Novel fusion proteins for the treatment of autoimmune diseases

Abstract

This invention provides a novel fusion protein that significantly reduces the expression of inflammatory cytokines without side effects and exhibits excellent therapeutic effects against various autoimmune diseases.

Description

[Technical Field]

[0001] The present invention relates to a novel fusion protein for the treatment of autoimmune diseases, specifically for regulating pathogenic Th17 cells in autoimmune diseases, and more specifically, to a novel fusion protein for the treatment or prevention of immune-related and inflammatory diseases mediated by IL-17A, IL-17F, or TNFα, by suppressing the IL-17A, IL-17F, and TNFα signaling pathways. [Background technology]

[0002] IL-17A and IL-17F induce the production of numerous inflammatory cytokines, chemokines, and adhesion molecules as inflammation is promoted, and can recruit neutrophils and macrophages to the site of inflammation. Many therapeutic antibodies targeting IL-17A (e.g., Secukinumab and Ixekizumab) or IL-17RA (Brodalumab) have demonstrated high clinical efficacy in patients with psoriasis, psoriatic arthritis, and ankylosing spondylitis and have been approved as therapeutic agents. Biologics such as bimekizumab and sonelokimab, which simultaneously target IL-17A and IL-17F, have also shown high clinical efficacy and have been approved as therapeutic agents. Furthermore, the interleukin-17 series can also form six distinct homodimeric cytokines (IL-17A, B, C, D, E, F) or the heterodimer IL-17A / F. All IL-17 receptors are type I transmembrane glycoproteins and consist of five families: IL-17RA, RB, RC, RD, and RE. IL-17 receptors, through their interaction with IL-17 ligands, mediate host defense while also contributing to inflammation and autoimmunity. IL-17RA, as a common receptor, can mediate distinct signaling with different IL-17 ligands. IL-17RA dimerizes with IL-17RC and can mediate signaling by homodimers of IL-17A and IL-17F, as well as heterodimers of IL-17A / F. IL-17RC and IL-17RA share 22% homology, and 55% homology in the ECD. IL-17RC has a longer ECD compared to IL-17RA, but its cytoplasmic domain is shorter and exists in a multiple splice configuration. IL-17RA has an affinity approximately 100 times weaker than IL-17F compared to IL-17A. IL-17RA and IL-17RC have different expression profiles and tissue distributions.In particular, the differing cytokine environments among TGFβ, IL-6, TNFα, IL-1β, and IL-23 affect the expression patterns of IL-17AA, IL-17FF homodimers, and IL-17AF heterodimers, and therefore also affect naive and differentiated Th17 cells.

[0003] On the other hand, TNFα is an inflammatory cytokine produced by activated macrophages and immune cells such as CD4+ T cells and NK cells. It is involved in the expansion of Th1 and Th17 cells, and plays a role in regulating the inflammatory response. When TNFα is an essential cytokine for a normal immune response, or when there is an imbalance such as overexpression of TNFα in the body, it can lead to various autoimmune diseases. Various TNFα inhibitors are used for the treatment and palliative care of autoimmune diseases, including infliximab, adalimumab, and the fusion protein etanercept. However, 30% of patients with autoimmune diseases such as rheumatoid arthritis do not respond to TNFα inhibitor treatment, and even in patients who do respond to TNFα inhibitor treatment, the response rate decreases as the duration of administration increases, and in many cases, drug changes are necessary due to relapse. Side effects of TNFα inhibitors include infections such as tuberculosis, lymphoma, and renal failure. However, if IL-17A and TNFα are released into the circulatory system for a long period due to RA synovitis, cardiovascular and systemic effects may also occur.

[0004] The exact causes of autoimmune diseases are still not fully understood, and various factors are intricately intertwined, making it difficult to expect effective treatment or a reduction in recurrence with a single target agent in the context of autoimmune diseases. Treatment targeting TNFα and IL-17A simultaneously has been shown to effectively reduce spondylitis and peripheral arthritis. Numerical reductions in bone proliferation, significant reductions in the severity of inflammation and edema were observed in the spine, and a decrease in RANK / Osteoprotegerin (OPG) markers was confirmed.

[0005] Autoimmune diseases such as rheumatoid arthritis (RA), osteoarthritis (OA), and ankylosing spondylitis are chronic autoimmune diseases in which abnormal regulation of immunity and inflammatory responses occurs due to complex genetic and environmental factors, leading to continuous proliferation of the synovial membrane within the joint and destruction of joint bone and cartilage. In particular, RA is approaching an aging society, with a rapidly increasing morbidity rate, and is classified into more than 120 detailed diseases such as degenerative arthritis, osteoarthritis, and rheumatoid arthritis due to chronic inflammation of the synovium. In the case of rheumatoid arthritis characterized by inflammation, there is infiltration of immune cells including CD4+ T cells in approximately 40-50% of synovial fluid cells, and structural changes such as thickening and proliferation of the synovial membrane due to inflammation by various cytokines and chemokines. As the main cause of RA, Th1 cells containing IFN-γ were focused on in initial studies, but blocking IFN-γ did not improve RA, and it has recently been confirmed that Th17 cells are the main cause of RA.

[0006] The structural damage in rheumatoid arthritis disease is related to pathogenic IL-17 resulting from the binding of Th17 cells and IL-17 receptors expressed from synovial fibroblasts, endothelial, and epithelial cells, or the destruction of cartilage and bone caused by this. The role of IL-17 in human RA has also been confirmed to induce the production of inflammatory cytokines, chemokines, and matrix metalloproteinases, and to amplify the inflammatory response in cooperation with other inflammatory driver cytokines, such as TNFα.

[0007] In anti-TNFα clinical studies, circulating Th17 cells and IL-17 levels were significantly increased in patients refractory to anti-TNFα (2.94% vs. 4.23% each; 92.1 pg / ml vs. 148.6 pg / ml, P <0.05). Meaningful therapeutic effects of anti-TNFα therapy were strongly associated with a decrease in both circulating Th17 cells and IL-17. Based on post-marketing clinical results of anti-TNFα, a Proof-of-Concept (PoC) study (NCT02430909) was conducted using combination therapy with bimekizumab and certolizumab pegol (CZP) to simultaneously target IL-17 and TNFα. As a result, compared to certolizumab pegol and placebo, the combination of certolizumab pegol and bimekizumab achieved faster remission with a greater advantage. However, the incidence of treatment emergent adverse events (TEAEs), specifically infections and infestations, was higher in the certolizumab pegol and bimekizumab combination group (50.0% (26 / 52)) compared to the certolizumab pegol and placebo group (22.2% (6 / 27)). Skin and subcutaneous disorders and gastrointestinal disorders were also higher in the certolizumab pegol and bimekizumab combination group compared to the certolizumab pegol and placebo group.

[0008] In an autoimmune disease environment, IL-17 induces the dimerization of IL-17RA and IL-17RC present on the cell membrane, and the inflammatory response is carried out through NF-κB, MAPK / ERK, p38, and JNK signaling by the adaptor protein Actl and TRAF6. Also, when IL-17 signaling is carried out through the Actl-TRAF2-TRAF5 complex, IL-17 mRNA stabilization and additional TNFα and IL-1β mRNAs are also stably expressed, and the inflammatory response can be further enhanced (Shadi Swidani et al., Front. Immunol. 10: 1293, 2019). The Th17 pathway of various inflammatory responses of pathogenic IL-17 signaling is different from the Th1 cell-dominant inflammatory response of TNFα. Therefore, when targeting other cytokines in addition to IL-17, the possibility of treatment is high. The inflammatory response caused by 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, when IL-17-TNFα-IL-36 simultaneously stimulates IL-17-TNFα, IL36r increases in a keratinocyte IkBζ-dependent manner, inducing a chain of psoriasis inflammatory responses (Manjeet K Paintlia, et al., J. Neurochem. 116(4): 508-521, 2012). SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0009] DETAILED DESCRIPTION OF THE INVENTION TECHNICAL PROBLEMS The present invention aims to provide a novel fusion protein for treating autoimmune diseases that significantly reduces the expression of inflammatory cytokines without side effects and exhibits excellent therapeutic efficacy against various autoimmune diseases in order to solve many problems including the above problems. However, these problems are exemplary and do not limit the scope of the present invention. MEANS FOR SOLVING THE PROBLEMS

[0010] Problem solving means According to another aspect of the present invention, 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 are provided, including amino acid substitutions selected from the group consisting of L9P, T24D, F59V, N88D, D122G, A156P and Q267R, and having at least 90% or more sequence homology to the amino acid sequence described in sequence number 69.

[0012] According to another aspect of the present invention, polynucleotides are provided for encoding the above-mentioned IL-17RA / RC mutant hybrid protein.

[0013] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0014] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0015] According to another aspect of the present invention, isomorphic or heterodimers comprising the above-mentioned IL-17RA / RC mutant hybrid protein are provided.

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

[0017] According to another aspect of the present invention, polynucleotides are provided that encode the above-mentioned IL-17RA / RC-Fc mutant hybrid protein.

[0018] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0019] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0020] According to another aspect of the present invention, isomorphic or heteromorphic dimers containing the above-mentioned IL-17RA / RC-Fc mutant hybrid protein are provided.

[0021] According to another aspect of the present invention, 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, vi i) a TNFR2 ECD variant hybrid protein is provided that has at least 90% sequence homology to the amino acid sequence described in sequence number 77, including amino acid substitutions selected from the group consisting of i) S33A, S36P, G75S, R119S, N164S, E210G, and F219I, viiii) G75S, and ix) S33A, S36P, G75S, R119S, and N164S.

[0022] According to another aspect of the present invention, polynucleotides are provided that encode the above-mentioned TNFR2 ECD mutant hybrid protein.

[0023] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0024] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0025] According to another aspect of the present invention, isomorphic or heterodimers containing the above-mentioned TNFR2 ECD mutant hybrid protein are provided.

[0026] According to another aspect of the present invention, a TNFR2 ECD-FC mutant hybrid protein is provided, wherein an antibody Fc domain is affixed to the N-terminus or C-terminus of the above TNFR2 ECD mutant hybrid protein.

[0027] According to another aspect of the present invention, polynucleotides are provided that encode the above-mentioned TNFR2 ECD-Fc mutant hybrid protein.

[0028] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0029] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0030] According to another aspect of the present invention, isomorphic or heteromorphic dimers containing the above-mentioned TNFR2 ECD-Fc mutant protein are provided.

[0031] According to another aspect of the present invention, 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 a tumor necrosis factor (TNF) 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, thereby providing a triple fusion protein.

[0032] According to another aspect of the present invention, a polynucleotide for encoding the above-mentioned triplicate fusion protein is provided.

[0033] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0034] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0035] According to another aspect of the present invention, isomorphic or heteromorphic dimers containing the above three fusion proteins are provided.

[0036] According to another aspect of the present invention, a pharmaceutical composition for the treatment of autoimmune diseases is provided, comprising the above-mentioned IL-17RA / RC mutant hybrid protein and the above-mentioned TNFR2 ECD mutant hybrid protein as active ingredients.

[0037] According to another aspect of the present invention, a pharmaceutical composition for the treatment of autoimmune diseases is provided, comprising the above-mentioned IL-17RA / RC-Fc mutant hybrid protein and the above-mentioned TNFR2 ECD-Fc mutant hybrid protein as active ingredients.

[0038] According to another aspect of the present invention, a pharmaceutical composition for treating autoimmune diseases is provided, comprising the above-mentioned triplicate protein or the above-mentioned isomorphic or heteromorphic dimer as an active ingredient. [Effects of the Invention]

[0039] Effects of the invention As described above, the novel fusion protein for the treatment of autoimmune diseases according to the present invention can effectively improve excessive inflammatory responses and irreversible inflammatory conditions mediated by the co-stimulation of IL-17A, IL-17F, and TNFα. Furthermore, it has the effect of reducing safety issues such as the risk of infection associated with the combined administration of single-target and single-target therapies, as well as the burden of treatment costs on patients with autoimmune diseases. Of course, these effects do not limit the scope of the present invention. [Brief explanation of the drawing]

[0040] [Figure 1]Figure 1 is a graph showing the results of productivity analysis using ProA affinity chromatography for the wild type and IL-17RA / RC-FC and IL-17RA / RC-Fc-TNFR2 constructs with each of the mutations described in Table 1.

[0041] [Figure 2a] Figure 2a shows the residues S33, S36, G75, R119, N164, E210, and F219 that were removed from the family consensus in the mammalian TNFR2 ECD (extracellular domain) arrangement, as described in Table 3.

[0042] [Figure 2b] Figure 2b is a graph showing the results of an analysis of the productivity of 11 types of IL-17RA / RC-Fc-TNFR2 into which the TNFR2 mutation of the present invention (Table 3) was introduced.

[0043] [Figure 2c] Figure 2c is a graph showing the results of analyzing the affinity (EC50) to TNFα of 11 types of IL-17RA / RC-Fc-TNFR2 introduced with the TNFR2 mutation of the present invention (Table 3). Column 12 shows the affinity (EC50) value for adalimumab to TNFα in the positive control group.

[0044] [Figure 3a] Figure 3a is a photograph of the SDS-PAGE gel of the IL-17RA / RC-FC protein of the present invention, purified using the Protein A column.

[0045] [Figure 3b] Figure 3b is a graph showing the protein fractions of two IL-17RA / RC-FC-TNFR2 mutants (T1-T4-14 and T1-T4-52) purified using the Protein A column.

[0046] [Figure 3c]Figure 3c is a photograph of an SDS-PAGE gel showing the fractionation of proteins from two IL-17RA / RC-FC-TNFR2 mutants (T1-T4-14 and T1-T4-52), purified using a protein A column, over time.

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

[0048] [Figure 4] Figure 4 is a graph showing the results of an analysis of the thermal stability of the IL-17RA-Fc mutant protein into which the mutation of the present invention described in Table 2 has been introduced.

[0049] [Figure 5a] Figure 5a is a graph showing the results of analyzing SEC after culturing IL-17RA / RC-Fc-TNFR2 and IL-17A proteins co-expressed in expiCHO cells as described in Table 10, along with TNFα produced from the cells, under conditions of 4°C and 24 hours.

[0050] [Figure 5b] Figure 5b is a gel photograph showing the results of confirming the proteins fractionated by SEC in Figure 5a, as described in Table 10, on a non-reducing gel and a reducing gel.

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

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

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

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

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

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

[0057] [Figure 8] Figure 8 is a graph showing the results of analyzing the anti-inflammatory efficacy of the fusion protein IL-17RA / RC-FC-TNFR2 of the present invention in a psoriasis-like environment using HaCaT cells. The graph shows the evaluation of anti-inflammatory efficacy by measuring the expression levels of cytokines and chemokines.

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

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

[0060] [Figure 10b] Figure 10b is a conceptual diagram showing the binding relationship of the fusion protein IL-17RA / RC-Fc-TNFR2 from tmTNFα-expressing cell lines.

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

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

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

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

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

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

[0067] [Figure 13] 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 the present invention from peripheral blood mononuclear cells derived from psoriasis patients.

[0068] [Figure 14a] Figure 14a is a graph showing the results of an analysis of human IL-17A & IL-17F double knock-in mice, confirming the expression of ML-17A and ML-17F, and the absence of mIL-17A and mIL-17F expression. The upper panel shows heterozygous mice, and the lower panel shows homozygous mice.

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

[0070] [Figure 14c] Figure 14c is a schematic diagram illustrating the in vivo experimental process using C57BL / 6 mouse experimental animals treated with IL-17RA / RC-Fc-mTNFR2.

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

[0072] [Figure 15a]Figure 15a is a 2D photograph observing the structure of the fusion protein IL-17RA / RC-FC-TNFR2 of the present invention. The 2D classification image collection shows the fusion protein from various angles.

[0073] [Figure 15b] Figure 15b shows the three-dimensional structure of the fusion protein IL-17RA / RC-FC-TNFR2 of the present invention, as captured by electron microscopy. It was reconstructed in 3D based on a 2D classification image.

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

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

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

[0077] [Figure 17a] Figure 17a is a schematic diagram showing the production schedule for glycansialylation of the IL-17RA / RC-Fc-TNFR2 hybrid protein.

[0078] [Figure 17b]Figures 17b, 17c, and 17d show SDS-PAGE images (Figure 17b) and SEC analysis results (Figure 17c) of proteins separated and purified in a culture medium of expi CHO-S transfused with both IL-17RA / RC-Fc-TNFR2 and GT6 using a protein A column, as well as a Western blot image (Figure 17d) confirming proteins that underwent sialylation and increased in size. [Figure 17c] Same as above. [Figure 17d] Same as above.

[0079] [Figure 17e] Figures 17e, 17f, and 17g show the results of ELISA analysis investigating the binding affinity of glycansialylated IL-17RA / RC-FC-TNFR2 hybrid fusion protein (T1-T4-14+GT6) to IL-17A, IL-17F, and TNFα. [Figure 17f] Same as above. [Figure 17g] Same as above.

[0080] [Figure 18a] Figures 18a and 18b are a series of graphs showing the results of analyzing the TNFR2 neutralizing ability of the IL-17RA / RC-Fc-TNFR2 hybrid fusion protein and the neutralizing ability of adalimumab in SW982 cells at IL-6 (top panel) and MMP3 (bottom panel) expression levels. [Figure 18b] Same as above.

[0081] [Figure 19] Figure 19 is a schematic diagram showing the three-dimensional structure of the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention. [Modes for carrying out the invention]

[0082] Best mode for carrying out the invention Definition of Terms

[0083] The terms "IL-17A and IL-17F" used in this document refer to the six families of IL-17, which exhibit more than 50% high homology to each other, forming isodimers (IL-17AA, IL-17FF) or heterodimers (IL-17AF), respectively, through IL-17RA / RC, via the signaling assembly, in which pathological IL-17 signaling proceeds. IL-17RA / RC heterodimers induce inflammatory responses through NFκB, MAPK, ERK, p38, and JNK signaling via adapter proteins Act1 and TRAF6. When IL-17 signaling proceeds via the Actl-TRAF2-TRAF5 complex, the inflammatory response may be further amplified by mRNA stabilization 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 of Th17 cells differs from that of the TNFα-mediated inflammatory response of Th1 cells. When IL-17 and TNFα act synergistically, inflammation can be irreversibly amplified. Therefore, when inflammation is irreversibly amplified, it may be difficult to improve autoimmune diseases with a single target. Therapeutic potential increases when IL-17 and TNFα are targeted simultaneously.

