KR20220139237A - Pharmaceutical composition for the prevention or treatment of diseases associated with immune dysregulation comprising oxidized immunoglobulin - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating immune dysregulation-related diseases having oxidized immunoglobulin as active ingredients, and a prevention or treatment method using the same. More specifically, the present invention provides a pharmaceutical composition for treating allergic diseases, chronic inflammatory diseases, autoimmune diseases or malignant tumor diseases having oxidized immunoglobulin, in which oxidation is artificially induced, as active ingredients and a novel immunoregulatory treatment method which is more differentiated and effective from existing immunoglobulin treatments by injecting the composition to patients with the diseases.

Description

A pharmaceutical composition for the prevention or treatment of diseases associated with immunomodulatory dysfunction comprising oxidized immunoglobulin as an active ingredient {Pharmaceutical composition for the prevention or treatment of diseases associated with immune dysregulation comprising oxidized immunoglobulin}

The present invention relates to a pharmaceutical composition comprising oxidized immunoglobulin as an active ingredient for the prevention or treatment of diseases related to immunomodulatory dysfunction, and a treatment method using the same.

In the pathogenesis of allergic diseases, chronic inflammatory diseases or autoimmune diseases, and malignant tumor diseases, hypersensitive immune response (allergic response) or excessive immune response to one's own antigen (autoimmune response) or decreased immune response to tumor cells ( It is well known that immune dysregulation (including in malignant diseases) plays a key role. Accordingly, immunomodulatory therapeutic effects of various types of immunoglobulin therapeutics including monoclonal antibody therapeutics have been confirmed in patients suffering from diseases related to immunomodulatory dysfunction and are actually being used in clinical practice.

Immunoglobulin therapeutics currently used for the prevention or treatment of allergic diseases, chronic inflammatory diseases or autoimmune diseases, and malignant tumor diseases are monoclonal antibodies or multiple Various immunomodulatory dysfunctions occurring in humans or mammals due to the production of two types of polyvalent or polyclonal immunoglobulin preparations isolated from plasma pools obtained from healthy blood donors of It is used for the prevention or treatment of diseases associated with immune dysregulation. However, the mechanism by which polyvalent or polyclonal immunoglobulin preparations isolated from the assorted plasma of a large number of healthy donors exhibit immunomodulatory therapeutic effects in the above diseases has not been clearly elucidated so far.

Immunoglobulin preparations currently used generally exhibit prophylactic and therapeutic effects in the above diseases when administered mainly by subcutaneous injection, intramuscular injection, or intravenous injection. In the case of a monoclonal antibody against a specific protein involved in the immune response, when injected into a diseased mammal, it specifically inhibits the activation of a specific immune pathway that the protein is involved in, thereby showing an immunomodulatory effect. is known Some researchers have found that polyvalent immunoglobulin preparations isolated from the blood of many healthy donors contain naturally occurring anti-idiotypes that react with antigen-binding sites of pathogenic antibodies that contribute to disease development. ) When antibodies are present and administered to patients, they react with immunoglobulin E (IgE) for allergens or immunoglobulin G (IgG) for autoantigens, which are pathological antibodies that play a key role in the development of disease, A hypothesis of passive anti-idiotype therapy has been proposed for the possibility of improving disease by inhibiting the function of pathological immune antibodies by forming an antibody immune complex.

The present inventor artificially induces oxidation of immunoglobulin protein to induce active anti-idiotype immunotherapy effect using immunoglobulin protein as an antigen, unlike the conventional hypothesis of passive anti-idiotype therapy. , the hypothesis of active anti-idiotype therapy was established and the present invention was devised. Currently, in the art, efforts are being made to block the oxidation of the immunoglobulin protein as much as possible in the production process of immunoglobulin therapeutics (especially monoclonal antibody therapeutics). The production of anti-idiotype antibodies (ie, anti-drug antibodies) to the antigen-reactive site is increased, thereby inhibiting the action of the therapeutic antibody, and through the formation of polymers, protein aggregation occurs, resulting in protein This is because, along with the loss, the risk of side effects, including inflammatory reactions due to complement activation, increases in patients receiving aggregated immunoglobulin.

Through the present invention, the present inventors have found that immunoglobulin preparations currently used for the treatment of diseases related to immune dysregulation in humans have an effect of blocking a specific immune response pathway or multivalent immunoglobulin using a targeted monoclonal antibody. In addition to passive immunotherapy, which induces the effect of blocking a number of pathological antibodies using It was intended to induce a response (active anti-idiotype immunotherapy). Accordingly, the present inventor proposes a novel pharmaceutical composition for immunomodulatory therapy that can prevent or treat diseases related to immunomodulatory dysfunction more effectively than existing immunoglobulin therapeutics using oxidized immunoglobulin in the present invention and a new treatment method using the same did.

Republic of Korea Patent Publication No. 10-2021-0122185 (published on Oct. 08, 2021)

The present invention provides a new pharmaceutical composition for immunomodulatory treatment that exhibits a superior immunomodulatory effect than conventional immunoglobulin when administered to patients suffering from diseases related to immunomodulatory dysfunction, and comprising the pharmaceutical composition An object of the present invention is to provide a method for preventing or treating diseases related to immunomodulatory dysfunction.

The present invention may provide a pharmaceutical composition for preventing or treating diseases related to immune dysregulation, which includes oxidized immunoglobulin as an active ingredient.

The oxidized immunoglobulin is prepared by mixing at least one oxidizing agent and immunoglobulin from the group consisting of an oxidizing agent such as ozone (ozone, O ₃ ), hydrogen peroxide (H ₂ O ₂ ), and sodium hypochlorite (NaClO). It may be characterized by reacting.

The oxidized immunoglobulin may be characterized by reacting by mixing 0.1 to 4 μg of ozone with respect to 1 mg of immunoglobulin.

The immunoglobulin may be any one of the group consisting of IgG, IgA, IgM, IgD and IgE.

The immunoglobulin may be characterized in that it is IgG.

The IgG may be characterized in that it is immunoglobulin G isolated from mammalian blood.

The IgG may be an IgG isolated from the mammal's own blood or an IgG isolated from the blood of other mammals.

The IgG may be characterized in that it is an IgG isolated from the culture solution of animal cells.

The immune dysregulation-related disease may be characterized in that it is any one from the group consisting of an allergic disease, a chronic inflammatory disease, an autoimmune disease, and a malignant tumor disease.

The allergic disease may be characterized in that it is any one from the group consisting of bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, urticaria, food allergy, anaphylaxis, and drug allergy.

The chronic inflammatory disease or autoimmune disease is degenerative arthritis, chronic inflammatory gastroenteritis, ankylosing spondylitis, chronic pustular dermatitis, rheumatoid arthritis, systemic lupus erythematosus, pemphigus, autoimmune thyroiditis, autoimmune hepatitis, chronic inflammatory bowel disease, autoimmune It may be characterized in that it is any one from the group consisting of nephritis, chronic inflammatory gastroenteritis, Sjogren's syndrome, scleroderma and psoriasis.

The malignant tumor disease is a solid cancer selected from the group consisting of lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, thyroid cancer, breast cancer, liver cancer, kidney cancer, prostate cancer and mesothelioma, or from the group consisting of leukemia, lymphoma and multiple myeloma. It may be characterized in that it is any one of the selected blood cancers.

The oxidized immunoglobulin may have an anticancer immunotherapeutic effect by immunomodulation through activation of cytotoxic T cells.

In addition, the present invention provides a novel pharmaceutical composition for immunomodulatory treatment, characterized in that the oxidized immunoglobulin is mixed with an adjuvant in addition to the oxidized immunoglobulin, and a method for preparing the same. The immune adjuvant included in the pharmaceutical composition is aluminum hydroxide, calcium phosphate, tyrosine, which are currently commonly used in humans for the purpose of enhancing immunity in current vaccine formulations or immunomodulatory therapeutics. , monophosphoryl lipid A (MPL), or histamine.

In addition, the present invention relates to a step of isolating immunoglobulin from the blood of a mammal or separating immunoglobulin from blood of other mammals (step 1), mixing the isolated immunoglobulin with an oxidizing agent and reacting the oxidized immunity Immune dysregulation comprising the steps of preparing globulin (step 2) and administering the oxidized immunoglobulin to an individual suffering from a disease related to immune dysregulation (step 3) ) may provide a method for preventing or treating a related disease.

The oxidizing agent may be characterized in that one selected from ozone, hydrogen peroxide, sodium hypochlorite, and the like.

The second step may be characterized in that 0.1 to 4 μg of ozone is mixed and reacted with 1 mg of the isolated immunoglobulin.

In addition, the present invention relates to a step of isolating immunoglobulin from the blood of a mammal or separating immunoglobulin from blood of other mammals (step 1), mixing the isolated immunoglobulin with an oxidizing agent and reacting the oxidized immunity It is possible to provide a method for preparing a pharmaceutical composition for preventing or treating diseases related to immune dysregulation, including the step of preparing globulin (second step).

The oxidizing agent may be characterized in that one selected from ozone, hydrogen peroxide, sodium hypochlorite, and the like.

The second step may be characterized in that 0.1 to 4 μg of ozone is mixed and reacted with 1 mg of the isolated immunoglobulin.

According to the present invention, the immunomodulatory dysfunction-related disease-related pharmaceutical composition of the present invention comprising oxidized immunoglobulin, which has artificially induced oxidation and increased immunogenicity, as an active ingredient, is administered to patients with immunomodulatory dysfunction-related diseases. When administered to a patient, it shows a significantly improved immunomodulatory therapeutic effect compared to the administration of an existing therapeutic agent containing immunoglobulin that has not been oxidized as an active ingredient, so that it has excellent preventive and therapeutic effects on diseases related to immunomodulatory dysfunction. compositions may be provided.

