New use of interferon epsilon

Abstract

The present invention relates to a new use of interferon epsilon, and more particularly, to a pharmaceutical composition for preventing or treating a disease, which comprises interferon epsilon as an active ingredient. The pharmaceutical composition has remarkably increased activity in a weakly acidic environment compared to a neutral environment, and thus can be systemically administered without a separate process such as targeting or masking for the purpose of controlling systemic toxicity, and thus can be effectively used for the treatment of a relevant disease.

Description

Technical Field

[0001] This application claims priority to Korean Patent Application No. 10-2023-0168553, filed on November 28, 2023, and Korean Patent Application No. 10-2024-0172093, filed on November 27, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] This invention relates to novel uses of interferon ε, and more specifically, to pharmaceutical compositions for the prevention or treatment of diseases comprising interferon ε as an active ingredient, wherein said interferon ε is activated in tissues with decreased pH due to the pathological state of the disease, to fusion proteins comprising antibodies or fragments thereof that bind directly or indirectly to interferon ε, and to pharmaceutical compositions comprising said fusion protein. Background Technology

[0003] Interferon (IFN) is a protein belonging to the cytokine family. Cytokines are molecules known to be used for intercellular communication to trigger the body's immune system. Interferon is named for its ability to "interfere" with viral replication by protecting cells from viral infection. In addition, it enhances the host's defense mechanisms by activating immune cells such as natural killer cells and macrophages, increasing the expression of major histocompatibility complex (MHC) antigens, and upregulating antigen presentation.

[0004] Interferons are classified into type I, including IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω; type II, including IFN-γ; and type III, including IFN-λ. Type I interferons, especially IFN-α and IFN-β, are widely used as therapeutic agents for various diseases. For example, IFN-α2a, IFN-α2b, and pegylated IFN-α2b are used to treat diseases such as blastocystic leukemia, melanoma, renal cell carcinoma, Kaposi's sarcoma, multiple myeloma, follicular and non-Hodgkin's lymphoma, and chronic myeloid leukemia. Human IFN-β has also been approved in Japan for the treatment of glioma, medulloblastoma, astrocytoma, and melanoma. Furthermore, IFN-β1 has been approved by the FDA for the treatment of multiple sclerosis.

[0005] Therefore, interferon has shown efficacy in numerous cancers. However, side effects may occur, such as fever, chills, myalgia, headache, nausea, and fatigue, which are similar to flu-like symptoms. In particular, the antitumor effects of systemic interferon therapy are often accompanied by serious side effects, including inflammation and direct tissue toxicity. Furthermore, due to the short half-life of interferon, the dosing intervals are inevitably shortened, which ultimately leads to increased side effects.

[0006] Methods for controlling systemic toxicity caused by interferon include: direct delivery of interferon to tumor sites using the tumor-targeting capabilities of monoclonal antibodies, conjugation of interferon with polyethylene glycol (PEG) to increase its half-life, and inhibition (masking) of its activity before it reaches the tumor. These methods are known in the art. However, tumor-targeting interferon may still be recognized by interferon receptors expressed systemically, leading to side effects. There is also the issue that the side effects of pegylated interferon are not substantially different from those of non-pegylated interferon. Furthermore, masked interferon has disadvantages, such as complex manufacturing processes, interferon activity depending on the activity of proteases in the tumor microenvironment, and the additional protein structures used for masking potentially inducing novel immune responses.

[0007] Therefore, a new strategy is needed that can suppress systemic toxicity caused by systemic administration of interferon while specifically inducing interferon activity in pathological tissues. Summary of the Invention

[0008] [The problem to be solved] The inventors have discovered that interferon ε (IFN-ε) exhibits a property of dramatically increased activity in low pH environments. Due to this property, it has been demonstrated that even when interferon ε is administered systemically to a mouse model of tumors, its non-specific immune activity in the blood and spleen is lower compared to the interferon β (IFN-β) administration group, while its immune activity within the tumor is similar, thus completing this invention.

[0009] Therefore, one object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease, comprising interferon ε as an active ingredient, wherein the interferon ε is activated in tissues where the pH is lowered due to the pathological state of the disease.

[0010] Another object of the present invention is to provide a fusion protein comprising interferon ε and an antibody or fragment thereof that binds directly or indirectly to interferon ε.

[0011] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease comprising the fusion protein, wherein the fusion protein is activated in tissues where the pH is lowered due to the pathological state of the disease.

[0012] Another object of the present invention is to provide a pharmaceutical composition consisting of interferon ε for the prevention or treatment of diseases.

[0013] Another object of the present invention is to provide a pharmaceutical composition substantially composed of interferon ε for the prevention or treatment of disease.

[0014] [Problem-solving methods] To achieve the above objectives, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases, comprising interferon ε as an active ingredient, characterized in that the interferon ε is activated in tissues where the pH is lowered due to the pathological state of the disease.