[0085] The term "TNFα (tumor necrosis factor-a)" used in this document refers to a membrane protein consisting of 233 amino acids. It is released via TACE as 157-amino acid soluble TNFα (sTNFα), which mediates biological activity. TNFα is involved in signal transduction mediated by two distinct TNFα receptors (TNFR1 and TNFR2), as well as in inflammatory responses mediated by transmembrane TNFα (tmTNFα), a precursor of sTNFα. TNFR1 is expressed in cells of most tissues, while TNFR2 is generally expressed in immune cells. TNFR2 is activated via tmTNFα rather than sTNFα. Generally, TNFR1 activation induces pro-inflammatory responses through cell death, while TNFR2 signaling from activated T cells can contribute to cell proliferation. Currently, five TNFα inhibitors (etanercept, infliximab, golimumab, adalimumab, and certolizumab pegol) are marketed as approved biological agents for the treatment of TNFα-induced diseases. Each antibody exhibits different efficacy in treating diseases. While they all neutralize sTNFα, other mechanisms of action suggest that determine the differences in treatment outcomes. For example, etanercept failed in clinical trials for Crohn's disease, Wegener's granuloma, and sarcoma, and was less effective in treating psoriasis compared to antibody-based TNFα inhibitors. Unlike etanercept, which is based on the TRAP protein, TNFα antibody therapies can stably bind to both sTNFα and tmTNFα. The activation of tmTNFα by TNFα antibody therapy is closely related to tmTNFα-induced TGF-β and IL-10 production, and it has been reported that this offers additional advantages over etanercept in some autoimmune diseases by activating regulatory T cells (S Bombardieri et al., Rheumatology, 46(7): 1191-1199. 2007).

[0086] Detailed description of the invention:

[0087] According to another aspect of the present invention, 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 are provided, including amino acid substitutions selected from the group consisting of L9P, T24D, F59V, N88D, D122G, A156P and Q267R, and having at least 90% or more sequence homology to the amino acid sequence described in sequence number 69.

[0089] In the above hybrid proteins, glycans may be added, and one or more of these glycans may be selected from the group consisting of N-glycans, O-glycans, and sialylated glycans.

[0090] In the above hybrid proteins, selection may also be made from the group consisting of sequence numbers 66 or 69.

[0091] According to another aspect of the present invention, polynucleotides are provided for encoding the above-mentioned IL-17RA / RC mutant hybrid protein.

[0092] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0093] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0094] According to another aspect of the present invention, isomorphic or heteromorphic dimers comprising the above-mentioned IL-17RA / RC mutant hybrid protein are provided.

[0095] According to another aspect of the present invention, an IL-17RA / RC-FC mutant hybrid protein is provided, wherein an antibody Fc domain is ligated to the N-terminus or C-terminus of the above IL-17RA / RC mutant hybrid protein.

[0096] In the above hybrid protein, the antibody Fc domain is also a hybrid Fc domain in which two or more isomorphic Fcs are mixed together, and the above Fc domain is selected from the group of sequence numbers 33 to 65.

[0097] According to another aspect of the present invention, polynucleotides are provided that encode the above-mentioned IL-17RA / RC-Fc mutant hybrid protein.

[0098] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0099] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0100] According to another aspect of the present invention, isomorphic or heteromorphic dimers comprising the above-mentioned IL-17RA / RC mutant hybrid protein are provided.

[0101] According to another aspect of the present invention, 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, v ii) A TNFR2 ECD variant hybrid protein is provided that has at least 90% sequence homology to the amino acid sequence described in sequence number 77, including amino acid substitutions selected from the group consisting of S33A, S36P, G75S, R119S, N164S, E210G, and F219I, viii) G75S, and ix) S33A, S36P, G75S, R119S, and N164S.

[0102] In the above mutant protein, it is a fusion linked to at least one linker-peptide from i or ix above, and may also have a glycan attached, the glycan being one or more selected from the group consisting of N-glycans, O-glycans and sialylated glycans, and selected from the group consisting of sequence number 70 or 81.

[0103] According to another aspect of the present invention, polynucleotides are provided that encode the above-mentioned TNFR2 ECD mutant hybrid protein.

[0104] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0105] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0106] According to another aspect of the present invention, isomorphic or heterodimers containing the above-mentioned TNFR2 ECD mutant hybrid protein are provided.

[0107] According to another aspect of the present invention, a TNFR2ECD-FC mutant hybrid protein is provided, wherein an antibody Fc domain is affixed to the N-terminus or C-terminus of the above-mentioned TNFR2 ECD mutant hybrid protein.

[0108] In the above mutant protein, the antibody Fc domain is also a hybrid Fc domain in which two or more isomorphic Fc domains are mixed together, and the Fc domain is selected from the group consisting of sequence numbers 33 or 65.

[0109] According to another aspect of the present invention, polynucleotides are provided that encode the above-mentioned TNFR2 ECD-Fc mutant hybrid protein.

[0110] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0111] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0112] According to another aspect of the present invention, isomorphic or heteromorphic dimers containing the above-mentioned TNFR2 ECD-Fc mutant protein are provided.

[0113] According to another aspect of the present invention, 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 a tumor necrosis factor (TNF) 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, thereby providing a triple fusion protein.

[0114] In the above three-type fusion protein, the IL-17RA / RC mutant hybrid protein is characterized by having at least 90% sequence homology to the amino acid sequence described in sequence number 69, including amino acid substitutions selected from the group consisting of the following.

[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 and

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

[0120] In the above-mentioned triplicate fusion protein, the tumor necrosis factor receptor 2 extracellular domain (ECD) is characterized by having at least 90% sequence homology with the amino acid sequence described in sequence number 77, including amino acid substitutions selected from the group consisting of the following.

[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] In the above three-type fusion protein, the fusion is linked to at least one linker-peptide from i or ix above, and a glycan may be attached, and the glycan is one or more selected from the group consisting of N-glycans, O-glycans, and sialylated glycans.

[0131] In the above triplicate fusion protein, the tumor necrosis factor receptor 2 may be selected from the group consisting of sequence numbers 83 or 94, and can simultaneously target IL-17A, IL-17F, and TNFα, and may also be selected from the group consisting of amino acid sequences described in sequence numbers 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

[0132] According to another aspect of the present invention, a polynucleotide for encoding the above-mentioned triplicate fusion protein is provided.

[0133] According to another aspect of the present invention, an expression vector containing the above polynucleotide is provided.

[0134] According to another aspect of the present invention, transformed cells into which the above expression vector has been introduced are provided.

[0135] According to another aspect of the present invention, isomorphic or heteromorphic dimers containing the above three fusion proteins are provided.

[0136] According to another aspect of the present invention, a pharmaceutical composition for the treatment of autoimmune diseases is provided, comprising the above-mentioned IL-17RA / RC-FC mutant hybrid protein or the above-mentioned TNFR2 ECD-Fc mutant protein as an active ingredient.

[0137] According to another aspect of the present invention, a pharmaceutical composition for treating autoimmune diseases is provided, comprising the above-mentioned triplicate protein or the above-mentioned isomorphic or heteromorphic dimer as an active ingredient.

[0138] In the above pharmaceutical composition, the above autoimmune diseases are rheumatoid arthritis, psoriasis, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), ankylosing spondylitis, pneumonia, asthma, atopic dermatitis, periodontitis (pyorrhea), conjunctivitis, keratitis, dry eye syndrome, fibromyalgia, lupus, systemic sclerosis, aphthous ulcers, Sjogren's syndrome, Guillain-Barré syndrome, and alopecia areata. The group may also consist of Areata, dermatomyositis, polyarteritis nodosa, relapsing polychondritis, thrombocytopenia, and multiple sclerosis, while the inflammatory bowel disease group may consist of ulcerative colitis, Crohn's disease, and Bethet's disease.

[0139] This invention utilizes amino acid sequence similarity information and domain similarity information between interspecies proteins for the introduction of mutations and reconstruction of IL-17RA / RC hybrids and TNFR2 to improve their stability and neutralizing ability. The bioinformatics-based back-to-consensus / ancestor technique allows for the analysis of the co-evolution of specific protein residues to enable long-term adaptation to the environment. In protein-protein interaction (PPI) analysis, a small gene library is generated based on an "evolutionary methodology" for improving the structural stability and function of proteins. Through simulation, residue substitution patterns are analyzed, and structural changes with different residues before and after substitution are confirmed in vitro to identify patterns that lead to structural stabilization, thereby confirming the mechanism that maintains high affinity and specificity. This is utilized in the production and addition of IL-7RA / RC hybrids and TNFR2 mutants.

[0140] A multi-specific fusion protein is a protein that can bind to two or more antigens simultaneously or sequentially. Specifically, a fusion protein specific to three targets has a first antigen-binding domain that can bind to the first antigen expressed on the cell, as well as to the secreted first antigen. The second or third antigen-binding domains are similar. It also has the characteristic of binding more effectively than when the first and second antigens are subjected to homomorphic or heteromorphic binding, respectively. Furthermore, by using a multi-specific fusion protein, it is possible to induce or block specific signal transduction by effectively inducing interaction between two cells towards a target. The multi-specific fusion protein provided in this invention is CD4 + T Cell, CD8 + It is useful in regulating the activation of various immune cells, such as T cells, monocytes, neutrophils, and macrophages, and can effectively improve the disease immune environment.

[0141] In one specific example, the multiple-specific fusion proteins provided at our hospital can bind to one or more targets, antigens, or epitopes with dissociation constants (KD) of approximately 1 μM or less, approximately 100 nM or less, approximately 40 nM or less, approximately 20 nM or less, approximately 10 nM or less, approximately 1 nM or less, approximately 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. 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] The present invention also exists in a chemically modified form using multiple specific fusion proteins. In some examples, the fusion proteins may be chemically modified by glycosylation, acetylation, PEGylation, amidation, derivatization by known protectors / barriers, proteolytic cleavage, and / or binding to intercellular ligands or other proteins. Many such chemical modifications can be carried out by known techniques.

[0143] In this document, the term "expression vector" refers to a vector prepared by inserting sections of an antibody, a protein receptor expression domain, or fragment thereof, amplified by PCR, into a restriction enzyme-cut vector, thereby enabling the expression of genetic material within cells.

[0144] In this invention, the expression vector may 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 may also be pBispecific or pcDNA3.4 vector.

[0145] One specific example of the fusion protein of the present invention also includes the following structural formulas (I) and (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 case, in structural formulas (I) and (II) above, N' is the N-terminus of the fusion protein, C' is the C-terminus of the fusion protein, A, B, and C are specific to the first, second, and third antigens, respectively, L1 to L3 are peptide hinges or linkers, and X is an Fc domain, representing an invariant domain known as "Fc" that connects from the immunoglobulin C-terminal region, including native Fc domains, recombined Fc domains, and mutant Fc domains, to effector-functioning proteins. In therapeutic proteins, the Fc region can provide an even longer half-life and integrate functions such as Fc receptor binding, protein A binding, complement fixation, and even placental transmission. In particular, such an even longer half-life of an Fc domain is based on its affinity for the neonatal Fc receptor (hereinafter abbreviated as "FcRn"). While increasing the half-life, it is possible to inactivate the antibody-dependent effects such as ADCC and CDC, which are side effects when antibody-based protein therapeutics are used, by inactivating the Fc domain-dependent effects of the antibody. However, the effects of the present invention are not limited to those described above.

[0148] A mutant Fc domain refers to a recombined antibody Fc domain protein produced by compounding components of the immunoglobulin's Fc domain, specifically those derived from different types of antibody molecules, such as IgG, IgD, IgE, and IgM, where all or part of the hinges (CH2 and CH3) of the Fc domain are derived from each other. An example of a modified IgG4 immunoglobulin is one of the following hyFc domain proteins (W0200814743A2; W02020102728A1; W02020102728A1).

[0149] To ensure a longer half-life for mutant Fc domains, increasing Fc affinity to FcRn under endozoan conditions (acidic pH) through Fc engineering utilizes an effective approach to extend the pharmacokinetics of monoclonal antibodies. (Atsuhiko Maeda et al., Mabs. 9(5): 844-853, 2017), 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 / S254TZT256E / H433K / N434F), EDHS mutation (V264E, L309D, Q311H, and N434S), EDHY mutation (V264E / L309D / Q311H / N434Y), DHS mutation (L309D / Q311H / N434S) Developed mutation sets of DHY mutations (L309D / Q311H / N434Y), IgG2-DHS mutations (V309D / Q311H / N434S), IgG3-DHS mutations (L309D / Q311H / N434S), or IgG4-DHS mutations (L309D / Q311H / N434S) can be prepared and utilized.

[0150] In the above structural formulas (I) and (II), L1 to L3 are peptide hinges or linkers, and in the fusion protein, the length of the linker-peptide is 2 or 60 aa (Amino acid), 4 or 55 aa, 5 or 50 aa, 5 or 46 aa, 5 or 45 aa, or 5 or 30 aa. Among the above linker-peptides, the linker-peptide that links an API (active pharmaceutical ingredient) to the N-terminus of the immunoglobulin Fc domain mutant protein may contain part or all of the hinge region of the IgG1 antibody heavy chain. If it contains part of the hinge region, it also means that an artificial linker-peptide has been added to the N-terminus or C-terminus of that part of the hinge region. In addition, the above hinge region is also a hybrid form of hinge region in which parts of the hinge regions of two or more antibody heavy chains are mixed.The above linker peptides are specifically GGGGSGGGGSGGGGSEKEKEEQEERTHTCPPCP (sequence number 83), RNTGRGGEEKKGSKEKEEQEERETKTPECP (sequence number 84), RNTGRGGEEKKSGKEKEEQEERETKTPECP (sequence number 85), RNTGRGGEEKKGGKEKEEQEERETKTPECP (sequence number 86), RNTGRGGEEKKSSKEKEEQEERETKTPECP (sequence number 87), RNTGRGGEEKKKEKEKEEQEERETKTPECP (sequence number 88), GGGGSGGGGSGGGGSLE (sequence number It is also one or more formulations selected from the group consisting of 89), TGIEGRMD (sequence number 90), TGKLSGSASAPKLEEGEFSEARV (sequence number 91), KLSGSASAPKLEEGEFSEARVLE (sequence number 92), TGGSGEGEGSEGSG (sequence number 93), GSGEGEGSEGSGLE (sequence number 94), AEAAAKEAAAAKA (sequence number 95), GGGGSGGGGSGGGGS (sequence number 96), A(EAAAK)4ALEA(EAAAK)4A (sequence number 97), EPKSSDKTHTCPPCP (sequence number 98), and EPKSCDKTHTCPPCP (sequence number 99).

[0151] Changes in the glycan composition and structure of Fc-fusions can alter structural domains, and specific glycation changes in therapeutic Fc-fusion proteins can also affect the pharmacokinetics (PK) and pharmacodynamics (PD) of the molecule. Glycans that significantly affect PK and PD include mannose, sialic acid, fucose, and galactose.

[0152] In some embodiments of the present invention, specific mutations can be considered to alter the glycosylation of a polypeptide. These mutations may be selected to introduce or remove one or more glycosylation sites, as well as O-linked or N-linked glycosylation sites. The means of increasing or decreasing the number of carbohydrate residues in a polypeptide are by chemical and / or enzymatic linking of glycosides to the polypeptide. In some embodiments, the polypeptide sequence can be appropriately adjusted depending on the type of expression system used, such as mammals or yeast. Different glycosidication patterns can be introduced from insect and plant cells, all of which can influence the amino acid sequence of the peptide. Generally, polypeptides of the present invention for human use can be expressed from mammalian cell lines that provide suitable glycosylation, such as HEK293 or CHO cell lines.

[0153] N-linked glycosylation means that the glycan structure is attached to the amide nitrogen of an asparagine residue in a protein. Glycans are branched, flexible chains of branched 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 IL-17RA and IL-17RC hybrid protein (sequence number 69), the N-glycan can attach to at least one amino acid residue selected from N17, N35, N173, N186, N213, N223, N309, N332, and N366. From the TNFR2 ECD (sequence number 77), the N-glycan can attach to at least one amino acid residue selected from N149 and N171.

[0154] As a partial embodiment of the present invention, since the in vivo half-life of a therapeutic protein can be increased by the glycan terminal residue, a method 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 producing sialylated glycoproteins at levels higher than endogenous levels through cell tissue manipulation of production cell lines, the half-life of the multi-target fusion protein of the present invention can be increased.

[0155] Rheumatoid arthritis (RA), osteoarthritis (OA), and ankylosing spondylitis are autoimmune diseases characterized by complex genetic and environmental factors that lead to dysregulation of immune and inflammatory responses, resulting in the persistent proliferation of the synovial membrane within the joints and the destruction of bone and cartilage. In particular, degenerative arthritis, which is rapidly increasing in prevalence due to the aging society, is classified into approximately 120 sub-diseases, including osteoarthritis and rheumatoid arthritis caused by chronic inflammation of the synovial membrane. In rheumatoid arthritis, which is characterized by inflammation, 40-50% of synovial cells are infiltrated by immune cells, including CD4+ T cells, and inflammation caused by various cytokines and chemokines leads to structural changes such as thickening and proliferation of the synovial membrane. Initial research focused on Th1 cells, including IFNγ, as the main cause of RA, but blocking IFNγ did not improve RA. Th17 cells have recently been identified as a major cause of rheumatoid arthritis (RA).

[0156] In patients with rheumatoid arthritis, a systemic pro-inflammatory response (PIR) mediated by IL-17 receptor binding is observed in articular fibroblasts, endothelial cells, and epithelial cells of structurally damaged tissue. IL-17 contributes to cartilage and bone destruction and induces and maintains the production of inflammatory cytokines, chemokines, and matrix metalloproteinases. IL-17 is a major pro-inflammatory cytokine, and its role in synergistic effects with TNFα has been studied in RA. Synergistic signaling between IL-17 and TNFα amplifies the mRNA inflammatory response of systemic inflammatory cytokines and chemokines. The inflammatory response resulting from 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, co-stimulation of IL-17 and TNFα with IL-36 leads to an increase in IL36γ in keratinocytes in an IκBζ-dependent manner, confirming a chain reaction of psoriatic inflammatory responses (Manjeet K Paintlia, et al., J. Neurochem. 116(4): 508-521, 2011).

