Novel fusion proteins for treatment of autoimmune diseases

Abstract

The present invention shows an excellent therapeutic effect on various autoimmune diseases by significantly reducing the expression of inflammatory cytokines (without side effects). The fusion protein is a novel fusion protein for treating autoimmune diseases.

Description

Technical Field

[0001] This invention relates to a novel fusion protein for the treatment of autoimmune diseases, which plays a role in regulating pathogenic Th17 cells in these diseases. More specifically, it is a novel fusion protein that inhibits the signaling pathways of IL-17A, IL-17F, and TNFα, and is used in the treatment and prevention of immune-related and inflammatory diseases mediated by IL-17A, IL-17F, or TNFα. Background Technology

[0002] IL-17A and IL-17F can induce the production of large amounts of inflammatory cytokines, chemokines, and adhesion molecules to promote inflammation, and can aggregate neutrophils and macrophages to the site of inflammation. Several therapeutic antibodies targeting IL-17A (e.g., Sekunumab and Ixekizumab) or IL-17RA (Brodalumab) have shown high clinical efficacy in patients with psoriasis, psoriatic arthritis, and ankylosing spondylitis, and have been approved as therapeutic agents. Meanwhile, biologics targeting IL-17A and IL-17F, such as Bimekizumab and Sonelokimab, have also shown high clinical efficacy and have been approved as therapeutic agents. Furthermore, the interleukin-17 family can form six different homodimeric cytokines (IL-17A, B, C, D, E, and F) or heterodimeric IL-17A / F. All IL-17 receptors are type I transmembrane glycoproteins, comprised of five families: IL-17RA, RB, RC, RD, and RE. IL-17 receptors mediate host defense through interactions with IL-17 ligands, and also play a role in inflammation and autoimmune responses. IL-17RA can act as a shared receptor, mediating different signaling pathways with different IL-17 ligands. IL-17RA and IL-17RC form a dimer, mediating signaling based on the IL-17A, IL-17F homodimer, and IL-17A / F heterodimer. IL-17RC and IL-17RA share 22% homology and 55% homology in the ECD. Compared to IL-17RA, IL-17RC has a longer ECD but a shorter cytoplasmic domain and exists in a multiple splice configuration. IL-17RA has an affinity for IL-17F that is approximately 100 times weaker than that for IL-17A. IL-17RA and IL-17RC have different expression profiles and tissue distributions. In particular, the different cytokine environments among TGFβ, IL-6, TNFα, IL-1β, and IL-23 affect the expression of IL-17AA, IL-17FF homodimer, and IL-17AF heterodimer, which, in the case of Th17 cells, affects both naive and differentiated Th17 cells.

[0003] On the other hand, TNFα is an inflammatory cytokine produced by activated immune cells such as macrophages, CD4+ T cells, and NK cells. It also participates in the expansion of extracellular Th17 cells and plays a role in regulating inflammatory responses. Although TNFα is an important cytokine in normal immune responses, imbalances such as overexpression of TNFα in the body can lead to various autoimmune diseases. To treat and alleviate autoimmune diseases, infliximab, adalimumab, and fusion protein etanercept are used as TNFα inhibitors. However, TNFα inhibitor treatment is ineffective in 30% of patients with autoimmune diseases such as rheumatoid arthritis. Even in patients who respond to TNFα inhibitor treatment, the response rate decreases with prolonged administration, and many patients need to switch medications due to relapse. Side effects of TNFα inhibitors include tuberculosis infection, lymphoma, and kidney failure. However, in RA synovitis, if IL-17A and TNFα are released into the bloodstream for a long period of time, they may have a systemic effect, especially on the cardiovascular system.

[0004] The exact causes of autoimmune diseases remain undetermined. Due to the complex interplay of various factors, it is difficult to expect effective treatment or reduced relapse rates with a single targeted therapy in the context of autoimmune diseases. However, treatments simultaneously targeting TNFα and IL-17A have proven effective in reducing spondylitis and peripheral arthritis. Decreased values ​​of spinal osteophytes, significantly reduced inflammation severity, reduced edema, and decreased RANK / Osteoprotegerin (OPG) markers were observed.

[0005] Rheumatoid arthritis (RA), osteoarthritis (OA), and ankylosing spondylitis, all autoimmune diseases, are chronic autoimmune diseases caused by complex genetic and environmental factors leading to dysregulation of immune and inflammatory responses. This results in the continuous proliferation of the synovium within the joints and the destruction of joint bone and cartilage. In particular, with the advent of an aging society, the incidence of RA has risen sharply. RA is divided into approximately 120 sub-diseases, including degenerative arthritis, osteoarthritis, and rheumatoid arthritis caused by chronic inflammation of the synovium. In rheumatoid arthritis, characterized by inflammation, approximately 40–50% of synovial cells are infiltrated by immune cells, including CD4+ T cells. Furthermore, due to inflammation caused by various cytokines and chemokines, the synovium exhibits structural changes such as hypertrophy and hyperplasia. Early studies suggested that Th1 cells (including IFN-γ) were the main cause of RA, but blocking IFN-γ did not improve RA. More recently, it has been confirmed that Th17 cells are the main cause of RA.

[0006] Structural damage in rheumatoid arthritis (RA) is associated with cartilage and bone destruction caused by pathogenic IL-17, which originates from the binding of IL-17 receptors to Th17 cells expressing articular fibroblasts, endothelial cells, and epithelial cells. In human RA, IL-17 can induce the production of inflammatory cytokines, chemokines, and matrix metalloproteinases, and also synergistically amplify the inflammatory response with other inflammatory driver cytokines and TNFα.

[0007] In clinical trials of anti-TNFα therapy, patients resistant to anti-TNFα showed a significant increase in circulating Th17 cells and IL-17 (2.94% vs. 4.23%; 92.1 pg / ml vs. 148.6 pg / ml, P<0.05, respectively). The significant therapeutic effect of anti-TNFα treatment was highly correlated with the simultaneous reduction of circulating Th17 cells and IL-17. Based on post-marketing clinical results of anti-TNFα therapy, a Proof-of-concept (PoC) study (NCT02430909) targeting both IL-17 and TNFα was performed using combination therapy with bimecrolimus and cetrozumab pegol (CZP). The results showed that, compared with the placebo group, the combination therapy of sertozumab and pimecrolimus achieved faster remission with greater advantages. However, treatment emergencies (TEAEs), such as infections and infestations, occurred more frequently in the combination therapy group (50.0% (26 / 52)) than in the placebo group (22.2% (6 / 27)). The incidence of skin and subcutaneous diseases and intestinal diseases was also higher in the combination therapy group compared with the placebo group.

[0008] In the context of autoimmune diseases, IL-17 induces dimerization of IL-17RA and IL-17RC on the cell membrane, leading to an inflammatory response via NF-κB, MAPK, ERK, p38, and JNK signaling by adapter proteins Act1 and TRAF6. Furthermore, IL-17 signaling via the Act1-TRAF2-TRAF5 complex may enhance the inflammatory response through IL-17 mRNA stabilization and the stable expression of additional TNFα and IL-1β mRNA (Shadi Swidani et al., Front. Immunol. 10:1293, 2019). The Th17 pathways involved in the multi-inflammatory response of pathogenic IL-17 signaling differ from the primary inflammatory response of Th1 cells to TNFα. Therefore, targeting cytokines other than IL-17 simultaneously holds significant therapeutic potential. The inflammatory response based on the combined action of IL-17 and TNFα amplifies the expression of CRP (C-reactive protein), IL-6, IL-8, CCL20, and MCP1. In psoriasis patients, regarding IL-17-TNFα-IL-36, IL-17-TNFα co-stimulation increases IL36r in keratinocytes in an IκBζ-dependent manner, leading to a chain of psoriatic inflammatory responses (Manjeet K Paintlia, et al., J. Neurochem. 116(4):508-521. 2012). Detailed Implementation Technical issues

[0009] This invention addresses several problems, including those described above, and aims to provide a novel fusion protein that exhibits excellent therapeutic efficacy against various autoimmune diseases by significantly reducing the expression of inflammatory cytokines (without side effects). However, this objective is merely illustrative, and the scope of the invention is not limited thereto. Technical solutions to solve technical problems

[0010] According to one embodiment of the present invention, the present invention provides an IL-17RA / RC mutant hybrid protein comprising amino acid substitutions selected from the group consisting of: i) Q267R; ii) T24D, N88D, D122G and Q267R;

[0011] iii) T24D, F59V, N88D, D122G and Q267R; iv) L9P, T24D, F59V, N88D, D122G and Q267R; and v) L9P, T24D, F59V, N88D, D122G, A156P and Q267R, share more than 90% sequence homology with the amino acid sequence of SEQ ID NO: 69.

[0012] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the IL-17RA / RC mutant hybrid protein.

[0013] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0014] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0015] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the IL-17RA / RC mutant mixed protein.

[0016] According to another embodiment of the present invention, the present invention provides an IL-17RA / RC-Fc mutant hybrid protein, wherein an antibody Fc domain is attached to the N-terminus or C-terminus of the IL-17RA / RC mutant hybrid protein.

[0017] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the IL-17RA / RC-Fc mutant hybrid protein.

[0018] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0019] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0020] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the IL-17RA / RC-Fc mutant mixed protein.

[0021] According to another embodiment of the present invention, a TNFR2 ECD mutant protein is provided, comprising amino acid substitutions selected from the group consisting of: i) S33A; ii) S33A and S36P; iii) S33A, S36P and R119S; iv) S33A, S36P, R119S and N164S; v) S33A, S36P, R119S, N164S and E210G; vi) S33A, S36P, R119S, N164S, E210G and F219I; vii) S33A, S36P, G75S, R119S, N164S, E210G and F219I; viiii) G75S; and ix) S33A, S36P, G75S, R119S and N164S, and SEQ ID. The sequence homology among the amino acid sequences of NO:77 is over 90%.

[0022] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the TNFR2 ECD mutant hybrid protein.

[0023] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0024] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0025] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the TNFR2 ECD mutant protein.

[0026] According to another embodiment of the present invention, the present invention provides a TNFR2 ECD-Fc mutant protein, wherein an antibody Fc domain is attached to the N-terminus or C-terminus of the TNFR2 ECD mutant protein.

[0027] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the TNFR2 ECD-Fc mutant protein.

[0028] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0029] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0030] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the TNFR2 ECD-Fc mutant protein.

[0031] According to another embodiment of the present invention, the present invention provides a triple fusion protein, wherein an IL-17RA / RC mutant hybrid protein is linked to the N-terminus of the antibody Fc domain and a tumor necrosis factor (TNF) receptor 2 extracellular domain (ECD) is linked to the C-terminus of the antibody Fc domain; or, the tumor necrosis factor receptor 2 extracellular domain (ECD) is linked to the N-terminus of the antibody Fc domain and the IL-17RA / RC mutant hybrid protein is linked to the C-terminus of the antibody Fc domain.

[0032] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the triple fusion protein.

[0033] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0034] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0035] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the triple fusion protein.

[0036] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for treating autoimmune diseases, comprising the IL-17RA / RC mutant mixed protein and the TNFR2 ECD mutant protein as active ingredients.

[0037] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for treating autoimmune diseases, comprising the IL-17RA / RC-Fc mutant mixed protein and the TNFR2 ECD-Fc mutant protein as active ingredients.

[0038] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for treating autoimmune diseases, comprising the triple fusion protein or the homodimer or heterodimer as an active ingredient. The effects of the invention

[0039] The present invention, formed as described above, is a novel fusion protein for treating autoimmune diseases, which can effectively improve the excessive inflammatory response and irreversible inflammatory disease environment mediated by the co-stimulation of IL-17A, IL-17F, and TNFα. Furthermore, it has the advantages of reducing safety issues such as the risk of infection associated with parallel administration of single-target therapeutic agents through single-target administration of multi-target therapeutic agents, and alleviating the treatment cost burden for patients with autoimmune diseases. However, the scope of the present invention is not limited to these effects. Attached Figure Description

[0040] Figure 1 This is a graph showing the productivity results of the wild-type IL-17RA / RC-Fc construct and the IL-17RA / RC-Fc-TNFR2 construct of the present invention described in Table 1, analyzed using ProA affinity chromatography.

[0041] Figure 2 a is a graph showing the mammalian TNFR2 ECD (extracellular domain) arrangement deviating from the residues of family consensus S33, S36, G75, R119, N164, E210, and F219 as described in Table 3.

[0042] Figure 2 b is a graph showing the results of analyzing the productivity of 11 IL-17RA / RC-Fc-TNFR2 variants with TNFR2 mutations introduced in this invention (Table 3).

[0043] Figure 2 c is a graph showing the results of analyzing the affinity (EC 50) of 11 IL-17RA / RC-Fc-TNFR2 variants with the TNFR2 mutation introduced in this invention (Table 3) for TNFα. 12 represents the affinity (EC 50) value of adalimumab as a positive control for TNFα.

[0044] Figure 3 a is an SDS-PAGE gel image of the IL-17RA / RC-Fc protein of the present invention purified by a protein A column.

[0045] Figure 3 b is a graph showing the separation of two proteins (T1-T4-14 and T1-T4-52) of the IL-17RA / RC-Fc-TNFR2 mutant by protein A column purification.

[0046] Figure 3c is an SDS-PAGE gel image of the proteins (T1-T4-14, T1-T4-52) of IL-17RA / RC-Fc-TNFR2 mutant 2 purified by protein A column, separated over time.

[0047] Figure 3 Table d is a graph showing the results of analyzing the productivity of 26 (Table 10) IL-17RA / RC-Fc-TNFR2 proteins newly cloned from the pBispec vector into the pcDNA3.4 vector. Proteins purified using the purification method described in Example 3 were used to analyze the yield.

[0048] Figure 4 This is a graph showing the results of analyzing the column stability of the protein of the IL-17RA-Fc mutant introduced by the present invention as described in Table 2.

[0049] Figure 5 a is a graph showing the results of SEC analysis after culturing expiCHO cells as described in Table 10 at 4°C for 24 hours by co-expressing the proteins of IL-17RA / RC-Fc-TNFR2 and IL-17A produced from E coli cells and TNFα produced from E coli cells.

[0050] Figure 5 b confirms the description in Table 10 using both non-reducing and reducing gels. Figure 5 Gel photograph of the results of SEC separation of proteins.

[0051] Figure 6 Figure a shows the results of the analysis of the neutralizing capacity of the hIL-17A fusion protein IL-17RA / RC-Fc-TNFR2, as measured by SEAP analysis.

[0052] Figure 6 b is a graph showing the results of the analysis of the neutralizing capacity of the hIL-17F fusion protein IL-17RA / RC-Fc-TNFR2, as measured by SEAP analysis.

[0053] Figure 6 c is a graph showing the results of the analysis of the neutralizing capacity of the TNFα fusion protein IL-17RA / RC-Fc-TNFR2, as measured by SEAP analysis.

[0054] Figure 7 a is a graph showing the results of the affinity analysis of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention for human IL-17A.

[0055] Figure 7b is a graph showing the results of the affinity analysis of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention for human IL-17F.

[0056] Figure 7 c is a graph showing the results of the affinity analysis of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention for human TNFα.

[0057] Figure 8 This is a graph showing the analysis results of the anti-inflammatory effect of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention in a psoriasis-like environment using HaCaT cells. The anti-inflammatory effect was evaluated by measuring the expression levels of cytokines and chemokines in the graph.

[0058] Figure 9 This is a graph showing the analysis results of IL-6 expression levels in hFLS-RA (rheumatoid fibroblast-like synovial cells) based on treatment with the fusion protein IL-17RA / RC-Fc-TNFR2.

[0059] Figure 10a shows cells expressing tmTNFα prepared for analysis of the transmembrane TNFα binding capacity of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention. This figure is a schematic diagram of the structure of the simplified tmTNFα expression vector (pLVXtmTNFα-IRES-ZsGreen1).

[0060] Figure 10b is a schematic diagram of the binding relationship of the fusion protein IL-17RA / RC-Fc-TNFR2 in the tmTNFα-expressing cell line.

[0061] Figure 10c is a fluorescence micrograph showing the tmTNFα binding ability of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention.

[0062] Figure 11a is a schematic diagram illustrating the MHC Class I / II epitope analysis steps for analyzing the immunogenicity of the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention.

[0063] Figure 11b is a graph showing the MHC Class II binding site of the fusion protein IL-17RA / RC-Fc-TNFR2 and the predicted immunogenicity level (red: high; orange: medium; yellow: low) for analyzing the immunogenicity of the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention.

[0064] Figure 12a is a graph showing the results of analyzing the in vitro immunogenicity of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention.

[0065] Figure 12b is a graph showing the results of analyzing the in vitro immunogenicity of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention.

[0066] Figure 12c is a graph showing the results of the analysis of the stimulation index (SI) for the immunogenicity of the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention.

[0067] Figure 13 is a graph showing the results of analyzing the expression of IL-17A and IL-17F cytokines in the fusion protein IL17-RA / RC-Fc-TNFR2 of this invention in peripheral blood mononuclear cells of psoriasis patients.

[0068] Figure 14a is a graph showing the confirmation of hIL-17A and hIL-17F expression and the analysis results of no expression of mIL-17A and mIL-17F in human IL-17A & IL-17F double knock-in mice. The upper end represents heterologous mice, and the lower end represents homologous mice.

[0069] Figure 14b is a graph showing the analysis results of the response of the fusion protein IL-17RA / RC-Fc-mTNFR2 of the present invention to hIL-17A and mTNFα.

[0070] Figure 14c is a schematic diagram of the in vivo experimental process based on IL-17RA / RC-Fc-mTNFR2 treatment using C57BL / 6 mice as experimental subjects.

[0071] Figure 14d is a graph showing the results of analyzing the neutralizing capacity of hIL-17A and mTNFα in C57BL / 6hIL-17A / 17F Double knock-in mice.

[0072] Figure 15a is a 2D photograph showing the structure of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention. It is a set of 2D classification photographs confirming the fusion protein from multiple perspectives.

[0073] Figure 15b shows the three-dimensional electron microscopy structure of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention. 3D reconstruction was performed based on the 2D classification image.

[0074] Figure 15c is an electron density map image analyzing the structure of the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention. The electron density map reconstructed in 3D based on the 2D classification image was superimposed using the IL-17RC-IL-17F complex (6HG9, PDB), IgG1, and the TNFα-TNFR2 complex (3ALQ, PDB).

[0075] Figure 16a is a graph showing the mass spectrometry analysis results of the trypsin hydrolase of the fusion protein IL-17RA / RC-Fc-TNFR2(T1-T4-14) of this invention.

[0076] Figure 16b is a graph showing the results of chymotrypsin hydrolase analysis of the fusion protein IL-17RA / RC-Fc-TNFR2(T1-T4-14) of the present invention.

[0077] Figure 17a is a schematic diagram of the production timeline of glycan sialylation of the IL-17RA / RC-Fc-TNFR2 mixed fusion protein.

[0078] Figures 17b, 17c, and 17d are SDS-PAGE images (Figure 17b) and SEC analysis results (Figure 17c) of proteins isolated and purified from expiCHO-S cell culture medium co-transfected with IL-17RA / RC-Fc-TNFR2 and GT6 using a protein A column, and Western blot images (Figure 17d) confirming that the protein increases in size through sialylation.

[0079] Figures 17e, 17f, and 17g are the results of ELISA analysis of the binding affinity of the glycan-sialyzed IL-17RA / RC-Fc-TNFR2 hybrid fusion protein (T1-T4-14+GT6) to IL-17A, IL-17F, and TNFα.

[0080] Figures 18a and 18b are a series of graphs showing the results of analyzing the TNFR2 neutralizing capacity of the IL-17RA / RC-Fc-TNFR2 mixed fusion protein and the neutralizing capacity of adalimumab in SW982 cells based on the expression levels of IL-6 (upper end) and MMP3 (lower end).