1 is autologous total immunoglobulin G (IgG) oxidized by reacting with ozone in patients with advanced solid cancer with distant metastases through Kaplan-Meier analysis, and autologous total immunoglobulin G (IgG) without oxidation treatment and intramuscular injection therapy. ) The graph is the result of comparing and analyzing the survival period and cumulative survival rate of the intramuscular injection treatment group and the survival rates at 1 month, 3 months, and 6 months after starting the above treatment in clinical trial subjects.
FIG. 2 shows peripheral blood CD8+ T cells at the start of the first cycle (week 0) and 4 weeks after the end of treatment (week 8) in 2 patients with advanced solid cancer who received intramuscular injection of oxidized autologous total IgG. This is the result of analyzing the change in the percentage (%) of IFN-γ-producing cells by flow cytometry.
FIG. 3 shows the results of confirming the oxidation of immunoglobulin by mixing it with ozone and sodium hypochlorite, and then confirming whether the immunoglobulin is oxidized by immunoblot method using an anti-DNP antibody.
4 is a result of confirming the oxidation of immunoglobulin using an anti-DNP antibody by immunoblot method after oxidizing commercialized human immunoglobulin for intramuscular injection and human immunoglobulin for intravenous injection with ozone.
Figure 5 shows the immune regulation in the case of intramuscular injection of IgG prepared by separating from the blood of a large number of commercially available healthy donors from one healthy normal person and intramuscular injection of IgG oxidized by mixing the above-mentioned IgG with ozone. The design of the clinical trial study conducted to confirm the difference in effect is shown.
6 is a graph comparing the IL-10 concentration in the culture solution obtained after reacting human peripheral blood mononuclear cells isolated from the peripheral blood of a healthy normal person (blood donor 1) with human IgG or oxidized human IgG.
7 is a graph comparing the concentration of IL-10 in the culture solution obtained after reacting human peripheral blood mononuclear cells isolated from the peripheral blood of a healthy normal person (blood donor 2) with human IgG or oxidized human IgG.
Figure 8 shows the IL-10 concentration in the culture medium according to the change in the degree of oxidation of human IgG when human IgG oxidized under various conditions is reacted with human peripheral blood mononuclear cells isolated from the peripheral blood of a healthy normal person (blood donor 2) and cultured; It is a graph analyzing the difference between
9 is a graph comparing the concentration of IL-8 in the culture solution obtained after reacting human peripheral blood mononuclear cells isolated and cultured from the peripheral blood of a healthy normal person (blood donor 1) with human IgG or oxidized human IgG.
10 is a graph comparing the IL-8 concentration in the culture solution obtained after reacting human peripheral blood mononuclear cells isolated from the peripheral blood of a healthy normal person (blood donor 2) with human IgG or oxidized human IgG.
11 is a graph showing the concentration of IL-8 in culture according to the change in the degree of oxidation of human IgG when human IgG oxidized under various conditions is reacted with human peripheral blood mononuclear cells isolated from the peripheral blood of a healthy normal person (blood donor 2) and cultured; FIG. This is a graph comparing the difference.
12 is a graph comparing OVA-specific IgG antibody titers in serum samples of mice obtained after subcutaneous injection of human IgG or oxidized human IgG in an Ovalbumin (OVA) allergy mouse model.
13 is a graph comparing and analyzing the difference in release rate (% release) of β-hexosaminidase secretion by IgE-antigen-mediated stimulation when human IgG and oxidized human IgG are treated in rat basophilic leukemia cells.

Hereinafter, the present invention will be described in more detail.

In the embodiments of the present invention, the present inventors isolated immunoglobulin from blood from patients suffering from an immunomodulatory dysfunction-related disease, artificially induced oxidation, and administering the oxidized immunoglobulin to the patient by intramuscular injection again. It was confirmed that it showed significantly superior disease treatment effect compared to the case of intramuscular administration of its own immunoglobulin without oxidation treatment, and oxidized immunoglobulin was found to be superior to that of conventional unoxidized immunoglobulin in normal humans and experimental animals. It was confirmed that a significantly improved immunomodulatory effect was induced by comparison, and that even in immune cells cultured in laboratory conditions, oxidized immunoglobulin could produce a significantly improved immunomodulatory therapeutic effect compared to the conventional non-oxidized immunoglobulin. By confirming that, the present invention was completed.

The present invention relates to a pharmaceutical composition for preventing or treating a disease related to an abnormality of immunomodulatory function comprising oxidized immunoglobulin as a main active ingredient.

The present invention relates to a method for preventing or treating an immunomodulatory dysfunction-related disease using oxidized immunoglobulin.

As used herein, the term “composition” is intended to include not only products comprising the specified ingredients, but also any products made directly or indirectly by combining the specified ingredients.

Each active ingredient used in the composition of the present invention may be present in any one or more of the composition of the present invention, an injectable formulation in which the composition of the present invention is dissolved, or in vivo. Or it may exist in the form of a non-covalently bound complex.

The composition of the present invention includes a composition in which one active ingredient is in a pharmaceutically or physiologically acceptable form, a composition in which all active ingredients are in the form of a pharmaceutically or physiologically acceptable salt, and one or more active ingredients are pharmaceutically or physiologically acceptable It may include a composition in the form of a salt, wherein the other active ingredients are in the form of a free base, or a composition in which a complex of one or more active ingredients is in the form of a pharmaceutically or physiologically acceptable salt.

The active ingredients may be intimately mixed with various types of pharmaceutically acceptable carriers depending on the type of preparation required for administration. The pharmaceutical composition of the present invention may preferably be in the form of a unit dosage, and it may be possible to dilute it so that it can be used by adjusting the dosage according to the judgment of the doctor.

"Immunoglobulin" of the present invention refers to a glycoprotein that plays an important role in immunity among serum components and can be defined by common characteristics such as specific physical, structural, and amino acid sequences that act as antibodies. The basic structure of immunoglobulin is that one pair of L chains (light chains) with a molecular weight of about 23,000 and a pair of H chains (heavy chains) with molecular weights of about 50,000 to 70,000 are connected by an S-S bond, and the types of H chains γ, α, It is classified into IgG, IgA, IgM, IgD, and IgE by μ, δ, and ε, respectively. The immunoglobulin used in the composition of the present invention may be IgG, IgA, IgM, IgD, IgE, or a mixture thereof, and may be a fragment thereof or a mixture thereof having a biologically equivalent activity.

In the present invention, the immunoglobulin may be whole immunoglobulin including IgG, IgA, IgM, IgD and IgE isolated from mammalian blood. Specifically, the immunoglobulin may be IgG isolated from the blood of a mammal suffering from an immunomodulatory dysfunction-related disease.

The immunoglobulin used in the composition, prophylaxis or treatment method of the present invention can be isolated using, for example, the following method. In order to separate whole immunoglobulins from mammalian blood or plasma, various methods including ethanol precipitation method, ion exchange resin adsorption chromatography method, or adsorption chromatography method using Protein A or Protein G column commonly used in the art can be separated. On the other hand, according to a method known in the art, after obtaining information on immunoglobulin from a cDNA library containing genetic information about antibody protein obtained from monocytes in peripheral blood of a mammal, cultured animal cells genetically engineered based on this information are used. It is also possible to use the one prepared by The recombinant immunoglobulin prepared in this way may include a genetically engineered recombinant immunoglobulin protein obtained by oxidizing human immunoglobulin by partially changing the amino acid sequence of mammalian immunoglobulin or the same. In addition, the immunoglobulin may be a part of an immunoglobulin that binds to an allergen, such as F(ab)′2 or a Fab fragment capable of reacting with an allergen among immunoglobulin proteins.

In addition, the immunoglobulin used in the composition of the present invention may be obtained from an animal of a species different from the mammal to which the composition of the present invention is to be administered. In particular, since immunoglobulins have high homology between different species, it is already known in the art that immunoglobulins obtained from one type of mammal can exhibit similar pharmacological effects even when administered to other types of mammals including humans. Therefore, the preventive or therapeutic effect of immune dysfunction disease by administering the composition of the present invention is obtained from a mammal of a different species from the animal to which the pharmaceutical composition of the present invention is finally administered for the purpose of suppressing the immunomodulatory dysfunction. In this case, the same may work.

As used herein, the term “mammal” refers to a mammal that is the subject of treatment, observation or experiment, and may preferably mean a human.

In the present invention, when the oxidized immunoglobulin is administered to a mammal, the oxidized immunoglobulin itself is used as an antigen, and anti-idiotypic antibodies that react with the antigen-binding site of a pathological immune antibody are naturally generated to form an antigen-antibody immune complex. It may mean an antibody that can do it.

In the present invention, the immunomodulatory dysfunction-related disease may be an allergic disease, a chronic inflammatory disease, an autoimmune disease, or a malignant tumor disease, but is not limited thereto.

The allergic disease is selected from the group consisting of bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, urticaria, food allergy, anaphylaxis and drug allergy, which are known to be caused by an allergic reaction to antigens present in the external environment. may be, but is not limited thereto.

The chronic inflammatory disease or autoimmune disease is degenerative arthritis, chronic inflammatory gastroenteritis, ankylosing spondylitis, chronic pustular dermatitis, rheumatoid arthritis, systemic lupus erythematosus, pemphigus, autoimmune thyroiditis, autoimmune hepatitis, chronic inflammatory bowel disease, autoimmune It may be selected from the group consisting of nephritis, chronic inflammatory gastroenteritis, Sjogren's syndrome, scleroderma, and psoriasis, but is not limited thereto.

The malignant tumor disease is a solid cancer selected from the group consisting of lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, thyroid cancer, breast cancer, liver cancer, kidney cancer, prostate cancer and mesothelioma, or from the group consisting of leukemia, lymphoma and multiple myeloma. It may be selected from selected blood cancers, but is not limited thereto.

The present invention provides a novel pharmaceutical composition for immunomodulatory therapy, characterized in that the oxidized immunoglobulin is mixed with an adjuvant, and a method for preparing the same.

The adjuvant included in the pharmaceutical composition is aluminum hydroxide, calcium phosphate, tyrosine, which are currently commonly used in humans for the purpose of enhancing immunity in current vaccine formulations or immunomodulatory therapeutics. monophosphoryl lipid A (MPL), or histamine.

The present invention can provide a pharmaceutical composition for the prevention or treatment of allergic diseases, chronic inflammatory diseases, autoimmune diseases, or malignant tumor diseases comprising oxidized immunoglobulin as an active ingredient.