[0015] To achieve another objective of the present invention, the present invention provides a fusion protein comprising interferon ε and an antibody or fragment thereof that binds directly or indirectly to interferon ε.

[0016] To achieve another objective of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of disease comprising the fusion protein, wherein the fusion protein is activated in tissues where the pH is lowered due to the pathological state of the disease.

[0017] To achieve another objective of the present invention, the present invention provides a pharmaceutical composition consisting of interferon ε for the prevention or treatment of diseases.

[0018] To achieve another objective of the present invention, the present invention provides a pharmaceutical composition substantially composed of interferon ε for the prevention or treatment of diseases.

[0019] The present invention will now be described in detail.

[0020] Furthermore, each description and embodiment disclosed in this invention can be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Moreover, the scope of this invention should not be considered limited to the specific descriptions provided below.

[0021] In this specification, the term "comprising" is used in the same sense as "including" or "characterized in" and does not exclude additional components not specifically mentioned in the compositions according to the invention. Furthermore, the term "consisting of" means excluding additional elements or components not described separately. The term "consisting substantially of" means that the composition may include, in addition to the described substances, substances that do not substantially affect its essential characteristics.

[0022] As used in this invention, the term "treatment" refers to the improvement of symptoms caused by a disease, including temporary or permanent relief of symptoms, elimination of the cause of symptoms, or prevention or delay of the manifestation of symptoms of a disease or pathological condition. This may include, but is not limited to, curing a disease, substantially preventing a disease or improving a condition, and reducing, curing or preventing one or more symptoms caused by a disease.

[0023] As used in this invention, the term "prevention" refers to any action that suppresses symptoms or delays the onset of disease by applying a pharmaceutical composition according to the invention.

[0024] As used in this invention, the term "improvement" refers to any behavior that reduces parameters associated with an abnormal condition, such as the severity of symptoms.

[0025] The words or terms used in the description and claims of this invention should not be construed as limited to their conventional or dictionary meanings. Rather, they should be interpreted in a manner consistent with the technical spirit of the invention, based on the principle that the inventors can appropriately define the terms and concepts to best interpret their invention.

[0026] The present invention provides a pharmaceutical composition for the prevention or treatment of diseases, comprising interferon ε as an active ingredient, characterized in that the interferon ε is activated in tissues where the pH is lowered due to the pathological state of the disease.

[0027] In this invention, interferon ε (IFN-ε) refers to a type I interferon family cytokine that activates the JAK-STAT pathway through its interaction with interferon α / β receptors (IFNAR1 and IFNAR2). Interferon ε is present in various organisms, including humans, mice, pigs, cattle, dogs, and horses, and is known to differ from interferon α (hereinafter referred to as IFN-α) and β (hereinafter referred to as IFN-β) in that it is primarily expressed in various mucosal tissues such as the lungs, small intestine, and reproductive organs. In this invention, interferon ε preferably refers to human interferon ε, but is not limited thereto. Furthermore, in this specification, interferon ε may be referred to as interferon ε, interferon-ε, interferon-e, IFN-ε, IFN-e, IFNε, or IFNe, all of which refer to the same interferon ε.