[0081] Figure 19 is a schematic diagram of the three-dimensional structure of the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention. Detailed Implementation

[0082] Definition of terminology:

[0083] The terms “IL-17A and IL-17F” used in this specification show more than 50% homology among the six families of IL-17, forming their own homodimers (IL-17AA, IL-17FF) or heterodimers (IL-17AF), and achieving pathological IL-17 signaling through IL-17RA / RC and signaling assembly. The IL-17RA / RC heterodimer induces an inflammatory response through NF-κB, MAPK, ERK, p38, and JNK signaling based on the adaptor proteins Act1 and TRAF6. When IL-17 signaling occurs via the Act1-TRAF2-TRAF5 complex, the inflammatory response may be further amplified by the stabilization of mRNAs of inflammatory cytokines and chemokines (e.g., IL-17A, TNFα, and IL-1β) (Shadi Swidani et al. Front. Immunol. 10:1293, 2019).

[0084] The pathogenic IL-17 signaling pathway in Th17 cells differs from the inflammatory response induced by TNFα in Th1 cells. Under the combined action of IL-17 and TNFα, inflammation can potentially escalate irreversibly. Therefore, in cases of irreversible inflammation, it may be difficult to treat autoimmune diseases with a single target. Simultaneously targeting both IL-17 and TNFα may improve the likelihood of successful treatment.

[0085] As used in this specification, "TNFα (tumor necrosis factor-α)" refers to a 233-amino acid membrane protein that, upon release via TACE, is released as a 157-amino acid soluble TNFα (sTNFα), thereby mediating its biological activity. In addition to mediating signaling by two different TNFα receptors (TNFR1 and TNFR2), TNFα also mediates inflammatory responses involving transmembrane TNFα (tmTNFα), a precursor to sTNFα. TNFR1 is expressed in cells of most tissues, while TNFR2 is typically expressed in immune cells. TNFR2 is activated by tmTNFα, not sTNFα. Typically, TNFR1 activation induces a pro-inflammatory response through apoptosis, while TNFR2 signaling in activated T cells promotes cell proliferation. Currently, five TNFα inhibitors (etanercept, infelximab, golimumab, adalimumab, and cetrus) are approved and marketed as biologics for the treatment of TNFα-induced diseases. Each antibody exhibits varying efficacy in disease diagnosis and treatment. Although both neutralize sTNFα, other effects can determine the difference in treatment efficacy. For example, etanercept failed in clinical trials for Crohn's disease, Wegener's granulomatosis, and sarcoidosis, and its efficacy in treating psoriasis was lower than that of antibody-based TNFα inhibitors. Unlike etanercept, which is a TRAP protein, TNFα antibody therapy can stably bind to both sTNFα and tmTNFα. It has been reported that the tmTNFα activity of TNFα antibody therapy is closely related to the production of TGF-β and IL-10 induced by tmTNFα, and by activating regulatory T cells, it has an advantage over etanercept in some autoimmune diseases (SBombardieri et al., Rheumatology, 46(7):1191-1199.2007).

[0086] Detailed description of the invention:

[0087] According to another embodiment of the present invention, the present invention provides an IL-17RA / RC mutant hybrid protein comprising amino acid substitutions selected from the group consisting of: i) Q267R; ii) T24D, N88D, D122G and Q267R;

[0088] iii) T24D, F59V, N88D, D122G and Q267R; iv) L9P, T24D, F59V, N88D, D122G and Q267R; and v) L9P, T24D, F59V, N88D, D122G, A156P and Q267R, share more than 90% sequence homology with the amino acid sequence of SEQ ID NO: 69.

[0089] According to the mixed protein, glycans may be added, which may be one or more selected from the group consisting of N-glycans, O-glycans and sialylated glycans.

[0090] Based on the said mixed protein, the group consisting of SEQ ID NO: 66 to SEQ ID NO: 69 can be selected freely.

[0091] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the IL-17RA / RC mutant hybrid protein.

[0092] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0093] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0094] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the IL-17RA / RC mutant mixed protein.

[0095] According to another embodiment of the present invention, the present invention provides an IL-17RA / RC-Fc mutant hybrid protein, wherein an antibody Fc domain is attached to the N-terminus or C-terminus of the IL-17RA / RC mutant hybrid protein.

[0096] According to the mixed protein, the antibody Fc domain can be a mixed Fc domain consisting of two or more homologous Fcs, and the Fc domain can be selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 65.

[0097] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the IL-17RA / RC-Fc mutant hybrid protein.

[0098] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0099] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0100] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the IL-17RA / RC mutant mixed protein.

[0101] According to another embodiment of the present invention, a TNFR2 ECD mutant protein is provided, comprising amino acid substitutions selected from the group consisting of: i) S33A; ii) S33A and S36P; iii) S33A, S36P and R119S; iv) S33A, S36P, R119S and N164S; v) S33A, S36P, R119S, N164S and E210G; vi) S33A, S36P, R119S, N164S, E210G and F219I; vii) S33A, S36P, G75S, R119S, N164S, E210G and F219I; viiii) G75S; and ix) S33A, S36P, G75S, R119S and N164S, and SEQ ID. The sequence homology among the amino acid sequences of NO:77 is over 90%.

[0102] According to the mutant protein, at least one of i to ix is ​​a fusion compound capable of being linked to a linker peptide, and a glycan may be added. The glycan may be one or more selected from the group consisting of N-glycans, O-glycans, and sialylated glycans, and may be selected from the group consisting of SEQ ID NO: 70 to SEQ ID NO: 81.

[0103] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the TNFR2 ECD mutant protein.

[0104] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0105] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0106] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the TNFR2 ECD mutant protein.

[0107] According to another embodiment of the present invention, the present invention provides a TNFR2 ECD-Fc mutant protein, wherein an antibody Fc domain is attached to the N-terminus or C-terminus of the TNFR2 ECD mutant protein.

[0108] According to the mutant protein, the antibody Fc domain can be a mixed Fc domain consisting of two or more homologous Fcs, and the Fc domain is selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO: 65.

[0109] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the TNFR2 ECD-Fc mutant protein.

[0110] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0111] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0112] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the TNFR2 ECD-Fc mutant protein.

[0113] According to another embodiment of the present invention, the present invention provides a triple fusion protein, wherein an IL-17RA / RC mutant hybrid protein is linked to the N-terminus of the antibody Fc domain and a tumor necrosis factor (TNF) receptor 2 extracellular domain (ECD) is linked to the C-terminus of the antibody Fc domain; or, the tumor necrosis factor receptor 2 extracellular domain (ECD) is linked to the N-terminus of the antibody Fc domain and the IL-17RA / RC mutant hybrid protein is linked to the C-terminus of the antibody Fc domain.

[0114] According to the triple fusion protein, the IL-17RA / RC mutant hybrid protein contains amino acid substitutions selected from the group consisting of amino acids, and is characterized by sequence homology of more than 90% with the amino acid sequence of SEQ ID NO: 69.

[0115] i)Q267R;

[0116] ii) T24D, N88D, D122G and Q267R;

[0117] iii) T24D, F59V, N88D, D122G and Q267R;

[0118] iv) L9P, T24D, F59V, N88D, D122G and Q267R;

[0119] And v)L9P, T24D, F59V, N88D, D122G, A156P and Q267R.

[0120] According to the triple fusion protein, the extracellular domain (ECD) of the tumor necrosis factor receptor 2 contains amino acid substitutions selected from the group consisting of amino acid sequences that share more than 90% sequence homology with the amino acid sequence of SEQ ID NO: 77.

[0121] i)S33A;

[0122] ii) S33A and S36P;

[0123] iii) S33A, S36P and R119S;

[0124] iv) S33A, S36P, R119S and N164S;

[0125] v)S33A, S36P, R119S, N164S and E210G;

[0126] vi) S33A, S36P, R119S, N164S, E210G and F219I;

[0127] vii) S33A, S36P, G75S, R119S, N164S, E210G and F219I;

[0128] viii)G75S; and

[0129] ix)S33A, S36P, G75S, R119S and N164S.

[0130] According to the triple fusion protein, one or more of i to ix can be a fusion compound linked to a linker peptide, and a glycan may be added, wherein the glycan may be one or more selected from the group consisting of N-glycans, O-glycans and sialylated glycans.

[0131] According to the triple fusion protein, the tumor necrosis factor receptor 2 can be selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO: 94, and can simultaneously target IL-17A, IL-17F and TNFα, and can be selected from the group consisting of the amino acid sequences of SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32.

[0132] According to another embodiment of the present invention, the present invention provides a polynucleotide that encodes the triple fusion protein.

[0133] According to another embodiment of the present invention, the present invention provides an expression vector comprising the polynucleotide.

[0134] According to another embodiment of the present invention, the present invention provides a transformed cell in which the expression vector is introduced.

[0135] According to another embodiment of the present invention, the present invention provides a homodimer or heterodimer comprising the triple fusion protein.

[0136] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for treating autoimmune diseases, comprising the IL-17RA / RC-Fc mutant mixed protein or TNFR2 ECD-Fc mutant protein as an active ingredient.

[0137] According to another embodiment of the present invention, the present invention provides a pharmaceutical composition for treating autoimmune diseases, comprising the triple fusion protein or the homodimer or heterodimer as an active ingredient.

[0138] According to the pharmaceutical composition for treating autoimmune diseases, the autoimmune diseases may be selected from rheumatoid arthritis, psoriasis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), ankylosing spondylitis, pneumonia, asthma, atopic dermatitis, periodontitis (Pyorrhea), conjunctivitis, keratitis, dry eye syndrome, fibromyositis, lupus, systemic sclerosis, aphthous ulcers, Sjogren's syndrome, Guillain-Barré syndrome, and alopecia areata. The group consists of Areata, polymyositis and dermatomyositis, relapsing polychondritis, thrombocytopenia, and multiple sclerosis, and the inflammatory bowel disease can be selected from the group consisting of ulcerative colitis, Crohn's disease, and intestinal Behcet's disease.

[0139] To achieve the introduction and reconstruction of mutations that enhance the stability and neutralizing capacity of IL-17RA / RC mixtures and TNFR2 used in this invention, sequence similarity and domain similarity information were utilized from the amino acid sequences of proteins from different species. The bioinformatics-based back-to-consensus / ancestor method can analyze the co-evolution of specific residues in proteins to enable long-term environmental adaptation. In protein-protein interaction (PPI) analysis, to improve protein structural stability and function, a small gene library was constructed based on evolutionary methodology. Residue substitution patterns were analyzed through simulation, and in vitro, the structural changes of residues before and after substitution to achieve structural stability were confirmed, thus confirming the mechanism for maintaining high affinity and specificity. This was then applied to the preparation and addition of IL-17RA / RC mixtures and TNFR2 mutants.

[0140] Multispecific fusion proteins are proteins that can bind to two or more antigens simultaneously or sequentially. Specifically, in fusion proteins that are specific to three targets, the first antigen-binding domain can bind to the first antigen expressed on the cell, or to the first antigen secreted. The second or third antigen-binding domains may also be the same. Furthermore, they possess the characteristic of more efficient binding when the first and second antigens bind homologously or heterologously. Moreover, when using multispecific fusion proteins, specific signal transduction can be induced or blocked by inducing effective targeting or interaction between the two cells. The multispecific fusion protein provided in this invention can facilitate the regulation of activation of various immune cells such as CD4+ T cells, CD8+ T cells, monocytes, neutrophils, and macrophages, effectively improving the disease immune environment.

[0141] In specific examples, the multispecific fusion protein provided in this application can bind to more than one target, antigen, or epitope with a dissociation constant (KD) of less than 1 μM, less than 100 nM, less than 40 nM, less than 20 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 50 pM, less than 10 pM, or less than 1 pM. The binding affinity of the first antigen-binding domain to the first antigen may be higher than the binding affinity of the second or third antigen-binding domain to the second or third antigen.

[0142] This invention can be in the form of multispecific fusion protein chemical modification. In some embodiments, the fusion protein can be chemically modified via glycosylation, acetylation, pegylation, amidation, derivatization based on known protecting / blocking groups, proteolytic enzyme cleavage, and / or binding to ligands or other proteins. Many such chemical modifications can be performed using known techniques.

[0143] As used in this specification, the term "expression vector" refers to a fragment prepared by inserting a fragment of an antibody, protein receptor expression domain, or a fragment thereof, amplified by PCR, into a vector that is digested with restriction enzymes to express genetic material in cells.

[0144] In this invention, the expression vector can be any expression vector system such as pcDNA, pBispecific, pTT, pTT3, pEFBOS, pBV, pJV, pcDNA3.4 TOPO, pEF6 TOPO, or pBJ. Specifically, the expression vector can be pBispecific or pcDNA3.4 vector.

[0145] A specific embodiment of the fusion protein of the present invention may include the following structural formula (I) and structural formula (II).

[0146] N'-A-L1-B-L2-X-L3-C-C'(I) or N'-C-L1-X-L1-B-L3-A-C'(II)

[0147] In this context, in structural formulas (I) and (II), N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, A, B, and C are specific for the first, second, and third antigens, respectively, L1-L3 are peptide hinges or linkers, and X is the Fc domain, which refers to the C-terminal region of an immunoglobulin that includes the natural Fc domain, recombinant Fc domain, and mutant Fc domain; that is, the constant domain of "Fc" as commonly known, linked to proteins with effector functions. As a therapeutic protein, the Fc region can provide a longer half-life or integrate functions such as Fc receptor binding, protein A binding, complement fixation, and placental transport. In particular, this longer half-life of the Fc domain is based on the binding affinity to the neonatal Fc receptor (hereinafter referred to as "FcRn"). While increasing the half-life, the invention can inactivate the Fc domain-dependent effector function of antibodies such as ADCC or CDC, which are side effects that occur when using antibody-based protein therapeutics. However, the effects of the invention are not limited to the above description.

[0148] A mutant Fc domain refers to a recombinant antibody Fc domain protein prepared from a combination of all or part of the hinge, CH2, and CH3 components that are the building blocks of the Fc domain of an immunoglobulin, derived from different types of antibody molecules (i.e., IgG, IgD, IgE, IgM, etc.). An example of a variant IgG4 immunoglobulin could be one of the hyFc domain proteins (WO2008147143A2; WO2020102728A1; WO2020102728A1).

[0149] To ensure a longer half-life for the Fc domain of mutants, Fc engineering was used to increase the Fc affinity of FcRn under endosome conditions (acidic pH), employing an effective method to prolong the pharmacokinetics of monoclonal antibodies (Atsuhiko Maeda et al., Mabs. 9(5):844-853, 2017). The mutations used included LS mutation (M428L / N434S, Xencor), YTE mutation (M252Y / S254T / T256E, Medimmune), HN mutation (H433K / N434F), QL mutation (T250Q / M428L). Paul R. Hinton et al. J. Biol. Chem. 279(8):6213-6216, 2004), ABDEG mutation (M252Y / S254T / T256E / H433K / N434F), and EDHS. mutations (V264E, L309D, Q311H and N434S), EDHY mutations (V264E / L309D / Q311H / N434Y), DHS mutations (L309D / Q311H / / N434S), DHY mutations (L309D / Q311H / N434Y), IgG2-DHS mutations (V309D / Q311H / N434S), IgG3-DHS mutations (L309D / Q311H / N434S), or developed mutation sets of IgG4-DHS mutations (L309D / Q311H / N434S) can be used alone or in combination.

[0150] In structural formulas (I) and (II), L1–L3 are peptide hinges or linkers. In the fusion protein, the length of the linking peptide can be 2–60 a.a. (Amino acid), 4–55 a.a., 5–50 a.a., 5–46 a.a., 5–45 a.a., or 5–30 a.a. In the linking peptide, the linking peptide that connects the API (active pharmaceutical ingredient) to the N-terminus of the immunoglobulin Fc domain mutant protein may include part or all of the hinge region of the IgG1 antibody heavy chain. If it includes part of the hinge region, an artificial linking peptide may be added to the N-terminus or C-terminus of a portion of the hinge region. Furthermore, the hinge region may also be a hybrid hinge region consisting of portions of the hinge regions of two or more antibody heavy chains. Specifically, the linker peptide may be selected from GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (SEQ ID NO: 83), RNTGRGGEEKKGSKEKEEQEERETKTPECP (SEQ ID NO: 84), RNTGRGGEEKKSGKEKEEQEERETKTPECP (SEQ ID NO: 85), RNTGRGGEEKKGGKEKEEQEERETKTPECP (SEQ ID NO: 86), RNTGRGGEEKKSSKEKEEQEERETKTPECP (SEQ ID NO: 87), RNTGRGGEEKKKEKEKEEQEERETKTPECP (SEQ ID NO: 88), GGGGSGGGGSGGGGSLE (SEQ ID NO: 89), TGIEGRMD (SEQ ID NO: 90), TGKLSGSASAPKLEEGEFSEARV (SEQ ID NO: 90), etc. One or more combinations of the group consisting of NO: 91), KLSGSASAPKLEEGEFSEARVLE (SEQ ID NO: 92), TGGSGEGEGSEGSG (SEQ ID NO: 93), GSGEGEGSEGSGLE (SEQ ID NO: 94), AEAAAKEAAAAKA (SEQ ID NO: 95), GGGGSGGGGSGGGGS (SEQ ID NO: 96), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 97), EPKSSDKTHTCPPCP (SEQ ID NO: 98), and EPKSCDKTHTCPPCP (SEQ ID NO: 99).

[0151] The glycan structure of Fc-fusion proteins, and changes in that structure, can affect structural domains. In therapeutic Fc-fusion proteins, changes in specific glycosylation can also affect the molecule's pharmacokinetics (PK) and pharmacodynamics (PD). Glycans that significantly influence PK and PD include mannose, sialic acid, fucose, and galactose.

[0152] In some embodiments of the invention, specific mutations may be considered to alter the glycosylation of the polypeptide. This mutation may be selected to introduce or remove more than one glycosylation site, such as an O-linked or N-linked glycosylation site. Methods for increasing or decreasing the number of carbohydrate residues in the polypeptide are based on the chemical and / or enzymatic binding of glycosides. In some embodiments, the sequence of the polypeptide may be appropriately modulated depending on the type of expression system used in mammals, yeast, etc. Different glycosylation patterns influenced by the amino acid sequence of the peptide may be introduced in insect and plant cells. Typically, polypeptides of the present invention for human use can be expressed in mammalian cell lines that provide appropriate glycosylation, such as HEK293 or CHO cell lines.

[0153] N-linked glycosylation refers to the attachment of a glycan structure to the amide nitrogen of an asparagine residue in a protein. Glycans are branches of branched carbohydrates, flexible chains of carbohydrates, and the exact structure of the glycan attached to the asparagine residue in a protein depends on the expression system used for glycoprotein production. In the mixed protein of IL-17RA and IL-17RC (SEQ ID NO: 69), N-glycans can attach to one or more amino acid residues selected from N17, N35, N173, N186, N213, N223, N309, N332, and N366. In TNFR2 ECD (SEQ ID NO: 77), N-glycans can attach to one or more amino acid residues selected from N149 and N171.

[0154] Since the therapeutic proteins of some embodiments of the present invention can increase their in vivo half-life based on glycan terminal residues, methods can be introduced to reduce the content of Neu5Gc (N-Glycolylneuraminic acid) and increase Neu5Ac (N-Acetylneuraminic acid) in glycoproteins expressed in CHO cells. By manipulating cell lines to produce sialylated glycoproteins at levels higher than endogenous levels, the half-life of the multi-targeting fusion protein of the present invention can be increased.

[0155] Rheumatoid arthritis (RA), osteoarthritis (OA), and ankylosing spondylitis (AS) are chronic autoimmune diseases caused by complex genetic and environmental factors that lead to dysregulation of the immune and inflammatory responses, resulting in the continuous proliferation of the synovium within the joints and the destruction of the joint's bone and cartilage. In particular, with the advent of an aging society, the incidence of degenerative arthritis has risen sharply, encompassing approximately 120 diseases, including osteoarthritis and rheumatoid arthritis caused by chronic inflammation of the synovium. In rheumatoid arthritis, characterized by inflammation, approximately 40–50% of the synovial cells are infiltrated with immune cells such as CD4+ T cells. Due to inflammation caused by various cytokines and chemokines, the synovium exhibits structural changes such as hypertrophy and hyperplasia. Early studies focused on Th1 cells, which contain IFN-γ, as a major cause of RA, but blocking IFN-γ did not improve RA. More recently, however, Th17 cells have been confirmed as the primary cause of RA.