In addition, the present invention provides a step of isolating immunoglobulin from the blood of a mammal or other mammals (step 1), mixing the isolated immunoglobulin with an oxidizing agent and reacting it to prepare oxidized immunoglobulin (step 2) ) and providing a method for preventing or treating a disease related to immune dysregulation, comprising administering the oxidized immunoglobulin to an individual suffering from an immune dysregulation-related disease (step 3) can do.

In the present invention, the oxidized immunoglobulin is prepared by reacting a mixture of immunoglobulin and an oxidizing agent, and the oxidizing agent is ozone (O ₃ ), which is known to generate oxygen free radicals or reactive oxygen species. ), hydrogen peroxide (H ₂ O ₂ ), and sodium hypochlorite (NaClO) may be selected from the group consisting of, but is not limited thereto.

According to an embodiment of the present invention, the oxidized immunoglobulin may be prepared by reacting liquid immunoglobulin with liquid hydrogen peroxide or sodium hypochlorite, or by mixing gaseous ozone gas. The reaction may be carried out for 1 minute to 24 hours at a temperature condition of 4 to 95 °C.

At this time, the amount of the ozone gas used may be 0.01 to 10 μg with respect to 1 mg of human IgG (hIgG), and specifically, 0.1 to 4 μg may be used.

The pharmaceutical composition of the present invention is preferably for subcutaneous injection. However, in an embodiment of the present invention, the composition is administered in a conventional manner also via intravenous, intraarterial, intramuscular, intraperitoneal, intrasternal, transdermal, intranasal, inhalational, topical, rectal, oral, intraocular or intradermal routes. can be administered.

The dosage of the pharmaceutical composition of the present invention may be determined in consideration of the dosage of the immunoglobulin used in treatment using the currently used immunoglobulin formulation. In the case of a general pharmaceutical composition, the dosage of the composition is determined according to the severity of symptoms, the age, weight, etc. of the patient, but in the treatment method of the present invention, it is determined according to the patient's sensitivity to oxidized immunoglobulin as well as the above-mentioned conditions. can

When the pharmaceutical composition of the present invention is administered once, the dosage of the oxidized immunoglobulin may be 0.001 to 1000 mg, specifically, 10 to 50 mg, but is not limited thereto. The composition may preferably be in the form of a solution or lyophilized powder, and may be used in a form contained in 0.5 to 2 ml of an injection buffer at one time of administration.

In addition, aluminum hydroxide, calcium phosphate, tyrosine, monophos, which are commonly used to increase immune response with immunoglobulin so that it can be used by dissolving it in an injection buffer contained in a separate vial. It may be formulated and provided in a mixed form with an immune adjuvant or histamine including monophosphoryl lipid A (MPL), etc. .

Injection buffers and other adjuvant components used to formulate the composition of the present invention into injectable preparations are known in the art. The injectable formulation for the composition of the present invention may contain other adjuvants such as, for example, a solubilizing agent, a pH adjusting agent, and a suspending agent in addition to the injection buffer. For example, the injection buffer may be physiological saline or the like.

It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration for the active ingredients of the present invention or a pharmaceutical composition comprising them will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined and will vary depending on the particular active ingredient used, the mode of administration, the effect of the agent, and the development of the disease state. In addition, it will be necessary to adjust the dosage according to the appropriate therapeutic level according to the factors of each patient to be treated, including the age, weight, diet, and time of administration of the patient.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example>

The inventors provide a method for preparing oxidized immunoglobulin through various examples below, and the oxidized immunoglobulin through immune cell culture experiments, animal experiments, and human clinical trials has superior immunity compared to conventional immunoglobulins. It has been demonstrated that the modulating effect and excellent immunomodulatory therapeutic effect can be exhibited for diseases with abnormal immunomodulatory function including allergic diseases, chronic inflammatory diseases, autoimmune diseases and malignant tumor diseases.

<Example 1> Clinical trial on the anticancer immunotherapeutic effect of oxidized immunoglobulin in patients with advanced solid cancer

The present inventors have reported that patients with advanced solid cancer with a life expectancy of less than 6 months after receiving approval for clinical trials from the institutional ethics committee, more specifically, metastasis of cancer to distant sites where the disease is not controlled by existing standard anticancer treatments Conducted a clinical trial to verify the anticancer immunotherapeutic effect and safety of 'autologous total IgG intramuscular injection therapy' for patients with solid cancer such as stage 4 lung cancer, colorectal cancer, ovarian cancer, mesothelioma, breast cancer, stomach cancer, or prostate cancer did.

Before starting treatment, 400 ml of venous blood was collected from the patients participating in the clinical trial using a double blood donation bag containing an anticoagulant, and then 200 ml of the patient's own plasma was separated by centrifugation. One that has a rest period of 4 weeks after administering intramuscular injection 8 times for 4 weeks at 50 mg per time of liquid autologous IgG obtained by separating the plasma from the plasma in an aseptic and nontoxic state by adsorption chromatography using Protein A beads 1 treatment cycle.

At the end of the first treatment cycle, the patient's clinical condition is checked, and if the patient's general health has not deteriorated to the extent that the clinical trial cannot be continued, and the patient agrees to continue treatment, the first treatment cycle Repeat clinical trial treatment for up to 4 cycles and observe the clinical status of solid cancer through examination findings, X-ray examination, computed tomography (CT), magnetic resonance imaging (MRI) examination, isotope examination, and blood test, etc. did.

Among the above patients, four patients who participated in the initial clinical trial administered the isolated autologous total IgG by intramuscular injection without oxidation treatment, but all four patients who participated in the initial clinical trial did not experience clinically significant therapeutic effects. He died within 6 months of initiation of study participation in a state where he failed to try the targeted 4 cycles of treatment.

Therefore, the inventor decided that it was necessary to modify the immunoglobulin, the material of immunotherapy, to have stronger antigenicity (immunogenicity) in order to obtain more effective anticancer immunotherapy than before for malignant tumor patients newly participating in clinical trials. .

Accordingly, the inventor devised the present invention to increase the effect of active anti-idiotype immunotherapy by artificially inducing oxidation of immunoglobulin protein as a method to increase the immunogenicity of immunoglobulin protein to increase the effect of active anti-idiotype immunotherapy. did. Meanwhile, in the current art, efforts are being made to block the oxidation of the immunoglobulin protein as much as possible in the production process of immunoglobulin therapeutics (especially monoclonal antibody therapeutics). The production of anti-idiotypic antibodies (ie, anti-drug antibodies) to the antigen-reactive site is increased, thereby inhibiting the action of the therapeutic antibody, and protein aggregation occurs through the formation of polymers. This is because the risk of side effects including loss and inflammatory response due to complement activation increases.

Therefore, from the 4 advanced solid cancer patients who participated in the initial clinical trial to 8 advanced solid cancer patients who newly participated in the clinical trial after the clinical trial, 50 mg of autologous total IgG in liquid state and high pressure pure oxygen for medical use were connected to an ozone gas generator. 200 μg of the obtained gaseous ozone gas was mixed in a syringe (that is, 4 micrograms of ozone gas per 1 mg of IgG was mixed) and reacted for 5 minutes. . In 8 solid cancer patients who received oxidized autologous IgG intramuscular injection, intramuscular oxidized autologous IgG injection was administered at the same injection dose, injection interval, and number of injections as in the 4 advanced solid cancer patients who participated in the initial clinical trial.

1-1. Analysis of survival period of patients with advanced solid cancer

As a result of the above clinical study, 8 patients with oxidized autologous IgG received intramuscular injection of oxidized autologous IgG, and 4 patients with autologous IgG received without oxidation treatment. group), it was confirmed that it showed statistically significantly higher survival period, 3-month survival rate, and 6-month survival rate (Table 1, FIG. 1). In addition, no severe adverse events related to the clinical study were observed during the study period in all 12 patients with advanced solid cancer who participated in the clinical study.

Number of patients participating in the clinical trial study (n) Median overall survival, Weeks (95% Ci) Number of survivors/survival rate (%) at 1 month Number of survivors/survival rate (%) at 3 months Number of survivors/survival rate (%) at 6 months Autologous IgG intramuscular injection group 4 14. 2 (5. 8, 22.5) 2/50% 1/25% 0/0 % Oxidized autologous IgG intramuscular injection group 8 33. 0 (28.5, 37.4)* 0/100 % 0/100 %** 7/86 %**

Kaplan-Meier analysis, *P<0.005; Fisher's exact test, **P<0.05

Table 1 above shows the results of analysis of the number of deaths and survival rates according to whether autologous total IgG oxidized or not administered intramuscularly to advanced solid cancer patients with distant metastasis. The oxidized autologous IgG used in FIGS. 1 and 1 was oxidized by reacting with ozone, and as a result of analyzing the number of deaths and survival rates at 1 month, 3 months, and 6 months after the start of autologous total IgG intramuscular injection, autologous IgG intramuscular injection It can be seen that the survival rate and survival period were statistically significantly increased in the patients who received intramuscular oxidized IgG compared to those who received the treatment.

1-2. Analysis of changes in the percentage of interferon-gamma (IFN-γ) producing cell fraction among peripheral blood CD8+ T cells before and after treatment in patients with advanced solid cancer who received oxidized autologous total IgG intramuscular injection therapy

In order to observe the changes in peripheral blood T cells before and after treatment from 8 patients with advanced solid cancer who received intramuscular oxidized autologous total IgG treatment, who participated in the above clinical trial study, peripheral blood mononuclear cells were isolated and flow cytometry analysis was performed. carried out. In a total of 5 patients, except for 3 patients who were unable to collect venous blood for flow cytomerty analysis due to deterioration of their systemic condition after participating in the clinical trial study, 1 treatment cycle immediately before the start (week 0) and at the end of the treatment cycle (week 8) Venous blood was collected and flow cytometry analysis was performed.