[0157] The present invention's triple-specific fusion protein IL-17RA / RC-L-Fc-L-TNFR2 (hereinafter abbreviated as IL-17RA / RC-Fc-TNFR2, where L is linker-) exhibits the same or greater target affinity compared to IL-17A monotarget antibody (e.g., secukinumab) or antibodies that simultaneously target IL-17A and IL-17F (e.g., bimekizumab), while increasing in some non-T cells, including pathogenic Th107 cells, NKT cells, γδT cells, monocytes, neutrophils, macrophages, and eosinophils. This protein therapy effectively improves the inflammatory environment by effectively controlling inflammatory signaling by IL-17A and IL-17F.

[0158] The fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention has an affinity equivalent to or greater than that of TNFα single-target antibodies or TNFα inhibitory fusion proteins such as adalimumab or etanercept, binding to soluble TNFα (sTNFα) and transmembrane TNFα (tmTNFα) of its ligand, effectively blocking inflammatory signaling by TNFR1 / 2, activating Treg (Regulatory T cells), and inducing an anti-inflammatory response through reverse signaling.

[0159] The fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention is a protein therapy agent that can treat autoimmune diseases by simultaneously or sequentially blocking inflammatory responses mediated by cytokines IL-17A, IL-17F, or TNFα, thereby effectively improving the systemic inflammatory immune environment, and can reduce disease recurrence by stably maintaining the improved immune environment.

[0160] The fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention additionally provides a method for treating inflammation or inflammatory disorders in experimental subjects, where administration includes intravenous or subcutaneous administration. However, the route of administration, dosage, and frequency of administration of IL-17RA / RC-Fc-TNFR2 of the present invention can be varied depending on the patient's condition and the presence or absence of side effects, and the optimal method of administration, dosage, and frequency can be selected within an appropriate range by a skilled technician. Furthermore, the fusion protein of the present invention can be administered in combination with other drugs or physiologically active substances whose therapeutic effects are known for treating the disease to be treated, or it can be standardized in the form of a compound formulation with other drugs.

[0161] The fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention provides a method for treating inflammatory or inflammatory autoimmune diseases and chronic inflammation-derived cancers in a subject requiring the therapeutic action of administering a therapeutically effective amount of the isolated peptide or a pharmaceutical composition containing the isolated peptide to the subject requiring the therapeutic action.

[0162] The present invention provides a therapeutic agent for treating inflammation or inflammatory disorders, comprising administering at least one anti-inflammatory agent to a therapeutically effective amount of an isolated peptide or a pharmaceutical composition containing the isolated peptide, wherein the anti-inflammatory agent is a corticosteroid. Specifically, cortisol, aldosterone, hydrocortisone, hydrocortisone acetate, cortisol acetate, thixocortolpivalate, prednisolone, methylprednisolone, prednisone, triamcinolone acetonide, salicylic acid resorcinol, sulfacetamide, urea, imidazole, betamethasone, and betamethasone sodium These can be immunosuppressant agents such as dexamethasone, dexamethasone sodium phosphate, nonsteroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitors, capsaicin, ibuprofen, chlorprednisone, or cyclosporine, cytokine synthesis inhibitors, tetracycline, minocycline, and doxycycline, or any combination thereof.

[0163] This invention provides antagonists for the systemic inflammatory cytokines IL-17A, IL-17F, and TNFα. IL-17A, IL-17F, and TNFα are cytokines that mediate the infectivity of autoimmune or chronic inflammatory diseases. In particular, when IL-17A, IL-17F, or TNFα cytokines act in conjunction, inflammatory activity is transiently induced, leading to an irreversible inflammatory disease environment. This invention provides antagonists that can regulate, for example, block, suppress, reduce, antagonize, neutralize, or inhibit the production of systemic inflammatory cytokines and / or chemokines mediated by IL-17A, IL-17F, and TNFα. These antagonists are superior to therapies that target only one or two of these cytokines and have the advantage of fundamentally blocking the exacerbation of inflammation caused by the increasing effects of IL-17A, IL-17F, and TNFα.

[0164] The present invention relates to a multiple target fusion protein comprising a hybrid protein of the extracellular domain (ECD) of IL-17RA and IL-17RC, which may contain mutations in the first IL-17A and IL-17F binding moiety, and a TNFR2 ECD, which may contain mutations in the second TNFα binding moiety. The IL-17RA and IL-17RC hybrid protein may have at least 95% homology to sequence number 66 or 69. For example, it may have at least 95% homology to IL-17RA / RC containing mutations such as L9P, T24D, F59V, N88D, D122G, A156P, or Q267R. Furthermore, TNFR2 ECDs can have at least 95% homology to sequence numbers 83 or 93, and can have at least 95% homology to TNFR2 ECDs containing mutations S33A, S36P, G75S, R119S, N164S, E210G, or F219I.

[0165] Specific embodiments of the multi-target fusion protein of 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 the above 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 to L3 are peptide hinges or linkers composed of 0-31, 2-10, or 3-10 amino acids, and X is the Fc domain, which includes a native Fc domain, a recombined Fc domain, or a mutant Fc domain. In therapeutic proteins, the Fc region can provide an even longer half-life. The half-life can be further increased by increasing the binding affinity to the neonatal Fc receptor (FcRn) through Fc engineering. To increase the half-life, the Fc domain can be an Fc mutant into which mutations have been introduced to increase affinity to FcRn, or mutations that minimize effector function such as ADCC or CDC.

[0168] In the above structural formulas (I) and (II), A is the first antigen-binding domain, which can be the human IL-17RA ECD polypeptide or an IL-17RA ECD polypeptide variant. B is the second antigen-binding domain, which can be the human IL-17RC ECD polypeptide or a human IL-17RC ECD polypeptide variant. The second antigen-binding domain is specific to the first antigen IL-17A and the second antigen IL-17F, respectively, and is also specific to both IL-17A and IL-17F (Rolf E. Kuestner et. al., J. Immunol. 46(7): 1191-1199, 2007). IL-17RA has a high affinity for IL-17A but a relatively low affinity for IL-17F. As a partial embodiment of the present invention, A can be bound in a hybrid form characterized by being the IL-17RAECD variant and B being the IL-17RC ECD variant 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, sequence number 114) and can become a polypeptide corresponding to wild-type ML-17RA exons 1-6 or a variant thereof. The B domain of the IL-17RA / RC hybrid protein corresponds to exons 8-16 of IL-17RC (wild type, sequence number 115) and can become a polypeptide corresponding to wild-type ML-17RC exons 8-16 or a variant thereof. From the above structural formulas (I) and (II), the A-Ll-B and B-L3-A IL-17RA / RC can be fused directly or via a linker to the C-terminus or N-terminus of Fc and have at least 95% homology to sequence numbers 66 or 69.The IL-17RA / RC hybrid protein of the present invention has a hybrid configuration in which the first antigen-binding domain corresponds to the A domain of IL-17RA and the second antigen-binding domain corresponds to the B domain of IL-17RC. However, the A-L1-B or B-L3-A domain as a whole is capable of binding to both the first antigen IL-17A and the second antigen IL-17F, and is a fusion domain with a new form and function that allows it to bind to the IL-17A / F heterodimer. However, the IL-17RA / RC of the present invention is not limited to the above.

[0169] In the above structural formulas (I) and (II) of the present invention, the third antigen-binding domain of C contains the human TNFR2 extracellular domain (ECD). 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 by having minimal side effects by having beneficial anti-inflammatory effects through soluble TNFα (sTNFα) neutralization, blocking the inflammatory response while blocking the signaling between soluble TNFα or transmembrane TNFα (tmTNFα) and TNFR1 / 2, or through reverse signaling of transmembrane TNFα. In structural formulas (I) and (II), the TNFR2 ECD of C can have at least 95% homology to sequence number 77.

[0170] As a partial embodiment of the present invention, the multi-target fusion protein includes the use of a dual-target fusion protein for the treatment, prevention, and / or diagnosis of diseases associated with the activity of IL-17A, IL-17F, and TNFα. As described herein, the present invention provides a therapeutic and preventive method that rapidly and sustainably improves the systemic inflammatory environment by simultaneously or sequentially suppressing the activity of IL-17A, IL-17F, and / or TNFα by binding to IL-17A, IL-17F, and TNFα simultaneously through a fusion protein, thereby suppressing the expression of cytokines and chemokines such as IL-8, IL-6, IL-lb, G-CSF, hBD2, DEFB4, IL-36r, IL-17C, CCL20, CCL23, CXCL10, LCN2, MMP3, E-selectin, and RANTES in immune or non-immune cells via the NFκB or MAPK pathway, while simultaneously inducing the secretion of anti-inflammatory cytokines.

[0171] The autoimmune diseases of the present invention include, for example, psoriasis, rheumatoid arthritis (RA), degenerative arthritis (osteoarthritis), ankylosing spondylitis, axial spondyloarthritis, juvenile idiopathic arthritis, rheumatoid arthritis osteoporosis, inflammatory fibrosis (e.g., scleroderma, pulmonary fibrosis, and cirrhosis), gingivitis, periodontosis, or periodontal diseases, and inflammatory bowel diseases. This includes administering the drug to patients with various inflammatory, immunological, and proliferative disorders, including, for example, Crohn's disease, ulcerative colitis, and inflammatory bowel disease, asthma (including allergic asthma), allergies, chronic obstructive pulmonary disease (COPD), multiple sclerosis, and cancer.

[0172] TNFα and IL-17, mentioned above, are the main cytokines involved in inflammation and the development of autoimmune diseases. Five biological agents—Etanercept, Infliximab, Golimumab, Adalimumab, and Certolizumab pegol—have been approved and marketed as TNFα inhibitors, which bind to TNFα and inhibit its biological activity. The therapeutic effects of each antibody vary depending on the disease, the progression of the disease, and the presence or absence of underlying conditions. While all TNFα inhibitors neutralize sTNFα, the response differs from patient to patient, with some patients not responding and many cases not showing sustained therapeutic effects. Specifically, 30% of patients with autoimmune diseases such as rheumatoid arthritis do not respond to TNFα inhibitors, and even in patients who do respond to TNFα inhibitors, the response rate decreases with longer administration periods, and drug changes due to relapse are often necessary (MH Buch et al. Rheumatology, 46, Issue 7, 1153-1156, 2007). Side effects of TNFα inhibitors have been reported, including infections such as tuberculosis, lymphoma, and renal failure. In particular, the increased incidence of cardiovascular disease and related mortality is closely associated with the prolonged release of IL-17A and TNFα into the circulatory system from RA synovitis (Anass Bouchnita et al. CR Biol. 340(11-12):456-473, 2017).

[0173] Clinical studies of TNFα inhibitors have confirmed a significant increase in circulating Th17 cells and IL-17 in patients who do not respond to TNFα inhibitors. Based on these clinical results, a Proof-of-Concept (PoC) study (NCT02430909) targeting both IL-17 and TNFα was conducted. In patients who did not respond to TNFα inhibitors, the combination administration of two biological agents, bimekizumab (anti-IL-17A / F) and certolizumab pegol (anti-TNFα), showed greater advantages compared to the certolizumab pegol monotherapy group, including faster remission and a numerically greater reduction in the mean swelling joint count (SJC) and tender joint count (TJC) compared to the monotherapy group. However, the combination therapy of two biological agents carries a higher risk of infection, and the incidence of treatment emergent adverse events (TEAEs) is higher compared to monotherapy or placebo groups, with skin and subcutaneous disorders and gastrointestinal disorders being more frequent (Sophie Glatt et al., Ann. Rheum. Dis .78(8):1033-1040, 2019).

[0174] Bi-antibodies ABT-122, JNJ-8104, and COVA322 that simultaneously target TNFα and IL-17 have been developed and clinically tested for rheumatoid arthritis (RA). Rapid response rates were observed in two clinical settings, but the relative advantages of anti-TNF antibodies (adalimumab) were difficult to confirm. Although high levels of anti-drug antibody (ADA) generation were observed, development is currently not underway (NCT01853033, NCT02141997, NCT02243787, NCT02349451, NCT02758392).

[0175] Based on the above research, to date, combination therapy using TNFα and IL-17 antibodies, or dual antibodies targeting both TNFα and IL-17 simultaneously, has been developed. While combination therapy offers flexibility in dosage and timing, it can be inconvenient for patients in terms of adaptation and pain. In the case of biological agents targeting cytokines simultaneously associated with innate immunity and proinflammatory autoimmune diseases, combination therapy carries a high risk of infection, making it difficult to achieve low disease activity and ultimately offering no therapeutic benefit. Recently developed co-formulations can also offer some flexibility in dosage, but finding formulation conditions that allow both antibodies to maintain chemical and physical stability based on their different molecular characteristics is not easy. Furthermore, combination therapy, or co-formulations, involves the additional cost of two different drug therapies, which increases the patient's treatment costs. Most biantibodies that simultaneously target TNFα and IL-17 have been reported to have a high potential for inducing immunogenicity (Mark A. Kroenke et al., Front. Immunol .12: 782788, 2021). High immunogenicity has serious implications for drug efficacy, pharmacokinetics, and stability, so development is currently suspended.

[0176] According to one example of the present invention, the manufactured multi-target fusion protein can simultaneously or sequentially bind to IL-17A, IL-17F, and TNFα, neutralizing all of them. This can effectively improve the excessive inflammatory response and irreversible inflammatory disease environment mediated by the co-stimulation of IL-17A, IL-17F, and TNFα, potentially reducing disease activity and achieving remission through sustained efficacy with low immunogenicity. Furthermore, single-agent administration of multi-target therapeutics is expected to reduce the safety concerns associated with the risk of infection associated with concomitant administration of single-target therapeutics, thereby lowering the treatment cost burden for patients with autoimmune diseases (Figure 19).

[0177] The pharmaceutical composition of the present invention may vary depending on the type of affected area in the patient, the application site, the number of treatments, the treatment time, the dosage form, the patient's condition, the type of adjuvant, etc. The dosage is not particularly limited, but it may be 0.01 μg / kg / day or 10 mg / kg / day. The above daily amount can be administered once a day, or divided into 2-3 doses per day at appropriate intervals, or intermittently over several days.

[0178] The pharmaceutical composition of the present invention contains compounds that can be administered orally or parenterally, and preferably, parenterally by intravenous injection, subcutaneous injection, intracerebral injection (intracerebrospinal fluid injection), intramuscular injection, and intraperitoneal injection.

[0179] The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. Solid or liquid pharmaceutical additives may also be used in the manufacture of the pharmaceutical composition. The pharmaceutical additives may be organic or inorganic. Examples of excipients include lactose, sucrose, sucrose, glucose, cornstarch, starch, talc, sorbitol, crystalline cellulose, dextrin, kaolin, calcium carbonate, and silicon dioxide. Examples of compounding agents 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. As colorants, any colorant permitted for addition to conventional pharmaceuticals may be used. These tablets and granules can be coated with sugar, gelatin, or other appropriate coatings as needed. Preservatives, antioxidants, etc., may also be added as needed. Furthermore, if the pharmaceutical composition is a drug, it may additionally contain one or more selected fillers, anti-agglutinants, lubricants, wetting agents, fragrances, emulsifiers, or preservatives. On the other hand, the dosage form of the pharmaceutical composition of the present invention should be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient, particularly after administration to mammals, as is desirable depending on the method of use.Examples of specific dosage forms include plasters, granules, lotions, liniments, lemonades, powders, syrups, liquids and solutions, aerosols, extracts, elixirs, fluid extracts, emulsions, suspensions, decoctions, infusions, tablets, suppositories, injections, sprits, and cataplasms. The product may be in one of the following forms: capsules, troches, tinctures, pastes, pills, or soft or hard gelatin capsules.

[0180] The pharmaceutical compositions of the present invention may further include components commonly used in other applications, such as stabilizers, solubilizers, and fragrances, as well as common auxiliary agents and carriers.

[0181] A composition according to one embodiment of the present invention may contain a pharmaceutically acceptable carrier, and may additionally contain pharmaceutically acceptable auxiliaries, excipients, or diluents in addition to the carrier.

[0182] As used in this document, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness, or similar reactions, when administered to humans. Examples of the carriers, excipients, and diluents mentioned above include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium hydrogen phosphate dihydrate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methyl hydroxybenzioate, profyl hydroxybenzioate, talc, magnesium stearate, and mineral oil. Additional ingredients such as fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may also be included.

[0183] Furthermore, compositions according to one embodiment of the present invention can be formulated using methods known in the art to allow for rapid release of the active ingredient, or sustained or delayed release. Dosage forms include powder, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions, and sterile powder forms.

[0184] A composition according to one embodiment of the present invention can be administered by a variety of routes, for example, orally, parenterally, such as suppositories, transdermally, intravenously, intraperitoneally, intramuscularly, intralesionally, nasally, or intraspinally, or by using an implantable device for sustained-release, continuous, or repeated release. The number of doses can be once a day or divided into several doses, within the range desired, and the duration of administration is not particularly limited.

[0185] A composition according to one embodiment of the present invention is generally administered systemically or topically, for example by intramuscular or intravenous injection, but if provided as a composition comprising a polynucleotide or an expression vector containing the same, it can most preferably be injected using an electroporator.

[0186] The administration route of a composition according to one embodiment of the present invention can be through a general route, as long as it can reach the target tissue. Such administration routes include, but are not limited to, parenteral administration, such as intraperitoneal, intravenous, intramuscular, subcutaneous, and synovial administration.

[0187] A composition according to one embodiment of the present invention can be formulated into a dosage form suitable for use with commonly used pharmaceutically acceptable carriers. Examples of pharmaceutically acceptable carriers include parenteral carriers such as water, suitable oils, saline solutions, aqueous glucose, and glycogen, and may additionally contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfate, sodium sulfate, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or profil-paraben, and chlorobutanol. Furthermore, depending on the method of administration and dosage form, the composition according to the present invention may appropriately contain suspending agents, solubilizers, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, analgesics, buffers, antioxidants, etc. 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 a composition according to one embodiment of the present invention for a patient varies depending on many factors, including the patient's height, body surface area, age, the specific compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The composition of the present invention can be administered in amounts of 100 ng / kg body weight to 10 mg / kg body weight, more preferably 1 μg / kg body weight or 1 mg / kg body weight, and most preferably 5 or 500 μg / kg body weight, although the dosage may be adjusted considering the above factors. Together, the pharmaceutical composition of the present invention is administered in a therapeutically effective amount.