[0156] In articular fibroblasts, endothelial cells, and epithelial cells of structurally damaged tissues in patients with rheumatoid arthritis (RA), a pro-inflammatory response induced by IL-17 receptor binding was confirmed. IL-17 leads to cartilage and bone destruction and induces and maintains the production of inflammatory cytokines, chemokines, and matrix metalloproteinases. IL-17 is a major driver of inflammation, and its synergistic effect with TNFα has been studied in RA. The synergistic signaling of IL-17 and TNFα amplifies the mRNA inflammatory response of pro-inflammatory cytokines and chemokines. The inflammatory response based on the combined action of IL-17 and TNFα amplifies the expression of CRP (C-reactive protein), IL-6, IL-8, CCL20, and MCP1. In patients with psoriasis, regarding IL-17-TNFα-IL-36, IL36r in keratinocytes increases in an IκBζ-dependent manner when IL-17 and TNFα are co-stimulated, and induces a chain of psoriatic inflammatory responses (Manjeet K Paintlia, et al., J. Neurochem. 116(4): 508-521, 2011).

[0157] The triple-specific fusion protein IL-17RA / RC-L-Fc-L-TNFR2 (hereinafter referred to as IL-17RA / RC-Fc-TNFR2, where L is a linker) of the present invention is a protein therapeutic agent that has the same or higher targeting affinity than a single IL-17A targeting antibody (e.g., secukinumab) or an antibody that simultaneously targets IL-17A and IL-17F (e.g., imigelizumab). It effectively controls the inflammatory signal transduction formed by increased IL-17A and IL-17F from some non-T cells, including pathogenic Th17 cells, NKT cells, γδT cells, monocytes, neutrophils, macrophages, and eosinophils, and can effectively improve the inflammatory environment.

[0158] The fusion protein IL-17RA / RC-Fc-TNFR2 of this invention is a protein therapeutic agent that has the same or higher affinity than TNFα single-targeting antibodies or adalimumab or etanercept as TNFα inhibitory fusion proteins. It effectively blocks the inflammatory signal transduction of TNFR1 / 2 by binding to soluble TNFα (sTNFα) and transmembrane TNFα (tmTNFα) as ligands, can activate Treg (Regulatory T cells), and can induce an anti-inflammatory response through reverse signaling.

[0159] The fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention is a protein therapeutic agent that simultaneously or sequentially blocks the inflammatory response caused by cytokines IL-17A, IL-17F or TNFα. It can not only treat autoimmune diseases by effectively improving the pro-inflammatory immune environment, but also reduce disease recurrence by stabilizing and maintaining the improved immune environment.

[0160] The fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention also provides a method of treating inflammation or inflammatory conditions in subjects, wherein administration includes intravenous or subcutaneous administration. However, regarding the route of administration, dosage, and frequency of administration, the IL-17RA / RC-Fc-TNFR2 of the present invention can be administered to subjects in various methods and dosages depending on the patient's condition and the presence or absence of side effects. Those skilled in the art to which this invention pertains can select the optimal method of administration, dosage, and frequency of administration within an appropriate range. Furthermore, the fusion protein of the present invention can be administered in combination with other known drugs or physiologically active substances that have therapeutic effects on the disease to be treated, or can be formulated into a combination formulation with other drugs.

[0161] According to the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention, a method for treating a subject with inflammation or an inflammatory autoimmune disease and chronic inflammation-induced cancer is provided, comprising the step of administering a therapeutically effective amount of the isolated peptide or a pharmaceutical composition containing the isolated peptide to the subject in need.

[0162] According to the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention, a method for treating inflammation or inflammatory conditions is provided, wherein the therapeutic agent is prepared by administering at least one anti-inflammatory agent, including corticosteroids, to a therapeutically effective amount of the isolated peptide or a pharmaceutical composition comprising said isolated peptide. Specifically, these could be cortisol, aldosterone, hydrocortisone, hydrocortisone acetate, cortisolacetate, tixocortolpivalate, prednisolone, methylprednisolone, prednisone, triamcinolone acetonide, salicylic acid resorcinol, sulfacetamide, urea, imidazole, betamethasone, betamethasone sodium phosphate, dexamethasone, and dexamethasone sodium phosphate. Immunosuppressant agents, including phosphate, nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, capsaicin, ibuprofen, chlorprednisone, cyclosporine, cytokine synthesis inhibitors, tetracycline, minocycline, and doxycycline, or any combination thereof.

[0163] This invention provides a neutralizer of the pro-inflammatory cytokines IL-17A, IL-17F, and TNFα. IL-17A, IL-17F, and TNFα are cytokines involved in pro-inflammatory responses in autoimmune diseases or chronic inflammatory diseases. In particular, when the cytokines IL-17A, IL-17F, or TNFα act synergistically, inflammatory activity can be over-induced, creating an irreversible inflammatory disease environment. This invention provides a neutralizer that can regulate (e.g., block, inhibit, reduce, antagonize, neutralize, or suppress) the production of pro-inflammatory cytokines and / or chemokines mediated by IL-17A, IL-17F, and TNFα, superior to therapeutic methods that target one or two of these cytokines, and has the advantage of fundamentally blocking the inflammatory exacerbation caused by the synergistic effects of IL-17A, IL-17F, and TNFα.

[0164] This invention relates to a multi-target fusion protein comprising: a hybrid protein of the extracellular domain (ECD) of IL-17RA and IL-17RC, which may contain mutations in the moiety binding to first IL-17A and IL-17F; and a TNFR2 ECD, which may contain mutations in the moiety binding to second TNFα. The IL-17RA and IL-17RC hybrid protein exhibits over 95% homology with SEQ ID NO: 66 to SEQ ID NO: 69. For example, it exhibits over 95% homology with IL-17RA / RC containing mutations of L9P, T24D, F59V, N88D, D122G, A156P, or Q267R. Furthermore, the homology between TNFR2 ECD and SEQ ID NO: 83 to SEQ ID NO: 93 is over 95%, and the homology between TNFR2 ECD containing mutations of S33A, S36P, G75S, R119S, N164S, E210G or F219I is over 95%.

[0165] A specific embodiment of the multi-target fusion protein in some embodiments of the present invention may include the following structural formulas (I) and (II).

[0166] N'-A-L1-B-L2-X-L3-C-C'(I) or N'-C-L1-X-L1-B-L3-A-C'(II)

[0167] In this case, in structural formulas (I) and (II), N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, A, B, and C are domains specific to the first, second, and third antigens, respectively, L1-L3 are peptide hinges or linkers composed of 0-31, 2-10, or 3-10 amino acids, and X is the Fc domain, including a natural Fc domain, a recombinant Fc domain, or a mutant Fc domain. In therapeutic proteins, the Fc region can provide a longer half-life. The half-life can be further increased by enhancing the binding affinity to the neonatal Fc receptor (FcRn) through Fc engineering. To increase the half-life, introducing mutations to increase affinity for FcRn or Fc mutants that minimize the effector function of ADCC or CDC can be equivalent to the Fc domain.

[0168] In structural formulas (I) and (II), A can be a human IL-17RAECD polypeptide or an IL-17RAECD polypeptide mutant as the first antigen-binding domain. B can be a human IL-17RC ECD polypeptide or a human IL-17RC ECD polypeptide mutant as the second antigen-binding domain. The second antigen-binding domain can be specific to the first antigen IL-17A and the second antigen IL-17F, respectively, and can be specific to both IL-17A and IL-17F (Rolf E. Kuestner et al., J. Immunol. 46(7):1191-1199, 2007). Although IL-17RA has a high affinity for IL-17A, it has a relatively low affinity for IL-17F. In some embodiments of the present invention, A is an IL-17RAECD mutant and B is an IL-17RC ECD mutant, which can bind into a mixed form with a linker length of "0". Furthermore, the A domain of the IL-17RA / RC hybrid protein (IL-17RA / RC) corresponds to exons 1-6 of IL-17RA (wild-type, SEQ ID NO: 114) and can be a polypeptide or a variant thereof corresponding to exons 1-6 of wild-type hIL-17RA. The B domain of the IL-17RA / RC hybrid protein corresponds to exons 8-16 of IL-17RC (wild-type, SEQ ID NO: 115) and can be a polypeptide or a variant thereof corresponding to exons 8-16 of wild-type hIL-17RC. In structural formulas (I) and (II), the respective A-L1-B and B-L3-A IL-17RA / RC can be directly fused to the C-terminus or N-terminus of Fc via a linker, achieving a homology of over 95% with SEQ ID NO: 66 to SEQ ID NO: 69. The first antigen-binding domain of the IL-17RA / RC hybrid protein of the present invention is IL-17RA, corresponding to the A domain, and the second antigen-binding domain is IL-17RC, corresponding to the B domain, forming a mixed binding structure. However, A-L1-B or B-L3-A as a whole can bind to both IL-17A as the first antigen and IL-17F as the second antigen, thus possessing a novel morphology and function capable of binding to the IL-17A / F heterodimer. However, the IL-17RA / RC of the present invention is not limited to the above description.

[0169] According to the present invention, in structural formulas (I) and (II), the third antigen-binding domain of C includes the extracellular region (ECD) of human TNFR2. It can be fused directly or via a linker to the C-terminus or N-terminus of Fc. The TNFR2 ECD of the present invention is characterized in that it can neutralize and block TNFR1 / 2 signaling between soluble TNFα (sTNFα) and transmembrane TNFα (tmTNFα), thereby blocking the inflammatory response, or it can produce a beneficial anti-inflammatory effect through reverse signaling of transmembrane TNFα, thus minimizing side effects. In structural formulas (I) and (II), the TNFR2 ECD of C has at least 95% homology with SEQ ID NO: 77.

[0170] In some embodiments of the present invention, multi-target fusion proteins include the use of dual-target fusion proteins in the treatment, prevention, and / or diagnosis of diseases related to the activity of IL-17A, IL-17F, and TNFα. The present invention provides a treatment and prevention method in which the fusion protein simultaneously or sequentially binds to IL-17A, IL-17F, and TNFα in the manner described above, thereby simultaneously or sequentially inhibiting the activity of IL-17A, IL-17F, and / or TNFα. This inhibits the expression of cytokines and chemokines such as IL-8, IL-6, IL-1b, G-CSF, hBD2, DEFB4, IL-36r, IL-17C, CCL20, CCL23, CXCL10, LCN2, MMP3, E-selectin, and RANTES on immune cells or non-immune cells via the NFκB or MAPK pathway, while simultaneously inducing the secretion of anti-inflammatory cytokines, thereby rapidly and sustainably improving the pro-inflammatory environment.

[0171] The autoimmune diseases of this invention include psoriasis, rheumatoid arthritis (RA), osteoarthritis, ankylosing spondylitis, axial spondyloarthritis, juvenile idiopathic arthritis, osteoporosis, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), gingivitis, periodontitis or periodontal diseases, inflammatory bowel diseases (e.g., Crohn's disease), and ulcerative colitis. This invention will be applied to patients suffering from a variety of inflammatory, immune, and proliferative diseases, including colitis, inflammatory bowel disease, asthma (including allergic asthma), allergies, chronic obstructive pulmonary disease (COPD), multiple sclerosis, and cancer.

[0172] TNFα and IL-17 are key cytokines in the pathogenesis of inflammatory and autoimmune diseases. Five biologics—etanercept, infliximab, golimumab, adalimumab, and certolizumab pegol—have been approved and marketed as TNFα inhibitors that inhibit TNFα biological activity by binding to it. The therapeutic efficacy of these antibodies varies depending on the type and severity of the disease, or the presence of underlying diseases. Although all TNFα inhibitors neutralize sTNFα, patient response differs; some patients do not respond, and sustained therapeutic effects are rare. Specifically, TNFα inhibitors are ineffective in 30% of patients with autoimmune diseases such as rheumatoid arthritis. Even in patients who respond to TNFα inhibitor treatment, the response rate decreases with prolonged administration, and many require medication changes due to relapse (M HBuch et al. Rheumatology, 46, Issue 7, 1153-1156, 2007). Side effects of TNFα inhibitors include infections such as tuberculosis, lymphoma, and renal failure. In particular, the increased cardiovascular disease and related mortality are closely related to the long-term release of IL-17A and TNFα into the bloodstream during RA synovitis (Anass Bouchnita et al. CRBiol. 340(11-12):456-473, 2017).

[0173] Clinical studies of TNFα inhibitors have demonstrated a significant increase in circulating Th17 cells and IL-17 in patients unresponsive to TNFα inhibitors. Based on these clinical findings, a Proof-of-concept (PoC) study (NCT02430909) targeting both IL-17 and TNFα was conducted. Results from combination therapy with two biologics, bimekizumab (anti-IL-17A / F) and cetuzumab (anti-TNFα), in patients unresponsive to TNFα inhibitors demonstrated greater advantages in achieving rapid resolution compared to cetuzumab monotherapy, with numerically greater reductions in mean swelling joint count (SJC) and tender joint count (TJC) compared to the monotherapy group. However, the combination therapy of the two biologics resulted in a higher risk of infection and treatment emergencies (TEAEs) than the monotherapy or placebo groups, with more frequent skin and subcutaneous diseases and gastrointestinal diseases (Sophie Glatt et al., Ann. Rheum. Dis. 78(8):1033-1040, 2019).

[0174] Bilateral antibodies ABT-122, JNJ-8104, and COVA322, which simultaneously target TNFα and IL-17, were developed and clinical trials were conducted for RA as an indication. Although a rapid response rate was demonstrated in the phase II clinical trial, it was difficult to demonstrate the relative advantages of the anti-TNF antibody (adalimumab). It was demonstrated that it produced high levels of anti-drug antibodies (ADA), and it is no longer under development (NCT01853033, NCT02141997, NCT02243787, NCT02349451, NCT02758392).

[0175] According to the above, to date, combination therapies targeting TNFα and IL-17, or dual antibodies simultaneously targeting TNFα and IL-17, have been developed. While combination therapy offers flexibility in dosage and timing, it can cause discomfort for patients in terms of compliance and pain. For biological agents targeting cytokines associated with both innate immune diseases and proinflammatory autoimmune diseases, combination therapy increases the risk of infection, making it difficult to achieve low disease activity and ultimately not beneficial for treatment. Recently developed combination therapies also offer some flexibility in dosage selection, but due to the different molecular characteristics of the two antibodies, achieving formulations that satisfy both antibody chemical and physical stability is not easy. Furthermore, combination therapy or combination formulations involve the additional cost of two different drug therapies, increasing treatment costs for patients. According to the report, most biantibodies that simultaneously target TNFα and IL-17 show a high probability of immunogenicity induction (Mark A. Kroenke et al., Front. Immunol. 12:782788, 2021). High immunogenicity may have a profound impact on efficacy, pharmacokinetics, and safety, and therefore research and development has been discontinued.

[0176] The multi-targeted fusion protein prepared according to an embodiment of the present invention can bind simultaneously or sequentially to IL-17A, IL-17F, and TNFα to neutralize all of them. This effectively improves the excessive inflammatory response and irreversible inflammatory disease environment caused by the co-stimulation of IL-17A, IL-17F, and TNFα. It can also maintain efficacy with low immunogenicity to reduce disease activity, thereby increasing the likelihood of achieving remission. Furthermore, single-drug administration of multi-targeted therapies reduces safety issues, such as the risk of infection caused by combined administration of single-targeted therapies, and reduces the treatment cost burden for patients with autoimmune diseases (Figure 19).

[0177] The pharmaceutical composition of this invention can be varied according to the type of lesion, applicable site, number of treatments, treatment time, dosage form, patient condition, and type of adjuvant. The dosage is not particularly limited and can range from 0.01 μg / kg / day to 10 mg / kg / day. The daily dose can be administered once a day or divided into 2-3 doses at appropriate intervals, or intermittently over several days.

[0178] In the pharmaceutical composition of the present invention, the compound can be administered orally or non-orally, preferably non-orally via intravenous injection, subcutaneous injection, intraventricular injection, intracerebrospinal fluid injection, intramuscular injection, and intraperitoneal injection.

[0179] The pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Furthermore, solid or liquid formulation additives may be used in the preparation of the pharmaceutical compositions. Formulation additives may be either organic or inorganic. Examples of excipients include lactose, sucrose, white sugar, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silica. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, calcium citrate, dextrin, and pectin. Examples of lubricants include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any colorant commonly approved for addition to pharmaceuticals may be used as a colorant. These tablets and granules may be coated with sugar coating, gelatin, or other suitable coating materials as needed. Furthermore, preservatives, antioxidants, etc., may be added as needed. Additionally, if the pharmaceutical composition is a pharmaceutical preparation, one or more of the following may be added: fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, or preservatives. On the other hand, the dosage form of the pharmaceutical composition of the present invention may be preferably adopted according to the method of use, particularly in a manner that provides rapid, sustained, or delayed release of the active ingredient upon administration to mammals, methods known in the art are preferred. Specific dosage forms may be selected from plasters, granules, lotions, liniments, lemonades, powders, syrups, liquids and solutions, aerosols, extracts, elixirs, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, succinates, catapults, capsules, troches, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0180] Commonly used ingredients may also be added to the pharmaceutical compositions of the present invention, such as stabilizers, solubilizers, and conventional adjuvants and carriers such as fragrances.

[0181] The composition of one embodiment of the present invention may contain a pharmaceutically acceptable carrier, and in addition to the carrier, may also contain a pharmaceutically acceptable adjuvant, excipient or diluent.

[0182] As used in this specification, the term "pharmaceuticalally acceptable" means a composition that is physiologically acceptable and, when applied to the human body, generally does not cause gastrointestinal discomfort, dizziness, or other allergic reactions or similar reactions. Examples of the carrier, excipients, and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition, it may contain fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

[0183] Furthermore, the compositions of one embodiment of the present invention are formulated using methods known in the art to achieve rapid, sustained, or delayed release of the active ingredient. Dosage forms include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injections, and sterile powders.

[0184] The composition of one embodiment of the present invention can be administered via various routes, such as oral, parenteral, suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, or intrathecal administration. It can also be administered using an implantable device for sustained, continuous, or repeated release. Within the desired range, the frequency of administration can be once daily or divided into several doses, and the dosing cycle is not particularly limited.

[0185] The composition of one embodiment of the present invention can be administered by general systemic or local administration, such as intramuscular or intravenous injection, but when provided as a composition containing a polynucleotide or including an expression vector containing such a polynucleotide, injection is most preferably performed using an electroporator.

[0186] The administration route of the composition of one embodiment of the present invention can be any conventional route, as long as it can reach the target tissue. Such administration routes may include parenteral administration, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, and intrasynovial administration, but are not limited thereto.

[0187] The compositions of one embodiment of the present invention can be formulated into suitable forms with commonly used pharmaceutically acceptable carriers. For example, pharmaceutically acceptable carriers include water, suitable oils, saline solutions, glucose solutions, and ethylene glycol, which are used for parenteral administration, and may also contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben, propylparaben, and chlorobutanol. Furthermore, the compositions of the present invention may, depending on the method of administration or dosage form, appropriately add suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, soothing agents, buffers, antioxidants, etc. The pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, latest edition].

[0188] The dosage of the composition of one embodiment of the present invention for a patient varies depending on many factors, including the patient's height, body surface area, age, specific compound administered, sex, time and route of administration, general health condition, and other concurrently administered drugs. The composition of the present invention can be administered at doses ranging from 100 ng / kg to 10 mg / kg, more preferably from 1 μg / kg to 1 mg / kg, and most preferably from 5 μg / kg to 500 μg / kg, with the dosage adjustable according to these factors. Furthermore, the pharmaceutical composition of the present invention will be administered at a therapeutically effective dose.

[0189] As used in this specification, the term "therapeutic effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment. The effective dose level can be determined based on factors including the type and severity of the disease, age, sex, drug activity, drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, concomitant drugs, and other factors well known in the medical field. The pharmaceutical compositions of the present invention can be administered at doses from 0.1 mg / kg to 1 g / kg, more preferably at doses from 1 mg / kg to 500 mg / kg. Furthermore, the dose can be appropriately adjusted according to the patient's age, sex, and condition.

[0190] The compositions of the present invention can be administered orally or parenterally. When administered parenterally, they can be administered via any systemic or local route. When administered systemically, they can be administered via intravenous injection, intraperitoneal injection, or intramuscular injection. When administered locally, they can be administered via intracranial administration, intracerebrospinal fluid administration, or subcutaneous injection. Implementation of the invention

[0191] The present invention will now be described in more detail through embodiments. However, the present invention is not limited to the embodiments disclosed below, and can be implemented through various different methods. The following embodiments are intended to more fully disclose the present invention and to fully inform those skilled in the art of the scope of the present invention.

