Novel use of interferon epsilon

Abstract

The present invention relates to novel use of interferon epsilon and, more specifically, to a pharmaceutical composition for the prevention or treatment of diseases, comprising interferon epsilon as an active ingredient. The pharmaceutical composition has significantly improved activity in a slightly acidic environment compared to a neutral environment, and thus can be administered systemically without separate treatment such as targeting or masking for controlling systemic toxicity, and therefore, can be effectively used for the treatment of related diseases.

Description

Novel uses of interferon epsilon

[0001] This application claims the benefit of 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 contents of which are incorporated herein by reference.

[0002] The present invention relates to a novel use of interferon epsilon, and more particularly, to a pharmaceutical composition for the prevention or treatment of a disease comprising interferon epsilon as an active ingredient, wherein the interferon epsilon is activated in a tissue whose pH is reduced due to a pathological state of the disease, a fusion protein comprising an antibody or a fragment thereof directly or indirectly bound to the interferon epsilon, and a pharmaceutical composition comprising the fusion protein.

[0003]

[0004] Interferon (IFN) is a protein belonging to a class of cytokines, molecules used in cell-to-cell communication to trigger the body's immune system. Interferon is named for its ability to protect cells from viral infection and "interfere" with viral replication. It also activates immune cells such as natural killer cells and macrophages, and upregulates antigen presentation by increasing the expression of major histocompatibility complex (MHC) antigens, thereby enhancing the host's defenses.

[0005] Interferons are classified into type I, which includes IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω; type II, which includes IFN-γ; and type III, which includes IFN-λ. Of these, type I, 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 as therapeutic agents for hairy cell leukemia, melanoma, renal cell carcinoma, Kaposi sarcoma, multiple myeloma, follicular and non-Hodgkin lymphoma, and chronic myelogenous leukemia, and human IFN-β has also been approved in Japan for the treatment of glioma, medulloblastoma, astrocytoma, and melanoma. In addition, IFN-β1 has been approved by the FDA for use as a therapeutic agent for multiple sclerosis.

[0006] While interferon has proven effective against numerous cancers, it also causes flu-like symptoms, including fever, chills, muscle pain, headache, nausea, and fatigue. Furthermore, the anti-tumor effects of systemic interferon therapy are often accompanied by serious side effects, including inflammation and direct tissue toxicity. Furthermore, interferon's short half-life necessitates shorter dosing cycles, ultimately increasing side effects.

[0007] Methods for controlling systemic toxicity by interferon include methods that utilize the tumor-targeting ability of monoclonal antibodies to directly deliver interferon to the tumor site, methods that increase the half-life of interferon by conjugating polyethylene glycol (PEG) to it, and methods that suppress (mask) the activity of interferon until it reaches the tumor. However, tumor-targeted interferon still has the problem that it can be recognized by interferon receptors expressed throughout the body, causing side effects, and there is virtually no difference in side effects between interferon with and without PEG attached. In addition, masked interferon has the disadvantage that the manufacturing process is complicated, the activity of interferon can vary depending on the activity of proteases in the tumor microenvironment, and the additional protein structure used for masking has the potential to induce a new immune response.

[0008] Therefore, a novel strategy is needed to induce interferon activity specifically in pathological tissues while suppressing systemic toxicity caused by systemic administration of interferon.

[0009]

[0010] Accordingly, the inventors of the present invention newly confirmed that the activity of interferon epsilon (IFN-ε) has a characteristic of rapidly increasing in a low pH environment, and due to this characteristic, when interferon epsilon was systemically administered to a tumor mouse model, non-specific immune activity in the blood and spleen was less than in the interferon beta (IFN-β) administration group, while the degree of immune activity within the tumor was similar, thereby completing the present invention.

[0011]

[0012] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease, which comprises interferon epsilon as an active ingredient, wherein the interferon epsilon is activated in a tissue whose pH is reduced due to the pathological state of the disease.

[0013]

[0014] Another object of the present invention is to provide a fusion protein comprising the interferon epsilon and an antibody or fragment thereof directly or indirectly bound to the interferon epsilon.

[0015]

[0016] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease, comprising the fusion protein, wherein the fusion protein is activated in a tissue whose pH is reduced due to the pathological state of the disease.

[0017]

[0018] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease comprising interferon epsilon.