Patients. No Interferon-gamma (IFN-γ) cells in CD8+ T cells (%) Baseline (week 0) Week 8 5 9.3 14.7 6 7.6 16.4 9 5.2 4.6 10 50 47.7 11 32.4 36.3

Table 2 shows that in 5 patients with advanced solid cancer who received intramuscular injections of autologous whole IgG oxidized by reaction with ozone, immediately before the start of treatment (week 0), 4 weeks, and 4 weeks after the end of 8 intramuscular injections of oxidized IgG (i.e., Data showing the fraction (%) of IFN-γ-producing cells among peripheral blood CD8+ T cells isolated from blood collected at 8 weeks). Referring to Table 2, the fraction (%) of IFN-γ-producing T cells, which is known to play a role in killing cancer cells, among total peripheral blood CD8+ T cells (cytotoxic T cells) in 3 of the 5 patients. ) was observed to increase. In addition, IFN-γ-producing cells among peripheral blood CD8+ T cells at the end of cycle 1 treatment (week 8) compared to baseline (week 0) among 5 patients receiving intramuscular injection of oxidized autologous total IgG. Two patients (No. 5 and No. 6 in Table 2) who showed an increase in the fraction (%) of Clinically significant in that the clinical course and general condition of the patient is very good, and the total tumor size and the number of metastasized lesions on imaging tests (CT and MRI scans) do not increase compared to before the start of treatment (stable disease state) One antitumor treatment effect (25% clinical response rate in 2 out of 8 patients) and a stable disease state in which such tumors do not progress further can be maintained continuously for the entire observation period, so that a total of 4 cycles of repeated treatment can be performed. there was.

2 is a flow cytometry analysis result of the percentage (%) of the IFN-γ-producing cell fraction among peripheral blood CD8+ T cells of two patients with advanced solid cancer who received intramuscular injection of oxidized autologous whole IgG. The above results were analyzed with blood collected immediately before the start of intramuscular injection of oxidized autologous IgG (baseline, 0 weeks) and after 1 treatment cycle (8 weeks).

Referring to Table 2 and FIG. 2, in the case of 2 patients (No. 5, No. 6) who could repeatedly administer the oxidized autologous IgG intramuscular injection treatment, intramuscular injection of oxidized autologous IgG 1 Cytotoxic T cells (CD8+) in which the proportion (%) of the IFN-γ-producing cell fraction among CD8+ T cells is increased by more than 50% at the end of the treatment cycle (week 8) compared to the time at the start of treatment (week 0) T cells) can be confirmed to show significant activation. This suggests that intramuscular injection of oxidized autologous whole IgG showed anticancer immunotherapeutic effect through the activation of IFN-γ secreting CD8+ T cells (ie, cytotoxic T cells) in advanced solid cancer patients.

Therefore, in the case of pembrolizumab (trade name, Keytruda, Merk, USA), an anti-PD1 monoclonal antibody treatment for anti-cancer immunotherapy, which is the most widely used in patients with advanced solid cancer worldwide, the size of the tumor in patients with various types of advanced solid cancer is reduced. The results reported that the clinical response rate was about 10%-30%, maintaining a stable disease that does not further increase or significantly reducing the size of the tumor (Chang E). , et al. The Oncologist 2021;26:e1786-e1799), oxidized autologous total IgG observed in Example 1 of the present invention In 8 advanced solid cancer patients who received intramuscular injection, the control group, non-oxidized autologous The fact that the 'pharmaceutical composition comprising oxidized immunoglobulin as an active ingredient' of the present invention showed a significantly longer survival period and significantly higher 3-month and 6-month survival rates compared to the 4 patients who received total IgG intramuscular injection treatment. It can be seen that it has clinical utility as an anticancer immunotherapy in patients with advanced solid cancer. Also, in the case of the existing Anti-PD1 antibody therapy, it is known that a significant number of patients who have been treated develop autoimmune diseases due to systemic side effects. However, in the case of intramuscular injection of oxidized immunoglobulin of the present invention, no systemic side effects were observed in all of the treated patients. Therefore, in patients with advanced solid cancer, the treatment using the pharmaceutical composition of the present invention is safer in terms of systemic side effects as well as a similar degree of anti-tumor treatment effect (clinical response rate) compared to the conventional anti-cancer immunotherapy using an Anti-PD1 antibody. It is considered to be a new anticancer immunotherapy with the advantage of

<Example 2> Preparation method 1 of a pharmaceutical composition containing oxidized immunoglobulin as a main component

Ozone (O ₃ ) and sodium hypochlorite (NaClO), which generate oxygen free radicals, which are the most used methods for oxidizing proteins, react with IgG to induce oxidation of proteins, and determine whether The degree of oxidation was confirmed by immunoblot. Intramuscular injection (Gamma globulin, Green Cross, Korea) or intravenous immunoglobulin (10% ivy globulin SN, Green Cross, Korea) isolated from the blood of many commercially available health donors, respectively, was prepared using ozone gas or liquid hypoglycemia, respectively. It was reacted with sodium chlorate at room temperature for 5 minutes. After the reaction, in order to detect whether immunoglobulin is oxidized, 2,4-dinitrophenylhydrazine (DNPH) reacts with the carbonyl group generated in the oxidized protein to chemically change to dinitrophenylhydrazone (DNP). An experiment to detect oxidized protein using an antibody against DNP using properties was performed by SDS-PAGE and immunoblot using Oxyblot ^TM Protein Oxidation Detection Kit (Millipore, Billerica, MA, USA).

2-1. Analysis of experimental materials

Immunoglobulin injections for intramuscular injection (Gammaglobulin, Green Cross, Korea) isolated from the blood of a number of health donors on the market are described in the manufacturer's drug manual as containing 165 mg/mL of human IgG, and the preparation is a solution. It is supplied in the form of a glass vial for injection.

In order to reconfirm the immunoglobulin component contained in the drug, intramuscular injection human immunoglobulin (Gammaglobulin, Green Cross, Korea) was diluted to 10 mg/mL with distilled water, and then IgG, IgM, IgA and albumin in the diluted solution were diluted. As a result of quantifying the concentration of nephelometry using a nephelometry measuring device (Cobas 8000 series, Roche Diagnostics, Mannheim, Germany), it was determined that 9.41 mg/mL of IgG was included, and IgA, IgM and albumin were measured by the measuring method using the device. It was not detected because it was present in concentrations lower than the minimum possible limit (IgA < 20 mg/dL, IgM < 13 mg/dL, albumin < 100 mg/dL). In addition, the concentration of IgE measured by a fluorescent enzyme immunoassay using the ImmunoCAP assay (Phadia US, Portage, MI, USA) system was measured to be 2.54 kU/L.

2-2. Validation of protein oxidation induction using commercialized human immunoglobulin for intramuscular injection

Figure 3 shows the results of confirming the oxidation of immunoglobulin using an anti-DNP antibody by immunoblot method after oxidizing commercialized human immunoglobulin for intramuscular injection with ozone and sodium hypochlorite. 1 mg of the immunoglobulin G was oxidized at room temperature for 5 minutes according to the following conditions, and during electrophoresis, 4 μg of IgG was added per lane. The conditions of each lane are: lane 1 is molecular weight marker, lane 2 is non-oxidized commercialized intramuscular human immunoglobulin G (hIgG), lane 3 is hIgG treated with 0.25 μg of ozone per 1 mg of IgG, lane 4 is IgG hIgG treated with ozone at 0.5 μg per 1 mg, lane 5 is hIgG treated with ozone at 1 μg per 1 mg of IgG, lane 6 is hIgG treated with ozone at 2 μg per 1 mg of IgG, lane 7 is 1 mg of IgG hIgG treated with 4 μg of ozone per sugar, lane 8 is hIgG treated with 8 μg of ozone per 1 mg of IgG, lane 9 is hIgG treated by reacting with 275 μg of liquid sodium hypochlorite per 1 mg of IgG for 5 minutes .

4 shows the results of confirming whether or not the immunoglobulin was oxidized by the immunoblot method using an anti-DNP antibody after oxidizing commercialized human immunoglobulin for intramuscular injection and human immunoglobulin for intravenous injection with ozone. 1 mg of immunoglobulin G was mixed with ozone for 5 minutes at room temperature and oxidized according to the following conditions, and 4 μg of IgG was added per lane during electrophoresis. The conditions of each lane are: lane 1 is molecular weight marker, lane 2 is non-oxidized commercial human immunoglobulin G for intramuscular injection, lane 3 is non-oxidized commercial human immunoglobulin G for intravenous injection, lane 4 is IgG per 1 mg Intramuscular injection human immunoglobulin G oxidized by reaction with 5 μg ozone, lane 5 is intravenous human immunoglobulin G oxidized by reaction with 5 μg ozone per 1 mg of IgG.

Referring to FIGS. 3 and 4 , it was confirmed that significant oxidation of immunoglobulins could be induced by both ozone and sodium hypochlorite, which are known to generate oxygen free radicals. In addition, it was confirmed that as the amount of treated oxygen free radicals increased, the oxidation of immunoglobulin was increased in a dose-dependent manner. As a result of the above results, it was confirmed that the commercially available immunoglobulin G formulation for intramuscular or intravenous injection uses non-oxidized immunoglobulin as a raw material and can effectively oxidize immunoglobulin when reacted with ozone or sodium hypochlorite. can

<Example 3> Method 2 of preparing a pharmaceutical composition containing oxidized immunoglobulin as a main component

In order to find an appropriate oxidizing condition that can minimize protein loss due to aggregation and precipitation caused by multimer formation according to the oxidation of immunoglobulin, the immunoglobulin is oxidized under various oxidizing conditions, and the oxidized immunoglobulin is The solution was centrifuged to quantify the protein concentration in the supernatant, and the amount of protein loss was calculated using <Equation 1> below. The experiment was carried out. Commercially available human IgG for intramuscular injection (gamma globulin, Green Cross, Korea) was diluted to 10 mg/ml with distilled water, mixed with ozone gas of various concentrations, and reacted at room temperature for 5 minutes. After the reaction, centrifugation was performed at 13,000 g for 15 minutes, and the concentration of immunoglobulin in the supernatant was measured using Bradford assay reagent (Biorad, USA). For reference, in these examples, oxidized IgG was mixed with liquid IgG and gaseous ozone in a syringe, reacted for 5 minutes, and then the gas was removed from the syringe. The experiment was carried out by performing 4 repetitions (quadruplicate) per ozone treatment condition, and the results were expressed as mean ± standard deviation, and are shown in [Table 3] below.