[0028] According to one embodiment of the invention, IFN-α and IFN-ω exhibit reduced activity under slightly acidic conditions (pH 6.0) compared to neutral conditions (pH 7.4), while IFN-β and IFN-ε exhibit increased activity under slightly acidic conditions, with IFN-ε showing a particularly significant difference. More specifically, IFN-ε shows a 5.02-fold increase in activity under slightly acidic conditions compared to neutral conditions, and even when treated with receptor concentrations 43.76 times lower in slightly acidic conditions, it still exhibits superior binding capacity compared to neutral conditions. Furthermore, it has been confirmed that pSTAT-1 signaling is activated in slightly acidic conditions, even at concentrations more than 1000 times lower than in neutral conditions. Therefore, the interferon ε according to the invention is characterized by its activity being exhibited in tissues with a pH below 7, i.e., tissues with a lower pH compared to normal tissues. The pH of the "pH-decreased tissue" is preferably, but not limited to, pH 5.0 to pH 6.9, pH 5.0 to pH 6.8, pH 5.0 to pH 6.7, pH 5.0 to pH 6.6, pH 5.0 to pH 6.5, pH 5.0 to pH 6.4, pH 5.0 to pH 6.3, pH 5.0 to pH 6.2, pH 5.0 to pH 6.1, pH 5.0 to pH 6.0, pH 5.0 to pH 5.9, pH 5.0 to pH 5.8, pH 5.0 to pH 5.7, pH 5.0 to pH 5.6, pH 5.0 to pH 5.5, pH 5.0 to pH 5.4, pH 5.0 to pH 5.3, pH 5.0 to pH 5.2, or pH 5.0 to pH 5.1. Furthermore, as described above, interferon ε exhibits increased activity in tissues with a lower pH compared to normal tissues, characterized by an increase of 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 10000, or more.Preferably, the activity increases by 2 to 10 times, 2 to 50 times, 2 to 100 times, 2 to 200 times, 2 to 300 times, 2 to 400 times, 2 to 500 times, 2 to 600 times, 2 to 700 times, 2 to 800 times, 2 to 900 times, 2 to 1000 times, 2 to 1500 times, 2 to 2000 times, 2 to 3000 times, 2 to 4000 times, 2 to 5000 times, 2 to 10000 times, or... More; or 3 to 10 times, 3 to 50 times, 3 to 100 times, 3 to 200 times, 3 to 300 times, 3 to 400 times, 3 to 500 times, 3 to 600 times, 3 to 700 times, 3 to 800 times, 3 to 900 times, 3 to 1000 times, 3 to 1500 times, 3 to 2000 times, 3 to 3000 times, 3 to 4000 times, 3 to 5000 times, 3 to 10000 times or more; Or 4 to 10 times, 4 to 50 times, 4 to 100 times, 4 to 200 times, 4 to 300 times, 4 to 400 times, 4 to 500 times, 4 to 600 times, 4 to 700 times, 4 to 800 times, 4 to 900 times, 4 to 1000 times, 4 to 1500 times, 4 to 2000 times, 4 to 3000 times, 4 to 4000 times, 4 to 5000 times, 4 to 10000 times or more; or 5 5 to 10 times, 5 to 50 times, 5 to 100 times, 5 to 200 times, 5 to 300 times, 5 to 400 times, 5 to 500 times, 5 to 600 times, 5 to 700 times, 5 to 800 times, 5 to 900 times, 5 to 1000 times, 5 to 1500 times, 5 to 2000 times, 5 to 3000 times, 5 to 4000 times, 5 to 5000 times, 5 to 10000 times or more, but not limited to these.

[0029] Therefore, the interferon ε according to the invention is characterized by its systemic administration without the need for special treatments such as targeting or masking. In particular, the pH of the tumor microenvironment is known to be approximately pH 5.6 to 6.8, and when using interferon ε according to the invention to treat cancer and / or tumors, it has the advantage of reduced side effects, even with systemic therapy. Indeed, according to one embodiment of the invention, when interferon β and ε are administered intraperitoneally to a mouse model of tumor induced using the B16F10 cell line constituting a low-pH tumor microenvironment, the interferon ε administration group showed less reduction in blood leukocyte count and myeloid cells compared to the interferon β administration group, and no excessive pSTAT-1 activation occurred. On the other hand, the level of intratumoral T cell infiltration and the reduction in myeloid cells were similar to those in the interferon β administration group.

[0030] Meanwhile, in this invention, "systemic application" means applying the composition according to the invention to the circulatory system to affect the whole body, and preferably includes intravenous, intramuscular, intraarterial, intramedullary, intrasheath, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intraintestinal, local, sublingual, rectal, or intratumoral application, but is not limited thereto.

[0031] The compositions of the present invention containing interferon ε as an active ingredient can be applied without limitation to diseases and / or pathological conditions that can be treated with currently known interferon ε.

[0032] Typically, STAT-1 signaling induced by interferon activation of IFNAR1 / IFNAR2 is known to promote the exposure of MHC class I molecules on the surface of cancer cells and, combined with the upregulation of tumor-associated antigens, to increase the overall antigenicity of the tumor, as is known in the art. Therefore, pathological conditions can refer to cancer and / or tumors.

[0033] In this invention, the cancer and / or tumor is preferably selected from at least one of ovarian cancer, papilloma, laryngeal papilloma, human papilloma, bladder cancer and cervical cancer, but there is no particular limitation on the cancer and / or tumor that can be treated with the composition according to the invention, including both solid tumors and hematologic cancers. Non-limiting examples of cancers and / or tumors include, but are not limited to, hairy cell leukemia, chronic myeloid leukemia, multiple myeloma, non-Hodgkin's lymphoma, skin cancers such as melanoma, Kaposi's sarcoma, kidney cancer, lung cancer, brain tumors such as glioma, breast cancer, colorectal cancer, liver cancer, hepatocellular carcinoma, stomach cancer, bronchial cancer, nasopharyngeal carcinoma, laryngeal cancer, pancreatic cancer, colon cancer, pancreatic cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, glioblastoma, neuroblastoma, and malignant mesothelioma.

[0034] Furthermore, interferon activates interferon-stimulated genes (ISGs) through the JAK-STAT pathway, and many ISGs are known in the art to directly target essential pathways and functions in the pathogen life cycle, thereby controlling viral, bacterial, and parasitic infections. Therefore, pathological conditions can refer to infectious diseases.

[0035] In this invention, the infectious disease preferably refers to a viral infection. Non-limiting examples of viral infections include, but are not limited to, HIV-induced AIDS, hepatitis B and C virus infections, and may also include, but are not limited to, one or more selected from influenza virus infection, respiratory syncytial virus infection, herpes virus infection, human papillomavirus infection, coronavirus infection, and other viral infections.