[0019]

[0020] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease, which consists essentially of interferon epsilon.

[0021]

[0022] In order to achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating a disease, which comprises interferon epsilon as an active ingredient, wherein the interferon epsilon is activated in a tissue whose pH is reduced due to a pathological state of the disease.

[0023]

[0024] In order to achieve another object of the present invention, the present invention provides a fusion protein comprising the interferon epsilon and an antibody or fragment thereof directly or indirectly bound to the interferon epsilon.

[0025]

[0026] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease, comprising the fusion protein, wherein the fusion protein is activated in a tissue whose pH is reduced due to a pathological state of the disease.

[0027]

[0028] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease comprising interferon epsilon.

[0029]

[0030] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a disease, which consists essentially of interferon epsilon.

[0031]

[0032] The present invention is described in detail below.

[0033]

[0034] Meanwhile, each description and embodiment disclosed in the present invention can also be applied to other descriptions and embodiments thereof. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0035] In this specification, the term "comprising" is used with the same meaning as "including" or "characterized by", and does not exclude additional components, etc. that are not specifically mentioned in the composition according to the present invention. In addition, the term "consisting of" means excluding additional elements, components, etc. that are not separately described. The term "essentially consisting of" means that, within the scope of the composition, it may include materials that do not substantially affect the basic characteristics thereof in addition to the materials described.

[0036] The term "treatment" as used herein comprehensively refers to improving symptoms caused by a disease, and includes temporarily or permanently alleviating symptoms, eliminating the cause of symptoms, or preventing or delaying the onset of symptoms of a disease or condition. This may include curing, substantially preventing, or improving the condition of a disease, and includes, but is not limited to, alleviating, curing, or preventing one or most symptoms resulting from a disease.

[0037] The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying their onset by administering a pharmaceutical composition according to the present invention.

[0038] The term "improvement" as used herein means any action that reduces a parameter associated with an abnormal condition, for example, the severity of a symptom.

[0039] The terms or words used in the description and claims of the present invention should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that conform to the technical idea of ​​the present invention, based on the principle that the inventor can appropriately define the concept of the term to explain his or her own invention in the best way.

[0040]

[0041] The present invention provides a pharmaceutical composition for the prevention or treatment of a disease, comprising interferon epsilon as an active ingredient, wherein the interferon epsilon is activated in a tissue whose pH is reduced due to a pathological state of the disease.

[0042]

[0043] In the present invention, the interferon epsilon (IFN-ε) refers to a cytokine belonging to type I interferon that is mediated through binding to IFNAR1 and IFNAR2, which are interferon alpha / beta receptors (IFN-α / β receptors), and plays a role in activating the JAK-STAT pathway through binding to the receptors. The interferon epsilon exists in various organisms including humans, mice, pigs, cows, dogs, horses, etc., and, unlike interferon alpha (hereinafter, IFN-α) and beta (hereinafter, IFN-β), is known to be mainly expressed in various mucosal tissues such as the lungs, small intestine, and reproductive organs. Meanwhile, in the present invention, the interferon epsilon preferably refers to human interferon epsilon, but is not limited thereto. Additionally, in the present specification, the interferon epsilon may be referred to as interferon epsilon, interferon-ε, interferon-e, IFN-ε, IFN-e, IFNε, or IFNe, but all of these refer to the interferon epsilon.

[0044]

[0045] According to one embodiment of the present invention, the activity of IFN-α and IFN-ω decreased under slightly acidic conditions (pH 6.0) compared to neutral conditions (pH 7.4), and the activity of IFN-ε along with IFN-β increased under slightly acidic conditions, but in particular, the difference was significant for IFN-ε. More specifically, IFN-ε was enhanced 5.02-fold in a slightly acidic environment compared to a neutral environment, and the binding ability was superior to that in a neutral environment even when treating a receptor at a concentration 43.76 times lower in a slightly acidic environment. In addition, it was confirmed that pSTAT-1 signaling was activated even at a concentration 1000 times lower than that in a neutral environment in a slightly acidic environment. Therefore, the interferon epsilon according to the present invention is characterized in that its activity appears in a tissue whose pH is less than pH 7, that is, a tissue whose pH is reduced compared to a normal tissue. The pH of the above "pH-reduced tissue" may preferably be 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. However, it is not limited thereto. In addition, as described above, the interferon epsilon has an activity of 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 1500 times, in tissues with reduced pH compared to normal tissues.Characterized in that the activity increases by 2000 times, 3000 times, 4000 times, 5000 times, 10000 times, or more, and preferably 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 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 may increase by more than this, but is not limited to this.