[0192] Example 1: Preparation of IL-17A and IL-17F neutralizing agents

[0193] To prepare a neutralizing agent that can effectively neutralize IL-17AA, IL-17AF, or IL-17FF, the inventors prepared the IL-17RA / RC-Fc protein. In particular, neutralizing agents that can bind simultaneously or sequentially to IL-17A or IL-17F can more effectively treat autoimmune diseases and inflammatory diseases (Sascha Gerdes & Joerg Albrecht., Br. J. Dermatol. Volume 186, Issue 4. 2022). The IL-17 receptor C (IL-17RC) can also bind to ligands IL-17F and IL-17A with high affinity (Rolf E. Kuestner et al., J. Immunol. 179(8):5462-5473. 2007). In IL-17RA / RC-Fc, the IL-17RA / RC hybrid protein utilizes the protein fragments involved in the binding of IL-17A or IL-17F in the extracellular domain (ECD) of IL-17RA and the ECD of IL-17RC as functional fragments. The hybrid protein (IL-17RA / RC fusion protein) was prepared by combining the functional fragments of each IL-17RA and IL-17RC. Furthermore, to ensure increased affinity for IL-17A and IL-17F, improved stability of the hybrid protein, or low immunogenicity, mutations were introduced into each of the IL-17RA and IL-17RC functional fragments using a back-to-consensus approach. Regarding the IL-17RA / RC mutant, the introduced mutations are as follows: leucine at position 9 is replaced with proline, threonine at position 24 is replaced with aspartic acid, histidine at position 52 is replaced with tyrosine, phenylalanine at position 59 is replaced with valine, glutamic acid at position 78 is replaced with lysine, asparagine at position 88 is replaced with aspartic acid, phenylalanine at position 89 is replaced with isoleucine, arginine at position 108 is replaced with lysine, aspartic acid at position 122 is replaced with glycine, alanine at position 156 is replaced with proline, and glutamic acid at position 267 is replaced with arginine.

[0194] Using wild-type IL-17RA / RC mixed protein as a control, various IL-17RA / RC-Fc-TNFR2 mutants were prepared as IL-17RA / RC mixed protein mutants or tri-specific fusion proteins to identify mutations that enhance the affinity, stability, and productivity of IL-17A and IL-17F. Example 2 of this invention is an experiment to screen for mutations that ensure affinity, do not affect stability, or further improve productivity of the IL-17RA / RC mixed protein when a TNFα-targeting moiety is added to the IL-17RA / RC mixture. The results, as shown in Table 1, indicate that compared to the control group wtIL-17RA / RC (wild-type) or wtIL-17RA / RC-Fc (mutant), the productivity of each IL-17RA / RC construct was significantly improved when multiple mutations were introduced. Figure 1 While improving the productivity, stability, affinity for ligands, and neutralization capacity are also greatly improved (Table 1). The complete sequence information of the fusion protein is summarized in Table 1, and the mutation information of the IL-17RA / RC mixed protein introduced into the IL-17RA / RC-Fc construct and the IL-17RA / RC-Fc-TNFR2 construct is summarized in Tables 2 and 3.

[0195] [Table 1] Complete sequence information of a fusion protein according to an embodiment of the present invention Serial Number name structure SEQ ID NO 1 T1-T4-01 T1Rf-Fc-T4wt 1 2 T1-T4-05 T1a-Fc-T4wt 2 3 T1-T4-06 T1b-Fc-T4wt 4 4 T1-T4-07 T1c-Fc-T4wt 6 5 T1-T4-08 T1c-Fc-T4a 8 6 T1-T4-09 T1c-Fc-T4b 10 7 T1-T4-10 T1c-Fc-T4c 12 8 T1-T4-11 T1c-Fc-T4d 14 9 T1-T4-12 T1c-Fc-T4e 16 10 T1-T4-13 T1c-Fc-T4f 18 11 T1-T4-14 T1c-Fc-T4g 20 12 T1-T4-30 T1c-Fc-T4h 22 13 T1-T4-31 T1c-Fca-T4g 24 14 T1-T4-32 T1c-Fcb-T4g 26 15 T1-T4-33 T1c-Fc-T4i 28 16 T1-T4-tandem T1c-Fc-T4TR 30 17 T1-T4-35 T1-Fc-mT4m 32 [Table 2]

[0196] IL-17RA / RC mutation information Serial Number Construct IL-17RA / RC mutation information 1 IL-17RA / RC-Fc(wt) 2 IL-17RA / RC-Fc Q267R 3 IL-17RA / RC-Fc T24D / N88D / Q267R 4 IL-17RA / RC-Fc T24D / N88D / D122G / Q267R 5 IL-17RA / RC-Fc T24D / F59V / N88D / D122G / Q267R 6 IL-17RA / RC-Fc L9P / T24D / F59V / N88D / D122G / Q267R 7 IL-17RA / RC-Fc L9P / T24D / F59V / N88D / D122G / A156P / Q267R 8 IL-17RA / RC-Fc Q267R 9 IL-17RA / RC-Fc L9P / T24D / F59V / N88D / D122G / Q267R 10 IL-17RA / RC-Fc L9P / T24D / F59V / N88D / D122G / A156P / Q267R

[0197] To induce IL-17RA / RC-Fc protein expression, primer pairs were prepared for polymerase chain reaction (PCR). Sufficient vectors were prepared by placing the mixture into a pBispec vector (Genexine, Inc., Korea). Regarding this expression vector, to generate the IL-17RA / RC-Fc fusion protein of this invention, a construct was prepared in which IL-17RA / RC, as a bioactive protein or active pharmaceutical ingredient, was linked to the N-terminus of an Fc domain mutant via a linker peptide. For the expression of the IL-17RA / RC-Fc fusion protein, ExpiCHO was used. TM (Gibco, Cat: A29127) cells and according to ExpiCHO TM The expression system kit was used to express the fusion protein. For preparation, the protein was expressed in ExpiCHO...TM Expression medium (Gibco, Cat: A29100-01) was used for one day under conditions of 8% CO2, 37°C, and 120 rpm. The DNA content reached 0.6–1.0 x 10⁻⁶ on the day of transfection using fresh medium. 7 Cells with a survival rate of over 95% were diluted to 1.0 x 10⁻⁶ cells / ml. 7 Prepare cells / ml. For transfection into the prepared mother cells, use ExpiFectamine. TM The CHO transfection kit (Gibco, Cat: A29129) was prepared with ExpiFectamine. TM CHO & plasmid DNA complex. Then, separately inject chilled OptiPRO. TM (Gibco, Cat: 12309019) Culture medium and inoculated with DNA prepared at appropriate concentrations and ExpiFectamine respectively. TM After applying the CHO reagent, the mixture was incubated at room temperature for 5 minutes, then inoculated and transfected into the mother cells before culturing. ExpiFectamine was applied 18 hours after transfection. TM The enhancer and feed contained in the CHO transfection kit were inoculated into transfected cells, and the cells were cultured for 7–10 days at 8% CO2, 37°C, and 120 rpm to complete the production. After the culture was completed, the cell culture medium was centrifuged at 4°C and 4000 rpm for 60 minutes, and the supernatant was separated and frozen at -80°C.

[0198] Example 2: Preparation of IL-17A, IL-17F and TNFα neutralizer

[0199] Pro-inflammatory cytokines interleukin-17 (IL-17) and tumor necrosis factor (TNFα) are therapeutic targets for various chronic inflammatory diseases. Furthermore, co-stimulation by IL-17 and TNF-α activates MAPK and NFκB, creating a pro-inflammatory environment and leading to a chain reaction of tissue damage. In particular, for patients with severe chronic autoimmune diseases, long-term drug use may activate compensatory mechanisms that inhibit the activity of certain drugs and induce adverse toxicities. To overcome this problem, it is necessary to target the inflammatory response pathways of both interleukin-17 (IL-17) and tumor necrosis factor (TNFα). To prepare a neutralizing agent that can simultaneously inhibit or neutralize IL-17A, IL-17F, and TNFα, a TNFα-binding moiety with an increased affinity for TNFα compared to wtTNFR2 was also ensured. In wtTNFR2, mutations selected from the back-to-consensus library were introduced into TNFR2 (Table 3). The IL-17RA / RC hybrid protein from Example 1 was linked to the N-terminus of Fc via a linker, and the TNFR2 mutant was linked to the C-terminus of Fc, thus completing the triple specific fusion protein (IL-17RA / RC-Fc-TNFR2).

[0200] Furthermore, to improve the stability, productivity, and neutralizing capacity of the TNFR2 ECD mutant of one embodiment of the present invention, after searching the NCBI database for interspecific sequences of the 258 amino acids containing the signal peptide in TNFR2 ECD, mutation positions were screened by arrangement. As a result, the selected mutations S33A, S36P, G75S, R119S, N164S, E210G, or F219I, and their various combinations, were selected as mutant combinations superior to the wild type based on evaluations of thermal stability, neutralizing capacity, and productivity. Figure 2 a). Compared to the wild type, the productivity of TNFR2 ECD mutants was generally improved. The TNFR2 mutant at number 10 in Table 3 (T4i, SEQ ID NO: 92) is a mutant with a partial deletion of the C-terminus of wtTNFR2, and the TNFR2 mutant at number 11 (T4TR, SEQ ID NO: 93) is a mutant with TNFR2 also linked via a linker; its productivity was slightly lower compared to other mutants. Figure 2 b).

[0201] Regarding the degree of improvement in the neutralizing ability of TNFα against TNFR2 mutants, the results were compared by comparing affinity (ECG) based on ELISA analysis. 50 ) value to observe ( Figure 2c). As a result, compared to wild-type TNFR2 ECD, the introduced TNFR2 ECD mutants mostly showed improved neutralizing capacity. Compared to wild-type TNFR2 ECD, the TNFR2 mutant at number 8 in Table 3 (T4g, SEQ ID NO: 77) showed a 40-fold increase in neutralizing capacity. This confirmed that the neutralizing capacity level was comparable to that of adalimumab (used as a positive control). Figure 2 The same as 12) of c. Mutations introduced into the TNFR2 ECD mutant, which improved both neutralizing capacity and productivity, were S33A, S36P, G75S, R119S, N164S, E210G, and F219I. Mutations introduced into the TNFR2 mutant (T4g, SEQ ID NO: 77) were S33A, S36P, G75S, R119S, N164S, E210G, and F219I. Table 3 summarizes the TNFR2 mutation information introduced into the IL-17RA / RC-Fc-TNFR2 construct described above. Regarding the TNFR2 mutant, the introduced mutations are as follows: alanine is introduced at amino acid serine at position 33, proline is introduced at amino acid serine at position 36, serine is introduced at amino acid glycine at position 75, serine is introduced at amino acid arginine at position 119, serine is introduced at amino acid asparagine at position 164, glycine is introduced at amino acid glutamic acid at position 210, or isoleucine is introduced at amino acid phenylalanine at position 219. [Table 3]

[0202] TNFR2 mutation information

[0203] The triple-specific fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention was prepared by linking the IL-17RA / RC mutant and the TNFR2 mutant prepared in Example 1 with multiple Fc and linkers. The triple-specific fusion protein of the present invention may include structural formula (I) and structural formula (II).

[0204] N'-A-L1-B-L2-X-L3-C-C'(I) or N'-C-L1-X-L1-B-L3-A-C'(II)

[0205] In this case, in structural formulas (I) and (II), N' is the N-terminus of the fusion protein, and C' is the C-terminus of the fusion protein. A is a partial domain of IL-17RAECD, and B is a partial domain of IL-17RCECD. Each domain corresponds to a protein that binds to IL-17A or IL-17F as a ligand via a functional fragment. However, A-L1-B or B-L3-A can bind to IL-17A as the first antigen, IL-17F as the second antigen, or the IL-17A / F heterodimer. L1 in "A-L1-B" acts as a linker and can be 0 to 31 amino acids, but can be "0" in IL-17RA / RC. IL-17RA / RC can be located at the N-terminus in Fc wild-type or mutant via the linker (L2). In the structural formulas (I) and (II), C is specific for TNFα as a third antigen and can be located at the C-terminus of Fc wild-type or mutant via linker L3.

[0206] The linker introduced into the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention can be one of the linkers shown in Table 4. The Fc domain introduced into the fusion protein is a recombinant antibody Fc domain protein, i.e., prepared by combining all or part of the hinge, CH2, and CH3 components of the Fc domain of an immunoglobulin derived from different types of antibody molecules such as IgG, IgD, IgE, and IgM. An example of a variant IgG4 immunoglobulin can be one of the hyFc domain proteins (WO2008147143A2; WO2020102728A1; WO2021046404A1). Table 5 of the present invention provides an example of an Fc domain.

[0207] To ensure a longer half-life, the Fc mutants in Table 5 introduce mutations that enhance Fc affinity for FcRn under endosomal conditions (acidic pH). Fc engineering using drugs that extend the in vivo duration of a monoclonal antibody was employed (AtsuhikoMaeda et al., Mabs. 9(5):844-853, 2017). The mutation sets used for this Fc engineering include LS mutation (M428L / N434S, Xencor), YTE mutation (M252Y / S254T / T256E, Medimmune), QL mutation (T250Q / M428L) (Paul R. Hinton et al., J. Biol. Chem. 279(8):6213-6216, 2004), or KF mutation (H433K / N434F), either alone or in combination. The linker sequence information of the triple specific fusion protein IL-17RA / RC-Fc-TNFR2 is summarized in Table 4. [Table 4]

[0208] IL-17RA / RC-Fc-TNFR2 linker sequence information connector amino acid sequence SEQ ID NO L1 TGIEGRMD 90 L2 GGGGSGGGGSGGGGSLE 89 L3 TGKLSGSASAPKLEEGEFSEARV 91 L4 KLSGSASAPKLEEGEFSEARVLE 92 L5 TGGSGEGEGSEGSG 93 L6 GSGEGEGSEGSGLE 94 [Table 5]

[0209] Fc sequence information

[0210] To prepare the fusion protein IL-17RA / RC-Fc-TNFR2 of this invention, various constructs were prepared, morphologically linked sequentially from the N-terminus to the C-terminus via linkers listed in Table 4, allowing the IL-17RA / RC mutant and the TNFR2 mutant to bind to the Fc mutant listed in Table 5. A mutation with high affinity for FcRn was introduced into the Fc domain of the IL-17RA / RC hybrid protein (Table 6). This nucleic acid was cloned into the pBispec vector. The IL-17RA / RC sequence information of the fusion protein IL-17RA / RC-Fc-TNFR2 is summarized in Table 6, and the sequence information of the TNFR2 mutant is summarized in Table 7. [Table 6]

[0211] IL-17RA / RC sequence information [Table 7]

[0212] TNFR2 sequence information

[0213] Regarding the expression of the IL-17RA / RC-Fc-TNFR2 fusion protein, ExpiCHO was used. TM (Gibco, Cat: A29127) cells and according to ExpiCHO TM The expression system kit was used to express the fusion protein. For preparation, the protein was expressed in ExpiCHO... TM Expression medium (Gibco, Cat: A29100-01) was used for one day under conditions of 8% CO2, 37°C, and 120 rpm. The DNA content reached 0.6–1.0 x 10⁻⁶ on the day of transfection using fresh medium. 7 Cells with a viability of over 95% and a density of 10 cells / ml were diluted to 1.0 x 10⁻⁶ cells / ml. 7 Prepare cells / ml. For transfection into the prepared mother cells, use ExpiFectamine. TM The CHO transfection kit (Gibco, Cat: A29129) was prepared with ExpiFectamine. TM CHO & plasmid DNA complex. Then, separately inject chilled OptiPRO. TM (Gibco, Cat: 12309019) Culture medium and inoculated with DNA prepared at appropriate concentrations and ExpiFectamine respectively. TM After applying the CHO reagent, the mixture was incubated at room temperature for 5 minutes, then inoculated and transfected into the mother cells before culturing. ExpiFectamine was applied 18 hours after transfection. TM The enhancer and feed contained in the CHO transfection kit were inoculated into transfected cells, and the cells were cultured for 7–10 days at 8% CO2, 37°C, and 120 rpm to complete the production. After the culture was completed, the cell culture medium was centrifuged at 4°C and 4000 rpm for 60 minutes, and the supernatant was separated and frozen at -80°C.

[0214] Example 3: Isolation and purification of IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2

[0215] The inventors analyzed the productivity and stability of the fusion proteins IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2, and found that they were improved. Mutants with improved productivity, stability, and neutralizing capacity were ensured by introducing the mutant combinations screened in Examples 1 and 2 into the wtIL-17RA (UniProt:humanIL-17RA:Q96F46), wtIL-17RC (UniProt:humanIL-17RC:Q8NAC3), and wtTNFR2 (UniProt:human TNFR2:Q9UIG9) proteins. Compared to the wild-type fusion proteins, these mutants showed significant improvements in productivity and stability. The IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2 fusion proteins were produced in CHO cells and purified using Protein A affinity. Subsequently, the supernatant of the IL-17RA / RC mixed fusion protein (IL-17RA / RC-Fc-TNFR2) was centrifuged for 60 minutes at 4°C and 4000 rpm, and the cell culture fluid (HCCF) was filtered to 0.45 μm. Next, a HiTrapMabSelect Sure column (Cytiva, 11-0034-95) was prepared. After stabilization with 10 column volumes (CV) of binding buffer (BB) (20 mM sodium phosphate, 150 mM NaCl (pH 7.2)), the sample (HCCF) was loaded, stabilized with 6 column volumes (CV) of binding buffer (BB), and then washed with washing buffer (WB, 100 mM sodium citrate, 150 mM NaCl (pH 5.0)). The column-bound protein was eluted with elution buffer (EB, 1M Glycine, 150mM NaCl (pH 3.0)). The elution was aliquoted into tubes containing 1M Tris buffer (pH 9.0) at 2.5 ml intervals. Protein elution was confirmed by SDS-PAGE. SDS-PAGE analysis of the IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2 (T1-T4-14) fusion proteins showed confirmation at approximately 77 kDa and 103 kDa under 10% reduction conditions. Figure 3 a to Figure 3c). The eluted protein was purified using a secondary purification process based on size exclusion chromatography (SEC) as follows: First, approximately 1 mL of the purified sample containing the IL-17RA / RC mixed fusion protein (IL-17RA / RC-Fc-TNFR2) was loaded onto the column at a flow rate of 1 mL / min (AKTATM pure25 (Cytiva, 29018224)). Specifically, the overpressure was set to 0.5 MPa and the flow rate to 1 mL / min, using a conformal column configuration. The column used was a Superdex 200 (Cytiva), and the elution buffer was a solution of 1 / 10 diluted LPS solution and CBP007B with Tween-20 (Cytiva, BR100054) added to achieve a concentration of 0.005%. Subsequently, separation was performed by observing the peak value based on the elution volume (HiLoad 16 / 600 Superdex 200pg 1x 120mL (Cytiva, 28989335)). For purification, two steps in size exclusion chromatography (SEC) were used to finally purify the eluent recovered from the Protein A column. The chromatographic yields of the fusion protein are summarized in Tables 8 and 9. [Table 8]

[0216] Yield of IL-17RA / RC-Fc purified by protein A column [Table 9]

[0217] Two yields of IL-17RA / RC-Fc-TNFR2 mutant T1-T4-14 T1-T4-52 TF vol 50mL 50mL Target MW Approximately 100kDa Approximately 100kDa time 7 days 7 days yield 24.7 mg / L 38.4 mg / L

[0218] Analysis of the productivity of the 26 IL-17RA / RC-Fc-TNFR2 strains listed in Table 10, recloned from the pBispec vector to the pcDNA3.4 vector, showed a significant increase in productivity even when only the vector was changed without construct mutations. Figure 3 d). Yields were measured using the purification method employed in Example 3. Information on the IL-17RA / RC-Fc-TNFR2 mutant construct of the present invention is summarized in Table 10. Linkers L1 and L2 in Table 10 are based on the linker sequence information in Table 4. To facilitate the synthesis of sialylated glycans, the IL-17RA / RC-Fc-TNFR2 mutant protein (serial number 26) was extracted from ExpiCHO-S...TM The system was introduced into a cell line containing galactosyltransferase-6 (GT6) to produce (Example 17). [Table 10]