[0036] The compositions according to the present invention comprise the aforementioned interferon ε as an active ingredient and can be formulated into suitable forms with pharmaceutically acceptable carriers, and may also contain excipients or diluents. The term "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and will not cause gastrointestinal disturbances, dizziness, or other allergic reactions when administered to humans. The pharmaceutically acceptable carrier may include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Furthermore, carriers for parenteral administration may include water, suitable oils, saline solutions, glucose solutions, and glycols, and may also include stabilizers and preservatives. Suitable stabilizers include antioxidants, such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben, or propylparaben, and chlorobutanol. The pharmaceutical compositions of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations can be found in the following literature.

[0037] As described above, the compositions according to the invention can be administered systemically and can be applied to mammals, including humans, by any method, such as oral or parenteral administration. Therefore, the compositions according to the invention can be formulated into oral or parenteral formulations according to the above-described routes of administration.

[0038] In the case of oral formulations, the compositions of the present invention can be formulated into powders, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, liquids, suspensions, etc., using methods known in the art. For example, oral formulations can be obtained by mixing the active ingredient with a solid excipient, grinding the mixture, adding suitable adjuvants, and processing it into a granular mixture to produce tablets or sugar-coated tablets. Examples of suitable excipients include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches such as corn starch, wheat starch, rice starch, and potato starch; celluloses such as cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose; and fillers such as gelatin and polyvinylpyrrolidone. In addition, disintegrants such as croscarmellose, agar, alginate, or sodium alginate can be added as needed. Furthermore, the pharmaceutical compositions of the present invention may also include anti-caking agents, lubricants, wetting agents, flavoring agents, emulsifiers, and preservatives.

[0039] In the case of parenteral preparations, the composition can be formulated into injections, creams, lotions, topical ointments, oils, moisturizers, gels, aerosols, and nasal inhalers using methods known in the art.

[0040] The total effective amount of the composition according to the invention can be administered to a patient as a single dose, or as multiple doses over a prolonged period via a fractionated treatment regimen. The content of the active ingredient in the composition according to the invention can be varied according to the severity of the disease. Preferably, the total preferred dose of the composition according to the invention can be from about 0.01 μg to 10,000 mg per kilogram of patient or subject body weight per day, most preferably from 0.1 μg to 500 mg. However, the dosage of the composition is determined by taking into account various factors such as formulation method, route of administration, treatment frequency, and the patient's age, weight, health status, sex, disease severity, diet, and excretion rate. Therefore, those skilled in the art will be able to determine the appropriate effective dose of the composition according to the invention by taking these factors into account. There are no particular limitations on the formulation, route of administration, or method of administration of the composition according to the invention, as long as it exhibits the effects of the invention.

[0041] The term "effective amount" in this invention refers to an amount that, when administered to a subject, exhibits effects such as improvement, treatment, detection, diagnosis, inhibition, or reduction of cancer or infectious diseases. The term "subject" can refer to an animal, preferably a mammal, particularly a human, and may also include cells, tissues, organs, etc., derived from an animal. The subject can be a patient who requires the aforementioned effects.

[0042] In this invention, the content of the composition is not significantly limited depending on the purpose or aspect of its use, and for example, it can be from 0.01 to 99% by weight, preferably from 0.5 to 50% by weight, more preferably from 1 to 30% by weight, based on the total weight of the composition. Furthermore, in addition to the active ingredient, the pharmaceutical composition according to the invention may further contain additives such as pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition of the invention may contain 0.1 to 99.9% by weight of interferon ε prepared by the method of the invention and 99.9 to 0.1% by weight of a carrier.

[0043] In addition, the present invention provides a fusion protein comprising the above-mentioned interferon ε and an antibody or fragment thereof that binds directly or indirectly to interferon ε.

[0044] In the fusion protein according to the present invention, the meaning of interferon ε is the same as that described in the above composition, and the specific configuration of the fusion protein will be described below.

[0045] In this invention, antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and fragments thereof. A complete antibody typically has a Y-shaped structure, consisting of two long heavy chains (H) and two short light chains (L). Each heavy and light chain is interconnected by disulfide bonds and is divided into a variable region (V) that reacts with the antigen and a constant region (C) that exhibits effector function. The variable region contains a complementarity-determining region (CDR), which determines the structure of the variable region to form a specific binding with the antigen and modulates the antibody binding strength. An antibody fragment refers to a specific portion that can react with an antigen and exhibit antigen-binding activity; examples include Fab fragments (papain digestion fragments), Fab' fragments (pepsin digestion and partial reduction fragments), F(ab')2 fragments (pepsin digestion fragments), Facb (fibrinolytic digestion fragments), Fd (pepsin digestion, partial reduction, and reaggregation fragments), scFv fragments (molecular biology technique fragments), etc. Preferably, the antibody is an antibody or fragment thereof that recognizes tumor-specific antigens and can be used as a targeted therapeutic agent for diseases such as cancer. Examples include trastuzumab, an antibody therapy targeting human epidermal growth factor receptor (HER-2); cetuximab, an antibody therapy targeting epidermal growth factor receptor (EGFR); atezolizumab, an antibody therapy targeting PD-L1 expressed on the surface of cancer cells; and cetuzumab, an antibody therapy targeting TROP2 expressed on the surface of cancer cells.