[0046]

[0047] Accordingly, the interferon epsilon according to the present invention has the characteristic of being able to be administered systemically without any special treatment such as targeting or masking. In particular, since the pH of the tumor microenvironment is known to be approximately pH 5.6 to 6.8, when the interferon epsilon according to the present invention is used to treat cancer and / or tumors, there is an advantage in that side effects can be reduced even when systemic administration therapy is used. In fact, according to one embodiment of the present invention, when interferon beta and epsilon were intraperitoneally administered to a tumor-induced mouse model using a B16F10 cell line that constitutes a low-pH tumor microenvironment, the interferon epsilon-administered group showed a smaller decrease in the number of white blood cells and myeloid cells in the blood compared to the interferon beta-administered group, and while excessive pSTAT-1 activity did not occur, it was confirmed that the level of increased T cell infiltration in the tumor and the level of reduced myeloid cells were similar to those of the interferon beta-administered group.

[0048] Meanwhile, in the present invention, the 'systemic administration' means administering the composition according to the present invention into the circulatory system to affect the entire body, and may preferably include intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, intrarectal, or intratumoral administration, but is not limited thereto.

[0049]

[0050] The composition comprising interferon epsilon according to the present invention as an active ingredient can be applied without limitation to any disease and / or pathological condition that can be treated using interferon epsilon known to date.

[0051] Generally, STAT-1 signaling, mediated by IFNAR1 / IFNAR2 activation by interferons, is known to promote the exposure of MHC class I molecules on the surface of cancer cells, which, combined with the upregulation of tumor-associated antigens, increases the overall antigenicity of the tumor. Therefore, the above-mentioned pathological condition may refer to cancer and / or tumors.

[0052] In the present invention, the cancer and / or tumor may preferably be at least one selected from ovarian cancer, papilloma, laryngeal papilloma, human papilloma, bladder cancer, and cervical cancer, but other cancers and / or tumors that can be treated with the composition according to the present invention are not particularly limited and include both solid cancers and blood cancers. Non-limiting examples of the above cancers and / or tumors include, but are not limited to, hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, non-Hodgkin's lymphoma, skin cancer such as melanoma, Kaposi's sarcoma, kidney cancer, lung cancer, brain tumors such as glioma, breast cancer, colon cancer, liver cancer, hepatocellular carcinoma, stomach cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colon cancer, pancreatic cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, glioblastoma, neuroblastoma, and malignant mesothelioma.

[0053] Additionally, interferons activate interferon-stimulated genes (ISGs) via the JAK-STAT pathway, and many ISGs are known to control viral, bacterial, and parasitic infections by directly targeting pathways and functions required for the life cycle of pathogens. Therefore, the above pathological condition may indicate an infectious disease.

[0054] In the present invention, the infectious disease may preferably mean a viral infection. Non-limiting examples of the viral infection may include, but are not limited to, AIDS caused by HIV, hepatitis B and C virus infections, and may also include, but are not limited to, at least one selected from the group consisting of influenza virus infection, respiratory syncytial virus infection, herpes virus infection, human papilloma virus infection, coronavirus infection, and other viral infections.

[0055]

[0056] The composition according to the present invention comprises the interferon epsilon described above as an active ingredient, and may be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may additionally contain an excipient or diluent. The term "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness when administered to humans. The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. The carrier for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may include water, a suitable oil, saline solution, aqueous glucose, glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, and the like. Other pharmaceutically acceptable carriers and formulations are described in the following references.

[0057]

[0058] Since the composition according to the present invention can be administered systemically as described above, it can be administered to mammals, including humans, by any method, for example, orally or parenterally. Accordingly, the composition according to the present invention can be formulated as a preparation for oral or parenteral administration, depending on the administration route described above.

[0059] In the case of preparations for oral administration, the composition of the present invention can be formulated into powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. For example, oral preparations can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture to obtain a tablet or dragee. Examples of suitable excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; cellulosics including cellulose, methylcellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; fillers such as gelatin and polyvinylpyrrolidone. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrating agent, depending on the case. Furthermore, the pharmaceutical composition of the present invention may additionally include an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like.