<Equation 1>

Loss ratio (%) of IgG protein = (concentration of unoxidized IgG protein - concentration of IgG protein present in the centrifuged supernatant after oxidation treatment)/concentration of unoxidized IgG protein x 100

Amount of ozone reacted with 1 mg of human IgG for 5 minutes IgG concentration in centrifugation supernatant after oxidation (mg/mL) Loss of IgG protein due to oxidation (%) 0 μg 10.9 ± 0.1 - 0.5 μg 10.8 ± 0.03 0.9 ± 0.9 1 μg 10.4 ± 0.1 4.4 ± 1.4 2 μg 10.2 ± 0.1 5.9 ± 1.9 4 μg 10.1 ± 0.2 7.6 ± 2.1 8 μg 9.0 ± 0.2 18.2 ± 0.3 16 μg 7.9 ± 0.1 27.1 ± 1.1

Data are presented as mean± standard deviation from quadruplicate experiments

Table 3 shows the results of calculating the IgG protein loss rate due to aggregation and precipitation of oxidized IgG by mixing an intramuscular injection solution of human IgG prepared by separating from the blood of many commercially available healthy donors with ozone gas. Referring to Table 3, as shown in the experimental results, it was confirmed that the more oxidation was induced (that is, the more the amount of reacted oxygen radicals increased), the more the immunoglobulins present in the solution were aggregated and precipitated. That is, it can be confirmed that there is a dose-dependent effect between the degree of oxidation and the aggregation and precipitation of immunoglobulins.

Therefore, it was confirmed that it is reasonable to properly oxidize immunoglobulin under conditions that induce oxidation of immunoglobulin but minimize loss due to aggregation of immunoglobulin to prepare a pharmaceutical composition for immunomodulatory treatment. The condition may be that the immunoglobulin loss rate is less than 10% or the amount of ozone treated per 1 mg of human IgG is 0.1 to 4 μg.

<Example 4> Whether oxidized immunoglobulin can exhibit an immunomodulatory effect when administered intramuscularly in a normal person was confirmed by a clinical test

A clinical trial confirmed whether oxidized immunoglobulin can exhibit a superior immunotherapeutic effect in healthy normal people compared to untreated immunoglobulin protein that has not been oxidized. A commercially available, non-oxidized, liquid, commercially available human IgG preparation (gamma globulin, Green Cross, Korea) was administered intramuscularly by intramuscular injection to one healthy adult 8 times for 4 weeks, followed by a rest period of 4 weeks. After a resting period, 50 mg of a commercially available human IgG preparation for intramuscular injection in liquid state was mixed with 400 µg of ozone in a syringe immediately before intramuscular injection (ie, 8 µg of ozone per 1 mg of human IgG) and reacted for 5 minutes, followed by a syringe In this study, intramuscular injection of oxidized IgG in the liquid phase from which ozone gas was removed was administered 8 times for 4 weeks, followed by a rest period of 4 weeks. The clinical study design for these treatments is shown in FIG. 5 . In addition, the serum obtained after collecting venous blood every 4 weeks from the time immediately before intramuscular injection of human IgG to the time point of 16 weeks for the first time was stored frozen at -20°C and then thawed again at room temperature to obtain Interleukin-10 (IL-10) in the serum. ) and IFN-γ concentrations were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) measuring kit (BD PharMingen, San Diego, CA, USA), and the results are shown in Table 4.

point of view Interleukin-10 concentration in serum (pg/mL) Interferon-gamma concentration in serum (pg/mL) Week 0 (baseline) 15.5 Undetectable (<4 pg/mL) 4 weeks 19.0 Undetectable (<4 pg/mL) 8 weeks 18.3 Undetectable (<4 pg/mL) 12 weeks 18.9 Undetectable (<4 pg/mL) 16 weeks 28.9 15.0

Table 4 shows the results of clinical trials confirmed by measuring the serum IL-10 and IFN-γ concentrations in order to compare the immunomodulatory effects of intramuscular injection of human IgG and oxidized human IgG in one healthy normal person.

5 shows the design of a clinical trial study conducted to confirm the difference in immunomodulatory effect between the intramuscular injection of commercially available human IgG and the intramuscular injection of oxidized human IgG in one healthy normal person. will be.

Referring to Tables 4 and 5 above, compared with the time point (8 weeks) after 50 mg of human IgG was administered intramuscularly, 50 mg of oxidized human IgG was intramuscularly injected 8 times for 4 weeks. It was found that the concentration of IL-10 and IFN-γ in the serum was significantly increased at the time point (16 weeks) elapsed. Therefore, it can be confirmed that the oxidized immunoglobulin exhibits a significantly higher immunomodulatory effect than the non-oxidized immunoglobulin even in normal persons through these clinical test results. In addition, no side effects related to the clinical trial were observed in the normal subjects during the clinical trial period.

<Example 5> Verification of immunomodulatory effect of oxidized immunoglobulin in human peripheral blood mononuclear cells

To verify the immunomodulatory effect of oxidized immunoglobulin In order to confirm the immunomodulatory efficacy of new drug candidates, a commonly used peripheral blood mononuclear cell culture model was used. Peripheral blood mononuclear cells (PBMCs) isolated from normal human venous blood by centrifugation using a density gradient are cultured, replaced with a culture medium containing immunoglobulin, and reacted, the culture medium is collected and present in the culture medium The immunomodulatory effects of oxidized and non-oxidized immunoglobulins were compared and analyzed by measuring the concentrations of cytokines, which are major mediators of the immune response.

PBMCs isolated from the venous blood of each donor by density gradient centrifugation using Cell Preparation Tubes ^TM (BD Biosciences, San Jose, CA) from 2 healthy normal adults (blood donor 1 and blood donor 2) were obtained from 10% fetal calf Serum, ₂ mM glutamate, 50 μg/ml penicillin strptomycin, and 10 mM HEPES in 1 ml of RPMI culture medium to make PBMCs 5 × 10 ⁶ cells. incubated in After culturing, after centrifuging the culture plate, remove the existing culture medium and treat only 1 ml of culture solution per well (negative control; Media only), use 1 ml of culture solution containing 80 μg of human IgG (human IgG; hIgG). When treated with 1 ml of a culture solution containing 80 μg of hIgG in which 1 μg of ozone per 1 mg of hIgG was reacted for 5 minutes (O ₃ -hIgG), 80 μg of human serum albumin (HSA) was added. When treated with 1 ml of culture solution, when treated with 1 ml of culture solution containing 80 μg of HSA reacted with 1 μg of ozone per 1 mg of HSA for 5 minutes (O ₃ -HSA), 275 μg of sodium hypochlorite per 1 mg of hIgG ( NaClO) was reacted for 5 minutes with 1 ml of a culture solution containing 80 μg of hIgG, 275 μg per 1 mg of HSA was reacted with sodium hypochlorite for 5 minutes and oxidized with 1 ml of a culture solution containing 80 μg of HSA ( NaClO-hIgG), as a positive control experiment, in the case of treatment with 1 ml of a culture solution containing 10 ng of Lipopolysaccharide (LPS, Sigma-aldrich Co., USA). After incubation again in a CO ₂ incubator at 37 ° C for 24 hours. After centrifuging the culture plate, the culture supernatant was collected. In the above experiment, the experiment was performed by performing quadruplicate 4 times for each condition. The supernatant obtained in the above experiment was aliquoted and stored frozen, thawed at room temperature, and the concentrations of IL-10 and IL-8 cytokines in the supernatant were measured using a cytokine ELISA set (BD PharMingen, San Diego, CA, USA). was used and measured according to the manufacturer's recommended method.

5-1. Confirmation of the effect of inducing increase in IL-10 concentration in culture medium by oxidized immunoglobulin in peripheral blood mononuclear cell culture laboratory model

Conditions Stimulants contained in 1ml of culture media/well Concentration of IL-10 (pg/mL) (mean±SEM) *Significant increase compared to media only (p<0.05) **p-value compared to condition 2 One Media only 11.4 ± 0.6 2 hIgG 80 μg 11.2 ± 0.4 - 3 O ₃ treated (1 ㎍ O ₃ / 1mg hIgG) hIgG 80 ㎍ 14.4 ± 0.3 * 0.001 4 HSA 80 μg 6.4 ± 0.2 - 5 O ₃ treated (1 μg O ₃ / 1 mg HSA) HSA 80 μg 7.0 ± 0.6 - 6 NaClO treated (275 μg NaClO / 1 mg hIgG) hIgG 80 μg 16.6 ± 0.5 * <0.001 7 NaClO treated (275 μg NaClO / 1 mg HSA) HSA 80 μg 5.3 ± 0.4 - 8 LPS 10 ng 108.8 ± 4.9 * <0.001

Interleukin-10 (IL-10); human immunoglobulin G (hIgG); Ozone (O ₃ ); Human serum albumin (HSA); sodium hypochlorite (NaClO); Lipopolysaccharide (LPS); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. * A statistically significant difference is considered when the p value is less than 0.05 when compared with the Student t-test (independent sample test). **p-value is when only the hIgG-treated groups were compared, the p-value was less than 0.05 when comparing the measured values of the hIgG group without oxidation (condition 2) and the Student t-test (independent sample test). cases were considered to show a statistically significant difference.

Table 5 above compares the concentration of IL-10 in the culture solution collected after reacting human peripheral blood mononuclear cells isolated and cultured from the peripheral blood of a healthy normal person (blood donor 1) with human IgG or oxidized human IgG. It is schematically shown in FIG. 5 .

Referring to Table 5 and Figure 6, as a result of an experiment using venous blood of a healthy normal person (blood donor 1), immune cells (peripheral blood mononuclear cells; PBMCs) isolated from the peripheral blood of a healthy normal person (blood donor 1) When reacted with human IgG oxidized with ozone or sodium hypochlorite (oxidized hIgG), the culture supernatant was compared to the case where only the negative control culture was treated (media only) or when reacted with human IgG without oxidation treatment. It can be seen that the concentration of IL-10 is significantly high (p<0.05). On the other hand, there was no significant increase in the IL-10 concentration of the culture supernatant in the condition treated with non-oxidized hIgG or oxidized HSA compared to the case where only the negative control culture was treated.