[0046] The fusion protein according to the present invention can be a fusion protein formed by linking interferon ε and an antibody or fragment thereof via a linker (preferably a peptide linker). A linker is a molecule that links two or more different substances together, consisting of short fragments of amino acids or amino acid analogs linked by peptide bonds. In this case, glycine, serine, alanine, etc., can be used as the main constituent amino acids, and glycine-serine linkers, glycine-serine-alanine linkers, etc., can be used. Such a linker can be attached to the C-terminus of the antibody heavy chain or the C-terminus of the antibody light chain, or the N-terminus of the linker can be attached to both the C-terminus of the antibody light chain and the C-terminus of the antibody heavy chain. In this case, the N-terminus of interferon ε can be attached to the C-terminus of the linker.

[0047] In the fusion protein according to the invention, since the antibody recognizes tumor-specific antigens and is intended for targeted therapy against diseases such as cancer, the pharmacological effects of the fusion protein will be substantially the same as those of the aforementioned interferon ε. Therefore, the present invention provides a composition comprising the fusion protein for tissue-specific treatment or prevention, wherein the tissue has a decreased pH due to a pathological condition.

[0048] In the compositions according to the invention, all terms including fusion protein, pathological condition, pH, and pH-decreased tissue have the same meaning as described in the above compositions, and the content described in the relevant parts can be directly applied to the compositions containing fusion proteins according to the invention.

[0049] [The effects of the invention] The interferon ε according to the present invention is characterized by significantly enhanced activity in a slightly acidic environment compared to a neutral environment. Therefore, it has the advantage of being able to be administered systemically without the need for additional processes such as targeting or masking to control systemic toxicity, and thus can be effectively used for the treatment of related diseases. Attached Figure Description

[0050] Figures 1a to 1f Results show the results of evaluating receptor binding capacity for each type I interferon (IFN-α2b, IFN-β, IFN-β R27T, IFN-β C17S / R27T, IFN-ω, and IFN-ε) in neutral and acidic environments.

[0051] Figure 2a and Figure 2b The receptor binding capacity for each type I interferon was shown in neutral and acidic environments, normalized to neutral conditions.

[0052] Figure 3a and Figure 3b The study showed changes in the binding ability of interferon ε to receptors IFNAR1 and IFNAR2 under acidic conditions compared to neutral conditions.

[0053] Figures 4a to 4c The results show the calculated binding kinetics and affinity of interferon ε with receptors IFNAR1 / 2, IFNAR1, and IFNAR2 under acidic conditions, compared to neutral conditions.

[0054] Figures 5a to 5c The results show the results of comparing pSTAT-1 expression levels under acidic and neutral conditions with interferon ε to confirm the extent of cell signal transduction.

[0055] Figure 6 This diagram illustrates an experiment used to evaluate the side effects and efficacy of interferon ε in an in vivo mouse model.

[0056] Figures 7a to 7g The results of blood analysis from in vivo mouse model experiments are shown.

[0057] Figures 8a to 8c The results of spleen analysis in an in vivo mouse model experiment are shown.

[0058] Figures 9a to 9c The results of tumor analysis in an in vivo mouse model experiment are shown.

[0059] The invention will now be described in detail.

[0060] However, the following embodiments are only for illustrating the present invention, and the scope of the present invention is not limited to the following embodiments.

[0061] Example 1. Evaluation of the receptor binding capacity of type I interferon under neutral and acidic conditions. To assess the receptor-binding capacity of type I interferon, IFNAR1 / 2 fusion proteins were produced in Expi293F cells. Human IFNAR1-Fc and IFNAR2-Fc proteins, using a knock-in-hole technique, were inserted into the pOptiVec-TOPO plasmid (Invitrogen) and transfected using the ExpiFectamine™ 293 transfection kit (Gibco #A14524). The transfected Expi293F cells were cultured for one week, and the supernatant was collected and purified using a Protein A column on an AKTA avant (Cytiva) system.