[0219] IL-17RA / RC-Fc-TNFR2 mutant construct information

[0221] Example 4: Thermal stability analysis of IL-17RA-Fc protein

[0222] For thermal stability analysis, IL-17RA-Fc protein and wtIL-17AR-Fc control protein were prepared as measurement samples at concentrations of 0.5–1 mg / ml. The samples were diluted three times with distilled water after diluting the 1000x dye in the Thermal Shift Assay Dye Kit (appliedbiosystems, 4461146) by 8x dilution. The melting curve was then measured by PCR (Quantstudio 6flex (appliedbiosystems, 4485689)) using the kit's reaction buffer, the protein, and the dye mixture (reaction buffer 7.5 μl; protein 10 μl; 8X dye 2.5 μl). The Tm and ΔTm values ​​were then confirmed using Thermal Shift Assay Software (Appliedbiosystems, 4466038). Tm is the temperature (°C) corresponding to the maximum value of the first derivative of the S / W-based DSF melting curve. The thermostability of IL-17RA-Fc with various mutations was measured, and the stability was maintained up to 65°C. Comparison of IL-17RA-Fc proteins in items 3, 6, and 7 of Table 11 showed that the IL-17RA-Fc protein with both T24D and N88D mutations exhibited greater thermostability compared to those with the T24D or N88D mutations. Figure 4 For sequences 2, 8, and 10, although T24D was added to the IL-17RA-Fc protein in combinations of F59V, R108K, and D122G mutations, the thermostability was not improved. To identify combinations of back-to-conssensus mutations that simultaneously affect the affinity and stability of IL-17A, thermostability and affinity analyses were performed. The results of the thermostability analysis of the IL-17RA-Fc fusion protein are summarized in Table 11. [Table 11]

[0223] Thermal stability analysis results

[0224] Example 5: Affinity analysis of ligands (IL-17A, IL-17F, TNFα) of IL-17RA / RC-Fc-TNFR2

[0225] For affinity analysis, IL-17AA (Peprotech, 200-17), IL-17FF (Peprotech, 200-25), IL-17AF (R&D systems, 5837-IL), TNFα (novusbio, NBP2-35076) microplates, 96-well / pure Grade, Black / F bottom, 350 μl (BRAND.781608), and an AHC2 biosensor (Sartorius, 18-5019) were prepared. Affinity measurements were performed using the Octet from Satorius AG. Hydration with 2x kinetic buffer was first performed using the AHC2 biosensor. The ligand concentrations were prepared such that IL-17RA / RC-Fc-TNFR2 could be attached to the biosensor at a concentration of 0.8–1 nm. The concentrations of the ligands used in the experiment were set to be at a moderate level within the expected concentration range, with 5 to 7 ranges defined. The hydrated biosensor was used, and a baseline was determined using a buffer solution for baseline determination. The biosensor was then transferred to a solution containing the IL-17RA / RC-Fc-TNFR2 to allow IL-17RA / RC-Fc-TNFR2 to bind at a concentration of 0.8–1 nm. The bound biosensor was then used to determine the baseline using the same buffer solution used for baseline determination during rebalancing. Next, association was performed at the site where the ligand was present, and dissociation was performed by transferring to a site containing the buffer solution used for baseline determination during dissociation.

[0226] The ligands (IL-17AA, IL-17AF, IL-17FF, and TNFα) of the octet using IL-17RA / RC-Fc-TNFR2 and their respective binding affinity values ​​are summarized in Table 12. The affinity of IL-17RA / RC-Fc-TNFR2 for the ligands (IL-17AA, IL-17AF, IL-17FF, and TNFα) was compared with that of bimekizumab as a positive control. Regarding the affinity for IL-17RA / RC-Fc-TNFR2 of IL-17AA, the measured affinity level was the same as that of imigelizumab. Regarding the affinity for IL-17AF, the measured affinity level was the same as or below that of imigelizumab. Regarding the affinity for IL-17FF, the measured affinity level was below that of imigelizumab. Furthermore, the affinity for the ligand TNFα of IL-17RA / RC-Fc-TNFR2 was compared with that of etanercept, which served as a positive control. Regarding the affinity for IL-17RA / RC-Fc-TNFR2, the measured affinity was above that of etanercept. Regarding the ligands (IL-17AA, IL-17AF, IL-17FF, and TNFα) of IL-17RA / RC-Fc-TNFR2 and their respective binding affinities, the measured affinities were all higher than those of wtIL-17RA, wtIL-17RC, and wtTNFR2. The affinity analysis results for each ligand (IL-17A, IL-17F, and TNFα) of IL-17RA / RC-Fc-TNFR2 are summarized in Table 12. [Table 12]

[0227] Affinity analysis results

[0228] *IL-17AA:IL-17RA / RC-Fc-TNFR2-Bimekizumab>IL-17RAwild type-IL-17RCwild type*IL-17AF:Bimekizumab>IL-17RA / RC-Fc-TNFR2-IL-17RC wild type>IL-17RAwild type

[0229] *IL-17FF:Bimekizumab>IL-17RA / RC-Fc-TNFR2>IL-17RC wild type>IL-17RAwild type

[0230] *TNFα:IL-17RA / RC-Fc-TNFR2>Etanercept

[0231] Example 6: Binding morphology analysis of IL-17RA / RC-Fc-TNFR2 with target ligands, IL-17A, and TNFα

[0232] The recombinant multi-targeting fusion protein of this invention is a protein fused with two or more protein domains. It is particularly important that the inherent biological activities of the constituent protein domains are not impaired. By selecting appropriate linkers between the various constituent domains and preparing suitable constructs, the activity of each constituent domain protein can be maintained; that is, activity can be maintained in a manner where the interaction between the ligand and receptor is not hindered by steric hindrance. Maintaining the activity of the recombinant multi-targeting fusion protein can achieve the stability of the fusion protein, prolong its plasma half-life, and improve its in vivo therapeutic effect.

[0233] It needs to be confirmed that the multi-targeting fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention can simultaneously bind to ligands IL-17AA, IL-17FF, IL-17AF, or TNFα. It needs to be confirmed that when IL-17RA / RC-Fc-TNFR2 binds to each ligand simultaneously, the neutralizing capacity is not significantly different compared to the neutralizing capacity of IL-17RA / RC or TNFR2 as individual components. IL-17RA / RC-Fc-TNFR2 and IL-17A are produced through co-expression based on co-transfection on expi-CHO cells, generating IL-17RA / RC-Fc-TNFR2 and IL-17A proteins. Human TNFα (hTNFα) is produced through overexpression in E. coli. Subsequently, the secured IL-17RA / RC-Fc-TNFR2, hIL-17A, and hTNFα proteins were purified using his-tag affinity, anion exchange, and size exclusion chromatography. The purified multi-target fusion proteins IL-17RA / RC-Fc-TNFR2, along with their targeting ligands, IL-17A, and TNFα, were incubated overnight at 4°C. To remove aggregation, the cultured proteins were centrifuged at 13,000 rpm for 10 minutes. Then, for the de-aggregated proteins, unbound and bound proteins were re-separated using SEC.

[0234] as a result, Figure 5 The first peak on the left of 'a' represents the binding of all three proteins. TNFα, which does not bind to IL-17A / RC-Fc-TNFR2, is confirmed in the peak on the right. SDS-PAGE gel analysis shows that TNFα forms a trimer based on hydrogen and ion binding, rather than through disulfide bonds, and is identified as a monomer in both non-reducing and reducing gels. For IL-17A, due to the formation of a dimer based on disulfide bonds, it is identified as a monomer in reducing gels and as a dimer in non-reducing gels. Furthermore, for IL-17A, because it was cultured in Expi-CHO cells (mammalian cells), the presence of glycosylation sites led to the identification of multiple bands based on glycosylation. Figure 5 b).

[0235] Co-culture (4°C, 24 hours) using proteins produced by the co-expression of IL-17RA / RC-Fc-TNFR2 and IL-17A, along with TNFα produced in *E. coli* cells, confirmed that the triple-specific fusion protein IL-17RA / RC-Fc-TNFR2 stably binds to both IL-17A (as the first ligand) and TNFα (as the second ligand) to form a complex. Furthermore, the simultaneous binding of the multi-target fusion protein IL-17RA / RC-Fc-TNFR2 to each target was confirmed, and the simultaneous or sequential binding of IL-17RA / RC-Fc-TNFR2 to each ligand did not produce significant differences in neutralizing capacity. Furthermore, BLI measurements (Example 5) confirmed that the affinity of the IL-17RA / RC-Fc-TNFR2 fusion protein for the ligand TNFα was <1.0E-12. After the IL-17RA / RC-Fc-TNFR2 fusion protein formed a complex with the first ligand and IL-17A, the measured affinity for the second ligand TNFα was 1.6E-11. Comparing the <1.0E-12 and 1.6E-11 values ​​confirmed that there was no significant difference in affinity between IL-17RA / RC-Fc-TNFR2 and the second ligand after binding to the first ligand.

[0236] Example 7: Effects of fusion proteins IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2 on the targeting ligand IL- Neutralization capacity analysis of IL-17AA, IL-17AF and IL-17FF

[0237] HEK-Blue was prepared for analysis of neutralization ability.TM Reporter cells (InvivoGen, hkb-IL-17, or HEK-Blue) TM TNFα), DPBS (Welgene, LB 001-02), FBS (Gibco, 16000044), DMEM (Welgene, LM 001-05), antibiotics (Welgene, LS203-01), HEK-Blue TM Selection (InvivoGen, hb-sel), Normocin TM (InvivoGen, ant-nr-1), QU anti-Blue TM Solution (InvivoGen, rep-qbs), Tween-20 (Junsei, 69295S1601), Autoclaved DW, Peprotech (200-17), IL-17A (Peprotech, cat no.200-17), IL-17F (Peprotech, cat no.200-25) and TNFa (Peprotech, cat no.300-01A).

[0238] In the inflammatory environment of autoimmune diseases, IL-17AA, IL-17AF, and IL-17FF ligands form a complex with IL-17RA / RC, inducing the formation of the IL-17 signalosome. This IL-17 signalosome complex amplifies inflammatory cytokines and chemokines through intracellular signal transduction (Arnaud Goepfert et al., Ann. Rheum. Dis. Cell Reports 41, 111489, 2022). The fusion protein IL-17RA / RC-Fc-TNFR2 selectively neutralizes ligands IL-17A, IL-17F, and IL-17A / F in an inflammatory environment, which is closely related to the therapeutic effect of autoimmune diseases.

[0239] To evaluate the neutralizing ability of the fusion proteins IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2 of the present invention for each ligand, the ligands of hIL-17AA, hIL-17AF, or hTNFα were treated according to their concentrations, and HEK-Blue... TM Reporter cells (InvivoGen, hkb-IL-17, or HEK-Blue) TMTNF-α secretes SEAP (secreted embryonic alkaline phosphatase) as a secreted reporter protein in a concentration-dependent manner. The inhibitory capacity of signal transduction pathways can be measured by quantifying SEAP in cell culture medium through treatment of the fusion proteins IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2 of this invention. HEK-Blue TM The cell isolation process was reported as follows: The culture medium was removed from the cultured cells, and the cells were isolated after washing twice with 1X PBS. The obtained cells were transferred to a 15 ml conical tube and centrifuged at 1200 rpm for 4 minutes. The isolated cells were then mixed with Test Media (DMEM, 10% FBS, 1% PS). Next, 10 μl of Trypan-Blue and 10 μl of resuspended cells were added and mixed. Then, 10 μl of a hemocytometer was added for further mixing, and the surviving cells were counted. The final concentration was 2.5 × 10⁻⁶ cells / mL. 5 Cells were mixed with test media and cultured overnight in 96-well plates at 100 μl per well. Then, a mixture of the fusion protein IL-17RA / RC-Fc (10–100 μg / ml) was prepared at a concentration of 0.005% Tween-20 / PBS. Next, three ligands (hIL-17AA, hIL-17AF, 10 ng / ml each) were prepared, and IL-17RA / RC-Fc and each ligand were treated separately in 96-well plates at 100 μl per well, and cultured for 24 hours. To evaluate the prepared HEK-Blue... TM The neutralizing ability of the IL-17RA / RC-Fc-TNFR2 protein in reporter cells to the ligand was measured by mixing 20 μl of QUANTI-Blue with the sample cultured for 24 hours. TMAfter incubating 180 μl of the solution for 10 minutes, the OD value was measured at 630 nm using a Bio-Tek microplate reader. A mixture of the fusion protein IL-17RA / RC-Fc-TNFR2 diluted in 0.005% Tween-20 / PBS (10–100 μg / ml) was prepared. Ligands (one of hIL-17AA, hIL-17FF, and TNFα) were also prepared at treatment concentrations of 10 ng / ml, 10 ng / ml, and 1 ng / ml, respectively. Subsequently, after performing the same procedure as described above, the OD value was measured at 630 nm using a Bio-Tek microplate reader. Bimecrolimus was used as a positive control for IL-17A and IL-17F neutralization capacity, and adalimumab was used as a positive control for TNFα neutralization capacity.

[0240] The results indicate the IC50 value representing the neutralizing ability of IL-17AA against the fusion protein IL-17RA / RC-Fc-TNFR2. 50 The concentration was 20.91 ng / ml, and the IC50 of the positive control group and memetizumab was... 50 The concentration was 19.87 ng / ml, showing a comparable level. The IC50 value represents the ability to neutralize TNFα in the fusion protein IL-17RA / RC-Fc-TNFR2. 50 The concentration was 14.94 ng / ml, and the IC50 of the positive control group and adalimumab was... 50 The concentration was 27.68 ng / ml, showing a significantly higher level in comparison. Furthermore, the IC50, representing the neutralizing capacity for IL-17FF, was [not specified in the original text]. 50 The concentration was 1152 ng / ml, and the IC50 of the positive control group and bimecrolimus was... 50 The concentration was 31.02 ng / ml, indicating low neutralizing capacity, confirming specificity for IL-17FF. The neutralizing capacity of the multi-target fusion protein IL-17RA / RC-Fc-TNFR2 for both ligands IL-17AA and TNFα was measured to be almost equivalent to that of the positive control group (mepiquat bromide and adalimumab). Figure 6 a to Figure 6 c). The results of the analysis of the neutralizing capacity of IL-17RA / RC-Fc-TNFR2 of the ligands (hIL-17AA, hIL-17FF, TNFα) by the SEAP analysis are summarized in Table 13. [Table 13]

[0241] Neutralizing capacity analysis results of IL-17RA / RC-Fc-TNFR2

[0242] Example 8: Affinity of the fusion protein IL-17RA / RC-Fc-TNFR2 to IL-17A, IL-17F, and TNFα ELISA

[0243] To analyze affinity, 50 ml of carbonate buffer (pH 9.6, Na₂CO₃ (Sigma, S7795, 0.1 M): NaHCO₃ (Sigma, S8875, 0.1 M) = 1:1) was prepared as the coating buffer. 1 μg / ml of the ligand recombinant protein (one of the following: HumanIL-17A PeproTech; 200-17, HumanIL-17F-PeproTech; 200-25, Human TNFα-PeproTech; 300-01A, MurineIL-17A-PeproTech; 210-17, MurineIL-17F-PeproTech; 210-17F, Murine TNFα-PeproTech; 315-01A) was added to the coating buffer and spread at 40 μl / well onto 96-well plates overnight at 4°C. On the second day, the wells were washed three times with 200 μl / well of ELISA TBS buffer (1X TBS and 0.1% Tween 20). After dilution in 100 μl / well of ELISA TBS buffer supplemented with 5% BSA, the plates were incubated at room temperature for 1 hour, followed by washing three times with 200 μl / well of ELISA TBS buffer. Then, 100 μl / well of IL-17RA / RC-Fc-TNFR2 protein was added to blocking buffer and incubated at room temperature for 2 hours. IL-17RA / RC-Fc-TNFR2 protein was treated at concentrations of 0 ng / ml, 0.05 ng / ml, 0.5 ng / ml, 5 ng / ml, 50 ng / ml, 500 ng / ml, 5000 ng / ml, and 50000 ng / ml. The HRP-bound secondary antibody (Abcam, Cat.Ab98624) was treated with 200 μl / well ELISATBS buffer three times and reacted at room temperature for 1 hour. Afterwards, the sample was washed five times with ELISATBS buffer, and after reacting with 100 μl TMB solution (Seracare KPL, Cat#5120-0080) at room temperature, 100 μl of stop solution (Cat#5150-0024) was added. The absorbance was measured at 650 nm using an ELISA reader. EC50 values ​​indicate the affinity of the fusion protein IL-17RA / RC-Fc-TNFR2 for hIL-17A. 50 The concentration was 9.237 ng / ml, and the positive control group had an ECG of 1,000 mg / ml compared to that of mepiquat. 50 The concentration was 6.197 ng / ml, which, in comparison, showed a similar level of EC indicating affinity for hIL-17F.50 The concentration was 130.3 ng / ml, and the positive control group had an ECG of 130.3 ng / ml compared to that of mepiquat rubella. 50 The concentration was 14.2 ng / ml, showing a comparable level. EC2000 exhibited an affinity for hTNFα from the fusion protein IL-17RA / RC-Fc-TNFR2. 50 The concentration was 43.08 ng / ml, and the positive control group and adalimumab EC were... 50 The value was 20.03, almost at the same level in comparison. In particular, the affinity of the multi-target fusion protein IL-17RA / RC-Fc-TNFR2 for the two ligands IL-17A and TNFα was measured to be almost at the same level as that of the positive control group, mepiquat citrate and adalimumab. Figure 7 a to Figure 7 c). The affinity measurements of the fusion protein IL-17RA / RC-Fc-TNFR2 for IL-17A, IL-17F, and TNFα are summarized in Table 14. [Table 14]

[0244] Affinity analysis results for L-17A, IL-17F, and TNFα

[0245] Example 9: Anti-inflammatory effects of the multi-targeted fusion protein IL-17RA / RC-Fc-TNFR2 in psoriasis patients under similar conditions Effect evaluation

[0246] The HaCaT cell line (human keratinocyte cell line) strongly expresses inflammatory cytokines and chemokines due to the combined stimulation of TNFα and IL-17. That is, as confirmed by ex vivo experiments in human psoriatic skin lesions, IL-6, IL-17C, IL-8, CXCL8, CXCL8, CCL20, BD2, LNC2 and S100A7, etc., can be identified as having increased production of multiple inflammatory cytokines, chemokines and antimicrobial peptide genes (Andrea Chiricozzi et al, J.Invest.Dermatol.131(3):677-687, 2011).

[0247] To confirm the expected therapeutic effects of IL-17RA / RC-Fc and TNFR2-Fc of one embodiment of the present invention in human psoriatic skin lesions, comparisons were made with a positive control antibody, imigelizumab, and adalimumab. Furthermore, the anti-inflammatory effects of the combined treatment with the IL-17RA / RC-Fc and TNFR2-Fc fusion protein or the treatment with IL-17RA / RC-Fc-TNFR2 alone were compared with those of the combined treatment with imigelizumab and adalimumab as a positive control group to confirm the therapeutic effects of IL-17RA / RC-Fc, TNFR2-Fc, and IL-17RA / RC-Fc-TNFR2 of the present invention. For the experiments, HaCaT (human keratinocytes) were maintained in 2% FBS and low-calcium DMEM (Gibco, 21068028) for more than 2 weeks. Subsequently, after treatment with 0.53 mM EDTA (10 min, 37°C), the cells were treated with TrypsinEDTA (2 min, 37°C), and the single-cell suspension was centrifuged (1200 rpm, 5 min, 4°C) with 2% FBS low-calcium DMEM. After counting the number of cells suspended in 2% FBS low-calcium DMEM, the cells were washed with PBS and then aliquoted into 12-well plates (1.0 x 10⁶ cells / well). 5 Cells were stabilized by culturing overnight (1 mL / mL), then placed in 0.9 mL of 0.5% FBS low-calcium DMEM and starved (24 hours, 37°C).