[0060] In the case of preparations for parenteral administration, they can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalers using methods known in the art.

[0061]

[0062] The total effective amount of the composition according to the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The composition according to the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the preferred total dosage of the composition according to the present invention may be about 0.01 ㎍ to 10,000 mg per 1 kg of body weight of the patient or subject per day, and most preferably 0.1 ㎍ to 500 mg. However, since the dosage of the composition is determined by taking into consideration various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, sex, severity of the disease, diet, and excretion rate, a person having ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The composition according to the present invention is not particularly limited in its formulation, administration route, or administration method as long as it exhibits the effects of the present invention.

[0063] The 'effective amount' of the present invention refers to an amount that, when administered to a subject, exhibits an effect of improving, treating, detecting, diagnosing, or inhibiting or reducing cancer or an infectious disease. The 'subject' may be an animal, preferably a mammal, particularly an animal including a human, and may also be a cell, tissue, organ, etc. derived from an animal. The subject may be a patient in need of the effect.

[0064] In the present invention, the content of the composition is not particularly limited depending on the purpose or aspect of use, and may be, for example, 0.01 to 99 wt%, preferably 0.5 to 50 wt%, and more preferably 1 to 30 wt%, based on the total weight of the composition. In addition, the pharmaceutical composition according to the present invention may further include additives such as pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient. The pharmaceutical composition of the present invention may include 0.1 to 99.9 wt% of interferon epsilon prepared by the method of the present invention, and 99.9% to 0.1 wt% of the carrier.

[0065]

[0066] In addition, the present invention provides a fusion protein comprising the interferon epsilon described above and an antibody or fragment thereof directly or indirectly bound to the interferon epsilon.

[0067]

[0068] In the fusion protein according to the present invention, the meaning of the interferon epsilon is the same as that described in the above-described composition, and the structure unique to the fusion protein is described below.

[0069]

[0070] In the present invention, the antibody includes a monoclonal antibody, a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), and fragments thereof. A complete antibody has an overall Y shape and is composed of two long heavy chains (H) and two short light chains (L). Each heavy chain and light chain are connected to each other by a sulfide bond and are divided into a variable region (V), which is a site that reacts with an antigen, and a constant region (C), which is a site that expresses an effector function. The variable region determines the structure of the variable region so that it can form a specific binding with an antigen and contains a complementarity-determining region (CDR), which controls antibody binding strength. The fragment of the above antibody represents a specific site that can react with an antigen and exhibit antigen-binding activity, and examples thereof include a Fab fragment (a fragment obtained by papain digestion), a Fab' fragment (a fragment obtained by pepsin digestion and partial reduction), a F(ab')2 fragment (a fragment obtained by pepsin digestion), a Facb (a fragment obtained by plasmin digestion), a Fd (a fragment obtained by pepsin digestion, partial reduction, and reaggregation), a scFv fragment (a fragment obtained by molecular biology techniques), and the like. Preferably, the antibody is an antibody or a fragment thereof that recognizes a tumor-specific antigen, and can be used as a targeted therapeutic agent for cancer, etc. For example, these may include Trastuzumab, an antibody treatment targeting human epidermal growth factor receptor (HER-2), Cetuximab, an antibody treatment targeting epidermal growth factor receptor (EGFR), Atezolizumab, an antibody treatment targeting PD-L1 expressed on the surface of cancer cells, and Sacituzumab, an antibody treatment targeting TROP2 expressed on the surface of cancer cells.

[0071]

[0072] The fusion protein according to the present invention may be a fusion protein formed by linking interferon epsilon and an antibody or a fragment thereof via a linker, preferably a peptide linker. The linker refers to a molecule that functions to link two or more separate substances, such as a short fragment of an amino acid or amino acid analogue, in which two or more amino acids or amino acid-like substances are linked to each other by a peptide bond. In this case, glycine, serine, alanine, etc. may be used as the main constituent amino acids, and thus a glycine-serine linker, a glycine-serine-alanine linker, etc. may be used. Such a linker may be linked to the C-terminus of the heavy chain of the antibody or the C-terminus of the light chain of the antibody, or the N-terminus of the linker may be linked to the C-terminus of the light chain of the antibody and the C-terminus of the heavy chain of the antibody. In this case, the N-terminus of the interferon epsilon may be linked to the C-terminus of the linker.