[0062] The receptor binding capacity of type I interferons under neutral and acidic conditions was assessed using an ELISA method. The type I interferons used in this study included Rebif (Merck), ABN101, ABN102 (Abion), IFN-α2b (BioLegend, #592704), IFN-ω (PBL, #11395-1), and IFN-ε (R&D Systems, #9667-ME-025 / CF). Interferon ε was diluted to 100 nM in PBS buffer and coated 96-well ELISA plates (Corning, #2592) with 100 μL per well, and incubated at 4 °C for 16 h. All buffers, except the coating buffer, were adjusted to pH 7.4 and pH 6.0 using 1 N HCl. The coated plates were washed three times with wash buffer (0.05% PBS-T, pH 7.4, pH 6.0), and then blocked for 1 hour at room temperature with 300 μL of blocking buffer (50 mg / mL BSA in PBS, pH 7.4, pH 6.0). After blocking, the plates were washed three times with wash buffer, and then IFNAR1 / 2 fusion protein was serially diluted in reagent dilution buffer (1 mg / mL BSA in 0.05% PBS-T, pH 7.4, pH 6.0) at a 1 / 3 dilution ratio, starting from 100 nM. 100 μL was added to each well, and the plates were incubated at room temperature for 1 hour. After washing three times with wash buffer, anti-human IgG-HRP (Jackson lab, #109-035-003) was diluted 1:5000, and 100 μL was added to each well, and the plates were incubated at room temperature for 1 hour. After washing three times with washing buffer, 100 μL of TMB solution (Surmodics, #TMBW 1000-01) was added, and the mixture was reacted at room temperature for 20 minutes. The absorbance was measured at 450 nm after the reaction.

[0063] Based on the results of pH assessment tests on the receptor binding capacity of type I interferon, such as Figures 1a to 1f As shown, Rebif, ABN101, ABN102 and IFN-ε exhibited superior receptor binding capacity under acidic conditions compared to neutral conditions, with IFN-ε showing the most prominent binding capacity under acidic conditions.

[0064] Example 2. Changes in the binding capacity of each type I interferon under acidic conditions compared to neutral conditions. The binding capacity of type I interferon, assessed using ELISA, was normalized and visualized under neutral conditions (see [link]). Figure 2a and 2bRebif, ABN101, ABN102, and IFN-ω exhibited superior receptor binding capacity under acidic conditions compared to neutral conditions. IFN-ε, at its highest concentration, showed a 5.02-fold increase in binding capacity under acidic conditions compared to neutral conditions, and also demonstrated excellent binding capacity even at receptor concentrations 43.76 times lower than those under neutral conditions.

[0065] This confirms that IFN-ε exhibits superior binding capacity under acidic conditions compared to neutral conditions.

[0066] Example 3. Changes in the binding affinity of interferon ε to IFNAR1 and IFNAR2 under acidic conditions compared to neutral conditions. To assess the receptor-binding capacity of type I interferon, human IFNAR1 and IFNAR2 fusion proteins were produced in Expi293F cells. The human IFNAR1-Fc and IFNAR2-Fc protein genes, using a knock-in-hole technique, were inserted into the pOptiVec-TOPO plasmid (Invitrogen) and transfected using the ExpiFectamine™ 293 transfection kit (Gibco #A14524). The transfected Expi293F cells were cultured for one week, and the supernatant was collected and purified using a Protein A column on an AKTA avant (Cytiva) system.

[0067] The receptor-binding capacity of type I interferon under neutral and acidic conditions was assessed using an ELISA method. Type I interferon was produced by inserting the human IFN-ε gene into the pOptiVec-TOPO plasmid (Invitrogen) and using the ExpiFectamine™ 293 transfection kit (Gibco, #A14524). Interferon ε was diluted to 100 nM in PBS buffer and coated 96-well ELISA plates (Corning, #2592) with 100 μL per well and incubated at 4 °C for 16 h. All buffers, except the coating buffer, were adjusted to pH 7.4 and pH 6.0 using 1N HCl. The coated plates were washed three times with washing buffer (0.05% PBS-T, pH 7.4, pH 6.0) and then blocked for 1 h at room temperature with 300 μL blocking buffer (50 mg / mL BSA in PBS, pH 7.4, pH 6.0). After blocking, the plate was washed three times with wash buffer. Then, the IFNAR1 / 2 fusion protein was serially diluted in reagent dilution buffer (1 mg / mL BSA in 0.05% PBS-T, pH 7.4, pH 6.0) at a 1 / 3 dilution ratio, starting at 100 nM, 100 μL per well, and incubated at room temperature for 1 hour. After washing three times with wash buffer, anti-human IgG-HRP (Jackson lab, #109-035-003) was diluted 1:5000, 100 μL per well, and incubated at room temperature for 1 hour. After washing three times with wash buffer, 100 μL of TMB solution (Surmodics, #TMBW 1000-01) was added, and the reaction was carried out at room temperature for 20 minutes. The absorbance was measured at 450 nm after the reaction.

[0068] Based on the results of pH assessment of the binding affinity of interferon ε to IFNAR1 and IFNAR2, such as Figure 3a and 3b As shown, this confirms that interferon ε exhibits stronger receptor binding via IFNAR2 in an acid-dependent manner under acidic conditions.