[0248] To induce a psoriasis model, IL-17AA, AF, FF, and TNFα cytokines were treated in 0.1 mL of 0.5% FBS low-calcium DMEM (24 h, 37 °C). Subsequently, the fusion proteins IL-17RA / RC-Fc-TNFR2, IL-17RA / RC-Fc, TNFR2-Fc, and the positive control antibodies imigituzumab (Antibody System, DHH28803) and adalimumab (Selleckchem, A2010) were treated in a concentration-dependent manner (15 h, 37 °C). The effects of the cytokines and inhibitors (fusion proteins IL-17RA / RC-Fc-TNFR2, IL-17RA / RC-Fc, TNFR2-Fc, and the positive control antibodies imigituzumab and adalimumab) on HaCaT cells, a psoriasis-like model, under the treatment conditions summarized in Table 15, are presented. [Table 15]

[0249] Treatment conditions for cytokines and inhibitors Concentration (ng / mL) / Treatment group 1 2 3 4 5 6 7 8 9 10 11 12 IL-17AA(1) - - + + + + + + + + + + IL-17AF(200) - - + + + + + + + + + + IL-17FF(10) - - + + + + + + + + + + TNFα(10) - + + + + + + + + + + + Inhibitors

[0250] *Inhibitor treatment groups (treatment groups 4–12) Treatment group 4: IL-17RA / RC-Fc 20 ng / mL

[0251] Treatment group 5: TNFR2-Fc 50 ng / mL

[0252] Treatment group 6: IL-17RA / RC-Fc 20 ng / mL + TNFR2-Fc 50 ng / mL

[0253] Treatment groups 7–9: IL-17RA / RC-Fc-TNFR2 5 ng / mL, 20 ng / mL, and 80 ng / mL, respectively.

[0254] Treatment group 10: Bimecrolimus 20 ng / mL

[0255] Treatment group 11: Adalimumab 50 ng / mL

[0256] Treatment group 12: Bimecrolimus 20 ng / mL + Adalimumab 50 ng / mL.

[0257] To perform qRT-PCR analysis for each treatment group, after cell lysis using Qiazol lysis reagent (Qiagen, 79306), chloroform was added, and the cells were centrifuged at 13200 rpm and 4°C for 15 minutes to separate the upper aqueous layer. Isopropanol was added to the separated organic layer to precipitate the RNA, followed by centrifugation at 13200 rpm and 4°C for 10 minutes. The particles were then washed with 75% DEPC-EtOH, centrifuged at 13200 rpm for 5 minutes, and the ethanol was evaporated. The particles were then dissolved in 30–50 μl of DEPC-water, resulting in a RNA concentration of 200–250 ng / μl. cDNA was obtained by reverse transcription using a cDNA synthesis kit (abm, G236). 1 μl of cDNA was mixed with target gene primers (Table 16) and 10 μl of SYBR Green, and then processed using QuantStudio. TM The 5 Real-Time PCR system performed RT-qPCR.

[0258] IL-8 (CXCL8) is a chemokine that recruits neutrophils and macrophages, while S100A7 (Psoriasin) is a protein overexpressed in psoriatic skin. Furthermore, CCL20 is also a chemokine that recruits neutrophils through inflammatory cytokines induced by LPS and IFNγ. BD2 is a skin-antimicrobial peptide produced in the epithelial cells of psoriasis patients. LCN2 (Lipocalin-2) is expressed in neutrophils and is a marker of skin lesions. IL-36γ (IL1F9) is an IL-1 lineage cytokine activated by NF-κB and is associated with psoriatic lesions, thus being used as a biomarker for psoriasis. When comparing HaCaT cells treated with IL-17 alone or TNF-α alone (columns 1 and 2, respectively) with those treated with both IL-17 and TNF-α (column 3), a significant increase in the expression of S100A7, CCL20, BD2, LCN2, CXCL8, and IL-36γ was confirmed. Figure 8 This means that, compared to treating IL-17 or TNFα alone, the synergistic effect of IL-17 and TNFα can have a significant impact on the excessive proliferation and pro-inflammatory response in psoriasis.

[0259] In the group treated with both IL-17 and TNFα, the expression of S100A7 and BD2 increased when treated with adalimumab. Furthermore, the inhibitory effect of pimecrolimus on CXCL8, IL-36γ, and CCL20 was not significant, but a significant inhibitory effect was confirmed when adalimumab and pimecrolimus were combined. The sequence information of the primers used in the RT-qPCR is summarized in Table 16. [Table 16]

[0260] Primer sequence information Gene name Base sequence (5'→3') SEQ ID NO hGAPDH F TCA CCA GGG CTG CTT TTA AC 100 hGAPDH R GAC AAG CTT CCC GTT CTC AG 101 hCXCL8 F AGC TCT GTG TGA AGG TGC AG 102 hCXCL8 R TCT CAG CCC TCT TCA AAA ACT TC 103 hS100A7 F CTT CCC CAA CTT CCT TAG TG 104 hS100A7 R GTA GTC TGT GGC TAT GTC TC 105 hCCL20F GCA AGC AAC TTT GAC TGC TG 106 hCCL20R CAA GTC CAG TGA GGC ACA AA 107 hBD2 F ATC AGC CAT GAG GGT CTT G 108 hBD2 R GCA GCA TTT TGT TCC AGG 109 hLCN2 F TGA GTG CAC AGG TGC CG 200 hLCN2 R TTT AGC AGA CAA GGT GGG GC 201 hIL-36γF CAG CCC ACA TTG CAG CTA AA 202 hIL-36γR AGG AGG CAA TGA ACC AGT CC 203

[0261] According to one embodiment of the present invention, IL-17RA / RC-Fc is a fusion protein targeting IL-17AA, IL-17AF, and IL-17FF, and TNFR2-Fc is a fusion protein targeting TNFα. The inhibitory abilities of IL-17RA / RC-Fc and TNFR2-Fc on their respective target ligands were compared with those of pimecrolimus and adalimumab as positive control antibodies. Cytokines and chemokines expressed by simultaneous treatment of IL-17 and TNFα were used as subjects. The inhibitory effects of single treatment and combined treatment of IL-17RA / RC-Fc and TNFR2-Fc on IL-17RA / RC-Fc-TNFR2 were compared, as well as the single and combined treatment of pimecrolimus and adalimumab as positive control antibodies. The results showed that when comparing the anti-inflammatory effects of IL-17RA / RC-Fc and pimecrolimus, (… Figure 8 (Columns 4 and 10) confirmed equivalent levels of inhibitory effect, and confirmed that the combined treatment group of IL-17RA / RC-Fc and TNFR2-Fc had a better inhibitory effect than the treatment groups alone. When comparing the anti-inflammatory effects of the TNFR2-Fc and adalimumab ( Figure 8 Columns 5 and 11) confirmed inhibitory effects at or above the same level. In particular, better inhibitory effects than adalimumab were confirmed for S100A7, BD2, and LCN2. The combined treatment group of IL-17RA / RC-Fc and TNFR2-Fc was confirmed to have better inhibitory effects than the single treatment group. When comparing IL-17RA / RC-Fc-TNFR2, a multi-targeting fusion protein that can simultaneously target IL-17AA, IL-17AF, and IL-17FF or TNFα prepared as an embodiment of the present invention, with the single or combined treatment groups of each inhibitor, a volume-dependent anti-inflammatory effect was confirmed in each psoriasis factor, including S100A7, CCL20, BD2, LCN2, CXCL8, and IL-36γ. Figure 8 (Columns 7-9), especially the combined treatment group of IL-17RA / RC-Fc and TNFR2-Fc ( Figure 8The treatment group (column 6) or the combination treatment group of limizumab and adalimumab (column 12) showed sufficient inhibitory effect on IL-17RA / RC-Fc-TNFR2 at 20 ng / ml, and a better inhibitory effect than the individual combination treatment groups was confirmed at IL-17RA / RC-Fc-TNFR2 at 80 ng / ml. When converting the molar concentration of the inhibitor treatment groups showing the same inhibitory effect, the fusion protein, IL-17RA / RC-Fc-TNFR2 at 80 ng / ml was 392 pM, and the combination treatment group of limizumab (20 ng / ml) and adalimumab (50 ng / ml) was 473 pM. When treating IL-17RA / RC-Fc-TNFR2 as described in this invention, an effect was achieved with a smaller volume.

[0262] In a psoriasis-like model induced by co-stimulation with IL-17A and ITNFα, single-target inhibitors of IL-17A or TNFα have limitations. Furthermore, adalimumab increased the expression of 100A7 and BD2, while the inhibitory effect of bimecrolimus on CXCL8, IL-36γ, and CCL20 was not significant. However, the combined treatment showed superior inhibitory effects on all cytokines compared to individual single-antibody treatments. Therefore, to effectively control the inflammatory response, simultaneous inhibition of IL-17A and TNFα is necessary.

[0263] The IL-17RA / RC-Fc-TNFR2 of this invention exhibits volume-dependent inhibitory effects on S100A7, CCL20, BD2, LCN2, CXCL8, and IL-36γ. Sufficient inhibitory effects were confirmed in the 20 ng / ml treatment group, and superior inhibitory effects were observed in the 80 ng / ml treatment group compared to the combination treatment group with a single inhibitor. Psoriasis is an immune cell-mediated chronic inflammatory skin disease characterized by the infiltration of inflammatory monocytes, macrophages, eosinophils, inflammatory Th17 and Th1 cells, and the excessive proliferation and abnormal differentiation of keratinocytes. Reducing the expression of inflammatory cytokines, chemokines, and antimicrobial peptides is crucial. The multi-targeting fusion protein IL-17RA / RC-Fc-TNFR2 of this invention can effectively control various cytokines and chemokines involved in the excessive proliferation and pro-inflammatory response induced by the synergistic effect of IL-17 and TNFα by simultaneously controlling IL-17 and TNFα, thus making it a promising therapeutic agent for psoriasis.

[0264] Example 10: Evaluation of the anti-inflammatory effect of IL-17RA / RC-Fc-TNFR2 in environments similar to rheumatoid inflammatory diseases price

[0265] Rheumatoid arthritis is an inflammatory disease of the synovium accompanied by systemic symptoms. The inflammation begins with synovial cells and progresses to the destruction of cartilage and soft tissues. The inflammatory response around synovial fibroblasts and leukocytes and stromal cells in the periphery of inflammation can be amplified by the combined stimulation of IL-17 and TFNα. In particular, the inflammatory induction effect of IL-17F alone is not significant, but if IL-17A and IL-17F act simultaneously, they will synergistically increase TNFα, IL-6, IL-8, CXCL1, CCL20, CXCL5, IL-23, E-selectin and EGR-1 through the MAPK family pathway. If TNFα is also present, it will induce a chronic inflammatory response through mRNA stabilization (Arnaud Hot and Pierre Miossec., Ann. Rheum. Dis. 70(5):727-32. 2011). In a rheumatoid arthritis (RA)-like cell model using the hFLS-RA (Cell application Inc.) cell line, to confirm the disease-improving effect of the multi-targeting fusion protein (IL-17RA / RC-Fc-TNFR2) of the present invention, IL-6 expression levels were evaluated by simultaneously treating hFLS-RA cells with TNFα and IL-17 (Bilal Osta et al. Front. Immunol. 6:151, 2015). Specifically, for rheumatoid hFLS, 2 x 10⁻⁶ cells were added to each well of a 96-well plate. 4 Cells were cultured in DMEM (Welgene, LM001-005) and 10% FBS (Gibco, 16000044) (24 h, 37°C). To induce a renal syndrome (RA) model on day two, cells were treated with IL-17A, TNFα cytokines, and single-targeting fusion proteins of IL-17RA / RC-Fc and TNFR2-Fc, as well as the multi-targeting fusion protein IL-17RA / RC-Fc-TNFR2. Bimecrolimus (Antibody System, DHH28803) and adalimumab (Selleckchem, A2010) were used as control drugs at the stated concentrations. Figure 9(24 hours, 37°C). The next day, to obtain cell culture medium and confirm IL-6 expression, an ELISA was performed as follows: Human IL-6 DuoSet ELISA (R&D systems, DY206-05), DuoSet ELISA Ancillary Reagent Kit 2 (R&D systems, DY008B). 50 mL of carbonate buffer (pH 9.6, Na2CO3 (sigma, S7795, 0.1M):NaHCO3 (sigma, S8875, 0.1M) = 1:1) was prepared as the coating buffer. 1 μg / mL of recombinant IL-6 protein was added to the coating buffer and spread at 40 μl / well onto 96-well plates overnight at 4°C. The next day, the plates were washed three times with 200 μl / well of ELISAATBS buffer (1X TBS and 0.1% Tween 20). After dilution in 100 μl / well of ELISATBS buffer supplemented with 5% BSA, the plate was incubated at room temperature for 1 hour, followed by washing three wells with 200 μl / well of ELISATBS buffer. Then, the plate was incubated for 2 hours after dispensing 100 μl / well of blocking buffer. The IL-6 standard curve concentrations were based on 9.38 pg / mL, 18.8 pg / mL, 37.5 pg / mL, 75 pg / mL, 150 pg / mL, 300 pg / mL, and 600 pg / mL. The HRP-bound secondary antibody was treated with 200 μl / well of ELISATBS buffer and reacted for 1 hour at room temperature. After washing five times with ELISATBS buffer, the plate was allowed to react with 100 μl of TMB solution at room temperature, followed by the addition of 100 μl of stop solution. For ELISA measurements, absorbance was measured at 540 nm and 450 nm using a BioTek Synergy HTX microplate reader. To evaluate the anti-inflammatory effect of IL-17RA / RC-Fc-TNFR2 protein, a concentration-dependent reduction in IL-6 expression patterns was observed after confirming the IL-6 expression pattern. Figure 9 (Columns 9, 10, and 11). Especially in the IL-17RA / RC-Fc-TNFR2 treatment group ( Figure 9 In column 11), the reduction in IL-6 was equivalent to that in the control group treated with a combination of bimecrolimus and adalimumab as control drugs, thus confirming the improvement in inflammation. 9, column 14).

[0266] Example 11: tmTNFα binding capacity of the fusion protein IL-17RA / RC-Fc-TNFR2

[0267] Adalimumab and infliximab, as antibody therapies, also bind to transmembrane TNFα (tmTNFα) to regulate inflammatory responses via MAP / ERK signaling. They can promote the proliferation of regulatory T cells (T reg) by facilitating cross-linking between tmTNFα and TNFR2, and can induce immune tolerance. TNFα inhibitors not only neutralize soluble TNFα (sTNFα), but also promote reverse signaling through tmTNFα binding, and further enhance therapeutic efficacy through T reg cell activation. Etanercept, a TNFR fusion protein, does not exert a greater effect through tmTNFα compared to antibody therapies, and therefore may be less effective than antibody therapies in some diseases.

[0268] In the IL-17RA / RC-Fc-TNFR2 fusion protein of this invention, to confirm that TNFR2, as a TNFR2 mutant with multiple mutant combinations introduced into wtTNFR2, possesses tmTNFα binding ability, tmTNFα-expressing cells were prepared. To prepare tmTNFα-IRES-puro lentivirus, HEK-293T (1.2 x 10⁻⁶) was injected into 90 mM culture dishes. 7Cells were cultured (24 hours, 37°C). The next day, after changing to fresh cell culture medium at 37°C, the cell lines were transfected with DNA vectors (pLVXtmTNFα-IRES-puro, pCMV VSVG, pCMV delta8.2 vector; 2:1:2, mixed with 15 μg PEI, source: Korea Atomic Energy Hospital) (2–4 hours, 37°C). Afterward, the culture medium was changed to 13 mL of fresh cell culture medium. The culture supernatant from the 72-hour mark was collected in a conical tube (50 mL) and centrifuged (500 g, 5 minutes) to obtain the virus. Impurities were removed using a 0.45 μm filter, and the virus was dispensed 1 mL into EP tubes and stored at -80°C. Subsequently, to confirm the binding capacity of IL-17RA / RC-Fc-TNFR2 and tmTNFα as test drugs, HeLa cell lines (human cervical cancer cell lines) were first prepared by culturing in DMEM (Welgene, LM001-05) and 10% FBS (Gibco, 16000044) (24–48 hours, 37°C). To passage the cells in 12-well plates, after removing the FBS from the cell culture medium with 10 mL of 1x DPBS, the cells were treated with 0.53 mM EDTA (10 min, 37°C) and trypsin-EDTA (2 min, 37°C), followed by the addition of 2% FBS and low-calcium DMEM. The single-cell suspension was then centrifuged (1200 rpm, 5 min, 4°C). During this process, coverslips conforming to each well were thoroughly sterilized using an alcohol lamp before being placed into the wells, using the 12-well plate as a reference. Subsequently, cells were resuspended in fresh DMEM cell culture medium to ensure cell rehydration and then dispensed into each well (1 x 10⁶ cells / well). 5Stabilization was achieved by adding cells / mL (24 hours, 37°C). Subsequently, to transfect the prepared tmTNFα-IRES-puro lentivirus, 8 μg / mL polybrene transfection reagent (Merck, TR-1003-G) was prepared as a 500 μl lentivirus mixture and mixed 1:1 with 500 μl of cell culture medium (total 1 ml) and placed into wells for incubation (24 hours, 37°C). The next day, the virus mixture was removed, and the cells were washed three times with 1X DPBS before being placed in 500 μl of cell culture medium for maintenance (24 hours, 37°C). As control drugs, adalimumab (Selleckchem, A2010), used as a TNFα antibody therapeutic agent, and IL-17RA / RC-Fc-TNFR2 (T1-T4-14), used as the test drug, were administered at 1 μg / mL and cultured for 6 hours (37°C). After stabilization in cell culture medium for 18 hours, for fluorescence staining, the cell culture medium was removed and the cells were washed three times with 1x DPBS. 4% PFA (paraformaldehyde) fixative was then added to each well and incubated at room temperature for 30 minutes. Next, the PFA treatment solution was removed and the cells were washed three times with 1x DPBS. The cells were then incubated at 4°C for 1 hour with blocking buffer (1% BSA and 0.1% NaN 3 in 1x DPBS). For fluorescence staining, a primary antibody (Adalimumab; IgG1, T1-T4-14; Fc antibody) diluted 500x in cell culture medium was added to the well (24 hours, 4°C). For secondary antibody treatment, the cell culture medium was removed and the cells were washed three times with 1x DPBS. A secondary antibody (Donkey anti-mouse IgG Alexa fluor 568) diluted 500x in cell culture medium was added to the well. After three washes with 1 mL of 1x DPBS, and after the coverslip containing the cells in the well plate adhered to the slide, the cells were treated with DAPI solution and antifluorescence quenching mounting media. Next, to fix the coverslip to the slide, nail polish was applied around the edges of the coverslip, and it was allowed to dry within a specified time. The results were then observed using a Lionheart FX Automated Microscope (BioTek). The results showed that, when the expression of tmTNFα (GFP) and the antibody (Alexa fluor 568; RFP) for IL-17RA / RC-Fc-TNFR2 was monitored using the Bicistronic vector shown in Figure 10a, the yellow color was confirmed. In the four columns of Figure 10c, the yellow fluorescence intensity was greater than that of adalimumab, the control drug.The results confirmed that the tmTNFα binding of IL-17RA / RC-Fc-TNFR2 was at the same level as that of the antibody therapy, and confirmed that it could achieve the same level of promotion of tmTNFα reverse signaling as the antibody therapy, as well as the induction of Treg cell activity and proliferation to achieve an additional inflammatory control response. The tmTNFα sequence information (SEQ ID NO: 82) inserted into the pLVX tmTNFα-IRES-ZsGreen1 vector is summarized in Table 17. [Table 17]

[0269]

[0270] Example 12: Immunotherapy using the fusion protein IL-17RA / RC-Fc-TNFR2 from human peripheral blood mononuclear cells (PBMCs) virulence evaluation

[0271] The phenomenon of inducing an immune response when high-molecular-weight protein drugs enter the body is called immunogenicity. For example, if antibodies act as immunogens in our bodies, they will induce both cellular and humoral immune responses. Anti-drug antibodies (ADAs) in cellular immune responses will affect the efficacy, stability, pharmacokinetics, and pharmacodynamic evaluation results of the drug. Therefore, when developing high-molecular-weight protein drugs, their analysis should be performed concurrently. In particular, for autoimmune disease treatments, which are chronically administered drugs, the generation and duration of ADAs and the clinical impact of antibodies must be considered. To confirm the immunogenicity potential of IL-17RA / RC-Fc-TNFR2, the multifusion protein of this invention, evaluations were performed using both in silico and in vitro methods, confirming the low immunogenicity of IL-17RA / RC-Fc-TNFR2.