[0073]

[0074] In the fusion protein according to the present invention, the antibody recognizes a tumor-specific antigen as described above and is intended for targeted treatment of cancer and the like. Therefore, it is clear that the pharmacological effects of the fusion protein will be substantially identical to those of the interferon epsilon described above. Accordingly, the present invention provides a tissue-specific treatment or prevention composition with a decreased pH due to a pathological condition, including the fusion protein.

[0075]

[0076] In the composition according to the present invention, the meanings of all terms including the fusion protein, pathological condition, pH and tissue with reduced pH, etc. are the same as those described in the composition described above, and the contents described in the corresponding items can be applied as is to the composition including the fusion protein according to the present invention.

[0077]

[0078] Interferon epsilon according to the present invention has the characteristic that its activity is greatly enhanced in a slightly acidic environment compared to a neutral environment, and therefore has the advantage of being able to be administered systemically without separate treatment such as targeting or masking to control systemic toxicity, and thus can be usefully used in the treatment of related diseases.

[0079]

[0080] Figures 1a to 1f show the results of evaluating the receptor binding ability in neutral and acidic environments by type of type 1 interferon (IFN-α2b, IFN-β, IFN-β R27T, IFN-β C17S / R27T, IFN-ω, and IFN-ε).

[0081] Figures 2a and 2b show the receptor binding capacity of each type of type 1 interferon in neutral and acidic environments, normalized to neutral conditions.

[0082] Figures 3a and 3b show changes in the binding capacity of interferon epsilon to the receptors IFNAR1 and IFNAR2 under acidic conditions compared to neutral conditions.

[0083] Figures 4a to 4c show the results of calculating the binding kinetics and affinity of interferon epsilon to the receptors IFNAR1 / 2, IFNAR1, and IFNAR2 under acidic conditions compared to neutral conditions.

[0084] Figures 5a to 5c show the results of comparing the expression levels of pSTAT-1 in order to confirm the degree of cell signaling under acidic conditions compared to neutral conditions of interferon epsilon.

[0085] Figure 6 illustrates a schematic diagram of an experiment to evaluate the side effects and efficacy of interferon epsilon in an in vivo mouse model.

[0086] Figures 7a to 7g show the blood analysis results of an in vivo mouse model experiment.

[0087] Figures 8a to 8c show the results of spleen analysis of an in vivo mouse model experiment.

[0088] Figures 9a to 9c show the tumor analysis results of an in vivo mouse model experiment.

[0089]

[0090] The present invention is described in detail below.

[0091] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0092]

[0093] Example 1. Evaluation of receptor binding capacity under neutral and acidic conditions for each type of type 1 interferon.

[0094] To evaluate the receptor-binding ability of type I interferon, IFNAR1 / 2 fusion proteins were produced in Expi293F cells. Human IFNAR1-Fc and IFNAR2-Fc proteins with knob-into-hole technology were inserted into pOptiVec-TOPO plasmid (Invitrogen) and transfected using ExpiFectamine™ 293 Transfection Kit (Gibco #A14524). Transfected Expi293F cells were cultured for one week, and the supernatant was collected and purified using a Protein A column in an AKTA avant (Cytiva) system.

[0095] The receptor binding capacity of type I interferons under neutral and acidic conditions was evaluated using ELISA experiments. Type I interferons used were Rebif (Merck), ABN101, ABN102 (Abion), IFN-α2b (biolegend, #592704), IFN-ω (PBL, #11395-1), and IFN-ε (R&D systems, #9667-ME-025 / CF). Type I interferons were diluted to 100 nM in PBS buffer, and 100 μl each was treated to a 96-well ELISA plate (Corning, #2592), and coated for 16 h at 4°C. All buffers except the coating buffer were adjusted to pH 7.4 and pH 6.0 with 1 N HCl. The coated plate was washed three times with washing buffer (0.05% PBS-T, pH 7.4, pH 6.0), and then treated with 300 μl of blocking buffer (50 mg / mL BSA in PBS, pH 7.4, pH 6.0) and incubated at room temperature for 1 hour. After blocking, the plate was washed three times with washing buffer, and the IFNAR1 / 2 fusion protein was serially diluted 1 / 3 from 100 nM in reagent diluent (1 mg / mL BSA in 0.05% PBS-T, pH 7.4, pH 6.0), and 100 μl of each was treated and incubated at room temperature for 1 hour. After washing three times with bashing buffer, 100 μl of anti-human IgG-HRP (Jackson lab, # 109-035-003) was diluted 1:5000 and incubated at room temperature for 1 hour. After washing three times with washing buffer, 100 μl of TMB solution (Surmodics, # TMBW 1000-01) was incubated at room temperature for 20 minutes. After incubation, the absorbance was measured at a wavelength of 450 nm.