[0069] Example 4. Receptor binding kinetics and affinity of interferon ε under acidic conditions compared to neutral conditions. To confirm the receptor binding kinetics of interferon ε under neutral and acidic conditions, analysis was performed using an Octet R8 (Sartorius) instrument. 20 nM each of hIFNAR1 / 2, hIFNAR1, and hIFNAR2 were coated onto the AHC biosensor (Sartorius) in 1x kinetic buffer. The 1x kinetic buffer was adjusted with 1N HCl according to the neutral and acidic analytical conditions.

[0070] Interferon ε was diluted in 1 / 2-fold steps starting from 200 nM to bind to the receptor coated on the AHC biosensor, and the kinetics were calculated using an Octet R8 instrument by means of the dissociation process.

[0071] As a result, similar to previous experimental results, it was confirmed that interferon ε has a lower KD value under acidic conditions compared to neutral conditions, and exhibits stronger binding under acidic conditions through AR2 compared to AR1.

[0072] Example 5. Activation of signal transduction by interferon ε was confirmed in Daudi cells and human CD3 T cells under acidic conditions compared to neutral conditions. To confirm the differences in cell signaling activation via interferon-ε receptor binding under neutral and acidic environments, human B lymphoblasts (Daudi cells) and CD3 T cells isolated from healthy human peripheral blood mononuclear cells (PBMCs) were used. CD3 T cells were isolated from human PBMCs using a human CD3 T cell isolation kit (Miltenyl Biotec).

[0073] Adjust the cell culture medium to pH 7.4 and pH 6.5 in RPMI 1640 using 1N HCl, then add 20 mM HEPES. Introduce Daudi cells and CD3 T cells at 2 x 10⁻⁶ cells per well. 5Cells were seeded in 96-well tissue culture plates. Interferon ε was diluted 100 nM in 1 / 10-fold increments in pH 7.4 and pH 6.5 media and treated for 24 hours. After 24 hours, cells were collected, isolated into single cells using cell separation buffer, and then lysed for 1 hour at 200 rpm and 4°C using RIPA buffer (BIOSESANG) containing protease and phosphatase inhibitors. The lysate was centrifuged at 15,000 rpm and 4°C for 15 minutes, and only the supernatant was collected. The dissolved protein was quantified using the BCA assay (ThermoFisher Scientific). 5X sample loading buffer (BIOSESANG) containing DTT was added to the quantified protein, and the mixture was boiled for 10 minutes to reduce all protein. Equal volumes of protein were loaded onto SDS-PAGE, separated by size, transferred to a PVDF membrane, and blocked with 5% BSA in TBS-T at 4°C for 1 hour. Anti-human pSTAT-1 (Cell Signaling Technology) was used as the primary antibody at a 1:1000 ratio and incubated overnight on a PVDF membrane. After washing three times with 0.05% TBS-T, secondary antibody (anti-rabbit Fc HRP antibody, Invitrogen) was added at a 1:5000 ratio and incubated for 30 minutes. After washing three times with 0.05% TBS-T, ECL solution (Bio-Rad) was added, and detection was performed using Chemidoc.

[0074] Based on the results of pH assessment tests on the activation of cell signaling by type I interferon, such as Figures 5a to 5c As shown, this confirms that under acidic conditions, interferon ε activates pSTAT-1 signaling in Daudi cells and human CD3 T cells at concentrations 1000 times lower than under neutral conditions.

[0075] Example 6. Evaluation of the side effects and efficacy of interferon ε in an in vivo mouse model. To evaluate the side effects and efficacy of interferon ε in an in vivo mouse model, human hIFNAR1 / 2 knock-in (hIFNAR1 / 2 KI) mice were used. This was achieved by using 5x10 cells from the B16F10 cell line expressing human hIFNAR1 / 2. 5 Subcutaneous injection of individual cells into hIFNAR1 / 2 KI mice induced a tumor model. When the tumor size reached 100-150 mm... 3 Mice were divided into a control group, IFN-β treatment group, and IFN-ε treatment group, and administered IFN-β intraperitoneally at a dose of 0.5 mg / kg for 5 days. A schematic diagram of the experiment is shown below. Figure 6 As shown in Figure A. One hour after the last administration, blood, spleen, and tumor samples were collected and analyzed using flow cytometry.

[0076] like Figures 7a to 7g As shown, blood analysis results confirmed that, compared to interferon β, interferon ε, whose activity increases under acidic conditions, resulted in a smaller decrease in white blood cell count, a marker of neutropenia as a side effect. It also increased CD8 T cells through T cell activation while decreasing immunosuppressive myeloid cells. Furthermore, it did not cause excessive activation of pSTAT-1, which is involved in downstream signaling in the blood.

[0077] like Figures 8a to 8c As shown, spleen analysis results confirmed that, compared with interferon β, interferon ε increased the proportion of T cells without causing excessive activation of pSTAT-1.