[0272] The fusion protein IL-17RA / RC-Fc-TNFR2 of this invention uses hIL-17RA, hIL-17RC, and hTNFR2 as binding mocies in a manner that neutralizes three targeting ligands (IL-17A, IL-17F, and TNFα). Mutant combinations were introduced into each binding moiety to improve the affinity for the ligands, thereby creating a triple-specific fusion protein. To evaluate the immunogenicity of the fusion protein IL-17RA / RC-Fc-TNFR2, the inventors conducted the evaluation in two steps: in silico immunogenicity prediction and in vitro analysis using five types of human peripheral blood cells. The in silico immunogenicity analysis utilized the IEMB (Immune Epitope Database). IEDB is a database operated with the support of the National Institute of Allergy and Infectious Diseases (NIAID), an institution under the National Institutes of Health (NIH) in Korea. It catalogs and continuously updates experimental data on antibodies and T-cell epitopes from studies in humans, primates, and other animal species, focusing on infectious diseases, allergies, autoimmune responses, and organ transplantation, ensuring access to the latest information. The IEDB server uses analytical methods that improve the accuracy of T-cell epitope predictions and are widely used worldwide for in silico immunogenicity analysis. T-cell MHC Class II epitopes were analyzed using a consensus method that represents the average of results from the "recommended methods" (NN-align, SMM-align, and CombLib / Sturniolo method) derived from IEDB (http: / / www.iedb.org / ) (Figure 11a). During the analysis, the input amino acid sequence is divided into all possible 15a.a. peptides (9 binding sequences and 6 side sequences). The predicted values ​​for the 15a.a. peptides by various analytical methods are then compared to the random score distribution of a large set of random peptides, thus transforming the results into statistical percentile rank. The median percentile rank of the analytical method becomes the final predicted value. A low percentile rank indicates a high predicted value, i.e., high MHC Class II receptor affinity.The MHCclassII epitope prediction system simplifies and visualizes the tabular results provided by IEDB. The X-axis represents the 27 HLA types, and the Y-axis represents the input amino acid sequence. The final prediction results of the comprehensive low analysis are represented by different colors (red: High; orange: Medium; yellow: Low; white: None) according to the degree of immunogenicity (Figure 11b).

[0273] According to the insilico immunogenicity analysis results, the two fusion proteins of IL-17RA / RC-Fc-TNFR2, T1-T4-14 and T1-T4-41, showed intermediate levels of immunogenicity at five sites (Figure 11b). The insilico immunogenicity analysis method predicts the likelihood of a specific peptide binding to MHC class II molecules and tends to over-predict. Therefore, to confirm whether the components predicted as highly immunogenic in the insilico immunogenicity analysis actually induce high immunogenicity in humans, an in vitro immunogenicity assay was performed to check for cell activation. The in vitro immunogenicity evaluation method uses human peripheral blood mononuclear cells (PBMCs) or dendritic cells (DCs) differentiated from PBMCs and T cells to predict the likelihood of clinical immunogenicity induction through cytokine analysis based on T cell proliferation or activation, including IFNγ and IL-2. To isolate cells from PBMCs, PBMCs were dissolved in an Automated Cell Thawing System (BioCision, 13-900-314) and then spin-down at 300g for 5 minutes in a centrifuge (Peripheral blood mononuclear cells (PBMCs), source: Catholic University Hematopoietic Cell Bank). After removing the supernatant, the cell particles were resuspended in AIM-V medium (GIBCO, 12055-083) and seeded in 100mM cell culture dishes. After DNase treatment, cells were cultured at 37°C in a 5% CO2 incubator. The following day, all cells were centrifuged (300g, 5 minutes) and resuspended in running buffer. FcRBlocking reagent (Miltenyi Biotec, 130-059-901) was added and the mixture was maintained at 4°C for 5 minutes. CD14 MicroBeads (Miltenyi Biotec, 130-050-20) were then added and the mixture was maintained at 4°C for 15 minutes. Finally, running buffer (Miltenyi Biotec, 130-091-221) was added, and the cells were centrifuged at 300g for 10 minutes at room temperature to ensure effective cell separation.After resuspending in cell culture medium, CD14- and CD14+ cells were obtained using an LS column. The collected CD14- cells were then placed into CD4 MicroBeads (Miltenyi Biotec, 130-045-101) and CD8 MicroBeads (Miltenyi Biotec, 130-045-201) and maintained at 4°C for 15 minutes. After adding electrophoresis buffer, the cells were gently spin-down for 10 minutes at room temperature and 300g, the supernatant was removed, and the cells were resuspended in electrophoresis buffer. Subsequently, the cells (CD4-CD8- cells) were collected via flowthrough using an LS column (Miltenyi Biotec, 130-042-401) and washed three times with electrophoresis buffer. Cells (CD4+CD8+ cells) present in the LS column were obtained using a plunger. The collected CD14-CD4-CD8- cells were loaded onto an LS column to collect the effluent (CD4+CD8+ cells). The collected CD4+ / CD8+ cells were then stored in liquid nitrogen for future use in CD4+ / CD8+ T cell activation experiments. CD14+ cells were cultured in GM-CSF (JW creagene, HGM-100) and AIM-V medium containing IL-4 (JW creagene, HI4-100) for 72 hours at 37°C. Afterwards, cells were cultured in AIM-V medium containing GM-CSF and IL-4 for an additional 3 days (72 hours at 37°C).

[0274] After a total of 6 days of dendritic cell (DC) differentiation, the resulting cells were supplemented with AIM-V medium containing GM-CSF and IL-4. Uninduced DCs were induced (24 h, 37°C) by treatment with T1-T4-14 and T1-T4-41 as test substances and by PPD (Thermo Fisher, 7600060) as a positive control. Antigen-induced DCs were also cultured (24 h, 37°C) in maturation medium (IL-6; JW Creagene, HIL6-100, IL-1β; JW Creagene, HIL-1B-100, TNFα; JW Creagene, HTNA-100, PGE2; Sigma, P0409, AIM-V medium). During DC cell culture, CD4+ / CD8+ cells stored in liquid nitrogen were thawed using an automated cell thawing system, followed by centrifugation at 300g for 5 minutes to obtain cells. After removing the supernatant, the cells were resuspended in AIM-V medium and aliquoted into 60mM culture dishes for culture (24 hours, 37°C). The next day, to confirm CD4+ / CD8+ T cell proliferation, 5μM CSFE (Invitrogen, C34554) was used for 10 minutes at 37°C. CFSE-stained CD4+ / CD8+ T cells were washed three times at room temperature (24 hours, 37°C) under AIM-V medium, 300g, 5 minutes. After obtaining antigen-induced DCs, they were washed three times at room temperature, 300g, 5 minutes using AIM-V medium. 2 x 103 5 CD4+ / CD8+ T cells and maturation 1x 10 4 DCs were aliquoted into 96-well plates. CFSE-stained CD4+ / CD8+ T cells were treated with anti-CD3 antibody (Biolegend, 300438) and anti-CD28 antibody (Biolegend, 302934). After 7 days of co-culture, cells were obtained for reaction in the dark at 4°C for 30 minutes after resuspending in antibody solutions (CD3 and CD4 or CD8 antibodies). Cells were washed with electrophoresis buffer and centrifuged at 300g for 5 minutes. After removing the supernatant, cells were resuspended in electrophoresis buffer, and CD4+ / CD8+ T cell proliferation was analyzed using a FACS Canto-II instrument (BD, 338962) (Figs. 12a, 12b, and 12c).

[0275] For in vitro immunogenicity evaluation, the positive control group used anti-CD3 / anti-CD28, and the internal control group used PPD. The test substance IL-17-RA / RC-Fc-TNFR2 (T1-T4-14, T1-T4-41) was treated three times at a single concentration (2 μg / ml) and the results were compared. The immunogenicity of the test substance IL-17-RA / RC-Fc-TNFR2 (T1-T4-14, T1-T4-41) is usually determined using the Stimulation Index (SI) value, which assesses the effect of the drug on the activity of immune cells. The SI value is ensured by dividing the proliferation rate of T cells confirmed under each condition by the proliferation rate of the control group. Generally, if the SI value reaches 2 or higher, which is the baseline value for measuring the effects of drug-based lymphocyte proliferation, it is considered to induce immunogenicity (Stimulation Index (SI) = test well / baseline; an SI ≥ 2 is considered ADA positive). As a positive control for ADA, for PPD, immunogenicity was confirmed if the SI value for MHC Class I and MHC Class II was above 2 in 5 PBMCs. Using the average SI value obtained after three repeated treatments of the test substance as a benchmark, IL-17-RA / RC-Fc-TNFR2 (T1-T4-14, T1-T4-41) had an SI value below 2 for MHC Class I and MHC Class II in the 5 PBMCs, ultimately confirming that immunogenicity was not induced.

[0276] Example 13: IL-17RA / RC-hybrid fusion protein (IL-17RA / RC-Fc) in peripheral blood mononuclear cells Validity evaluation

[0277] Autoimmune diseases are chronic illnesses. The number and characteristics of immune cells in PBMCs differ from those in healthy individuals. The lesion sites and PBMCs in patients also differ from those in healthy individuals. In psoriasis and psoriatic arthritis, IL-17A is associated with these conditions. + CD8 + The severity of T-cell disorders is correlated with the level of IL-17A. Furthermore, IL-17A is present in damaged tissues or joints. + CD8 +The proportion of T cells is locally elevated (Xiaofei Xu, et al., Arthritis Rheumatol. 72(8):1303-1313, 2020). IL-17A and IL-17F are expressed in psoriasis and psoriatic arthritis lesions, with IL-17F being approximately 32 times higher than IL-17A in psoriatic lesions (Helena Iznardo et al., Ther. Adv. Chronic. Dis. 12:1-16, 2021). IL-17F plays a role in amplifying pro-inflammatory responses; inflammatory cytokines are amplified when IL-17F is expressed in conjunction with IL-17A or IL-17A in conjunction with ITNFα. In a clinical efficacy study of bimecrolimus in the joints and skin of patients with psoriatic arthritis (NCT01087788), simultaneous neutralization of L-17A and IL-17F resulted in more effective suppression of the inflammatory response compared to neutralization of IL-17A alone. Bimecrolimus demonstrated a better ACR20 response (8 weeks) than secukinumab (54% vs. 15%) and icorizumab (62% vs. 30%), which neutralized IL-17A only in 80% of cases compared to placebo (Glatt S etal. Ann. Rheum. Dis. 77(4):523-532, 2017). In psoriasis patients' PBMCs, the regulatory function of cytokine production was analyzed based on treatments with the experimental substances IL-17RA / RC-Fc (IL-17A / F inhibition), TNFR2-Fc (TNFα inhibition), IL-17RA / RC-Fc and TNFR2-Fc combined treatment, IL-17RA / RC-Fc-TNFR2 (IL-17A / F and TNFα simultaneous inhibition), and control drugs (Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL control group).

[0278] Patients with psoriasis (n=4) were recruited and blood donations were accepted to obtain peripheral blood mononuclear cells (Seoul St. Mary's Hospital, Catholic University of Medicine, Approval No. KC21TNSI0375). 2 μg / mL of anti-CD3 was applied to 48-well plates at 5 x 10⁻⁶ cells / well. 5The isolated PBMCs were aliquoted into cells / well / 500 μl. Additionally, the test samples were treated with 100 ng / mL LPS according to the respective treatment conditions and incubated (72 h, 37°C). Subsequently, the culture supernatant was separated to perform IL-17A (human IL-17A Duoset ELISA, R&D system DY317) and IL-17F (human IL-17F Duoset, R&D system DY1335B) ELISAs. (Absorbance measurements: Molecular Devices) The results showed that the production of IL-17A in both the experimental substance group and the control drug group was reduced in terms of the average analysis of PBMC Ps2-5 results and the percentage change in production in psoriasis patients. Specifically, the reduction was 96.3% for IL-17RA / RC-Fc (0.5 μg / mL) + TNFR2-Fc (0.5 μg / mL), and for IL-17RA / RC-Fc-TNFR2 at levels of 0.1, 0.5, 1.0, and 2.5 μg / mL. The reductions of 91.9%, 89.85%, 88.3%, and 96.5% were observed, with a 97.4% reduction confirmed in the combined treatment group of the control drug Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL (Figure 13). Regarding the evaluation and analysis of PBMC Ps2–5 results and the percentage change in production in psoriasis patients, IL-17F production showed a reduction effect in all groups, including the test substance group and the control drug group. In the IL-17RA / RC-Fc-TNFR2 1.0 and 2.5 μg / mL groups, reductions of 67.3% and 100% were observed, respectively, with a 100% reduction confirmed in the repeated treatment group of the control drug (Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL) (Figure 13). Furthermore, in IL-17RA / RC-Fc-TNFR2 (simultaneous inhibition of IL-17A / F and TNFα), IL-17A, IL-17F, IL-21, IL-22, and TNFα can be identified. The production of IFN-γ was reduced. Specifically, in IL-17F inhibition, compared to single inhibition of IL-17RA / RC-Fc, an effect was confirmed in simultaneously inhibiting L-17RA / RC-Fc-TNFR2, which simultaneously inhibits IL-17A / F and TNFα. Regarding TNFα inhibition, an effect was also confirmed in simultaneously inhibiting IL-17RA / RC-Fc-TNFR2, which simultaneously inhibits IL-17A / F and TNFα. In Example 7 of the present invention (SEAP analysis), the IL-17F neutralization capacity of IL-17RA / RC-Fc-TNFR2 was lower than that of bimekizumab, but the inhibitory capacity against IL-17F in PBMCs of psoriasis patients was reduced by 100%, showing the same level of reduction as the 100% reduction in the control drug (Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL) combined treatment group. Compared to the combination treatment group with imijezumab and adalimumab, which have high affinity for IL-17A, IL-17F, or TNFα co-expressed in PBMCs of psoriasis patients, this treatment achieved equivalent inhibitory effects by simultaneously inhibiting IL-17RA / RC-Fc-TNFR2 dehumanization of IL-17A, IL-17F, or TNFα. This indicates that by screening for and inhibiting pathogenic cytokines in the psoriasis and autoimmune disease environment, inflammatory diseases can be effectively improved with minimal side effects.

[0279] Example 14: IL-17A and TNFα neutralization of IL-17RA / RC-Fc-mTNFR2 in a psoriasis-induced animal model Capability Analysis

[0280] To evaluate the neutralizing capacity in animal models inducing psoriasis, the following experiments were performed. Humanized double-knock-in mice expressing human IL-17A and IL-17F were prepared by removing mouse IL-17A and IL-17F using CRISPR / Cas9 technology (Figure 14a). The IL-17RA / RC-Fc-TNFR2 of this invention is a multi-targeting fusion protein targeting human IL-17A, human IL-17F, and human TNFα. The C57BL / 6hIL-17A / 17F humanized double-knock-in mouse expresses human IL-17A and human IL-17F, but the TNFα is mouse TNFα (mTNFα). Therefore, in a psoriasis model using humanized double-knock-in mice, it is difficult to evaluate the effect of simultaneously inhibiting all three hIL-17A, hIL-17F, and hTNFα ligands, and the resulting therapeutic effect. Therefore, in order to evaluate the effect of IL-17RA / RC-Fc-TNFR2 in double knock-in mice, the inventors replaced the hTNFR2 ECD, which serves as the hTNFα binding moiety of IL-17RA / RC-Fc-TNFR2, with an mTNFR2 ECD to target hIL-17A, hIL-17F, and mTNFα expressed in humanized double knock-in mice, thereby preparing IL-17RA / RC-Fc-mTNFR2. To confirm that IL-17RA / RC-Fc-mTNFR2 and T1-mT4-35 (SEQ ID NO: 32) respond to hIL-17A, hIL-17F, and mTNFα, the experiments in Example 8 were performed. The IL-17RA / RC-Fc-mTNFR2 of the present invention achieved EC50 values ​​of 80.31 ng / ml for human IL-17A and 90.74 ng / ml for human IL-17F, and 71.01 ng / ml for mouse TNFα, confirming similar affinity for each cytokine (Figure 14b).

[0281] To induce psoriasis, hair was shaved (from the neck to the buttocks with scissors, and depilatory cream was applied; after 40 seconds, it was wiped dry with a towel). 60 mg of Imiquimod (IMQ; Aldera Cream) was applied to the shaved areas (back) of each group in the overall experimental group (G1–G4) shown in Figure 14c (once daily for 5 treatments). As an additional disease model test substance, 200 μl of LPS (lipopolysaccharide of Escherichia coli, O111:B4) was administered intraperitoneally once on the last day of the experiment (day 5). Subsequently, IL-17RA / RC-Fc-mTNFR2, the test substance of this invention, was administered at 1 mg / kg (once or twice daily, starting the day after IMQ application). Blood was collected from each individual and allowed to stand at room temperature for 10–20 minutes, followed by centrifugation (2500 RPM, 15 minutes) to separate plasma from the supernatant, which was then cryopreserved. Blood samples were collected twice, once before IMQ application and once on the last day of application (day 5). Standard curves for IL-17A and TNFα were obtained using ELISA (Duoset ELISA, R&D system), thus confirming the neutralizing ability of hIL-17RA / RC-Fc-mTNFR2 on cytokines (hIL-17A and mTNFα). Regarding the confirmation of in vivo neutralizing capacity in mice, ELISA results from mouse blood samples at the end of the experiment (day 5) confirmed that the neutralizing capacity against mouse TNFα was most significantly inhibited in G4 (C57BL / 6hIL-17A / 17F double KI mice + hT1-mT4-35) mice (G3; 7.30 pg / mL, G4; 0.0 pg / mL). In terms of neutralizing human IL-17A, compared to G3 (C57BL / 6hIL-17A / 17F double KI mice), the neutralizing capacity in G4 (C57BL / 6hIL-17A / 17F double KI mice) was significantly lower. The inhibition was significantly reduced in IL-17RA / RC-Fc-mTNFR2 (hT1-mT4-35). (G3: 140.79 pg / mL, G4: 47.89 pg / mL) (Figure 14d). The affinity analysis results of IL-17RA / RC-Fc-mTNFR2 (hT1-mT4-35) for IL-17A and mTNFα are summarized in Table 18, and the G1 to G4 information analysis in the psoriasis analysis of C57BL / 6hIL-17A / 17F double Knock-In mice is summarized in Table 19. [Table 18]

[0282] Analysis results of mouse psoriasis [Table 19]

[0283] Experimental conditions (G1 to G4) information Classification Experimental conditions G1 C57BL / 6 wild-type mice G2 C57BL / 6wild-type mice+"hIL-17RA / RC-Fc-mTNFR2" G3 C57BL / 6hIL-17A / 17F double KI mice G4 C57BL / 6hIL-17A / 17F double KI mice+"hIL-17RA / RC-Fc-mTNFR2"

[0284] Example 15: Cryo-EM observation of IL-17RA / RC-Fc-TNFR2 mixed fusion protein

[0285] Three-dimensional transmission electron microscopy (3-dimensional TEM) is a technique for studying the structure of protein complexes through biological image analysis. High-resolution three-dimensional structural analysis allows for the analysis of the structures of individual domains of the IL-17RA / RC-mixed fusion protein, as well as their interactions with binding domains and with target proteins. De novo structural analysis confirmed the interaction between the IL-17RA / RC-Fc-TNFR2 fusion protein and its target protein. Specifically, for negative staining, 5 μl of the IL-17RA / RC-mixed fusion protein sample was added to a carbon-coated grid fixed with automated closed clamps (Dumont, Swiss) and stained negatively with 1% uranyl acetate. Excess staining solution was removed by wiping the grid edges with filter paper (Whatman, UK), and the grid was dried for 10 seconds before experiments were performed using a Technai 10TEM (FEI, USA) running at 100 kV. Images were recorded at a magnification of 0.32 nm / pixel using an Ultrascan 1000 CCD (Gatan, US). Image analysis of the automated data for negative staining utilized a total of 7084 IL-17RA / RC-Fc-TNFR2 particles. Image alignment, reconstruction, 3D volume rendering, and visualization were performed, with the average grade calculated using the RELION 3.1 program. In the initial classification, images that were not compact or poorly stained were excluded; folded images that could be viewed from the left / right side were selected for IL-17RA / RC-Fc-TNFR2 hybrid fusion protein particle analysis. The results, as shown in Figure 15a, confirm the successful particle imaging of the IL-17RA / RC-Fc-TNFR2 protein from multiple angles, based on which 3D reconstruction was performed (Figure 15b). In the electron density plot, binding models for IL-17RC and IL-17F, and binding models for TNFR2 and TNFα, stored in the current PDB (Protein Data Bank), were fitted using superimposition (Figure 15c). The binding models for IL-17RC and IL-17F, and the binding models for TNFR2 and TNFα, were obtained using PDBID:6HG9 and PDBID: 3ALQ. Due to the longer, softer linkers of the TNFR2 and TNFα binding regions, the electron density maps are thin, resulting in even thinner structures in 3D reconstruction. Furthermore, for the TNFR2 ECD, the unbound substrate morphology is difficult to visualize in the electron density map due to the soft linkers; however, as shown in Figure 15c, the binding of TNFR2 and TNFα is well-fitted into the electron density map. Therefore, the IL-17RA / RC-mixture and TNFR2 ECD of the IL-17RA / RC-Fc-TNFR2 mixed fusion protein were confirmed to bind to the substrates in a 1:1 ratio. Using Fc as a baseline, the longer IL-17RC portion was identified as the upper part.