In addition, when reacted with human IgG oxidized with ozone or sodium hypochlorite (oxidized hIgG), it was confirmed that the IL-10 concentration of the culture supernatant was significantly higher than that of hIgG without oxidation (p<0.001). When LPS used as a positive control was treated, a significantly higher concentration of IL-10 was measured in the culture supernatant compared to other conditions (p<0.05), so this experiment using cultured human peripheral blood mononuclear cells (PBMCs) It can be verified that this has been done properly.

Conditions Stimulants contained in 1ml of culture media/well Concentration of IL-10 (pg/mL) (mean±SEM) *Significant increase compared to media only (p<0.05) **p-value compared to condition 2 One Media only 7.7 ± 0.6 2 hIgG 80 μg 7.6 ± 0.7 - 3 O ₃ treated (1 μg O ₃ / 1 mg hIgG) hIgG 80 μg 17.8 ± 0.5 * <0.001 4 HSA 80 μg 4.9 ± 0.4 - 5 O ₃ treated (1 μg O ₃ / 1 mg HSA) HSA 80 μg 3.7 ± 0.2 - 6 LPS 10 ng 455.4 ± 11.2 * <0.001

Interleukin-10 (IL-10); human immunoglobulin G (hIgG); Ozone (O ₃ ); Human serum albumin (HSA); sodium hypochlorite (NaClO); Lipopolysaccharide (LPS); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. * A statistically significant difference is considered when the p value is less than 0.05 when compared with the Student t-test (independent sample test). **p-value is when only the hIgG-treated groups were compared, the p-value was less than 0.05 when comparing the measured values of the hIgG group without oxidation (condition 2) and the Student t-test (independent sample test). cases were considered to show a statistically significant difference.

Table 6 above compares the IL-10 concentration in the culture solution collected after reacting human peripheral blood mononuclear cells isolated and cultured from the peripheral blood of another healthy normal person (blood donor 2) with IgG or oxidized IgG. It is schematically shown in FIG. 7 .

Referring to Tables 6 and 7, as a result of an experiment using peripheral blood of a healthy normal person (blood donor 2), immune cells (peripheral blood mononuclear cells; PBMCs) isolated from a healthy normal person reacted with human IgG oxidized with ozone. In the case of oxidized hIgG, the concentration of IL-10 in the supernatant of the culture was significantly higher (p< 0.05). On the other hand, there was no significant increase in the IL-10 concentration of the culture supernatant when reacted with non-oxidized human IgG compared to the case where only the negative control culture was treated.

In addition, when reacted with ozone-oxidized human IgG (oxidized hIgG), it was confirmed that the IL-10 concentration in the culture supernatant was significantly higher than that of hIgG without oxidation (p<0.001). When LPS used as a positive control was treated, it was confirmed that a significantly higher concentration of IL-10 was measured in the culture supernatant compared to other conditions (p<0.05), and the cultured human peripheral blood mononuclear cells (PBMCs) were It can be confirmed that this experiment used was properly performed.

5-2. Changes in IL-10 concentration in culture according to changes in ozone oxidizing dose of immunoglobulin in peripheral blood mononuclear cell culture laboratory model

PBMCs isolated from the venous blood of a healthy normal person (2 blood donors) were cultured in the same manner as above, and then treated with 1 mL of a culture solution containing 80 μg of hIgG in which 1 μg of ozone per 1 mg of hIgG per well was reacted with ozone for 5 minutes. In one case, in the case of treatment with 1 mL of a culture solution containing 80 μg of hIgG in which 4 μg of ozone per 1 mg of hIgG was reacted for 5 minutes, 1 mL of a culture solution containing 80 μg of hIgG in which 16 μg of ozone per 1 mg of hIgG was reacted for 5 minutes When treated with 1 mL of a culture solution containing 80 μg of HSA reacted with 1 μg of ozone per 1 mg of HSA for 5 minutes, culture solution containing 80 μg of HSA reacted with 4 μg of ozone per 1 mg of HSA for 5 minutes In the case of treatment with 1 mL, after incubation again in a CO ₂ incubator at 37 ° C. for 24 hours under the conditions of treatment with 1 mL of a culture solution containing 80 μg of HSA reacted with ozone at 16 μg per 1 mg of HSA for 5 minutes. After centrifuging the culture plate, the culture supernatant was collected. In the above experiment, the experiment was performed by performing quadruplicate each 4 times per one condition. The supernatant obtained in the above experiment was dispensed and stored frozen at -20 °C, thawed at room temperature, and the concentration of IL-10 cytokine present in the supernatant was measured using the cytokine ELISA set (BD, USA) as recommended by the manufacturer. was measured according to

Amount of O ₃ reacted with 1mg of hIgG or HSA Concentration of IL-10 (pg/mL) (mean±SEM) P value* hIgG 80 μg treatment (1mL/well) HSA 80 μg treatment (1mL/well) 1 μg 17.8 ± 0.5 3.7 ± 0.2 <0.001* 4 μg 4.5 ± 0.9 5.1 ± 1.3 0.754 16 μg 1.5 ± 0.4 1.1 ± 0.03 0.320

Interleukin-10 (IL-10); human immunoglobulin G (hIgG); Ozone (O ₃ ); Human serum albumin (HSA); Lipopolysaccharide (LPS); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. * A statistically significant difference is considered when the p value is less than 0.05 when compared with the Student t-test (independent sample test).

Table 7 above shows the concentration of IL-10 in culture according to the change in the degree of oxidation of human IgG by reacting human peripheral blood mononuclear cells isolated from the peripheral blood of a healthy normal person (blood donor 2) and cultured with human IgG oxidized under various conditions. The difference is compared, and it is shown in FIG. 8 as a graph.

Referring to Tables 7 and 8, as a result of the above experiment, between 80 μg of hIgG treated with 1 μg of ozone per 1 mg of human IgG and 80 μg of HSA reacted with 1 μg of ozone per 1 mg of HSA for 5 minutes, IL-10 in the culture medium A significant difference in concentration was observed (*p<0.001, Inter-group differences were analyzed by the Student t-test).

In the case of oxidized human IgG (hIgG), 80 µg of hIgG treated with 1 µg of ozone per mg of human IgG was 80 µg of hIgG treated with 4 µg of ozone per 1 mg of hIgG, or 16 per mg of hIgG. It can be seen that the immunomodulatory effect of stimulating IL-10 secretion is reduced as the ozone concentration is higher than that of hIgG 80 μg treated with ozone (p<0.05, Student t-test). compared with ozone treatment of 1 μg per mg of human IgG).

These results show that when the amount of ozone reacted with 1 mg of hIgG reacted with human immune cells cultured under the conditions for oxidizing immunoglobulin confirmed in Table 3 of Example 3 of the present invention is 4 μg or more (ie, too much oxidation) It can be seen that can rather reduce the immunomodulatory effect of the protein.

5-3. Effect of Inducing Increased IL-8 Secretion by Oxidized Immunoglobulin in a Laboratory Model of Peripheral Blood Mononuclear Cell Culture

Conditions Stimulants contained in 1ml of culture media/well Concentration of IL-8 (pg/ml) (mean±SEM) *Significant increase compared to media only (p<0.05) p-value compared to media only **p-value compared to condition 2 One Media only 107.6 ± 0.8 2 hIgG 80 μg 83.9 ± 2.0 - <0.001 3 O ₃ treated ( 1 ㎍ O ₃ / 1 mg hIgG) hIgG 80 ㎍ 132.5 ± 0.7 * <0.001 <0.001 4 HSA 80 μg 94.3 ± 2.0 - 0.001 5 NaClO treated (275 μg NaClO / 1 mg hIgG) hIgG 80 μg 252.7 ± 2.8 * <0.001 <0.001 6 LPS 10 ng 253.9 ± 2.8 * <0.001 <0.001

Interleukin-10 (IL-10); human immunoglobulin G (hIgG); Ozone (O ₃ ); Human serum albumin (HSA); sodium hypochlorite (NaClO); Lipopolysaccharide (LPS); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. * A statistically significant difference is considered when the p value is less than 0.05 when compared with the Student t-test (independent sample test). **p-value is when only the hIgG-treated groups were compared, the p-value was less than 0.05 when comparing the measured values of the hIgG group without oxidation (condition 2) and the Student t-test (independent sample test). cases were considered to show a statistically significant difference.

Table 8 above compares the concentration of IL-8 in the culture solution collected after reacting human peripheral blood mononuclear cells isolated from the peripheral blood of a healthy normal person (blood donor 1) with human IgG or oxidized human IgG. It is schematically shown in FIG. 9 .

Referring to Tables 8 and 9, as a result of an experiment using the peripheral blood of a healthy normal person (blood donor 1), immune cells (peripheral blood mononuclear cells; PBMCs) isolated from the peripheral blood of a healthy normal person (blood donor 1) were Compared to the case of reacting with human IgG oxidized with ozone or sodium hypochlorite (oxidized hIgG), the culture medium compared to the case of reacting with the negative control culture medium only (media only) or non-oxidized human IgG It can be seen that the concentration of IL-8 was significantly increased (p<0.05). On the other hand, it was confirmed that there was no significant increase in the IL-8 concentration of the cell culture supernatant under the conditions in which hIgG and HSA were treated without oxidation compared to the case where only the negative control culture was treated.

In addition, when LPS used as a positive control was treated, it was confirmed that a significantly higher concentration of IL-8 was secreted in the cell culture supernatant compared to other conditions (p<0.05), and the cultured human peripheral blood mononuclear cells (PBMCs) ), it can be confirmed that this experiment was performed properly.