[0078] like Figures 9a to 9c As shown, tumor analysis results confirm that interferon ε increases T cell infiltration into tumors and reduces immunosuppressive myeloid cells through anti-tumor immune activation similar to that of interferon β.

[0079] In an in vivo mouse model, interferon ε exhibited less nonspecific immune activation in blood and spleen compared to interferon β, while showing similar levels of immune activation within tumors. Based on this, it was confirmed that interferon ε, with its increased activity under acidic conditions, possesses superior antitumor efficacy compared to other interferon subtypes.

[0080] [Industrial Applicability] As described above, the interferon ε according to the present invention is characterized by significantly enhanced activity in a slightly acidic environment compared to a neutral environment. Therefore, it has the advantage of being able to be administered systemically without the need for additional processes such as targeting or masking to control systemic toxicity, and thus can be effectively used for the treatment of related diseases.

Claims

1. A pharmaceutical composition for the prevention or treatment of a disease, comprising interferon ε as an active ingredient, wherein said interferon ε is activated in tissues where the pH is lowered due to the pathological state of the disease.

2. The pharmaceutical composition according to claim 1, wherein the composition is for systemic administration.

3. The composition according to claim 1, wherein the disease is cancer or an infectious disease.

4. The composition according to claim 1, wherein the pH of the pH-decreased tissue is 5.0 to 6.

8.

5. The composition according to claim 1, wherein the activity of interferon ε increases by 2 to 1000 times at pH 6.0 compared to pH 7.

4.

6. The composition according to claim 2, wherein the systemic administration is intravenous, intramuscular, intraarterial, intramedullary, intrasheath, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intraintestinal, local, sublingual, rectal, or intratumoral administration.

7. The composition according to claim 3, wherein the cancer is at least one selected from the group consisting of: ovarian cancer, papilloma, laryngeal papilloma, human papilloma, bladder cancer, cervical cancer, hairy cell leukemia, chronic myeloid leukemia, multiple myeloma, non-Hodgkin's lymphoma, skin cancer such as melanoma, Kaposi's sarcoma, kidney cancer, lung cancer, brain tumor such as glioma, breast cancer, colorectal cancer, liver cancer, hepatocellular carcinoma, gastric cancer, bronchial cancer, nasopharyngeal carcinoma, laryngeal cancer, pancreatic cancer, colon cancer, pancreatic cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, glioblastoma, neuroblastoma, and malignant mesothelioma.

8. The composition of claim 3, wherein the infectious disease is at least one selected from the group consisting of: AIDS caused by HIV, hepatitis B and C virus infection, influenza virus infection, respiratory syncytial virus infection, herpes virus infection, human papillomavirus infection, and coronavirus infection.

9. A fusion protein comprising interferon ε and an antibody or fragment thereof that binds directly or indirectly to interferon ε.

10. The fusion protein of claim 9, wherein the fusion protein is characterized in that interferon ε is linked to an antibody or a fragment thereof via a linker.

11. A pharmaceutical composition for the prevention or treatment of a disease, comprising the fusion protein according to claim 9 or 10, wherein the fusion protein is activated in tissues where the pH is lowered due to the pathological state of the disease.

12. The pharmaceutical composition according to claim 11, wherein the composition is for systemic administration.

13. The composition of claim 11, wherein the disease is cancer or an infectious disease.

14. The composition of claim 11, wherein the pH of the pH-decreased tissue is 5.0 to 6.

8.

15. The composition of claim 11, wherein the activity of interferon ε increases by 2 to 1000 times at pH 6.0 compared to pH 7.

4.

16. The composition of claim 12, wherein the systemic administration is intravenous, intramuscular, intraarterial, intramedullary, intrasheath, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intraintestinal, local, sublingual, rectal, or intratumoral administration.

17. The composition according to claim 13, wherein the cancer is at least one selected from the group consisting of: ovarian cancer, papilloma, laryngeal papilloma, human papilloma, bladder cancer, cervical cancer, hairy cell leukemia, chronic myeloid leukemia, multiple myeloma, non-Hodgkin's lymphoma, skin cancer such as melanoma, Kaposi's sarcoma, kidney cancer, lung cancer, brain tumor such as glioma, breast cancer, colorectal cancer, liver cancer, hepatocellular carcinoma, gastric cancer, bronchial cancer, nasopharyngeal carcinoma, laryngeal cancer, pancreatic cancer, colon cancer, pancreatic cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, esophageal cancer, bile duct cancer, testicular cancer, rectal cancer, head and neck cancer, cervical cancer, ureteral cancer, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, glioblastoma, neuroblastoma, and malignant mesothelioma.

18. The composition of claim 13, wherein the infectious disease is at least one selected from the group consisting of: AIDS caused by HIV, hepatitis B and C virus infection, influenza virus infection, respiratory syncytial virus infection, herpes virus infection, human papillomavirus infection, and coronavirus infection.

Images