[0096]

[0097] As a result of evaluating the receptor binding ability of the tested type 1 interferons according to pH, as can be seen in Figures 1a to 1f, Rebif, ABN101, ABN102, and IFN-ε showed superior receptor binding ability under acidic conditions than under neutral conditions, and IFN-ε showed the best binding ability under acidic conditions.

[0098]

[0099] Example 2. Changes in binding capacity under acidic conditions compared to neutral conditions for each type of type 1 interferon.

[0100] The binding capacity of type I interferons evaluated using ELISA experimental method was standardized to neutral conditions and graphically represented (see Fig. 2a and Fig. 2b). Rebif, ABN101, ABN102, and IFN-ω showed superior receptor binding capacity under acidic conditions than under neutral conditions, and the binding capacity of IFN-ε at the highest concentration was enhanced 5.02-fold under acidic conditions compared to neutral conditions, and it was superior to the binding capacity under neutral conditions even when treating a receptor concentration 43.76 times lower.

[0101] Through this, it was confirmed that IFN-ε has better binding ability under acidic conditions than under neutral conditions.

[0102]

[0103] Example 3. Changes in the binding ability of interferon epsilon to IFNAR1 and IFNAR2 under acidic conditions compared to neutral conditions.

[0104] To evaluate the receptor-binding ability of type I interferon, human IFNAR1, IFNAR2 fusion proteins were produced in Expi293F cells. The human IFNAR1-Fc, IFNAR2-Fc protein genes, which had knob-into-hole technology, were inserted into pOptiVec-TOPO plasmid (Invitrogen), and then 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 in an AKTA avant (Cytiva) system.

[0105] The receptor binding capacity of type I interferon under neutral and acidic conditions was evaluated using an ELISA experiment. Type I interferon was produced by inserting the human IFN-ε gene into the pOptiVec-TOPO plasmid (Invitrogen) using the ExpiFectamine™ 293 Transfection Kit (Gibco, #A14524). 100 μl of interferon epsilon diluted to 100 nM in PBS buffer was added to a 96-well ELISA plate (Corning, #2592) and coated for 16 h at 4°C. All buffers except the coating buffer were adjusted to pH 7.4 and 6.0 with 1 N HCl. The coated plate was washed three times with washing buffer (0.05% PBS-T, pH 7.4, pH 6.0), and then treated with 300 μl of blocking buffer (50 mg / mL BSA in PBS, pH 7.4, pH 6.0) and incubated at room temperature for 1 hour. After blocking, the plate was washed three times with washing buffer, and the IFNAR1 / 2 fusion protein was serially diluted 1 / 3 from 100 nM in reagent diluent (1 mg / mL BSA in 0.05% PBS-T, pH 7.4, pH 6.0), and treated with 100 μl of each, and incubated at room temperature for 1 hour. After washing three times with washing buffer, 100 μl of anti-human IgG-HRP (Jackson lab, #109-035-003) was diluted 1:5000 and incubated at room temperature for 1 hour. After washing three times with washing buffer, 100 μl of TMB solution (Surmodics, #TMBW 1000-01) was incubated at room temperature for 20 minutes. After incubation, the absorbance was measured at a wavelength of 450 nm.

[0106] As a result of evaluating the binding ability of the tested interferon epsilon to IFNAR1 and IFNAR2 according to pH, it was confirmed that interferon epsilon's receptor binding was strengthened in an acid-dependent manner by IFNAR2, as can be seen in Figures 3a and 3b.

[0107]

[0108] Example 4. Receptor binding kinetics and affinity of interferon epsilon under acidic versus neutral conditions.