Conditions Stimulants contained in 1ml of culture media/well Concentration of IL-8 (mean±SEM) (pg/mL) *Significant increase compared to media only (p<0.05) **p-value compared to condition 2 One Media only 556.6 ± 24.2 2 hIgG 80 μg 563.7 ± 20.4 - 3 O ₃ treated ( 1 ㎍ O ₃ / 1 mg hIgG) hIgG 80 ㎍ 1079.7 ± 26.3 * <0.001 4 HSA 80 μg 552.0 ± 19.6 - 5 O ₃ treated ( 1 ㎍ O ₃ / 1 mg HSA) HSA 80 ㎍ 581.2 ± 8.5 - 6 LPS 10 ng 1528.3 ± 68.8 * <0.001

Interleukin-8 (IL-8); human immunoglobulin G (hIgG); Ozone (O ₃ ); Human serum albumin (HSA); Lipopolysaccharide (LPS); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. * A statistically significant difference is considered when the p value is less than 0.05 when compared with the Student t-test (independent sample test). **p-value is when only the hIgG-treated groups were compared, the p-value was less than 0.05 when comparing the measured values of the hIgG group without oxidation (condition 2) and the Student t-test (independent sample test). cases were considered to show a statistically significant difference.

Table 9 above compares the IL-8 concentration in the culture solution collected after reacting human peripheral blood mononuclear cells isolated and cultured from the peripheral blood of another healthy normal person (blood donor 2) with human IgG or oxidized human IgG. This was schematically illustrated in FIG. 10 .

Referring to Table 9 and Figure 10, the experimental procedure using the peripheral blood of a healthy normal person (blood donor 2) and immune cells (peripheral blood mononuclear cells; PBMCs) isolated from the peripheral blood of a healthy normal person (blood donor 2) were In the case of reaction with ozone-oxidized human IgG (oxidized hIgG), the concentration of IL-8 in the culture medium was significant compared to the case of treatment with only the negative control culture medium (media only) or treatment with human IgG without oxidation treatment. It can be seen that the increase was significantly increased (p<0.05).

In addition, it was confirmed that a significantly higher concentration of IL-8 was secreted in the culture medium treated with LPS used as a positive control compared to other treatment conditions (p<0.05). It can be confirmed that this experiment was performed properly.

5-4. Changes in the amount of IL-8 secretion in the culture medium according to the change in the ozone dose that oxidizes immunoglobulins in the peripheral blood mononuclear cell culture laboratory model

When PBMCs isolated from the venous blood of a healthy normal person (blood donor 2) were cultured in the same way as above, and then treated with 1 mL of a culture solution containing 80 μg of hIgG in which 1 μg of ozone was reacted for 5 minutes per 1 mg of hIgG per well. , When treated with 1 mL of a culture solution containing 80 μg of hIgG in which 4 μg of ozone per 1 mg of hIgG was reacted for 5 minutes, treated with 1 mL of a culture solution containing 80 μg of hIgG in which 16 μg of ozone per 1 mg of hIgG was reacted for 5 minutes In one case, in the case of treatment with 1 mL of a culture solution containing 80 μg of HSA in which 1 μg of ozone per 1 mg of HSA was reacted for 5 minutes, 1 mL of a culture solution containing 80 μg of HSA reacted with 4 μg of ozone per 1 mg of HSA for 5 minutes In the case of treatment with 1 mL of a culture solution containing 80 μg of HSA reacted with 16 μg of ozone per ₁ mg of HSA for 5 minutes, the culture plate After centrifugation, the culture supernatant was collected. In the above experiment, the experiment was performed by performing quadruplicate 4 times for each condition. The supernatant obtained in the above experiment was aliquoted, stored frozen at -20 °C, thawed at room temperature, and the concentration of IL-8 cytokine present in the supernatant was measured using the cytokine ELISA set (BD, USA) as recommended by the manufacturer. was measured according to

Amount of O ₃ reacted with 1mg of hIgG or HSA Concentration of IL-8 (pg/mL) (mean±SEM) P value* hIgG 80 μg treatment (1 mL/well) HSA 80 μg treatment (1 mL/well) 1 μg 1079.9 ± 26.3 581.2 ± 8.5 <0.001 4 μg 975.3 ± 20.8 583.6 ± 12.4 <0.001 16 μg 943.8 ± 49.2 597.4 ± 10.3 <0.001

Interleukin-8 (IL-8); human immunoglobulin G (hIgG); Ozone (O ₃ ); Human serum albumin (HSA); Lipopolysaccharide (LPS); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. *Statistically significant when the IL-8 concentration in the culture supernatant between the hIgG-treated group and the HSA-treated group treated with different ozone doses is less than 0.05 when compared with the student t-test (independent sample test). considered to be different.

Table 10 shows the IL-8 concentration in the culture medium according to the change in the degree of oxidation of human IgG by reacting human peripheral blood mononuclear cells isolated and cultured from the peripheral blood of a healthy normal person (blood donor 2) with human IgG oxidized under various conditions. It is a comparison, and it is shown in FIG. 11 schematically.

Referring to Table 10 and FIG. 11, the results of comparing the same three conditions (treatment of 1 μg, 4 μg, and 16 μg to 1 mg of IgG or HSA 1 mg, respectively) treated with different doses of ozone to IgG or HSA , it was confirmed that the concentration of IL-8 in the culture medium was significantly higher in the case of treatment with oxidized IgG under all three different conditions compared with the case of treatment with oxidized HSA (*p<0.001, Inter -group differences were analyzed by the Student t-test).

Also, in the case of oxidized IgG, the immunomodulatory effect of stimulating the secretion of IL-8 was found in 80 μg of IgG treated with 1 μg of ozone per 1 mg of IgG compared with 80 μg of IgG treated with 4 μg of ozone per 1 mg of IgG. It can be seen that there is a significant increase (p<0.05).

These results show that when the amount of ozone reacted with 1 mg of hIgG under the conditions of oxidizing the immunoglobulin of the present invention is 4 μg or more, the immunomodulatory effect when the amount of ozone treated with the immunoglobulin is too large as the amount of ozone increases. It can be seen that can be rather reduced.

Through this Example 5, it can be seen that the oxidized immunoglobulin exhibits a significant immunomodulatory effect. Specifically, oxidized immunoglobulin exhibits an immunomodulatory effect of secreting significantly higher amounts of IL-10 from human peripheral blood immune cells compared to non-oxidized immunoglobulin, human serum albumin, or oxidized human serum albumin. can be found

The IL-10 has an immunomodulatory effect known to improve immune dysregulation diseases, including autoimmune diseases, cancer, chronic inflammatory diseases, and autoimmune diseases in a number of studies, It is an important immunomodulatory substance that has been confirmed to exhibit anti-inflammatory, anti-allergic and anti-cancer effects (Saralva M, et al. J Exp Med. 2020;217:e20190418, Akdis CA, et al. J Clin Invest 2014;124; 4678-80). In particular, clinical trial research results were announced that the administration of recombinant IL-10 showed clinically meaningful improvement in patients suffering from advanced solid cancer and chronic inflammatory diseases including chronic inflammatory bowel disease, rheumatoid arthritis, and psoriasis. It is being developed as a therapeutic agent for the above diseases. On the other hand, when pegylated recombinant human IL-10 treated with PEGylation, a technology that increases the half-life of protein drugs in the body, is administered by subcutaneous injection daily in patients with advanced solid cancer, IFN-γ from serum samples collected before and after treatment It has been reported that a systemic immunostimulatory effect of significantly increasing the concentration of , a significant increase in the number of CD8+ T cells infiltrating cancer tissues, and an effect of prolonging the progression-free survival period were reported (Naing A, et al. J Clin Oncol 2016, Naing A, et al. Cancer Cell 2018;34:775-79). Therefore, this Example 5 shows the immunomodulatory therapeutic effect of oxidized human immunoglobulin increasing the secretion of IL-10 from immune cells that can improve patients suffering from chronic inflammatory diseases, malignant tumors, and allergic diseases, anticancer immunotherapy It can be proven to have anti-allergic and immunomodulatory effects.

In addition, through the immunomodulatory effect of increasing the secretion of IL-10 from immune cells of the oxidized immunoglobulin confirmed in Example 5 of the present invention, the oxidized oxidized immunoglobulin confirmed through clinical trials in patients with advanced solid cancer in Example 1 of the present invention It shows that intramuscular injection of immunoglobulin induces a systemic immunomodulatory effect that increases the fraction of IFN-γ-secreting cells among peripheral blood CD8+ T cells (cytotoxic T cells), thereby exhibiting anticancer immunotherapeutic effects.

In addition, the oxidized immunoglobulin in Example 5 has an immunomodulatory effect of secreting significantly higher IL-8 than non-oxidized immunoglobulin, human serum albumin, or oxidized human serum albumin in human peripheral blood immune cells. was shown. It has been reported that IL-8 exhibits an antiallergic immunomodulatory effect by inhibiting the release of histamine from basophils, which plays an important role in the pathogenesis of allergic diseases (Kuna P et al. J Immunol 1991;147: 1920-1924, Alam R, et al. Am J Respir Cell Mol Biol 1992;7:427-433). Therefore, it can be seen from Example 5 that the oxidized immunoglobulin exhibits an anti-allergic immunomodulatory effect capable of treating allergic diseases.

<Example 6> Confirmation of anti-allergic immunomodulatory effect of oxidized immunoglobulin in egg albumin allergy mouse model

Currently, the most commonly used animal model, ovalbumin (OVA) allergy mouse model (Ayoub M, et al. Int lmmunopharmacol 2003), is the most commonly used animal model to verify the anti-allergic effect of drugs in developing new drugs for allergic diseases ;3:523-53) to verify the antiallergic immunomodulatory effect of the oxidized immunoglobulin of the present invention.

To create an allergy mouse model for OVA, OVA (Sigma Chemical Co., St. Louis, Mo) 0.2 mg/mL was added to BALB/c female mice with 20 mg/mL of aluminum hydroxide (Thermo scientific, Imject Alum, 77161) and 1 Allergy to OVA was induced by mixing at a ratio of :1 (v/v) and administering 0.2 mL by intraperitoneal injection for a total of 3 times on days 0, 7, and 14. Formation of antibodies to OVA was determined by enzyme-linked immunosorbent assay (ELISA) using serum obtained by centrifuging blood from the heart while sacrificing mice on day 21 by using serum OVA-specifically IgG. Antibodies were measured. The purpose of this study was to determine whether the immunoglobulin protein oxidized in the animal model of allergy exhibited a higher antiallergic immunomodulatory effect to a significantly different degree than that of human immunoglobulin that was not oxidized as a control.