[0189] As used in this document, the term "therapeutably effective dose" means a sufficient amount to treat a disease with a reasonable recipient-to-risk ratio applicable to medical treatment. The effective dose level will be determined by factors including the individual's species and severity, age, sex, drug activity, sensitivity to the drug, administration time, route of administration and elimination ratio, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered in doses of 0.1 mg / kg or 1 g / kg, more preferably 1 mg / kg or 500 mg / kg. On the other hand, the above doses will be appropriately adjusted depending on the patient's age, sex, and condition.

[0190] The composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered via either systemic or local route. Systemic administration can be done by intravenous injection, intraperitoneal injection, or intramuscular injection. Local administration can be done by intracerebral administration, intracerebral rebrospinal administration, subcutaneous injection, etc.

[0191] Embodiments of the Invention The present invention will be described in more detail below through examples. However, the present invention is not limited to the examples disclosed below and can be embodied in a variety of different forms, and the following examples are provided to ensure that the disclosure of the present invention is complete and that a person of ordinary skill will be fully informed of the scope of the invention. [Examples]

[0192] Example 1: Production of IL-17A and IL-17F antagonists

[0193] The inventors have produced IL-17RA / RC-Fc proteins for the manufacture of neutralizing agents that can effectively neutralize IL-17AA, IL-17AF, or IL-17FF. In particular, antagonists that can bind IL-17A or IL-17F simultaneously or sequentially can more effectively treat autoimmune and inflammatory diseases (Sascha Gerdes & Joerg Albrecht., Br. J. Dermatol. Volume 186, Issue 4. 2022). 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 the IL-17RA / RC-Fc hybrid protein, the extracellular domain (ECD) of IL-17RA and the protein portion of IL-17RC ECD that are involved in IL-17A or IL-17F binding are utilized as functional fragments. Functional fragments of each IL-17RA and IL-17RC are compounded to produce the hybrid protein (IL-17RA / RC fusion protein). In addition, mutations are introduced into each of the IL-17RA and IL-17RC functional fragments via a back-to-consensus method to ensure increased affinity for IL-17A and IL-17F, increased stability of the hybrid protein, or low immunogenicity. The above-mentioned IL-17RA / RC variants may also introduce mutations into the following amino acids: proline from the 9th amino acid lysine, aspartate from the 24th amino acid threonine, tyrosine from the 52nd amino acid histidine, valine from the 59th amino acid phenylalanine, lysine from the 78th amino acid glutamic acid, aspartate from the 88th amino acid asparagine, isoleucine from the 89th amino acid phenylalanine, lysine from the 108th amino acid arginine, glycine from the 122nd amino acid aspartate, proline from the 156th amino acid alanine, or arginine from the 267th glucose.

[0194] Using the wild-type IL-17RA / RC hybrid protein as a control group, experiments were conducted to search for mutations that could improve affinity, stability, and productivity of IL-17A and IL-17F. These mutations included various IL-17RA / RC hybrid protein variants or the tri-specific fusion protein IL-17RA / RC-Fc-TNFR2. Example 2 of the present invention is an experiment to select mutations that can improve or at least enhance the affinity, stability, and productivity of the IL-17RA / RC hybrid protein when a TNFα-targeting moiety is added to the IL-17RA / RC hybrid. As shown in Table 1 below, the productivity of each IL-17RA / RC construct was greatly improved when various mutations were introduced compared to the control group wtIL-17RA / RC (wild-type) or wtIL-17RA / RC-Fc (mutant) (Figure 1). Along with the above-mentioned improvements in productivity, stability, ligand affinity, and neutralizing ability also increased significantly (Table 1). The overall sequence information of the above fusion proteins is summarized in Table 1 below, and the mutation information of the IL-17RA / RC hybrid proteins introduced into the IL-17RA / RC-FC construct and the IL-17RA / RC-Fc-TNFR2 construct is summarized in Tables 2 and 3 below.

[0195] [Table 1]

[0196] [Table 2]

[0197] To induce protein expression of IL-17RA / RC-Fc, each primer pair was prepared and polymerized via a chain reaction. This was then incorporated into a pBispec vector (Genexine, Inc., South Korea) to produce a recombined vector. This expression vector was used to produce a construct in which the biologically active protein or active pharmacological component IL-17RA / RC was linked to the N-terminus of the Fc domain variant via a linker peptide for the production of the IL-17RA / RC-FC fusion protein of the present invention. The expression medium for the above IL-17RA / RC-Fc fusion protein was ExpiCHO® (Gibco, Cat: A29127) cells, and the expression of the above fusion protein was carried out in accordance with the protocol of the ExpiCHO® expression system kit. For manufacturing, cells were cultured in ExpiCHO® expression medium (Gibco, Cat: A29100-01) at 8% CO2, 37°C, and 120 rpm for 1 day. On the day of DNA plasma injection, cells were grown to 0.6-1.0 x 10⁷ cells / ml with a viability of 95% or more. These cells were then transplanted into fresh medium at a rate of 1.0 x 10⁷ cells / ml. 7 Dilute to cells / ml and prepare. For transfusion into the prepared mother cells, prepare the ExpiFectamine® CHO & plasmid DNA complex using the ExpiFectamine® CHO Transfusion Kit (Gibco, Cat: A29129). Then, separate the cells into cold OptiPRO® SFM® (Gibco, Cat: 12309019) medium, inoculate with the DNA and ExpiFectamine® CHO reagent prepared at the appropriate concentrations, mix, incubate at room temperature for 5 minutes, inoculate into the mother cells for transfusion, and start culturing. 18 hours after transfusion, inoculate with the enhancer and feed contained in the ExpiFectamine® CHO Transfusion Kit, and culture for 7-10 days under 8% CO2, 37°C, and 120 rpm conditions to complete production. After culturing, the cell culture medium was centrifuged at 4°C and 4000 rpm for 60 minutes to separate the superior solution, which was then stored frozen at -80°C. [Examples]

[0198] Example 2: Production of IL-17A, IL-17F, and TNFα antagonists

[0199] The systemic inflammatory cytokines interleukin-17 (IL-17) and tumor necrosis factor (TNFα) are targets for the treatment of many chronic inflammatory diseases. Furthermore, co-stimulation by IL-17 and TNFα leads to the activation of MAPK and NF-KB, creating a systemic inflammatory environment and resulting in a chain reaction of tissue damage. In particular, in patients with severe chronic autoimmune diseases, chronic drug administration can activate a compensation mechanism that partially suppresses activity and induces undesirable toxicity. To overcome these problems, it is necessary to simultaneously target the inflammatory response pathways of interleukin-17 (IL-17) and tumor necrosis factor (TNFα). To manufacture antagonists that can simultaneously suppress or neutralize IL-17A, IL-17F, and TNFα, we secured a TNFα binding moiety that has improved affinity for TNFα compared to wtTNFR2. Mutations selected from the back-to-consensus library were introduced into wtTNFR2 (Table 3), and the IL-17RA / RC hybrid protein from Example 1 was linked to the N-terminus of Fc and the above TNFR2 variant to the C-terminus of Fc via linkers to complete the triple-specific fusion protein (IL-17RA / RC-FC-TNFR2).

[0200] Furthermore, to improve the stability, productivity, and neutralizing ability of the TNFR2ECD mutant in one embodiment of the present invention, the interspecific sequence of the 258 amino acids of TNFR2 ECD, including the signal peptide, was searched and aligned in the NCBI database, and the mutation locations were selected. As a result, the selected S33A, S36P, G75S, R119S, N164S, E210G, or F219I mutations and their various formulations were selected as mutant formulations superior to the wild type through evaluation of thermal stability, neutralizing ability, and productivity (Figure 2a). The TNFR2ECD mutant showed significantly improved productivity compared to the wild type. The TNFR2 mutant number 10 in Table 3 (T4i, sequence number 92) is a mutant in which the C-terminus of wtTNFR2 has been partially deleted, and the TNFR2 mutant number 11 (T4TR, sequence number 93) is a mutant in which TNFR2 has been additionally linked via a linker, resulting in a slight decrease in productivity compared to the other mutants (Figure 2b).

[0201] The degree of improvement in the neutralizing ability of TNFR2 mutants towards TNFα was determined by ELISA analysis, and the affinity (EC) was measured. 50The values ​​were compared (Figure 2c). As a result, the TNFR2 ECD mutants with introduced mutations showed improved neutralizing ability in most cases compared to wild-type TNFR2ECD. The TNFR2 mutant number 8 in Table 3 below (T4g, sequence number 77) showed an improvement of approximately 40 times compared to wild-type TNFR2 ECD. This was confirmed to be at a level of neutralizing ability equivalent to that of the positive control group adalimumab (number 12 in Figure 2c). The mutations introduced into the TNFR2 ECD mutants, which showed improved neutralizing ability and productivity in all aspects, were selected as S33A, S36P, G75S, R119S, N164S, E210G, and F219I. The mutations introduced into the TNFR2 mutant (T4g, sequence number 77) were S33A, S36P, G75S, R119S, N164S, E210G, and F219I. Information on TNFR2 mutations introduced into the above IL-17RA / RC-Fc-TNFR2 construct is summarized in Table 3 below. TNFR2 mutants may also introduce mutations into alanine from amino acid 33 (serine), proline from amino acid 36 (serine), serine from amino acid 75 (glycine), proline from amino acid 119 (serine), serine from asparagine at 164, glycine from amino acid 210 (glutamic acid), and isoleucine from phenylalanine at 219.

[0202] [Table 3]

[0203] The triple-specific fusion protein IL-17RA / RC-FC-TNFR2 of the present invention is produced by linking the IL-17RA / RC variant produced in Example 1 above with the TNFR2 variant above via various Fc and linkers. The triple-specific fusion protein of the present invention may also include the following structural formulas (I) and (II): The triple-specific fusion protein of the present invention may also include the following structural formulas (I) and (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 the above 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 subdomain of IL-17RAECD, and B is a subdomain of IL-17RC ECD. Each domain is a functional fragment and corresponds to a protein that binds to the ligand IL-17A or IL-17F. However, A-L1-B or B-L3-A as a whole can bind to all of the primary antigens, IL-17A and the secondary antigen, IL-17F, and can also bind to the IL-17A / F heterodimer. L1 in "A-L1-B" is a linker and can have 0 to 31 amino acids, but can also be "0" from IL-17RA / RC. IL-17RA / RC can also be located at the N-terminus via a linker (L2) in Fc wild-type or mutant forms. In the above structural formulas (I) and (II), C is specific to the third antigen TNFα and can also be located at the C-terminus of the 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 below. The Fc domain introduced into the above fusion protein refers to a recombined antibody Fc domain protein produced by compounding antibody molecules of different types, i.e., IgG, IgD, IgE, IgM, etc., in which all or part of the hinges, CH2 and CH3, of the Fc domain components of the immunoglobulin. An example of a modified IgG4 immunoglobulin is one of the hyFc domain proteins (W0200814 group 43A2; W02020102728A1; W02021046404A1). Table 5 of the present invention shows examples of Fc domains.

[0207] The Fc variants in Table 5 below have mutations introduced to increase Fc affinity to FcRn under endosomal conditions (acidic pH) to ensure an even longer half-life. Fc engineering is utilized to extend the drug's residence period in the body for single-clon antibodies (Atsuhiko Maeda et al., Mabs. 9(5):844-853, 2017). LS mutations (M428L / N434S, Xencor), YTE mutations (M252Y / S254T / T256E, Medimmune), QL mutations (T250Q / M428L) (Paul R. Hinton et al., J. Biol. Chem. Chem. 279(8): 6213-6216, 2004), or KF mutations (H433K / N434F) can be utilized individually or as a combination of mutation sets that utilize the corresponding Fc engineering. The linker sequence information for the above-mentioned triple-specific fusion protein IL-17RA / RC-Fc-TNFR2 is summarized in Table 4 below.

[0208] [Table 4]

[0209] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0210] To produce the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention, various constructs were constructed in which the IL-17RA / RC mutant and the TNFR2 mutant were linked to the Fc mutant shown in Table 5 via the linker shown in Table 4, in an N-terminus to C-terminus order. Mutations with high affinity for FcRn were introduced into the Fc domain of the IL-17RA / RC hybrid protein (Table 6). The nucleic acid was cloned into a pBispec vector. The IL-17RA / RC sequence information for the above fusion protein IL-17RA / RC-Fc-TNFR2 is summarized in Table 6 below, and the sequence information for the TNFR2 mutant is summarized in Table 7 below.

[0211] [Table 6-1] [Table 6-2]

[0212] [Table 7-1] [Table 7-2] [Table 7-3]

[0213] Expression of the IL-17RA / RC-Fc-TNFR2 fusion protein was performed using ExpiCHO® (Gibco, Cat: A29127) cells, following the protocol of the ExpiCHO® Expression System Kit. Preparation involved culturing cells in ExpiCHO® Expression Medium (Gibco, Cat: A29100-01) at 8% CO2, 37°C, and 120 rpm for 1 day, followed by DNA plasma injection. The cells were 0.6-1.0 x 10⁶ on the day of injection. 7 Cells / ml, viability of over 95% of cells grown in fresh culture medium, 1.0 x 10 7Prepare by diluting to cells / ml. To perform transfection on the prepared mother cells, an ExpiFectamine (registered trademark) CHO transfection kit (Gibco, Cat: A29129) was used to prepare an ExpiFectamine (registered trademark) CHO & plasmid DNA complex. Cold OptiPRO (registered trademark) SFM (registered trademark) (Gibco, Cat: 12309019) medium was aliquoted respectively, and after inoculating the appropriately concentrated DNA and ExpiFectamine (registered trademark) CHO reagent, they were mixed and left standing at room temperature for 5 minutes, then inoculated into the mother cells to start transfection culture. 18 hours after transfection, the enhancer and feed contained in the ExpiFectamine (registered trademark) CHO transfection kit were inoculated into the transfected cells, and cultured under the conditions of 8% CO2, 37 °C and 120 rpm for 7 to 10 days to complete production. After culturing, the above cell culture solution was centrifuged at 4 °C and 4000 rpm for 60 minutes, then the supernatant was separated and stored frozen at -80 °C.

Example

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

[0215] The inventors analyzed improvements in the productivity and safety of the fusion proteins IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2. Mutant formulations selected in Examples 1 and 2 were introduced into the wtIL-17RA (UniProt:human IL-17RA:Q96F46), wtIL-17RC (UniProt:human IL-17RC:Q8NAC3), and wtTNFR2 (UniProt:human TNFR2:Q9UIG9) proteins to obtain mutants with improved productivity, stability, and neutralizing ability. These mutants showed significant improvements in productivity and stability compared to the wild-type fusion protein. The IL-17RA / RC-FC and IL-17RA / RC-Fc-TNFR2 fusion proteins were produced in CHO cells and purified based on Protein A affinity. Subsequently, the IL-17RA / RC hybrid fusion protein (IL-17RA / RC-Fc-TNFR2) supernatant was centrifuged at 4°C and 4000 rpm for 60 minutes, and the cell culture medium (HCCF) was filtered through a 0.45 μm filter. Next, a HiTrap MabSelect Sure column (Cytiva, 11-0034-95) was prepared. After stabilizing 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, and then the column was subsequently washed with wash buffer (WB) (100 mM sodium citrate, 150 mM NaCl (pH 5.0)). The bound proteins were eluted using elution buffer (EB) (1M Glycine, 150 mM NaCl (pH 3.0)), and 2.5 ml of each were separated into tubes containing IM Tris buffer (pH 9.0). After protein elution, the proteins were confirmed by SDS-PAGE. SDS-PAGE analysis of the IL-17RA / RC-FC and IL-17RA / RC-Fc-TNFR2(Tl-T4-14) fusion proteins revealed them to be approximately 77 kDa and 103 kDa under 10% reduction conditions (Figure 3a or 3c).The eluted protein was purified using a secondary purification process with size exclusion chromatography (SEC) as follows. First, a purified sample containing the IL-17RA / RC hybrid fusion protein (IL-17RA / RC-FC-TNFR2) was loaded onto the column at a flow rate of 1 ml / min (AKTATM pure 25 (Cytiva, 29018224)) to a volume of about 1 mL. Specifically, the overpressure was set to 0.5 MPa and the flow rate was set to 1 ml / min according to the column. The column used above was Superdex200 (Cytiva), and the buffer used for elution was LPS solution, a solution in which CBP007B was diluted 1 / 10 and Tween-20 (Cytiva, BR100054) was added to a concentration of 0.005 %. Then, separation was carried out while observing the peak according to the elution volume of the buffer (HiLoad 16 / 600 Superdex 200 pg lx 120 ml (Cytiva, 28989335)). The order of purification was the final purification in two steps of size exclusion chromatography (SEC) of the eluate recovered from the Protein A chromatography column. The yields of the chromatography of the above-performed fusion protein are summarized in Tables 8 and 9 below.

[0216]

Table 8

[0217]

Table 9

[0218] Analysis of the productivity of 26 IL-17RA / RC-Fc-TNFR2 variants (listed in Table 10 below), newly cloned from pBispec vectors to pcDNA3.4 vectors, showed that production increased significantly even when only the vector was changed without construct mutations (Figure 3d). Yields were measured using proteins purified using the purification method used in Example 3 above. The IL-17RA / RC-Fc-TNFR2 variant construct information of the present invention is summarized in Table 10 below. Linkers L1 and L2 in Table 10 are based on the linker sequence information in Table 4. The IL-17RA / RC-Fc-TNFR2 variant protein number 26 was produced in a cell line in which galactosyltransferase-6 (GT6) was introduced into the ExpiCHO-S® system to promote the synthesis of sialylated glycans (Example 17).

