 μl of 1 M ethanolamine was added to the plate, and the biosensor plate and sample plate were placed in an Octet tube. (登録商標) The plate was then loaded onto the -K2 instrument and the degree of CD40L binding on the amine sensor was measured. After the measurement was completed, the 96-well black sample plate was removed from the instrument and mixed with 200 μl of 1X Kinetics buffer to set the baseline. The analytes PG-410 and PG-400 were serially diluted with 1X Kinetics buffer to concentrations ranging from 200 to 12.5 nM, and then 200 μl of each solution was dispensed into the 96-well black sample plate. The sample plate was then transferred to the Octet (登録商標) After loading onto the -K2 instrument, the degree of CD40L binding was measured by BLI analysis.

[0175] [Table 16]

[0176] As a result, as can be seen from FIG. 38 and Table 16, the fusion protein (PG-410) according to one embodiment of the present invention has a higher K value for CD40L than PG-400. on The value was about 4.3 times lower, but K DThe values ​​were below fM (<1E-12), indicating very high binding affinity. Conclusively, PG-400 and PG-410 proteins were found to bind well to human CD40L. PG-410, a fusion protein (anti-CD40L-NTIG-IL-10Vm) according to one embodiment of the present invention, was found to bind to CD40L with very high binding affinity.

[0177] Experimental Example 7: Pharmacodynamic analysis of PG-110 The present inventors have investigated the half-life, area under the curve (AUC), and maximum blood concentration (C max The pharmacodynamic (PK) profile was confirmed by comparing the drug's pharmacokinetics (PD) and pharmacodynamics (PK) profiles.

[0178] Specifically, three male Sprague Dawley rats per group were administered the test fusion protein (PG-110) at a dose of 1 mg / kg via subcutaneous (SC), intravenous (IV), intraperitoneal (IP), or intramuscular (IM) routes. Blood samples were collected before and 0.5, 1, 5, 10, 24, 48, 72, 120, 168, 240, and 336 hours after injection and stored at room temperature for 30 minutes to allow for coagulation. The coagulated blood samples were centrifuged at 3,000 rpm for 10 minutes, and serum from each sample was collected and stored in a deep freezer. The serum concentration of the fusion protein according to one embodiment of the present invention was analyzed using an IgG-Fc quantitation set (Bethyl, Cat# E80-104, Lot# E80-104-30), which specifically detects Fc in administered proteins.

[0179] As a result, as can be seen in Figure 39, it was found that the physiologically active protein fused with NTIG was maintained in the body for a long period of time regardless of the route of administration.

[0180] Although the present invention has been described with reference to the above-mentioned embodiments and experimental examples, these are merely illustrative, and a person skilled in the art would understand that various modifications and equivalent embodiments are possible. Therefore, the true technical scope of the present invention should be determined by the technical spirit of the appended claims.

[0181] The NTIG protein according to one embodiment of the present invention can be used as a fusion partner for various biologically active proteins to effectively increase the half-life of the biologically active proteins, thereby enhancing the therapeutic efficacy of the biologically active proteins, and also minimizes effector functions such as ADCC and CDC, which are drawbacks of Fc fusion proteins, thereby enabling safer drug delivery of proteins. Therefore, the NTIG protein according to one embodiment of the present invention can be effectively used in the production of protein pharmaceuticals.

Claims

1. An immunoglobulin Fc domain mutant protein having the amino acid sequence of SEQ ID NO: 1 or 2, in which the 18th amino acid, threonine (T), is substituted with glutamine (Q) and the 196th amino acid, methionine (M), is substituted with leucine (L), and which is capable of forming a heterodimer due to the inclusion of a Knobs-into-Holes structure.

2. The immunoglobulin Fc domain mutant protein of claim 1, wherein the Knobs-into-Holes structure comprises a Knobs structure in which the 10th amino acid, serine (S), is substituted with cysteine ​​(C) based on SEQ ID NO: 77 of the CH3 domain of the immunoglobulin Fc domain protein and the 22nd amino acid, threonine (T) is substituted with tryptophan (W), based on SEQ ID NO: 77 of the CH3 domain, or the 22nd amino acid, threonine (T) is substituted with tryptophan (W), based on SEQ ID NO: 77 of the CH3 domain, or the 22nd amino acid, threonine (T) is substituted with tyrosine (Y) based on SEQ ID NO: 77 of the CH3 domain.

3. The immunoglobulin Fc domain variant protein of claim 1, wherein the Knobs-into-Hole structure is one in which the 5th amino acid, tyrosine (Y), is substituted with cysteine ​​(C), the 22nd amino acid, threonine (T) is substituted with serine (S), the 24th amino acid, leucine (L) is substituted with alanine (A), and the 63rd amino acid, tyrosine (Y) is substituted with valine (V) based on SEQ ID NO: 77 of the CH3 domain of the immunoglobulin Fc domain protein; one in which the 22nd amino acid, threonine (T) is substituted with serine (S), the 24th amino acid, leucine (L) is substituted with alanine (A), and the 63rd amino acid, tyrosine (Y) is substituted with valine (V) based on SEQ ID NO: 77 of the CH3 domain; or one in which the 63rd amino acid, tyrosine (Y) is substituted with threonine (T) based on SEQ ID NO: 77 of the CH3 domain.

4. An immunoglobulin Fc domain variant protein according to any one of claims 1 to 3, consisting of the amino acid sequence of SEQ ID NO: 8 or 9.

5. A fusion protein in which one or more biologically active proteins (APIs, active pharmaceutical ingredients) are linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein according to any one of claims 1 to 4.

6. The fusion protein of claim 5 , wherein the biologically active protein is a cytokine, an enzyme, a blood clotting factor, an extracellular domain of a membrane receptor, a growth factor, a peptide hormone, or an antibody mimetic.

7. A heterodimer comprising an immunoglobulin Fc domain variant protein according to any one of claims 1 to 4 or a fusion protein according to claim 5 or 6.

8. A polynucleotide encoding an immunoglobulin Fc variant protein according to any one of claims 1 to 4 or a fusion protein according to claim 5 or 6.

9. A recombinant vector comprising the polynucleotide of claim 8.

10. A composition comprising an immunoglobulin Fc domain variant protein according to any one of claims 1 to 4, a fusion protein according to claim 5 or 6, or a heterodimer according to claim 7.

11. A method for treating a disease in a mammalian subject, other than a human, comprising administering to said subject a therapeutically effective amount of the fusion protein of claim 5 or 6 or the heterodimer of claim 7.

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