[0109] Interferon epsilon receptor binding kinetics were analyzed under neutral and acidic conditions using an Octet R8 (Satorious) instrument. AHC biosensors (Satorious) were coated with 20 nM each of hIFNAR1 / 2, hIFNAR1, and hIFNAR2 using 1x kinetics buffer. Depending on the neutral and acidic assay conditions, 1x kinetics buffer was titrated with 1 N HCl.

[0110] Interferon epsilon was diluted 1 / 2-fold from 200 nM and bound to the coated receptor in the AHC biosensor, and the kinetics were calculated on the Octet R8 instrument through the dissociation process.

[0111] As a result, similar to the previous experimental results, it was confirmed that interferon epsilon had a lower KD value under acidic conditions compared to neutral conditions, and had stronger binding affinity under acidic conditions for AR2 than for AR1.

[0112]

[0113] Example 5. Confirmation of signaling activation of Daudi cells and human CD3 T cells under acidic conditions compared to neutral conditions by interferon epsilon.

[0114] To determine the differences in the degree of cell signaling in neutral and acidic environments through receptor binding of interferon epsilon, human B lymphoblast Daudi cells and CD3 T cells isolated from healthy donor human peripheral blood mononuclear cells (PBMCs) were used. CD3 T cells from human PBMCs were isolated using a human CD3 T cell isolation kit (Miltenyl biotec).

[0115] The cell culture medium was adjusted to pH 7.4 and pH 6.5 using 1N HCl in RPMI1640, and 20 mM HEPES was added. Daudi cells and CD3 T cells were seeded in 96-well tissue culture plates at a density of 2 x 10 5After seeding cells individually, interferon epsilon was diluted 1 / 10-fold from 100 nM in pH 7.4 and pH 6.5 media and treated for 24 hours. After 24 hours, the cells were collected and first dissociated into single cells using cell dissociation buffer, and then lysed by reacting with RIPA buffer (BIOSESANG), protease inhibitor, and phosphatase inhibitor at 200 rpm, 4℃, for 1 hour. After centrifugation at 15,000 rpm, 4℃, for 15 minutes, only the supernatant was collected, and the dissolved proteins were quantified using BCA assay (ThermoFisher Scientific). The quantified proteins were added to 5X sample loading buffer (BIOSESANG) containing DTT and boiled for 10 minutes to reduce all proteins. This was loaded with an equal amount of protein through SDS-PAGE, separated by size, and transferred to a PVDF membrane, blocked with 5% BSA in TBS-T at 4℃ for 1 hour, and anti-human pSTAT-1 (Cell signaling technology) was used as the primary antibody and attached to the PVDF membrane overnight at a ratio of 1:1000. After washing three times with 0.05% TBS-T, the secondary antibody, anti-rabbit Fc HRP antibody (Invitrogeon), was treated at a ratio of 1:5000 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.

[0116] As a result of evaluating the activation of cell signaling according to pH of the tested type 1 interferon, as can be seen in Figures 5a to 5c, it was confirmed that interferon epsilon activates the pSTAT-1 signal in Daudi cells and human CD3 T cells in an acidic environment at a concentration that is more than 1000 times lower than in a neutral environment.

[0117]

[0118] Example 6. Evaluation of side effects and efficacy of interferon epsilon in an in vivo mouse model.

[0119] To evaluate the side effects and efficacy of interferon epsilon in an in vivo mouse model, human hIFNAR1 / 2 knock-in (hIFNAR1 / 2 KI) mice were used. 5x10 B16F10 cell lines expressing human hIFNAR1 / 2 were injected into hIFNAR1 / 2 KI mice. 5 Tumor models were induced by subcutaneous injection of cells. Tumor sizes ranged from 100 to 150 mm. 3 When administered, the animals were divided into control, IFN-β, and IFN-ε administration groups and administered intraperitoneally at a dose of 0.5 mg / kg for 5 days. The experimental schematic is as shown in Figure 6A. One hour after the last administration, blood, spleen, and tumor were collected and analyzed using a flow cytometer.