[0219] [Table 10-1]

[0220] [Table 10-2] [Examples]

[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 in the sample at concentrations of 0.5-1 mg / ml. The 1000x dye from the Thermal Shift Assay Dye Kit (Applied Biosystems, 4461146) was diluted to 8x, and the sample was placed in the solution and diluted with tertiary distilled water. Then, the reaction buffer from the above kit, the protein, and the dye mixture were mixed (7.5 μl reaction: 10 μl protein: 2.5 μl 8X dye), and the melting curve was measured by PCR (Quantstudio 6 flex (Applied Biosystems, 4485689)). Subsequently, the Tm and ΔTm values ​​were confirmed using Thermal Shift Assay Software (Applied Biosystems, 4466038). Tm corresponds to the maximum value of the first derivative of the DSF melting curve determined by S / W in °C. The thermal stability of IL-17RA-Fc proteins with various mutations was measured, and stability was maintained up to 65°C. Comparing the IL-17RA-FC proteins numbered 3, 6, and 7 in Table 11, the IL-17RA-Fc protein with both T24D and N88D mutations introduced showed increased thermal stability compared to the IL-17RA-Fc protein with the T24D mutation or the IL-17RA-Fc protein with the N88D mutation. According to Figures 4, numbers 2, 8, and 10, the IL-17RA-Fc protein, with the addition of T24D to the F59V, R108K, and D122G mutation formulations, did not show increased thermal stability. To determine which formulations among the diverse back-to-consensus mutation formulations simultaneously affect IL-17A affinity and stability, thermal stability and affinity analysis experiments were performed. The results of the thermal stability analysis of the IL-17RA-Fc fusion proteins are summarized in Table 11 below.

[0223] [Table 11] [Examples]

[0224] Example 5: Analysis of the affinity of IL-17RA / RC-Fc-TNFR2 to ligands (IL-17A, IL-17F, TNFα)

[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), Microplate, 96-well / pureGrade, Black / F bottom, 350 μl (BRAND.781608), and AHC2 biosensor (Sartorius, 18-5019) were prepared. Affinity measurement was performed using Sartorius Octet, and the AHC2 biosensor was first hydrated with 2x kinetic buffer. Ligand concentrations were prepared so that IL-17RA / RC-Fc-TNFR2 would adhere to the biosensor at a density of 0.8-1 nm. Five to seven ligand concentration intervals were set for the experiment, and the expected KD value was determined at a concentration approximately midway between these intervals. A baseline was established using the hydrated biosensor and a buffer solution. Subsequently, the biosensor was transferred to a solution containing IL-17RA / RC-FC-TNFR2, and the IL-17RA / RC-FC-TNFR2 was bound to a density of 0.8-1 nm. The bound biosensor was then re-equilibrated using the buffer solution used to establish the baseline. After that, association was performed by transferring it to a solution containing the ligand, and then dissociation was performed by transferring it to a solution containing the buffer solution used to establish the baseline.

[0226] The octet of IL-17RA / RC-Fc-TNFR2 for ligands (IL-17AA, IL-17AF, IL-17FF, and TNFα) and their respective binding affinity values are the same as Table 12 below. The affinity of IL-17RA / RC-Fc-TNFR2 for ligands (IL-17AA, IL-17AF, IL-17FF, and TNFα) was compared with bimekizumab in the positive control group. The affinity of IL-17RA / RC-Fc-TNFR2 for IL-17AA was measured to have an affinity comparable to that of bimekizumab. The affinity of IL-17RA / RC-Fc-TNFR2 for IL-17AF was measured to have an affinity equal to or lower than that of bimekizumab. The affinity of IL-17RA / RC-Fc-TNFR2 for IL-17FF was measured to have an affinity lower than that of bimekizumab compared to bimekizumab. Also, the affinity of IL-17RA / RC-Fc-TNFR2 for the ligand TNFα was compared with etanercept in the positive control group. The affinity of IL-17RA / RC-Fc-TNFR2 was measured to have an affinity equal to or higher than that of etanercept compared to etanercept. All the binding affinity values of IL-17RA / RC-Fc-TNFR2 for ligands (IL-17AA, IL-17AF, IL-17FF, and TNFα) were measured to have higher affinity values than wtIL-17RA, wtIL-17RC, and wtTNFR2. The results of the affinity analysis of each ligand (IL-17A, IL-17F, TNFα) of IL-17RA / RC-Fc-TNFR2 are summarized in Table 12 below.

[0227]

Table 12

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

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

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

[0231] Example 6: Analysis of the binding morphologies of IL-17RA / RC-Fc-TNFR2 and target ligand, and IL-17A and TNFα.

[0232] The recombined multiple target fusion protein of the present invention is a protein in which two or more protein domains are fused, and it is important to ensure that the intrinsic biological activity of each component protein domain is not damaged. If appropriate linkers are selected between each component domain and the protein is manufactured with appropriate constructs, the activity of each component protein, i.e., the ligand-receptor interaction, can be maintained without steric hindrance. Maintaining the activity of the recombined multiple target fusion protein allows for improved stability, extended plasma half-life, and enhanced in vivo efficacy of the fusion protein.

[0233] This invention aims to confirm that the multi-target fusion protein IL-17RA / RC-Fc-TNFR2 simultaneously binds to each ligand, IL-17AA, IL-17FF, IL-17AF, or TNFα. It also aims to confirm that when IL-17RA / RC-Fc-TNFR2 binds to each ligand simultaneously, the neutralizing ability of each ligand is not significantly different from that of the individual components of IL-17RA / RC or TNFR2. IL-17RA / RC-Fc-TNFR2 and IL-17A were co-expressed in expi-CHO cells via co-transfection, producing IL-17RA / RC-Fc-TNFR2 and IL-17A proteins. Human TNFα (hTNFα) was produced in Escherichia coli (E. coli) through overexpression. Subsequently, the secured IL-17RA / RC-Fc-TNFR2, hIL-17A, and hTNFα proteins were purified through his-tag affinity, anion exchange, and size exclusion chromatography. The purified multi-target fusion protein IL-17RA / RC-Fc-TNFR2, along with the target ligands IL-17A and TNFα, were incubated overnight at 4°C. The cultured proteins were then centrifuged at 13,000 rpm for 10 minutes to remove aggregation. The proteins from which aggregation had been removed were then separated again using SEC to separate the unbound proteins from the bound proteins.

[0234] As a result, the first peak on the left side of Figure 5a represents the form in which all three proteins are bound, while IL-17A and TNFα are shown, and TNFα not bound to IL-17RA / RC-Fc-TNFR2 is confirmed in the peak on the right side. In the SDS-PAGE gel analysis results, TNFα formed a trimer via hydrogen and ionic bonds, not disulfide bonds, and was confirmed in monomer form on both non-reducing and reducing gels. In the case of IL-17A, it formed a dimer via disulfide bonds, and was confirmed at the size of a unit on reducing gels and at the size of a dimer on non-reducing gels. Furthermore, in the case of IL-17A, since it was cultured in mammalian Expi-CHO cells, it had a glycosylation site, which was confirmed by many bands due to glycosylation (Figure 5b).

[0235] When TNFα produced in E. coli cells different from the protein produced by the co-expression of IL-17RA / RC-Fc-TNFR2 and IL-17A was co-cultured (4°C, 24 hours), it was confirmed that the triple-specific fusion protein IL-17RA / RC-Fc-TNFR2 stably binds to the first ligand IL-17A and the second ligand TNFα, forming a complex. Furthermore, by confirming that the multiple-target fusion protein IL-17RA / RC-FC-TNFR2 can bind to each target simultaneously, it can be predicted that there is no significant difference in neutralizing ability when IL-17RA / RC-Fc-TNFR2 binds to each ligand simultaneously or sequentially. In addition, BLI measurement (Example 5) confirmed that the affinity of the IL-17RA / RC-Fc-TNFR2 fusion protein to its ligand TNFα was <1.0E-12. When the IL-17RA / RC-Fc-TNFR2 fusion protein was made to form a complex with the first ligand, IL-17A, and the affinity to the second ligand TNFα was measured, the affinity was measured at 1.6E-11. Comparing the above <1.0E-12 and 1.6E-11 values, it was confirmed that there was no significant difference in affinity between IL-17RA / RC-Fc-TNFR2 binding to the first ligand and then to the second ligand. [Examples]

[0236] Example 7: Analysis of the neutralizing ability of fusion proteins IL-17RA / RC-FC and IL-17RA / RC-Fc-TNFR2 against target ligands IL-17AA, IL-17AF, and IL-17FF.

[0237] For neutralizing capacity analysis, HEK-Blue® reporter cells (InvivoGen, hkb-IL-17 or HEK-Blue® TNFα), DPBS (Welgene, LB 001-02), FBS (Gibco, 16000044), DMEM (Welgene, LM 001-05), antibiotic (Welgene, LS 203-01), HEK-Blue® Selection (InvivoGen, hb-sel), Normocin® (InvivoGen, ant-nr-1), QU anti-Blue® Solution (InvivoGen, rep-qbs), Tween-20 (Junsei, 69295S1601), Autoclaved DW, Peprotech (200-17), IL-17A (Peprotech, cat no. 200-17), IL- We have prepared 17F (Peprotech, cat no. 200-25) and TNFα (Peprotech, cat no. 300-01A).

[0238] In the inflammatory environment of autoimmune diseases, IL-17AA, IL-17AF, and IL-17FF ligands form complexes with IL-17RA / RC, inducing the formation of IL-17 signalosomes. These IL-17 signalosome complexes then amplify inflammatory cytokines and chemokines through intracellular signaling (Arnaud Goepfert et al, Ann. Rheum. Dis. CellReports 41, 111489, 2022). The selective neutralization of ligands IL-17A, IL-17F, and IL-17A / F by the fusion protein IL-17RA / RC-Fc-TNFR2 in the inflammatory environment is closely associated with the therapeutic effects 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 to their respective ligands, HEK-Blue® reporter cells (InvivoGen, hkb-IL-17, or HEK-Blue® TNF-α) will secrete secreted embryonic alkaline phosphatase (SEAP) in a concentration-dependent manner when treated with ML-17AA, ML-17AF, or hTNFα ligands according to their respective concentrations. By treating the fusion proteins IL-17RA / RC-Fc and IL-17RA / RC-Fc-TNFR2 of the present invention and quantifying SEAP in the culture medium, the inhibitory ability on signal transduction pathways can be measured. During the isolation process of HEK-Blue® reporter cells, the culture medium was removed from the cultured cells, washed twice with 1XPBS, and the cells were isolated. The harvested 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). Subsequently, 10 μl of Trypan-blue and 10 μl of resuspended cells were added and mixed. After adding 10 μl using a hemocytometer and mixing, the viable cells were counted. Final concentration: 2.5 x 10⁻⁶ 5Mix with the test medium to obtain cells / ml cells, and after subculturing 100 μl of cells per well of a 96-well plate, culture overnight in an incubator. Then, prepare a mixture (10 - 100 μg / ml) of the fusion protein IL-17RA / RC-Fc protein of the present invention diluted in 0.005% Tween-20 / PBS at different concentrations. Subsequently, prepare three kinds of ligands (hIL-17AA, hIL-17AF, treatment concentration 10 ng / ml each), and treat the subcultured cells with 100 μl per well of IL-17RA / RC-Fc and each ligand in a 96-well plate, and culture in an incubator for 24 hours. To evaluate the neutralizing ability of the prepared HEK-Blue® reporter cells' IL-17RA / RC-Fc-TNFR2 protein against each ligand, mix 180 μl of QUANTI-Blue® solution with 20 μl of the sample cultured for 24 hours above, culture for 10 minutes, then place it in a microplate reader (Bio-Tek microplate reader), and measure the OD value at a wavelength of 630 nm. Prepare a mixture (10 - 100 μg / ml) of the fusion protein IL-17RA / RC-FC-TNFR2 diluted in 0.005% Tween-20 / PBS at different concentrations. Also prepare one of the ligands (hIL-17AA, hIL- FF, TNFα) at treatment concentrations of 10 ng / ml, 10 ng / ml, and 1 ng / ml respectively. Thereafter, after performing in the same manner as the above method, place it in a microplate reader (Bio-Tek microplate reader), and measure the OD value at a wavelength of 630 nm. Use bimekizumab for the positive control group for IL-17A and IL-17F neutralizing ability, and use adalimumab for the positive control group for TNFα neutralizing ability.

[0240] As a result, the IC representing the neutralizing ability of the fusion protein IL-17RA / RC-Fc-TNFR2 against IL-17AA 50 concentration is 20.9 ng / ml, and the IC of the positive control group, bimekizumab 50This is at a comparable level to 19.87 ng / ml. IC2 represents the neutralizing ability of the fusion protein IL-17RA / RC-FC-TNFR2 against TNFα. 50 The concentration was 14.94 ng / ml, and the positive control group and adalimumab IC were evaluated. 50 This is a high level compared to 27.68 ng / ml. Furthermore, the IC, which represents the neutralizing ability against IL-17FF, is also high. 50 The concentration was 1152 ng / ml, and the positive control group and the IC of bimekizumab 50 Although its neutralizing ability was confirmed to be lower compared to 31.02 ng / ml, it was confirmed to have specificity for IL-17FF. The neutralizing ability of the multi-target fusion protein IL-17RA / RC-Fc-TNFR2 against its two ligands, IL-17AA and TNFα, was measured at approximately the same level as that of bimekizumab and adalimumab in the positive control group (Figure 6a or 6c). The results of the analysis of the neutralizing ability of IL-17RA / RC-Fc-TNFR2 against ligands (hIL-17AA, hIL-17FF, TNFα) measured in the above SEAP analysis are summarized in Table 13 below.

[0241] [Table 13] [Examples]

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

[0243] For affinity analysis, 50 ml of carbonate buffer (pH 9.6, Na2CO3 (Sigma, S7795, 0.1M):NaHCO3 (Sigma, S8875, 0.1M) = l:l) was prepared in coating buffer. One of the ligand-recombined proteins (Human IL-17A PeproTech; 200-17, Human IL-17F - PeproTech; 200-25, Human TNFα - PeproTech; 300-01A, Murine IL-17A - PeproTech; 210-17, Murine IL-17F - PeproTech; 210-17F, Murine TNFα-PeproTech; 315-01A) at 1 μg / ml was added to the coating buffer, and 96-well plates were coated at 40 μl / well overnight at 4°C. On day 2, the plates were washed three times with 200 μl / well of ELISA TBS buffer (IXTBS and 0.1% Tween20). After dilution with 100 μl / well of ELISA TBS buffer with 5% BSA added, the plates were left at room temperature for 1 hour, and then washed three times with 200 μl / well of ELISA TBS buffer. Subsequently, 100 μl / well of IL-17RA / RC-Fc-TNFR2 protein was added to blocking buffer and left at room temperature for 2 hours. The IL-17RA / RC-Fc-TNFR2 protein was treated at concentrations of 0 / 0.05 / 0.5 / 5 / 50 / 500 / 5000 / 50000 ng / ml. The plates were washed three times with 200 μl / well of ELISA TBS buffer, treated with HRP-conjugated secondary antibody (Abeam, Cat.Ab98624), and reacted at room temperature for 1 hour. Subsequently, the sample was washed five times with ELISA TBS buffer, reacted with 100 μl of TMB solution (Seracare KPL, Cat#5120-0080) at room temperature, then 100 μl of stop solution (Stop solution, Cat#5150-0024) was added, and the absorbance was measured at 650 nm using an ELISA reader. This represents the EC50, which indicates the affinity of the fusion protein IL-17RA / RC-Fc-TNFR2 to ML-17A. 50 The positive control group had a concentration of 9.237 ng / ml, while the EC group of bimekizumab 50This is at a comparable level to 6.197 ng / ml and represents the EC of affinity for ML-17F. 50 A concentration of 130.3 ng / ml was the positive control group, and bimekizumab EC 50 This is at approximately the same level as 14.2 ng / ml. EC represents the affinity of the fusion protein IL-17RA / RC-Fc-TNFR2 for hTNFα. 50 The concentration was 43.08 ng / ml, in the positive control group and in the adalimumab EC group. 50 The value was 20.03, which is at a nearly equivalent level. In particular, the affinity of the multi-target fusion protein IL-17RA / RC-Fc-TNFR2 to the two ligands IL-17A and TNFα was measured at a level nearly equivalent to that of the positive control group bimekizumab and adalimumab (Figure 7a or 7c). The measurement results of the affinity of the above fusion protein IL-17RA / RC-Fc-TNFR2 to IL-17A, IL-17F, and TNFα are summarized in Table 14 below.

[0244] [Table 14] [Examples]

[0245] Example 9: Evaluation of the anti-inflammatory efficacy of the multi-target fusion protein IL-17RA / RC-Fc-TNFR2 in an environment similar to that of a psoriasis patient.

[0246] HaCaT cell lines (human epidermal keratinocyte lines) express strong pro-inflammatory cytokines and chemokines in response to co-stimulation by TNFα and IL-17. In other words, the production and amplification of numerous inflammatory cytokines and chemokines, as well as antimicrobial peptide genes, which are also observed in human psoriatic skin lesions in ex vivo experiments, can be confirmed (Andrea Chiricozzi et al., J. Invest. Dermatol. 131(3):677-687, 2011).

[0247] To confirm the expected therapeutic efficacy of IL-17RA / RC-Fc and TNFR2-Fc in human psoriatic lesions according to one embodiment of the present invention, a comparison was made between positive control antibodies, bimekizumab and adalimumab. Furthermore, the anti-inflammatory efficacy of IL-17RA / RC-Fc, TNFR2-Fc, and IL-17RA / RC-Fc-TNFR2 was sought by comparing the anti-inflammatory efficacy of groups treated with a combination of IL-17RA / RC-Fc and TNFR2-Fc fusion protein or IL-17RA / RC-Fc-TNFR2 monotherapy with the combination of positive control bimekizumab and adalimumab. For the above experiment, HaCaT (human epidermal keratinocyte cell line) was prepared by maintaining it in 2% FBS and low-calcium DMEM (Gibco, 21068028) for more than two weeks. Subsequently, after treatment with 0.53 mM EDTA (10 min, 37°C), the cells were treated with Trypsin EDTA (2 min, 37°C), 2% FBS low-calcium DMEM was added, and the single-cell suspension was centrifuged (1200 rpm, 5 min, and 4°C). After counting the number of cells suspended in 2% FBS low-calcium DMEM, the cells were placed on a 12-well plate (1.0 x 10⁶). 5 Cells were divided into 1 mL cells and cultured overnight. After stabilization, the cells were washed in PBS medium and starvation was carried out by adding 0.9 mL of 0.5% FBS low-calcium DMEM (24 hours, 37°C).