[0286] Example 16: Attachment of N-glycans to the IL-17RA / RC-Fc-TNFR2 mixed fusion protein

[0287] The glycan structure of Fc-fusions and structural changes can alter structural domains. In therapeutic Fc-fusion proteins, changes in specific glycosylation can also affect the pharmacokinetics (PK) and pharmacodynamics (PD) of the molecule. Therefore, the inventors investigated the possibility of constructing an expression system to improve productivity by confirming whether the IL-17RA / RC-Fc-TNFR2 mixed fusion protein (T1-T4-14, SEQ ID NO: 1) is attached to N-glycans. In the sequence of the Fc fusion protein prepared in the described examples, N-glycan site determination was performed using peptide mapping to confirm whether N-glycans were theoretically attached. After adding the prepared Fc fusion protein to achieve a concentration of 10% 50% TCA (trichloroacetic acid) solution, the reaction was carried out at -20°C for 30 minutes. After centrifugation at 13000 rpm for 10 minutes, the supernatant was removed and a desalting step was performed. The desalted Fc fusion protein was cleaved using different hydrolases (trypsin, chymotrypsin), and the attachment of the cleaved peptides was confirmed by the attachment rate (%) of the N-glycan site using a combined high-performance liquid chromatography-essential liquid chromatography-mass spectrometry (UPLC-ESI-MS) analysis apparatus (Figures 16a and 16b). The cleaved peptides were separated by C18 reverse gradient chromatography, performed for 70 minutes using a gradient condition of mobile phase A (purified water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid). The gradient condition of mobile phase B was performed as follows: 2% from 0 to 5 minutes, 30% from 5 to 50 minutes, 95% from 51 to 61 minutes, and 2% from 62 to 70 minutes. The separated peptides were ionized by a mass analyzer, and the molecular weight of the ionized peptides was monitored and analyzed in the range of 200 m / z to 2000 m / z. The results of N-glycosylation site and percentage analysis performed using UPLC-ESI-MS were integrated with the results of two hydrolases, identifying a total of 12 N-glycosylation sites. Only the N22 site of the IL-17RA / RC-Fc-TNFR2 mixed fusion protein was not identified. The results of N-glycosylation site identification for different hydrolases are summarized in Table 20. [Table 20]

[0288] N-glycan site determination results

[0289] Example 17: Glycan sialylation of IL-17RA / RC-Fc-TNFR2 mixed fusion protein

[0290] A significant portion of biopharmaceuticals are recombinant proteins, with over 70% being glycoproteins. Human-like glycosylation is crucial for therapeutic efficacy, particularly in reducing immunogenicity and increasing serum half-life. The degree of sialylation of glycosylated proteins (glycoproteins) can be an important characteristic affecting the protein's plasma half-life or safety. In drug-producing cells, recombinant expression of sialylated proteins may be difficult in organisms lacking enzymes for sialylation or in naturally occurring proteins with non-human sialylation patterns (Xiaotian Zhong et al. Methods. Mol. Biol. 12(3):53. 2022). If in ExpiCHO-S TM The introduction of galactosyltransferase-6 (GT6) into the system promotes the synthesis of sialylated glycans. For IL-17RA / RC-Fc-TNFR2, Expi CHO-S was used in the same manner as in Example 1, according to ExpiCHO. TM The expression system kit was followed to induce the expression of the fusion protein. For this experiment, the ratio of IL-17RA / RC-Fc-TNFR2 fusion protein DNA to IGT6 DNA was 5:4. The GT6 DNA was Beta-1,4-galactosyltransferase 6 (B4GALT6), using a Lenti-ORF clone of B4GALT6(mGFP-tagged)-Human UDP-Gal (origene CAT#:RC210213L4). IL-17RA / RC-Fc-TNFR2 fusion protein DNA and IGT6 DNA (T1-T4-14: 50 μg, GT6: 40 μg) were transfected, and ExpiFectae was added 18 hours later. TMThe CHO transfection kit includes an enhancer and feed, with additional additions of 200mM MUridine (GLENTHAM, Cat: GP7250-5G), 0.4mM MnCl2 (sigma, Cat: 244589-50G), 1M galactose (GLENTHAM, Cat: GC7360-500G), and 1.55ml of ManNAc (65mM N-acetylmannosamine, Glentham, Cat: GK3319). The mixture was then cultured at 8% CO2, 32°C, and 120rpm. On day 4, 2mL and 1.55mL of UMnG and ManNAc were added along with the enhancer and feed, and the mixture was cultured at 8% CO2, 32°C, and 120rpm. Harvesting and production were completed on day 7. Figure 17a shows the IL-17RA / RC-Fc-TNFR2 hybrid. A schematic diagram of the production timeline for the glycan sialylation of the fusion protein. The IL-17RA / RCFc-TNFR2 fusion protein, which underwent glycan sialylation in the same manner as in Example 3, was purified using Protein A affinity. The glycan-sialylated IL-17RA / RCFc-TNFR2 fusion protein and the IL-17RA / RCFc-TNFR2 fusion protein were loaded into a Superdex 200 (Cytiva) for separation and purification (HiLoad16 / 600 Superdex 200pg 1x120ml (Cytiva, 28989335), Buffer: 1x PBS, 0.005% tween). 20). Due to the change in protein size in the SEC results, the peak shifted forward (Fig. 17b, Fig. 17c). Western blot results confirmed that the protein performing glycan sialylation was larger (Fig. 17d). The affinity of IL-17RA / RC-Fc-TNFR2 performing glycan sialylation to ligands IL-17A, IL-17F, and TNFα was analyzed. In the control group, the affinity of T1-T4-14 (SEQ ID NO: 1) generated from pBispec, T1-T4-14 generated from pcDNA3.4, and T1-T4-14 performing glycan sialylation to each ligand was analyzed by ELISA. The affinity of T1-T4-14 generated from pBispec, pcDNA3.4, or pcDNA3.4+GT6 to IL-17A, IL-17F, and TNFα was measured (Fig. 17e, Fig. 17f, and Fig. 17g).In IL-17A, the EC50 values ​​of T1-T4-14 were confirmed to be 75.18 ng / ml, 60.65 ng / ml, and 54.32 ng / ml, respectively. In IL-17F, the EC50 values ​​of T1-T4-14 were confirmed to be 70.20 ng / ml, 45.48 ng / ml, and 62.06 ng / ml, respectively. In TNFα, the EC50 values ​​of T1-T4-14 were confirmed to be 68.00 (ng / ml), 87.26 (ng / ml), and 44.34 (ng / ml).

[0291] To confirm the stability of the IL-17RA / RCFc-TNFR2 fusion protein in vivo, the amount of residual protein was measured after 4 hours of treatment in mouse (C57BL / 6) serum. The results showed that the introduction of GT6 increased the stability of the introduced sialylated T1-T4-14 by two times (14.99 μg / ml vs 28.94 μg / ml).

[0292] Example 18: Affinity of IL-17RA / RC-Fc-TNFR2 mixed fusion protein to TNFα

[0293] To confirm the disease-improving effect of TNFR2 neutralization based on the multi-targeting fusion protein (IL-17RA / RC-Fc-TNFR2) in a rheumatoid arthritis (RA)-like cell model using SW982 cells, the expression levels of IL-6 and MMP3 were evaluated by treating SW982 cells with human TNFα. In RA, inflammatory cells such as neutrophils and macrophages are recruited to the joints, leading to overexpression of inflammatory cytokines such as IL-1β, TNFα, IL-6, and CXCL8, resulting in joint structural damage. The cells were processed in 96-well plates with a density of 1 × 10⁻⁶ cells per well. 4Cells were cultured overnight (37°C) in DMEM (Welgene, LM001-005) and 10% FBS (Gibco, 16000044). The next day, a renal syndrome (RA) model was induced by treatment with 10 ng / ml TNFα cytokine. IL-17RA / RC-Fc-TNFR2 (T1-T4-14) protein and adalimumab (Selleckchem, A2010) as a control were simultaneously treated with TNFα at concentrations of 2.5 ng / ml, 10 ng / ml, 40 ng / ml, and 160 ng / ml (48 h, 37°C). After 48 h of treatment, the cell culture medium was removed, and ELISA was performed as follows to confirm IL-6 and MMP3 levels. The ELISA products used are as described below, and all materials were prepared according to the product descriptions: HumanIL-6 DuoSet ELISA (R&D systems, DY206-05), Human TotalmMP3 Duoset ELISA (R&D systems, DY513-05), and DuoSet ELISA Ancillary Reagent Kit 2 (R&D systems, DY008B). In the ELISA plates, each ELISA capture antibody was diluted in 1X Reagent Diluent (1% PBS in PBS, pH 7.2-7.4, 0.2 μm filtered, R&D systems, Catalog#DY995) at 100 μl / well and plated overnight at room temperature. The plates were washed three times with 300 μl / well of Yi1X ELISA wash buffer (0.05% Tween 20 in PBS, pH 7.2-7.4, R&D systems, Catalog#WA126). Add 100 μl / well of reagent diluent and incubate at room temperature for at least 1 hour to achieve blocking. After washing with 300 μl / well of ELISA wash buffer, add 100 μl / well of IL-6, MMP3 standard protein, and cell culture medium sample respectively, and incubate at room temperature for 2 hours (IL-6 is used after a 20-fold dilution, and MMP3 is used directly). Regarding the concentrations for the standard curve, according to the information provided, IL-6 concentrations are 9.38, 18.8 pg / mL, 37.5 pg / mL, 75 pg / mL, 150 pg / mL, 300 pg / mL, and 600 pg / mL, and MMP3 concentrations are 31.3 pg / mL, 62.5 pg / mL, 125 pg / mL, 250 pg / mL, 500 pg / mL, 1000 pg / mL, and 2000 pg / mL.The sample was washed three times with 300 μl / well of ELISA wash buffer. Detection antibody was added at 100 μl / well for each wash, and the reaction was allowed to proceed for 2 hours. After washing three times with 300 μl / well of ELISA wash buffer, the HRP-bound secondary antibody was treated and reacted at room temperature for 20 minutes. The sample was then washed five times with ELISA wash buffer. After allowing 100 μl of TMB solution to react at room temperature, 50 μl of stop solution was added. ELISA measurements were performed using a BioTek Synergy HTX Microplate reader, measuring absorbance at 540 nm and 450 nm. The results show that the TNFR2 neutralizing capacity of IL-17RA / RC-Fc-TNFR2 of this invention inhibits IL-6 and MMP3 at levels comparable to adalimumab (Figures 18a and 18b).

[0294] This invention has been described with reference to the above embodiments, but these are merely examples. Those skilled in the art will understand that various modifications and equivalent embodiments can be implemented. Therefore, the true scope of protection of this invention should be determined according to the technical concept outlined in the appended claims.

Claims

1. An IL-17RA / RC mutant hybrid protein, characterized in that, It contains amino acid substitutions selected from the group consisting of amino acid sequences that have more than 90% sequence homology with the amino acid sequence of SEQ ID NO:

69. i)Q267R; ii) T24D, N88D, D122G and Q267R; iii) T24D, F59V, N88D, D122G and Q267R; iv) L9P, T24D, F59V, N88D, D122G and Q267R; and v)L9P, T24D, F59V, N88D, D122G, A156P and Q267R.

2. The IL-17RA / RC mutant hybrid protein according to claim 1, characterized in that, Add sugar chains.

3. The IL-17RA / RC mutant hybrid protein according to claim 2, characterized in that, The glycan is selected from one or more of the group consisting of N-glycans, O-glycans, and sialylated glycans.

4. The IL-17RA / RC mutant hybrid protein according to claim 1, characterized in that, Select the group consisting of SEQ ID NO: 66 to SEQ ID NO:

69.

5. A polynucleotide, characterized in that, The IL-17RA / RC mutant hybrid protein according to claim 1 is encoded.

6. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 5.

7. A transformed cell, characterized in that, Introduce the expression vector according to claim 6.

8. A homodimer or heterodimer, characterized in that, It contains the IL-17RA / RC mutant hybrid protein as described in claim 1.

9. An IL-17RA / RC-Fc mutant hybrid protein, characterized in that, An antibody Fc domain is attached to the N-terminus or C-terminus of the IL-17RA / RC mutant hybrid protein according to claim 1.

10. The IL-17RA / RC-Fc mutant hybrid protein according to claim 9, characterized in that, The antibody Fc domain is a mixed Fc domain consisting of two or more homologous Fc cells.

11. The IL-17RA / RC-Fc mutant hybrid protein according to claim 10, characterized in that, The hybrid Fc domain is selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO:

65.

12. A polynucleotide, characterized in that, The IL-17RA / RC-Fc mutant hybrid protein according to claim 9 is encoded.

13. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 12.

14. A transformed cell, characterized in that, The expression vector according to claim 13 is introduced.

15. A homodimer or heterodimer, characterized in that, It contains the IL-17RA / RC-Fc mutant hybrid protein as described in claim 9. 16.i) A TNFR2 ECD mutant protein, characterized in that, It contains amino acid substitutions selected from the group consisting of amino acid sequences that have more than 90% sequence homology with the amino acid sequence of SEQ ID NO:

77. ii)S33A; iii) S33A and S36P; iv) S33A, S36P and R119S; v)S33A, S36P, R119S and N164S; vi) S33A, S36P, R119S, N164S and E210G; vii) S33A, S36P, R119S, N164S, E210G and F219I; viii) S33A, S36P, G75S, R119S, N164S, E210G and F219I; ix)G75S; and x)S33A, S36P, G75S, R119S and N164S.

17. The TNFR2 ECD mutant protein according to claim 16, characterized in that, At least one of i to ix is ​​a fusion compound interconnected by linking peptides.

18. The TNFR2 ECD mutant protein according to claim 16, characterized in that, Add sugar chains.

19. The TNFR2 ECD mutant protein according to claim 18, characterized in that, The glycan is selected from one or more of the group consisting of N-glycans, O-glycans, and sialylated glycans.

20. The TNFR2 ECD mutant protein according to claim 16, characterized in that, Select the group consisting of SEQ ID NO: 83 to SEQ ID NO:

94.

21. A polynucleotide, characterized in that, The TNFR2 ECD mutant protein according to claim 16 is encoded.

22. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 21.

23. A transformed cell, characterized in that, The expression vector according to claim 22 is introduced.

24. A homodimer or heterodimer, characterized in that, It contains the TNFR2 ECD mutant protein as described in claim 16.

25. A TNFR2 ECD-Fc mutant protein, characterized in that, The antibody Fc domain is attached to the N-terminus or C-terminus of the TNFR2 ECD mutant protein according to claim 16.

26. The TNFR2 ECD-Fc mutant protein according to claim 25, characterized in that, The antibody Fc domain is a mixed Fc domain consisting of two or more homologous Fc cells.

27. The TNFR2 ECD-Fc mutant protein according to claim 26, characterized in that, The hybrid Fc domain is selected from the group consisting of SEQ ID NO: 33 to SEQ ID NO:

65.

28. A polynucleotide, characterized in that, The TNFR2 ECD-Fc mutant protein according to claim 25 is encoded.

29. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 28.

30. A transformed cell, characterized in that, Introduce the expression vector according to claim 29.

31. A homodimer or heterodimer, characterized in that, It contains the TNFR2 ECD-Fc mutant protein as described in claim 25.

32. A triple fusion protein, characterized in that, The IL-17RA / RC mutant hybrid protein is linked to the N-terminus of the antibody Fc domain and the tumor necrosis factor (TNF) receptor 2 extracellular domain (ECD) is linked to the C-terminus of the antibody Fc domain. Alternatively, the tumor necrosis factor receptor 2 extracellular domain (ECD) is linked to the N-terminus of the antibody Fc domain and the IL-17RA / RC mutant hybrid protein is linked to the C-terminus of the antibody Fc domain.

33. The triple fusion protein according to claim 32, characterized in that, The IL-17RA / RC mutant hybrid protein contains amino acid substitutions selected from the group consisting of amino acid sequences that share more than 90% sequence homology with the amino acid sequence of SEQ ID NO:

69. i)Q267R; ii) T24D, N88D, D122G and Q267R; iii) T24D, F59V, N88D, D122G and Q267R; iv) L9P, T24D, F59V, N88D, D122G and Q267R; and v)L9P, T24D, F59V, N88D, D122G, A156P and Q267R.

34. The triple fusion protein according to claim 32, characterized in that, The tumor necrosis factor receptor 2 extracellular domain (ECD) comprises amino acid substitutions selected from the group consisting of amino acid sequences that share more than 90% sequence homology with the amino acid sequence of SEQ ID NO:

77. i)S33A; ii) S33A and S36P; iii) S33A, S36P and R119S; iv) S33A, S36P, R119S and N164S; v)S33A, S36P, R119S, N164S and E210G; vi) S33A, S36P, R119S, N164S, E210G and F219I; vii) S33A, S36P, G75S, R119S, N164S, E210G and F219I; viii)G75S; and ix)S33A, S36P, G75S, R119S and N164S.

35. The triple fusion protein according to claim 34, characterized in that, At least one of i to ix is ​​a fusion compound interconnected by linking peptides.

36. The triple fusion protein according to claim 32, characterized in that, Add sugar chains.

37. The triple fusion protein according to claim 36, characterized in that, The glycan is selected from one or more of the group consisting of N-glycans, O-glycans, and sialylated glycans.

38. The triple fusion protein according to claim 32, characterized in that, The tumor necrosis factor receptor 2 is selected from the group consisting of SEQ ID NO: 83 to SEQ ID NO:

94.

39. The triple fusion protein according to claim 32, characterized in that, Simultaneously targets IL-17A, IL-17F, and TNFα.

40. The triple fusion protein according to claim 32, characterized in that, The group consisting of the amino acid sequences of SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32 was selected.

41. A polynucleotide, characterized in that, The triple fusion protein according to claim 32 is encoded.

42. An expression carrier, characterized in that, It contains the polynucleotide as described in claim 41.

43. A transformed cell, characterized in that, Introduce the expression vector according to claim 42.

44. A homodimer or heterodimer, characterized in that, It includes the triple fusion protein according to claim 32.

45. A pharmaceutical composition for treating autoimmune diseases, characterized in that, It contains the IL-17RA / RC mutant hybrid protein according to claim 1 and the TNFR2 ECD mutant protein according to claim 16 as active ingredients.

46. ​​A pharmaceutical composition for treating autoimmune diseases, characterized in that, It contains the IL-17RA / RC-Fc mutant hybrid protein according to claim 9 and the TNFR2 ECD-Fc mutant protein according to claim 25 as active ingredients.

47. A pharmaceutical composition for treating autoimmune diseases, characterized in that, It contains the triple fusion protein according to any one of claims 32 to 40 or the homodimer or heterodimer according to claim 44 as an active ingredient.

48. The pharmaceutical composition for treating autoimmune diseases according to any one of claims 45 to 47, characterized in that, The autoimmune diseases mentioned are selected from the group consisting of rheumatoid arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease (IBD), and ankylosing spondylitis.

49. The pharmaceutical composition for treating autoimmune diseases according to claim 48, characterized in that, The inflammatory bowel disease was selected from the group consisting of ulcerative colitis, Crohn's disease, and intestinal Behcet's disease.

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