[0120] As shown in Figures 7a to 7g, blood analysis results showed that interferon epsilon, which is active in acidic conditions, showed a smaller decrease in white blood cell counts, which is an indicator of neutropenia side effects, compared to interferon beta, and increased CD8 T cells through T cell activation while reducing immunosuppressive myeloid cells. In addition, it did not cause excessive activation of pSTAT-1, which corresponds to downstream signaling, in the blood.

[0121] As can be seen in Figures 8a to 8c, the spleen analysis results confirmed that interferon epsilon further increased the ratio of T cells compared to interferon beta and did not cause excessive pSTAT-1 activity.

[0122] As a result of tumor analysis, Figs. 9a to 9c confirmed that interferon epsilon increases infiltration of T cells into tumors and reduces immunosuppressive myeloid cells through anti-tumor immune activity to a degree similar to interferon beta.

[0123] In an in vivo mouse model, interferon epsilon induced less nonspecific immune activity in the blood and spleen than interferon beta, while exhibiting similar levels of intratumoral immune activity. Based on this, we confirmed that interferon epsilon exhibits enhanced activity in acidic conditions and exhibits superior anticancer efficacy compared to other interferon subtypes.

[0124]

[0125] As described above, the interferon epsilon according to the present invention has the characteristic that its activity is greatly enhanced in a slightly acidic environment compared to a neutral environment, and therefore has the advantage of being able to be administered systemically without separate treatment such as targeting or masking to control systemic toxicity, and thus can be usefully used in the treatment of related diseases.

Claims

1. A pharmaceutical composition for the prevention or treatment of a disease, comprising interferon epsilon as an active ingredient, wherein the interferon epsilon is activated in a tissue whose pH is reduced due to the pathological state of the disease.

2. A pharmaceutical composition according to claim 1, characterized in that the composition is for systemic administration.

3. A composition according to claim 1, characterized in that the disease is cancer or an infectious disease.

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

8.

5. A composition according to claim 1, wherein the activity of interferon epsilon is increased 2 to 1000 times at pH 6.0 compared to pH 7.

4.

6. A composition characterized in that the systemic administration in paragraph 2 is intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intrarectal, or intratumoral administration.

7. A composition according to claim 3, characterized in that 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 myelogenous leukemia, multiple myeloma, non-Hodgkin's lymphoma, skin cancer such as melanoma, Kaposi's sarcoma, kidney cancer, lung cancer, brain tumors such as glioma, breast cancer, colon cancer, liver cancer, hepatocellular carcinoma, stomach cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colon cancer, pancreatic cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, glioblastoma, neuroblastoma, and malignant mesothelioma.

8. A composition according to claim 3, characterized in that 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 papilloma virus infection, and coronavirus infection.

9. A fusion protein comprising interferon epsilon and an antibody or fragment thereof directly or indirectly bound to said interferon epsilon.

10. In claim 9, the fusion protein is a fusion protein characterized in that interferon epsilon and an antibody or a fragment thereof are linked by a linker.

11. A pharmaceutical composition for preventing or treating a disease, comprising a fusion protein according to claim 9 or 10, wherein the fusion protein is activated in a tissue whose pH is reduced due to a pathological state of the disease.

12. A pharmaceutical composition according to claim 11, characterized in that the composition is for systemic administration.

13. A composition according to claim 11, wherein the disease is cancer or an infectious disease.

14. A composition according to claim 11, wherein the pH of the tissue with decreased pH is 5.0 to 6.

8.

15. A composition according to claim 11, wherein the activity of interferon epsilon is increased 2 to 1000 times at pH 6.0 compared to pH 7.

4.

16. A composition according to claim 12, characterized in that the systemic administration is intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intrarectal, or intratumoral administration.

17. A 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 myelogenous leukemia, multiple myeloma, non-Hodgkin's lymphoma, skin cancer such as melanoma, Kaposi's sarcoma, kidney cancer, lung cancer, brain tumors such as glioma, breast cancer, colon cancer, liver cancer, hepatocellular carcinoma, stomach cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, colon cancer, pancreatic cancer, prostate cancer, bone cancer, thyroid cancer, parathyroid cancer, esophageal cancer, biliary tract cancer, testicular cancer, rectal cancer, head and neck cancer, cervical spine cancer, ureteral cancer, neuroblastoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, glioblastoma, neuroblastoma, and malignant mesothelioma.

18. A composition according to claim 13, characterized in that 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 papilloma virus infection, and coronavirus infection.

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