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

[0249] [Table 15]

[0250] *Inhibitor treatment groups (Treatment groups 4 to 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 at concentrations of 5 ng / mL, 20 ng / mL, and 80 ng / mL, respectively.

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

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

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

[0257] To perform qRT-PCR analysis of each treatment group, cells were disrupted using the cell Qiazol lysis reagent (Qiagen, 79306), chloroform was added, and the mixture was centrifuged at 13,200 rpm at 4°C for 15 minutes to separate the upper aqueous layer. Isopropanol was added to the separated organic layer to precipitate the RNA, and the mixture was centrifuged at 13,200 rpm at 4°C for 10 minutes. Subsequently, the pellet was washed with 75% DEPC-EtOH, centrifuged at 13,200 rpm for 5 minutes, and the ethanol from the pellet was evaporated. The pellet was then dissolved in 30-50 μl of DEPC-water, and the concentration of the dissolved RNA was 200-250 ng / μl. cDNA was obtained by reverse transcription using a cDNA synthesis kit (abm, G236), and 1 μl of cDNA was mixed with the target gene primer (Table 16) and 10 μl of SYBR green. RT-qPCR was then performed using a QuantStudio® 5 Real-Time PCR system instrument.

[0258] IL-8 (CXCL8) is a chemokine that recruits neutrophils and macrophages, while S100A7 (Psoriasin) is a protein overexpressed in psoriatic skin. CCL20 is also an inflammatory cytokine induced by LPS, IFNγ, etc., and is a chemokine that recruits neutrophils. BD2 is a skin-antimicrobial peptide produced from epithelial cells of psoriasis patients. LCN2 (Lipocalin-2) is expressed by neutrophils and is a marker of skin damage. IL-36γ (IL1F9) is an IL-1 series cytokine that is activated by NFκB, is associated with psoriatic lesions, and is used as an infection biomarker. When comparing groups of HaCaT cells treated with IL-17 alone or TNF-α alone (Rain 1 and 2, respectively) with groups treated with IL-17 and TNFα simultaneously (Rain 3), a significant increase in the expression of S100A7, CCL20, BD2, LCN2, CXCL8, and IL-36γ was observed (Figure 8). This suggests that the combined effect of IL-17 and TNFα, rather than IL-17 or TNFα alone, significantly impacts psoriasis overgrowth and systemic inflammatory responses.

[0259] In the group treated simultaneously with IL-17 and TNFα, adalimumab treatment actually increased the expression of S100A7 and BD2. Furthermore, while bimekizumab did not significantly suppress CXCL8, IL-36γ, and CCL20, combined treatment with adalimumab and bimekizumab revealed significant expression suppression and sclerosis. The sequencing information of the primers used in the above RT-qPCR is summarized in Table 16 below.

[0260] [Table 16]

[0261] IL-17RA / RC-Fc, produced by one embodiment of the present invention, is a fusion protein that targets IL-17AA, IL-17-AF, and IL-17FF, while TNFR2-FC is a fusion protein that targets TNFα. Through the present invention, the inhibitory activity of IL-17RA / RC-FC and TNFR2-Fc against their respective target ligands was compared with the positive control antibodies bimekizumab and adalimumab, targeting cytokines and chemokines expressed by simultaneous treatment with IL-17 and TNFα. The inhibitory efficacy of IL-17RA / RC-Fc-TNFR2 was compared between groups treated with IL-17RA / RC- and TNFR2-Fc alone, groups treated with TNFR2-Fc in combination, and groups treated with the positive control antibodies bimekizumab and adalimumab alone and in combination. As a result, when comparing the anti-inflammatory effects of IL-17RA / RC-Fc and bimekizumab (Figure 8, Rain 4 and 10), the same level of inhibitory efficacy was confirmed, and the combination treatment group of IL-17RA / RC-Fc and TNFR2-Fc showed even better inhibitory efficacy than the mono-treatment group. When comparing the anti-inflammatory effects of TNFR2-Fc and adalimumab (Figure 8, Rain 5 and Rain 11), equivalent or better inhibitory efficacy was confirmed. In particular, for SW0A7, BD2, and LCN2, even better inhibitory efficacy than adalimumab was confirmed. The combination treatment group of IL-17RA / RC-Fc and TNFR2-Fc showed even better inhibitory efficacy than the mono-treatment group. When IL-17RA / RC-FC-TNFR2, a multi-target fusion protein that simultaneously targets IL-17AA, IL-17AF, and IL-17FF or TNFα, produced according to one embodiment of the present invention, was compared with treatment groups treated with each inhibitor alone or in combination, dose-dependent anti-inflammatory effects were confirmed from the psoriasis factors S100A7, CCL20, BD2, LCN2, CXCL8, and IL-36γ (Figure 8, Rain 7-9). In particular, the combination treatment group of IL-17RA / RC-Fc and TNFR2-Fc (Figure 8, Rain 6) or the combination treatment group of bimekizumab and adalimumab (Rain 12) showed sufficient inhibitory efficacy at 20 ng / ml of IL-17RA / RC-Fc-TNFR2, and superior inhibitory efficacy was confirmed at 80 ng / ml of IL-17RA / RC-Fc-TNFR2 compared to each of the combination treatment groups.When the inhibitor treatment groups exhibiting the same inhibitory effect were converted to molar concentration, the fusion protein IL-17RA / RC-Fc-TNFR2 at 80 ng / ml was at 392 pM, while the group treated with bimekizumab (20 ng / ml) and adalimumab (50 ng / ml) was at 473 pM. The present invention demonstrated efficacy at an even smaller volume when treating with IL-17RA / RC-Fc-TNFR2.

[0262] In psoriasis-like models induced by simultaneous stimulation of IL-17A and TNFα, single-target inhibitors of either IL-17A or TNFα have limitations. Furthermore, adalimumab actually increased the expression of 100A7 and BD2, and bimekizumab did not significantly suppress CXCL8, IL-36γ, and CCL20. However, combination therapy showed superior inhibitory efficacy compared to single-antibody treatments for all cytokines. Therefore, simultaneous inhibition of IL-17A and TNFα may be necessary to effectively control the inflammatory response.

[0263] The IL-17RA / RC-Fc-TNFR2 of the present invention showed dose-dependent sclerosis with S100A7, CCL20, BD2, LCN2, CXCL8, and IL-36. Sufficient inhibitory effects were confirmed in the 20 ng / ml treatment group, and at 80 ng / ml, it showed even better inhibitory efficacy than the group treated with a single inhibitor. Psoriasis is an immune cell-mediated chronic inflammatory skin disease in which infiltration of inflammatory mononuclear cells, macrophages, eosinophils, and inflammatory Th17 and Th1 cells, along with hyperproliferation and abnormal differentiation of keratinocytes, occurs simultaneously. It is important to simultaneously reduce the expression of inflammatory cytokines, chemotactic chemokines, and antimicrobial peptides. The multi-target fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention can be used as an excellent psoriasis treatment agent by simultaneously regulating IL-17 and TNFα, thereby effectively controlling various cytokines and chemokines involved in hyperproliferation and systemic inflammatory responses due to the elevated effects of IL-17 and TNFα. [Examples]

[0264] Example 10: Evaluation of the anti-inflammatory efficacy of IL-17RA / RC-Fc-TNFR2 in a rheumatoid inflammatory disease-like environment.

[0265] Rheumatoid arthritis is a synovial inflammatory disease accompanied by systemic symptoms. Inflammation begins in synovial luminal cells and progresses to the destruction of cartilage and soft tissue. The inflammatory response in synovial fibroblasts and surrounding leukocytes and stromal cells is amplified by the co-stimulation of IL-17 and TFNα. In particular, while the inflammatory effect of IL-17F alone is not significant, when IL-17A and IL-17F act simultaneously, TNFα, IL-6, IL-8, CXCL1, CCL20, CXCL5, IL-23, E-selectin, and EGR-1 are elevated through the MAPK family pathway. When TNFα is added, chronic inflammation is induced through mRNA stabilization (Arnaud Hot and Pierre Miossec., Ann. Rheum. Dis .70(5):727-32.2011). To confirm the disease-improving effect of the multi-fusion protein (IL-17RA / RC-Fc-TNFR2) of the present invention in a rheumatoid arthritis (RA)-like cell model using the hFLS-RA (Cell application Inc.) cell line, TNFα and IL-17 were simultaneously treated in hFLS-RA cells, and IL-6 expression levels were evaluated (Bilal Osta et al. Front. Immunol. 6: 151, 2015). Specifically, rheumatoid hFLS cells were treated with 2 x 10⁶ cells in each well of a 96-well plate. 4Cells were cultured in DMEM (Welgene, LM001-005) and 10% FBS (Gibco, 16000044) for 24 hours at 37°C. On day 2, to induce an RA model, cells were treated with IL-17A, TNFα cytokines, and IL-17RA / RC-Fc, TNFR2-Fc single-target fusion proteins and the single-target fusion protein IL-17RA / RC-Fc-TNFR2, and treated with the above concentrations of the control drugs bimekizumab (Antibody System, DHH28803) and adalimumab (Selleckchem, A2010) (Figure 9) for 24 hours at 37°C. The following day, the culture medium was obtained, and to 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). The coating buffer was prepared as 50 ml of carbonate buffer (pH 9.6, Na2CO3 (sigma, S7795, 0.1M):NaHCO3 (sigma, S8875, 0.1M) = l:l). 1 μg / mL of reconstituted IL-6 protein was added to the coating buffer, and 96-well plates were coated at 40 μl / well overnight at 4°C. On the second day, the above plates were washed three times with ELISA TBS buffer (1x TBS and 0.1% Tween20) at 200 μl / well. Subsequently, the solution was diluted with 100 μl / well of ELISA TBS buffer containing 5% BSA, left at room temperature for 1 hour, and then washed three times with 200 μl / well of ELISA TBS buffer. After that, the cells were separated with 100 μl / well of blocking buffer and left for 2 hours. The concentrations of the IL-6 standard curve were set to 9.38, 18.8, 37.5, 75, 150, 300, and 600 pg / mL as instructed. The solution was washed three times with 200 μl / well of ELISA TBS buffer, treated with HRP-conjugated secondary antibody, and reacted at room temperature for 1 hour. After that, the solution was washed five times with ELISA TBS buffer, reacted with 100 μl of TMB solution at room temperature, and then 100 μl of stop solution was added.ELISA measurements were performed using a BioTek Synergy HTX microplate, measuring absorbance at 540 nm and 450 nm. To evaluate the anti-inflammatory efficacy of the IL-17RA / RC-Fc-TNFR2 protein, IL-6 expression was examined and found to decrease in a concentration-dependent manner (Figure 9, Rain 9, 10, 11). In particular, in the IL-17RA / RC-Fc-TNFR2 treatment group (Figure 9, Rain 11), the IL-6 level decreased more significantly in the control group treated with bimekizumab and adalimumab in combination with the control drug formulation, thus confirming the anti-inflammatory effect (Figure 9, Rain 14). [Examples]

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

[0267] The antibody therapies adalimumab and infliximab also bind to transmembrane TNFα (tmTNFα), modulate the inflammatory response through MAP / ERK signaling, and promote cross-linking of tmTNFα and TNFR2, thereby regulating T cells (T). reg ) can promote proliferation and induce immune tolerance. The efficacy of TNFα inhibitors is not only in neutralizing soluble TNFα (sTNFα), but also in promoting reverse signaling through the binding of tmTNFα and T reg Therapeutic efficacy can be further improved through cell activation. Etanercept, a TNFR fusion protein, does not exert as much efficacy through tmTNFα as antibody-based therapies, and therefore, in some diseases, its efficacy may be lower than that of antibody-based therapies.

[0268] The fusion protein of this invention is IL-17RA / RC-Fc-TNFR2. To confirm that TNFR2 mutants, in which various mutations have been introduced into wtTNFR2, have tmTNFα binding, tmTNFα-expressing cells were produced. For the production of the tmTNFα-IRES-puro lentivirus, HEK-293T (1.2 x 10) was used in a 90 mm culture dish. 7Cells were separated and cultured (24 hours, 37°C). The next day, the culture medium was changed to fresh medium at 37°C, and the DNA vector (pLVX tmTNFα-IRES-puro, pCMV VSVG, pCMV delta8.2 vector; 2:1:2, mixed with 15 μg PEI, Source: Korea Nuclear Hospital) was plasma injected into the cell line (2-4 hours, 37°C). Subsequently, the culture medium was changed to 13 ml of fresh medium, and the superior culture solution was collected in a conical tube (50 mL) after 72 hours. The virus was then obtained using a centrifuge (500 g, 5 mins), impurities were removed using a 0.45 μm filter, and the cells were separated into 1 mL portions using an EP tube and stored at -80°C. Subsequently, to confirm the binding ability of the test drugs IL-17RA / RC-FC-TNFR2 and tmTNFα, HeLa cell lines (human cervical cancer cell lines) were first cultured in DMEM (Welgene, LM001-05) and 10% FBS (Gibco, 16000044) (24-48 hours, 37°C). To subculture the above cells into a 12-well plate, the FBS in the culture medium was removed with 10 mL of 1x DPBS, followed by treatment with 0.53 mM EDTA (10 min, 37°C) and trypsin EDTA (2 min, 37°C). Then, 2% FBS low-calcium DMEM was added, and the single-cell suspension was centrifuged (1200 rpm, 5 min, 4°C). During this time, cover glasses appropriate for each well of the 12-well plate were thoroughly disinfected (sterilized) using an alcohol lamp before being placed in the wells. Then, the secured cells were suspended in a fresh DMEM culture medium, and cells (1x10) were placed in each well. 5Cells (1 / mL) were separated and stabilized (24 hours, 37°C). Then, to transmote the cell culture with the pre-prepared tmTNFα-IRES-puro lentivirus, 8 μg / mL polyblen transmote infection reagent (Merck, TR-1003-G) was prepared in 500 μl lentivirus mixture, mixed with 500 μl culture medium in a 1:1 ratio (total 1 ml), and added to each well for culture (24 hours, 37°C). The next day, the virus mixture was removed, and after three washing steps using IXDPBS, 500 μl was added and maintained (24 hours, 37°C). The control drug, the TNFα antibody therapy agent adalimumab (Selleckchem, A2010), and the test drug, IL-17RA / RC-Fc-TNFR2 (Tl-T4-14), were each treated at 1 μg / mL and cultured for 6 hours (37°C). Subsequently, the culture medium was stabilized for 18 hours, and then, for fluorescent staining, the culture medium was removed, the wells were washed three times with 1x DPBS, and 4% PFA (paraformaldehyde) fixative was added to each well and maintained at room temperature for 30 minutes. Next, the PFA treatment solution was removed, the wells were washed three times with IX DPBS, and then the wells were maintained at 4°C for 1 hour in barrier buffer (1% BSA and 0.1% NaN3in 1X DPBS). For fluorescent staining, the primary antibody (Adalimumab; IgG1, T1-T4-14; Fc antibody) was diluted in the culture medium at 500x and added to the corresponding wells (24 hours, 4°C). For secondary antibody treatment, the culture medium was removed, the wells were washed three times with 1x DPBS, and the secondary antibody (Donkey anti-mouse IgG Alexa fluor 568) was diluted in the culture medium at 500x and added to the corresponding wells. Afterward, the cells in the well plate were washed three times with 1 mL x DPBS, and the cover glass was rubbed against the slide glass. Then, the cells were treated with DAPI solution and fluorescent staining mounting medium (Antifade mounting media). Next, the cover glass was coated with nail polish to fix it to the slide glass, and after drying for a certain period of time, it was observed using a Lionheart FX Automated Microscope (BioTek).As a result, when both tmTNFα (GFP) and an antibody (Alexa fluor 568; RFP) capable of sensing IL-17RA / RC-Fc-TNFR2 are expressed using the bicistronic vector shown in Figure 10a, they are identified as yellow. In Figure 10c, column 4 shows that the degree of yellow luminescence is stronger than that of the control drug adalimumab. Through these results, it is confirmed that IL-17RA / RC-FC-TNFR2 binds to tmTNFα at the same level as antibody therapy, enabling additional inflammatory control responses through the promotion of tmTNFα reverse signaling and induction of Treg cell activity and proliferation at a level equivalent to antibody therapy. The tmTNFα sequencing information (sequence number 82) inserted into the above pLVX tmTNFα-IRES-ZsGreen 1 vector is summarized in Table 17 below.

[0269] [Table 17] [Examples]

[0270] Example 12: Evaluation of the immunogenicity of the fusion protein IL-17RA / RC-FC-TNFR2 using human peripheral blood mononuclear cells (PBMCs)

[0271] In the case of high molecular weight protein pharmaceuticals, the ability to induce an immune response upon ingestion into the body is called immunogenicity. For example, if an antibody acts as an immune source in the human body, it induces cellular and humoral immune responses, and anti-drug antibodies (ADAs) in the cellular immune response will affect the efficacy stability of the drug and the results of pharmacokinetic and pharmacodynamic evaluations. Therefore, when developing high molecular weight protein pharmaceuticals, analysis of this aspect should also be carried out. 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-inducing potential of the fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention, and a two-step in vitro evaluation was performed, confirming the low immunogenicity of IL-17RA / RC-Fc-TNFR2.

[0272] The fusion protein IL-17RA / RC-Fc-TNFR2 of the present invention utilizes hIL-17RA, hIL-17RC, and hTNFR2 as binding sites to neutralize all three target ligands (IL-17A, IL-17F, and TNFα). Mutagenic formulations that improve ligand affinity, stability, and productivity of the fusion protein are introduced at each binding site, and the protein is constructed as a triple-specific fusion protein. To evaluate the immunogenicity of the above fusion protein IL-17RA / RC-Fc-TNFR2, the inventors evaluated its immunogenicity in two stages: in silico immunogenicity prediction and in vitro analysis using five types of human peripheral blood cells. The IEDB (Immune Epitope Database) was used for the above in silico immunogenicity analysis. The IEDB, operated with the support of the National Institute of Allergy and Infectious Diseases (NIAID), an agency under the National Institutes of Health (NIH), is a database that catalogs and continuously updates experimental data on antibodies and T-cell epitopes studied in humans, primates, and other animal species from the perspectives of infectious diseases, allergies, autoimmune responses, and organ transplantation, making it a database that allows access to the latest information. The IEDB server uses analytical methods that improve the accuracy of T-cell epitope prediction and is the most widely used in silico immunogenicity analysis worldwide. T-cell MHC Class II epitopes were analyzed using the consensus method, which represents the average value of results from the "recommended methods" (NN-align, SMM-align, and CombLib / Stumiolo method) of the IEDB (http: / / www.iedb.org / ) (Figure 11a).The analysis divides all input amino acid sequences by 15 possible aa peptides (9 binding sequences and 6 lateral sequences), and then converts the predicted values ​​for each analysis method for the 15 aa peptides into statistical percentile ranks by comparing them to the random score distribution of a large set of random peptides. The median percentile rank of the analysis method becomes the final predicted value. A lower percentile rank indicates a lower predicted value, i.e., a higher affinity for the MHC Class II receptor. The MHC Class II epitope prediction system simplified and visualized the results provided by IEDB in Excel format. The X-axis represents 27 HLA types, and the Y-axis represents the input amino acid sequences. The final predicted results, analyzed comprehensively, are shown in different colors (red: High; orange, medium: yellow, low: white, none) according to the degree of immunogenicity (Figure 11b).

[0273] The in silico immunogenicity analysis results above predicted that the two fusion proteins of IL-17RA / RC-Fc-TNFR2, T1-T4-14 and T1-T4-41, would each show intermediate (medkm) levels of immunogenicity at five sites (Figure 11b). In silico immunogenicity analysis is a method that predicts the likelihood of a specific peptide binding to an MHC class II molecule, and tends to over-predict. Therefore, to confirm whether the parts predicted to be highly immunogenic in in silico immunogenicity analysis induce high immunogenicity in the human body, the presence or absence of T cell activation was determined through in vitro immunogenicity experiments as described below. In vitro immunogenicity evaluation is a method that predicts the possibility of inducing immunogenicity in clinical settings by analyzing cytokines such as IFNγ and IL-2 due to T cell proliferation and activation using human peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) differentiated from PBMCs and T cells. To separate cells from PBMCs, the PBMCs were thawed in an Automated Cell Thawing System (BioCision, 13-900-314), then centrifuged at 300g for 5 minutes (Peripheral Blood Mononuclear Cells (PBMCs). Source: Catholic University Hematopoietic Cell Bank). After removing the superior solution, the cell pellet was resuspended in AIM-V medium (GIBCO, 12055-083) and seeded in a 100 mm cell dish. After treatment with DNase, the cells were cultured at 37°C in a 5% CO2 incubator. The following day, all cells were centrifuged (300g, 5 minutes) and harvested. After harvesting, the cells were suspended in running buffer, FcR Blocking reagent (Miltenyi Biotec, 130-059-901) was added, and the cells were maintained at 4°C for 5 minutes. Then, CD 14 MicroBeads (Miltenyi Biotec, 130-050-20) were added, and the cells were maintained at 4°C for 15 minutes. Subsequently, running buffer (Miltenyi Biotec, 130-091-221) was added, and the cells were centrifuged at room temperature at 300g for 10 minutes to secure the cells. After suspension in culture medium, CD14 was added using an LS column.- and CD14 + Cells were harvested. Collected CD14 - After adding CD4 microBeads (Miltenyi Biotec, 130-045-101) and CD8 MicroBeads (Miltenyi Biotec, 130-045-201) to the cells, maintain at 4°C for 15 minutes. After adding execution buffer, spin down gently at room temperature and 300g for 10 minutes to remove the supernatant, and resuspend the cells in execution buffer. Then load with an LS column (Miltenyi Biotec, 130-042-401) and flow through (CD4). - CD8 + After collecting the cells, wash the LS column three times with execution buffer. Use a plunger to remove the cells (CD4) from the LS column. - CD8 + Harvested cells. Collected CD14 - CD4 - CD8 - Cells are loaded into an LS column, and the permeate (CD4 + CD8 + Collect cells. Then, secure the CD4 + / CD8 + The cells will now use CD47 + / CD8 + For T cell activity experiments, store in liquid nitrogen before use. CD14 + Cells were cultured in AIM-V medium containing GM-CSF (JW Creagen, HGM-100) and IL-4 (JW Creagen, HI4-100) for 72 hours at 37°C. Next, AIM-V Medium containing GM-CSF and IL-4 was placed in a culture dish, and an additional 3 days of culture were carried out (72 hours at 37°C).

[0274] Cells harvested after undergoing dendritic cell (DC) differentiation for a total of 6 days were added to AIM-V medium containing GM-CSF and IL-4. Unsensitized DCs were treated with the test substances T1-T4-14 and T1-T4-41, and the positive control group was treated with PPD (Thermo Fisher, 7600060) for sensitization (24 hours, 37°C). Antigen-sensitized DCs were added to maturation medium (IL-6; JW Cleargen, HIL6-100, IL-1β; JW Cleargen, HIL-1B-100, TNFα; JW Cleargen, HTNA-100, PGE2; Sigma, P0409, AIM-V medium) for additional culture (24 hours, 37°C). CD4 was stored in liquid nitrogen while the DC cells were cultured. + / CD8 + After thawing the cells using an automated cell thawing system, the cells were centrifuged at 300g for 5 minutes to obtain the cells. After removing the superior solution, the cells were resuspended in AIM-V medium, separated into 60 mm culture dishes, and cultured (24 hours, 37°C). CD4 was collected the following day. + / CD8 + To confirm T cell proliferation, the cells were treated with 5 μM CSFE (Invitrogen, C34554) and incubated at 37°C for 10 minutes. (CFSE-stained CD4) + / CD8 + T cells were washed three times with 300 g of AIM-V medium for 5 minutes at room temperature (24 hours, 37°C). After harvesting the antigen-sensitized DCs, they were washed three times with 300 g of AIM-V medium for 5 minutes at room temperature. 2x10 5 CD47 + / CD8 + T cells and matured 1x10 4 Divided two DCs into 96-well plates. CFSE-stained CD47 + / CD8 +T cells were treated with anti-CD3 antibody (Biolegend, 300438) and anti-CD28 antibody (Biolegend, 302934). After co-culture for 7 days, the cells were harvested, resuspended in antibody solution (CD3 and CD4 or CD8 antibodies), and reacted at 4°C in the dark for 30 minutes. Washed in run buffer and centrifuged at 300g for 5 minutes to obtain the cells. After removing the supernatant, the cells were resuspended in run buffer and denatured using a FACS Canto-II equipped (BD, 338962) for CD4 + / CD8 + T cell proliferation was analyzed (Figures 12a, 12b, and 12c).

[0275] In vitro immunogenicity evaluation was performed using anti-CD3 / anti-CD28 in the positive control group and PPD in the internal control group. The test substance was IL-17-RA / RC-Fc-TNFR2 (T1-T4-14, T1-T4-41), and three replicates were performed under a single concentration condition (2 μg / ml) for comparative analysis. The immunogenicity of the test substance IL-17-RA / RC-Fc-TNFR2 (T1-T4-14, T1-T4-41) was determined using the Stimulation Index (SI) value, which is generally used to assess the effect of a drug on the activity of immune cells. The SI value was obtained by dividing the T cell proliferation rate observed under each condition by the proliferation rate of the control group. The SI value is a generally known reference value for measuring the efficacy of drugs in lymphocyte proliferation, and an SI value of 2 or higher is considered to induce immunogenicity (Stimulation Index (SI) = testwell / reference line, SI > 2 is considered ADA positive). In the ADA-positive control group, immunogenicity is confirmed when the SI value for MHC Class I and MHC Class II from all five types of PBMCs is 2 or higher. Based on the average SI value obtained from treating the above test substance in three replicates, it was finally confirmed that IL-17-RA / RC-Fc-TNFR2 (Tl-T4-14, T1-T4-41) does not induce immunogenicity when the SI value for MHC Class I and MHC Class II from all five types of PBMCs is 2 or lower. [Examples]

[0276] Example 13: Evaluation of the efficacy of IL-17RA / RC-hybrid fusion protein (IL-17RA / RC-Fc) in peripheral blood mononuclear cells

[0277] Autoimmune diseases are chronic diseases, and the number of immune cells (population) and the characteristics of each immune cell in PBMCs are not the same as in healthy individuals. The lesion site tissue and PBMCs in patients are also not the same as in healthy individuals. In the case of psoriasis and psoriatic arthritis, IL-17A + CD8 + It correlates with the severity of T-cell disease. Also, IL-17 is locally present in damaged tissue or joint tissue. + A + CD8 + The T cell ratio is high (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 systemic inflammatory responses, and inflammatory cytokines are amplified when IL-17F is expressed together with IL-17A or IL-17A and TNFα. A clinical efficacy study of bimekizumab for joints and skin in patients with psoriatic arthritis (NCT01087788) verified that simultaneous neutralization of IL-17A and IL-17F more effectively suppressed inflammatory responses compared to neutralization of IL-17A alone. The ACR20 response (at 8 weeks) of bimekizumab showed superior efficacy compared to placebo, with 80% versus 17% of patients receiving bimekizumab, compared to secukinumab (54% versus 15%) and ixekizumab (62% versus 30%), which neutralized only IL-17A (Glatt S et al. Ann. Rheum.Dis .77(4):523-532, 2017). In psoriasis patients with PBMCs, the cytokine production regulatory efficacy of treatment with the test substances IL-17RA / RC-Fc (IL-17A / F inhibition), TNFR2-Fc (TNFα inhibition), a combination of IL-17RA / RC-Fc and TNFR2-Fc, and IL-17RA / RC-Fc-TNFR2 (simultaneous inhibition of IL-17A / F and TNFα), as well as a control drug (Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL combination treatment group) was analyzed.

[0278] Psoriasis patients (n=4) were recruited, and peripheral blood mononuclear cells were obtained from donated blood (Catholic University Seoul St. Mary's Hospital, approval number KC21TNSI0375). PBMCs were isolated by coating 48-well plates with 2 μg / mL anti-CD3 and then 5x10. 5Cells were separated into 500 l cells per well. Additionally, 100 ng / mL LPS was added for stimulation, and the test material was treated and cultured according to each treatment condition (72 hours, 37°C). Subsequently, the superior culture solution was separated, and IL-17A (human IL-17A Duoset ELISA, R&D system DY317) and IL-17F (human IL-17F Duoset, R&D system DY1335B) ELISAs were performed (absorbance measurement: molecular devices). As a result, IL-17A production was reduced in all groups of the test substance and control drugs, with a 96.3% reduction in IL-17RA / RC-Fc (0.5 μg / mL) + TNFR2-Fc (0.5 μg / mL), and a 91.9% reduction, 89.85% reduction, 88.3% reduction, and 96.5% reduction in IL-7RA / RC-Fc-TNFR2 (0.1, 0.5, 1.0, 2.5 μg / mL), respectively. In the control drug (Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL combined treatment group), a 97.4% reduction was confirmed (Figure 13). IL-17F production was also reduced in all groups of the test substance and control drugs, with a 97.4% reduction in IL-17A production. The analysis results showed a reduction effect in both the test substance and the control drug group. IL-17RA / RC-Fc-TNFR2 at 1.0 μg / mL and 2.5 μg / mL showed reductions of 67.3% and 100%, respectively, while the combined control drug (Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL) group showed a 100% reduction (Figure 13). Furthermore, IL-17RA / RC-Fc-TNFR2 (simultaneous inhibition of IL-17A / F and TNFα) showed a reduction in the production of IL-17A, IL-17F, IL-21, IL-22, TNFα, and IFN-γ. In particular, efficacy was confirmed for IL-17F inhibition with IL-17RA / RC-Fc-TNFR2, which simultaneously inhibits IL-17A / F and TNFα, compared to single inhibition with IL-17RA / RC-Fc. In TNFα inhibition, IL-17A / F and TNFα were also inhibited. Efficacy was confirmed with IL-17RA / RC-Fc-TNFR2, which simultaneously suppresses TNFα.In Example 7 of the present invention (SEAP analysis), the neutralizing ability of IL-17RA / RC-Fc-TNFR2 against IL-17F was found to be slightly lower than that of bimekizumab. However, the inhibitory effect on IL-17F in psoriasis patient PBMCs was 100% reduction, which is the same level as the 100% reduction in the control drug (Bimekizumab 0.5 μg / mL + Adalimumab 0.5 μg / mL) combined treatment group. Compared to the combined treatment group of bimekizumab and adalimumab, which have high affinity for IL-17A, IL-17F, or TNFα, respectively, which were elevated in psoriasis patient PBMCs, IL-17RA / RC-Fc-TNFR2, which simultaneously suppresses IL-17A, IL-17F, and TNFα, showed an equivalent level of inhibitory efficacy. This means selectively suppressing pathogenic cytokines in the psoriasis and autoimmune disease environment, and improving side effects with minimal intervention. [Examples]

[0279] Example 14: Analysis of IL-17A and TNFα neutralizing ability of IL-17RA / RC-Fc-mTNFR2 from a psoriasis-inducing animal model.

[0280] In animal models in which psoriasis models were induced, experiments were conducted as follows to evaluate neutralizing ability. Using CRISPR / Cas 9 technology, mouse IL-17A and mouse IL-17F were removed to produce humanized double Knock-In mice expressing human IL-17A and IL-17F (Figure 14a). The IL-17RA / RC-Fc-TNFR2 of the present invention is a multi-target fusion protein that targets human IL-17A, human IL-17F, and human TNFα. The C57BL / 6 mIL-17A / 17F humanized mouse expresses human IL-17A and human IL-17F, but TNFα is mouse TNFα (mTNFα). Therefore, it is difficult to evaluate the efficacy of simultaneously inhibiting three types of mIL-17A, mIL-17F, and hTNFα ligands, and the therapeutic effect thereof, in a psoriasis disease model using humanized mice. Therefore, in order to evaluate the efficacy of IL-17RA / RC-Fc-TNFR2 in double Knock-In mice, the inventors replaced the hTNFR2 ECD in the hTNFα binding site of IL-17RA / RC-Fc-TNFR2 with an mTNFR2 ECD to produce IL-17RA / RC-Fc-mTNFR2 so that it could target hIL-17A, hIL-17F, and mTNFα expressed in humanized mice. To confirm that the above IL-17RA / RC-Fc-mTNFR2, Tl-mT4-35 (sequence number 32), reacts with mIL-17A, mIL-17F, and mTNFα, the experiment according to Example 8 was performed. The IL-17RA / RC-Fc-mTNFR2 of the present invention reacts with human IL-17A and human IL-17F, respectively, with EC 50 The values ​​were 80.31 ng / ml, 90.74 ng / ml, and 71.01 ng / ml for mouse TNFα, confirming similar affinity for each cytokine (Figure 14b).

[0281] To induce psoriatic disease, hair removal was performed (shaving a wide area from the neck to the buttocks using hair clippers, applying hair removal cream, and wiping it off with tissue paper 40 seconds later). Imiquimod (IMQ; Aldera Cream) 60 mg was applied to the skin of each hair removal area (back area) in the overall test group (G1-G4) (Figure 14c) (once a day, for a total of 5 times). On the last day of the test (day 5), 200 μl of LPS (lipid polysaccharide of Escherichia coli, O111:B4) was administered once intraperitoneally to the test material of the additional disease model. Subsequently, 1 mg / kg of the test material of the present invention, IL-17RA / RC-Fc-mTNFR2, was administered (once every 2 days starting the day after IMQ application). Blood samples were collected from each individual, incubated at room temperature for 10-20 minutes, centrifuged (2,500 RPM, 15 minutes), and plasma was separated from the superior solution and then stored frozen. Blood samples were collected a total of two times: before IMQ application and on the final day of application (day 5). Standard curves for IL-17A and TNFα were presented via ELISA (Duoset ELISA, R&D system), and the neutralizing ability of hIL-17RA / RC-Fc-mTNFR2 against cytokines (hIL-17A and mTNFα) was confirmed. ELISA results of mouse blood samples on the final day (day 5) of the mouse in vivo neutralizing ability confirmation test showed that the neutralizing ability to neutralize mouse TNFα was most significantly suppressed in G4 (C57BL / 6 hIL-17A / 17F double KI mice + hT1-mT4-35) (G3: 7.30 pg / mL, G4: 0.0 pg / mL). Similarly, the neutralizing ability to neutralize human IL-17A was significantly suppressed in G4 (C57BL / 6 hIL-17A / 17F double KI mice + hT1-mT4-35) compared to G3 (C57BL / 6 hIL-17A / 17F double KI mice) (G3: 140.79 pg / mL, G4: 47.89 pg / mL) (Figure 14d). The affinity analysis results for the above IL-17RA / RC-Fc-mTNFR2(hTl-mT4-35) to IL-17A and mTNFα are shown in Table 18 below, and G1 or G4 information from the C57BL / 6 hIL-17A / 17F double Knock-In mouse psoriasis analysis are shown in Table 19.

[0282] [Table 18]

[0283] [Table 19] [Examples]

[0284] Example 15: Electron microscopy (cryo-EM) observation of IL-17RA / RC-FC-TNFR2 hybrid fusion protein

[0285] Three-dimensional transmission electron microscopy (3-dimensional TEM) is a technique for elucidating the structure of protein complexes through biological image analysis. Through high-resolution 3D structural analysis, the structure of each domain of the IL-17RA / RC hybrid fusion protein and the interaction between the binding domain and the target protein can be analyzed. De novo structural analysis confirmed the interaction between the IL-17RA / RC-Fc-TNFR2 fusion protein and the target protein. Specifically, for negative staining, 5 μl of the IL-17RA / RC hybrid fusion protein sample was added to a carbon-coated grid on a glow-discharged (Harrick Plasma, US) grid fixed with automated closure forceps (Dumont, Swiss), and lattice-negative staining was performed with 1% uraneil acetate. The edge of the grid was brought into contact with filter paper (Whatman, UK) to remove excess staining solution, and the grid was dried for 10 seconds before being experimented on with a Technai 10 TEM (FEI, USA) operating at 100 kV. Images were recorded using an Ultrascan 1000CCD (Gatan, US) at a magnification of 0.32 nm / pixel. For negative staining, automated image analysis used a total of 7,084 IL-17RA / RC-FC-TNFR2 particles. Image alignment, reconstruction, 3D volume rendering, and visualization were performed, and class mean values ​​were calculated using the RELION 3.1 program. Initial classification excluded images that were not compacted and did not stain well, and folded images that could be viewed from the left / right side were selected to perform particle analysis of the IL-17RA / RC-Fc-TNFR2 hybrid fusion protein. As a result, as shown in Figure 15a, 2D classification was performed using raw data particles, confirming that the IL-17RA / RC-Fc-TNFR2 protein was captured as particles from all angles, and 3D reconstruction was performed based on this (Figure 15b).In the electron density map, the binding models of IL-17RC and IL-17F, the binding model of TNFR2 and TNFα, and Fc, currently stored (deposition) in the PDB (Protein Data Bank), were fitted using superimposition (Figure 15c). The binding models of IL-17RC and IL-17F and TNFR2 and TNFα were utilized from PDB ID: 6HG9 and PDB ID: 3ALQ, respectively. The portion bound to TNFR2 and TNFα is longer and has a more flexible linker, so the electron density map appears thinner, and may be even thinner in 3D reconstruction. In the case of TNFR2 ECD, when the substrate is not bound, it can be difficult to confirm the electron density map due to the flexible linker, but as shown in Figure 15c, it was confirmed that the electron density map bound to TNFR2 and TNFα was successfully fitted. Therefore, it was confirmed that the IL-17RA / RC-hybrid and TNFR2 ECD in the IL-17RA / RC-Fc-TNFR2 hybrid fusion protein bind to their respective substrates in a 1:1 ratio. It was also confirmed that they bind to their respective substrates in a 1:1 ratio relative to the intermediate Fc, and that the longer IL-17RC structure is located at the top relative to the intermediate Fc. [Examples]

[0286] Example 16: Attachment of N-glycan to IL-17RA / RC-FC-TNFR2 hybrid fusion protein

[0287] The glycan configuration and structure of Fc-fusion proteins can alter structural domains, and specific glycation changes in therapeutic Fc-fusion proteins can affect the pharmacokinetics (PK) and pharmacological activity (PD) of the molecule. Therefore, the inventors investigated the feasibility of constructing an expression system necessary for improving productivity by confirming the ability of N-glycans to attach to the IL-17RA / RC-Fc-TNFR2 hybrid fusion protein (T1-T4-14, sequence number 1). To theoretically confirm the ability of N-glycans to attach to the Fc-fusion proteins produced in the above examples, N-glycan site determination was performed using peptide mapping. The above-produced Fc-fusion proteins were added to a 50% TCA (Trichloroacetic acid) solution to a concentration of 10%, and reacted at -20°C for 30 minutes. Subsequently, the supernatant was removed after centrifugation at 13,000 rpm for 10 minutes to carry out desalting. The desalted Fc fusion protein was cleaved with different hydrolytic enzymes (trypsin, chymotrypsin), and the attachment of the cleaved peptides was confirmed by the percentage of N-glycan attachment using an analytical system combining ultrahigh performance liquid chromatography and mass spectrometry (UPLC-ESI-MS) (Figures 16a and 16b). The cleaved peptides were separated via C18 reversed-phase gradient chromatography and performed for 70 minutes under gradient conditions of mobile phase A (purified water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid). The gradient conditions for mobile phase B were 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 using a mass spectrometer, and the ionized molecular weight was detected and analyzed within the range of 200 m / z to 2000 m / z. Using UPLC-ESI-MS analysis equipment, N-glycosylation site and occupancy percentage analysis was performed. Combining the results from the two hydrolases, a total of 12 N-glycosylation sites were identified, with only the N22 position of the IL-17RA / RC-Fc-TNFR2 hybrid fusion protein not being identified.The results of determining the N-glycan moieties of different hydrolytic enzymes are summarized in Table 20 below.

[0288] [Table 20] [Examples]

[0289] Example 17: Glycansyl oxidation of IL-17RA / RC-Fc-TNFR2 hybrid fusion protein

[0290] A significant portion of biopharmaceuticals are recombined proteins, of which over 70% are glycoproteins. Human-like glycosylation is important because it is associated with therapeutic efficacy, reduces immunogenicity, and increases plasma half-life. The degree of sialylation of glycosylated proteins (glycoproteins) can be an important property that affects the plasma half-life or safety of the protein. In protein drug production cells, the recombination and expression of sialylated proteins can be difficult if the organism lacks the enzyme for sialylation or if it naturally produces proteins with non-human sialylation patterns (Xiaotian Zhong et al. Methods. Mol. Biol. 12(3):53.2022). Introducing galactosyltransferase-6 (GT6) in the ExpiCHO-S® system can promote the synthesis of sialylated glycans. Expression of the IL-17RA / RC-Fc-TNFR2 fusion protein was induced using Expi CHO-S, in accordance with the ExpiCHO® expression system kit protocol, as in Example 1. For the above experiment, the ratio of IL-17RA / RC-Fc-TNFR2 fusion protein DNA to GT6 DNA was 5:4. The GT6 DNA used was Lenti-ORF clone of B4GALT6(mGFP-tagged)-Human UDP-Gal(origene CAT#:RC210213L4) with Beta-1, 4-galactosyltransferase 6 (B4GALT6).DNA of the IL-17RA / RC-Fc-TNFR2 fusion protein and GT6 DNA were transfused (T1-T4-14: 50 μg, GT6: 40 μg). After 18 hours, enhancers and feeds from the ExpiFectamine® CHO transfusion kit were added. Additionally, 2 ml and 1.55 ml each of UMnG (200 mM Uridine (GLENTHAM, Cat: GP7250-5G), 0.4 mM MnCl2 (sigma, Cat: 244589-50G), 1 M Galactose (GLENTHAM, Cat: GC7360-500G), and ManNAc (65 mM N-acetylmannosamine, Glentham, Cat: GK3319) were added. Subsequently, the cells were incubated at 8% CO2, 32°C, and 120°C. The cells were cultured under rpm conditions, and on day 4, 2 ml each of UMnG and ManNAc were added along with the enhancer and feed. The cells were cultured under 8% CO2, 32°C, and 120 rpm conditions, and harvested on day 7 to complete production. Figure 17a is a schematic diagram showing the production schedule for glycansialylation of IL-17RA / RC-Fc-TNFR2 hybrid fusion protein. The IL-17RA / RC-Fc-TNFR2 fusion protein, which underwent glycansialylation in the same manner as in Example 3, was purified by Protein A affinity. The glycansialylated IL-17RA / RC-Fc-TNFR2 fusion protein was loaded into a Superdex200 (Cytiva) and separated and purified (HiLoad 16 / 600 Superdex 200 pg 1xl 20ml (Cytiva, 28989335), Buffer: 1x PBS, 0.005% tween 20). SEC results showed the peak due to the change in protein size shifted further forward (Figure 17b, Figure 17c), and Western blotting results confirmed that the size of the glycansialylated protein was even larger (Figure 17d). Analysis was also performed to examine the effect of glycansialylated IL-17RA / RC-Fc-TNFR2 on its affinity for ligands IL-17A, IL-17F, and TNFα.The control group underwent ELISA analysis of ligand-specific affinity for T1-T4-14 (sequence number 1) produced by pBispec, T1-T4-14 produced by pcDNA3.4, and glycansialylated T1-T4-14. The affinity for IL-17A, IL-17F, and TNFα of T1-T4-14 produced by pBispec, pcDNA3.4, or pcDNA3.4+GT6 was measured, respectively (Figures 17e, 17f, and 17g). For IL-17A, the EC of T1-T4-14 was measured. 50 The values ​​observed were 75.18 ng / ml, 60.65 ng / ml, and 54.32 ng / ml, respectively, and in IL-17F, the EC levels were T1-T4-14. 50 The values ​​observed were 70.20 ng / ml, 45.48 ng / ml, and 62.06 ng / ml, respectively, and in TNFα, the EC levels of T1-T4-14 were observed. 50 The values ​​were confirmed to be 68.00 (ng / ml), 87.26 (ng / ml), and 44.34 (ng / ml).

[0291] To confirm the in vivo stability of the IL-17RA / RC-Fc-TNFR2 fusion protein, the amount of residual protein was measured after 4 hours of treatment in mouse (C57BL / 6) serum. The results showed that T1-T4-14, which was sialyzed by GT6 introduction, had more than twice the stability (14.99 μg / ml vs 28.94 μg / ml). [Examples]

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

[0293] In a rheumatoid arthritis (RA)-like cell model using SW982 cells, the disease-improving effect of the multi-target fusion protein (IL-17RA / RC-Fc-TNFR2) through its TNFR2 neutralizing ability was confirmed by treating SW982 cells with human TNFα and evaluating the expression levels of IL-6 and MMP3. In RA, inflammatory cells such as neutrophils and macrophages recruit to the joints, and joint structure is destroyed by the overexpression of inflammatory cytokines such as IL-Iβ, TNFα, IL-6, and CXCL8. SW982 cells were placed in each well of a 96-well plate at a rate of 1 x 10⁶ 4Cells were cultured overnight in DMEM (Welgene, LM001-005) and 10% FBS (Gibco, 16000044) at 37°C. The following day, an RA model was induced by treatment with TNFα cytokine at 10 ng / ml, and IL-17RA / RC-Fc-TNFR2 (T1-T4-14) protein and the control drug adalimumab (Selleckchem, A2010) were simultaneously treated with TNFα at concentrations of 2.5, 10, 40, and 160 ng / ml (48 hours, 37°C). After 48 hours of treatment, the culture medium was collected, and ELISA was performed as follows to confirm IL-6 and MMP3. The ELISA products used are as follows, and all materials were prepared according to the product instructions: Human IL-6 DuoSet ELISA (R&D systems, DY206-05), Human Total MMP3 Duoset ELISA (R&D systems, DY513-05), and DuoSet ELISA Ancillary Reagent Kit 2 (R&D systems, DY008B). Each ELISA capture antibody was diluted in IX Reagent Diluent (1% PBS in PBS, pH 7.2-7.4, 0.2 μm filtered, R&D systems, Catalog #DY995), and 100 μl / well was added to the ELISA plate, and the plates were coated overnight at room temperature. The plates were washed three times with 300 μl / well of IX 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 allow to stand at room temperature for at least 1 hour to block the reaction. After washing with 300 μl / well of ELISA wash buffer, add 100 μl / well each of IL-6, MMP3 reference protein, and culture sample, and allow to react at room temperature for 2 hours (IL-6 is used diluted 20-fold, MMP3 is used as is). Follow the instructions for the standard curve concentrations: IL-6 9.38, 18.8, 37.5, 75, 150, 300, 600 pg / mL and MMP3 31.3, 62.5, 125, 250, 500, 1000, 2000 pg / mL. Wash three times with 300 μl / well of ELISA wash buffer.Detection antibody was added at a rate of 100 μl / well and reacted for 2 hours. After washing three times with 300 μl / well of ELISA wash buffer, the HRP-conjugated secondary antibody was treated and reacted at room temperature for 20 minutes. Subsequently, the plates were washed six times with ELISA wash buffer. After reacting 100 μl of TMB solution at room temperature, 50 μl of presentation solution was added. ELISA measurements were performed using a BioTek Synergy HTX Microplate reader, measuring absorbance at 540 nm and 450 nm. As a result, the TNFR2 neutralizing ability of the present invention's IL-17RA / RC-Fc-TNFR2 was confirmed to suppress IL-6 and MMP3 at a level comparable to adalimumab (Figures 18a and 18b).

[0294] Although the present invention has been described with reference to the embodiments described above, these are merely illustrative, and a person with ordinary skill in the art will understand that a variety of modifications and equivalent other embodiments are possible. Accordingly, the true scope of technical protection of the present invention should be determined by the technical idea of ​​the appended claims.

Claims

1. i) T24D, N88D, D122G and Q267R, ii) T24D, F59V, N88D, D122G and Q267R, iii) L9P, T24D, F59V, N88D, D122G and Q267R, and iv) An IL-17RA / RC mutant hybrid protein having at least 90% sequence homology to the amino acid sequence described in Sequence ID No. 69, including amino acid substitutions selected from the group consisting of L9P, T24D, F59V, N88D, D122G, A156P and Q267R.

2. In paragraph 1, A hybrid protein of the IL-17RA / RC mutant with added glycosylation.

3. In paragraph 2, The above glycans are one or more IL-17RA / RC mutant hybrid proteins selected from the group consisting of N-glycans, O-glycans, and sialylated glycans.

4. In paragraph 1, An IL-17RA / RC mutant hybrid protein selected from the group consisting of sequence numbers 66 to 69.

5. A polynucleotide that encodes the IL-17RA / RC mutant hybrid protein of item 1.

6. An expression vector containing the polynucleotide described in item 5.

7. Transformed cells into which the expression vector described in item 6 has been introduced.

8. Isomorphic or heterodimers containing the IL-17RA / RC mutant hybrid protein of item 1.

9. An IL-17RA / RC-FC mutant hybrid protein, in which an antibody Fc domain is attached to the N-terminus or C-terminus of the IL-17RA / RC mutant hybrid protein of item 1.

10. In paragraph 9, The above antibody Fc domain is an IL-17RA / RC-Fc mutant hybrid protein in which two or more identical Fc domains are mixed together.

11. In paragraph 10, the above-mentioned hybrid Fc domain is an IL-17RA / RC-FC mutant hybrid protein selected from the group consisting of sequence numbers 33 to 66.

12. Polynucleotides that encode the IL-17RA / RC-FC mutant hybrid protein of item 9.

13. An expression vector containing the polynucleotide of item 12.

14. Transformed cells into which the expression vector described in item 13 has been introduced.

15. Isomorphic or heterodimers containing the IL-17RA / RC-FC mutant hybrid protein of item 9.

16. 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) A TNFR2 ECD mutant protein having at least 90% sequence homology to the amino acid sequence described in sequence number 77, including amino acid substitutions selected from the group consisting of S33A, S36P, G75S, R119S and N164S.

17. In paragraph 16, a TNFR2 ECD mutant protein is a fusion of at least one linkapeptide from i to ix above.

18. In paragraph 16, A TNFR2 ECD mutant protein with added sugar chains.

19. In paragraph 18, One or more TNFR2 ECD mutant proteins composed of the above-mentioned sugar chain N-glycans, O-glycans, and sialylated sugar chains.

20. In paragraph 16, TNFR2 ECD mutant proteins selected from the group consisting of sequence numbers 83 to 94.

21. A polynucleotide that encodes the TNFR2 ECD variant protein in item 16.

22. An expression vector containing the polynucleotide described in item 21.

23. Transformed cells into which the expression vector described in item 22 has been introduced.

24. Isomorphic or heterodimers containing the TNFR2 ECD mutant protein described in item 16.

25. The TNFR2ECD-FC mutant protein is a variant of the TNFR2 ECD mutant protein described in item 16, in which an antibody Fc domain is attached to the N-terminus or C-terminus.

26. In paragraph 25, The above antibody Fc domain is a TNFR2 ECD-Fc variant protein with a hybrid domain in which two or more identical Fc units are mixed together.

27. In paragraph 26, The above hybrid Fc domain is a TNFR2 ECD-Fc variant protein selected from the group consisting of sequence numbers 33 to 65.

28. Isomorphic or heterodimers containing the TNFR2 ECD-Fc mutant protein described in item 25.

29. An expression vector containing polynucleotides as described in item 28.

30. Transformed cells into which the expression vector described in item 29 has been introduced.

31. Isomorphic or heterodimers containing the TNFR2 ECD-Fc mutant protein described in item 25.

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

33. In paragraph 32, the above IL-17RA / RC mutant hybrid protein is a triple fusion protein characterized by containing amino acid substitutions selected from the group consisting of the following, and having at least 90% sequence homology with the amino acid sequence described in sequence number 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. In paragraph 32, The above tumor necrosis factor receptor 2 extracellular domain (ECD) is a triplicate protein characterized by having at least 90% sequence homology to the amino acid sequence described in sequence number 77, including amino acid substitutions selected from the group consisting of the following: 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.

35. In paragraph 34, A three-type fusion protein in which at least one of the above i to ix is ​​linked to each other by a linker peptide.

36. In paragraph 32, A triple-fusion protein with added sugar chains.

37. In paragraph 36, The above-mentioned glycans are one or more triplicate proteins selected from the group consisting of N-glycans, O-glycans, and sialylated glycans.

38. In paragraph 32, The tumor necrosis factor receptor 2 described above is a triplicate fusion protein selected from the group consisting of sequence numbers 83 to 94.

39. In paragraph 32, A triplicate fusion protein that simultaneously targets IL-17A, IL-17F, and TNFα.

40. In paragraph 32, A triplicate fusion protein selected from the group consisting of amino acid sequences described by sequence numbers 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32.

41. A polynucleotide that encodes the triplicate fusion protein of item 32.

42. An expression vector containing the polynucleotide described in item 41.

43. Transformed cells into which the expression vector described in item 42 has been introduced.

44. An isomorphic or heterodimer containing the three fusion proteins described in item 32.

45. A pharmaceutical composition for the treatment of autoimmune diseases comprising the IL-17RA / RC mutant hybrid protein of item 1 and the TNFR2 ECD mutant protein of item 16 as active ingredients.

46. A pharmaceutical composition for the treatment of autoimmune diseases comprising the IL-17RA / RC-FC mutant hybrid protein described in item 9 and the TNFR2 ECD-Fc mutant protein described in item 25 as active ingredients.

47. A pharmaceutical composition for the treatment of autoimmune diseases, comprising as an active ingredient a triplicate fusion protein from one of the items 32 to 40, or an isomorphic or heteromorphic dimer from item 44.

48. In one of the paragraphs 45 through 47, The above autoimmune disease is a composition selected from the group consisting of rheumatoid arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease (IBD), and ankylosing spondylitis.

49. In paragraph 48, The above-mentioned inflammatory bowel disease is a composition selected from the group consisting of ulcerative colitis, Crohn's disease, and Behçet's disease.

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