Specifically, for the progress of the animal experiment, the OVA-allergic mouse model was used to divide the animals into 3 groups, and the following treatments were performed 3 times over a period of 0 days, 7 days, and 14 days from 18 animals in each group, 6 mice in each group. At this time, the first group was subcutaneously administered with 0.2 mL of saline as a negative control group, and the second group was subcutaneously injected with 10 mg of intramuscular human immunoglobulin (gamma globulin, Green Cross, Korea) (hIgG). In the third group, 4 μg of ozone gas per 1 mg of immunoglobulin was mixed in a syringe, reacted for 5 minutes, the gas was removed, and 10 mg of liquid oxidized oxidized human immunoglobulin (O ₃ -hIgG) was administered. It was administered by subcutaneous injection. To compare the immunomodulatory effects of subcutaneous injection of oxidized human immunoglobulin and subcutaneous injection of human immunoglobulin without oxidation treatment in the OVA allergy mouse model, serum was collected by centrifugation from blood collected on day 21 from mice in each group. It was then aliquoted and stored frozen at -20 °C. The titer of IgG reacting specifically with OVA in serum samples thawed at room temperature was measured by enzyme linked immune-sorbent assay (ELISA). In a 96-well ELISA plate, 0.25 µg of OVA per well was diluted in carbonate buffer and reacted at 4 °C for 16 hours, then with 3% BSA-0.1% Tween-20 in phosphate buffered saline (PBS) at room temperature. to block the non-specific reaction, and 100 μl of mouse serum diluted at 1:500 (v:v) with the same buffer was dispensed as quadruplicate in each well and reacted overnight at 4°C. After reacting with alkaline phosphase-attached anti-mouse IgG antibody for 2 hours, color was developed with p-Nitrophenyl phosphate and absorbance was measured at 405 nm. Between each reaction step, it was washed 5 times with PBS (PBST) containing 0.1% Tween-20. And the titer of the OVA-specific IgG antibody was expressed as the absorbance value measured at 405 nm.

Groups Treatments
(subcutaneous injection) Titer of OVA-specific IgG (Absorbance value) (mean±SEM) *p-value compared to condition 1 (Saline treated OVA-sensitized mice) **ANOVA p-value
(statistical differences among 3 groups) One Saline 0.295 ± 0.025 0.001
(Tukey's post hoc analysis
Group 1-3: 0.013
Group 2-3: 0.001) 2 hIgG 10 mg 0.252 ± 0.016 0.186 3
O ₃ treated (4 μg O ₃ / 1 mg hIgG) hIgG 10 mg 0.395 ± 0.022 0.013

human immunoglobulin G (hIgG); ovalbumin (OVA); Ozone (O ₃ ); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. *p-value is a statistically significant difference when comparing the OVA-specific IgG antibody measurement result of the negative control mouse group subcutaneously injected with physiological saline and the student t-test (independent sample test) when the p value is less than 0.05. considered visible. **p-value is one-way ANOVA test, and when the p value is less than 0.05, it means that there is a statistically significant difference in the mean value of OVA-specific IgG antibody levels between the 3 groups.

Table 11 shows the results of verifying the OVA-specific IgG antibody induction effect (ie, anti-allergic effect) by hIgG in the Ovalbumin (OVA) allergy mouse model, and is schematically shown in FIG. 12 .

Referring to Table 11 and FIG. 12 , as a result of the above experiment, a significant difference was observed in the average value of the OVA-specific IgG antibody level between the three groups. As a result of Tukey's post-statistical analysis to determine in which group a significant difference in the mean value of OVA-specific IgG antibody levels occurred, in the mouse group administered with 10 mg of oxidized hIgG, physiological saline as a negative control was administered. It can be seen that the average value of the OVA-specific IgG antibody level significantly increased compared to the group administered with only the mouse group or the group administered with 10 mg of non-oxidized hIgG (One-way ANOVA test with Tukey's post hoc test, p=0.013, p=0.001, respectively). In particular, there was no statistically significant difference between the group administered with non-oxidized hIgG 10 mg and the negative control group administered with only physiological saline (p=0.186, Table 11, FIG. 12 ). Therefore, through this Example 6, it can be seen that the anti-allergic immunomodulatory effect of increasing the OVA-specific IgG antibody titer occurs when oxidized IgG is administered compared to non-oxidized IgG.

It has been known that an allergen-specific IgG antibody acts as a blocking antibody that competitively blocks an allergen-specific IgE antibody from reacting with an allergen. In past animal model studies and recent clinical trial studies for allergy patients, when allergen-specific IgG is administered by injection, it has a significant antiallergic effect in suppressing allergic reactions caused by the allergen and IgE binding and a reduction in allergic diseases. It has been clearly demonstrated and reported to exhibit a therapeutic effect to reduce symptoms (Flicker S, et al. Curr Top Microbiol Immunol 2011; Gevaert P, et al. J Allergy Clin Immunol 2022). Therefore, it can be seen that the observed allergen-specific IgG production induction effect of oxidized hIgG as shown in Example 6 can be applied to the treatment of allergic diseases by inducing a significant anti-allergic immunomodulatory effect of oxidized-treated IgG. .

<Example 7>

Formulation Example 7-1

Oxidized immunoglobulin (or IgG) 12mg

Contains one or more adjuvants among adjuvants (aluminum hydroxide 0.001-2 mg, calcium phosphate appropriate amount, tyrosine 1 mg, MPL 1 mg)

Sodium Chloride 4mg

Aminoacetic acid 45mg

D-mannitol 4mg

sodium hydroxide appropriate amount

0.8-2ml water for injection

(Water for injection is supplied in a separate vial from the above other ingredients)

Formulation Example 7-2

Oxidized immunoglobulin (or IgG) 12mg

Histamine dihydrochloride 0.15㎍

Sodium Chloride 4mg

Aminoacetic acid 45mg

D-mannitol 4mg

sodium hydroxide appropriate amount

0.8-2ml water for injection

(Water for injection is supplied in a separate vial from the above other ingredients)

In order to enhance the immunomodulatory effect of the oxidized immunoglobulin as in Formulation Examples 7-1 or 7-2, a pharmaceutical composition is prepared by additionally mixing an immune adjuvant or histamine as commonly practiced in the art. can do.

<Example 8> Confirmation of inhibitory effect of oxidized IgG on immediate-type allergic reaction caused by antigen binding to IgE in cultured rat basophilic leukemia cell line of oxidized IgG

The RBL-2H3 cell line (rat basophilic leukemia cell line) is a cell commonly used to check the antiallergic effect of new drug candidates. The RBL-2H3 cell line has an IgE antibody receptor (FcεRI) on its surface, and when activated with IgE and antigen, degranulation is induced and is known to secrete a mediator of an allergic reaction, beta-hexosaminidase ( β-hexosaminidase) is known as an indicator of degranulation (Planta Med. 1998;64:577-578).

To determine whether oxidized IgG can inhibit the immediate-type allergic reaction caused by antigen binding to IgE from basophil cells, the release of β-hexosaminidase by antigen stimulation after IgE sensitization in RBL-2H3 cell line was lowered. was measured by the experimental method of

RBL-2H3 cells were added to Dulbecco's modified eagle medium with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin added to 0.5 mL per well in a humidified CO ₂ incubator at 37°C. (5% CO ₂ /95% air). RBL-2H3 cells were aliquoted in a 48-well cell culture plate at 2 × 10 ⁵ cells/well and cultured for 24 hours, followed by a specific IgE antibody (DNP-sIgE; Sigma, St) against 20 ng/mL dinitropheny (DNP) per well. Louis, MO, USA) was replaced with 0.5 mL of medium containing the cells and cultured for 16 hours to sensitize DNP-sIgE. Cells in each well were treated with Siraganian buffer (119 mM NaCl, 5 mM KCl, 1 mM CaCl ₂ , 0.4 mM MgCl ₂ , 25 mM PIPES, 5.6 mM glucose, 0.1% bovine serum albumin, pH 7.2p; hereafter abbreviated as S buffer). ), after washing twice with the inhibitor, human IgG 2mg (hIgG; liquid commercially available human IgG for intramuscular injection, gamma globulin, Green Cross, Korea) or oxidized human IgG 2mg (O ₃ -treated IgG 2mg; liquid After mixing 1 μg of ozone gas per 1 mg of commercially available human IgG for intramuscular injection in a syringe and reacting for 5 minutes, the gas was removed and 20 μl of S buffer containing oxidized human IgG in liquid state) In the negative control and positive control experimental conditions, only 20 μl of S buffer was treated, and then all four conditions were reacted at 37° C. for 50 minutes. Afterwards, 180 μl of S buffer was added to the negative control experimental conditions, and for the remaining 3 experimental conditions, S buffer containing 20 ng of dinitrophenyl-human serum albumin (DNP-HSA, Sigma, St. Louis, MO, USA) per well. 180 μl was added (finally, 200 μl of buffer is included in each well), and after stimulation for 40 minutes, the reaction was stopped by cooling the 48-well cell culture plate on ice for 10 minutes. The above four treatment conditions were all four repeated experiments (quadruplicated) per one condition to measure the results.

In order to measure the amount of β-hexosaminidase liberated in the supernatant in each well after antigen stimulation, 50 μL of the supernatant is transferred to a 96-well plate, and 50 μL of substrate buffer (4-p-nitrophenyl-N- Add acetyl-β-D-glucosaminide, 3.3 mM, sodium citrate, 0.1 M, pH 4.5) and react at 37°C for 70 minutes, then add 100 μL of stop solution (0.1 M Na ₂ CO ₃ , pH 10) to react was stopped and absorbance was measured at 405 nm. In addition, in order to measure the amount of β-hexosaminidase remaining in the cell pellet, after removing all the supernatant from each well after the antigen stimulation, 1% Triton X-100 buffer was added to the cell pellet at 200 per well. After processing μl, 50 μL of the cell lysate obtained by mixing it using a pipette is transferred to a 96-well plate, and 50 μL of substrate buffer (4-p-nitrophenyl-N-acetyl-β-D-glucosaminide, 3.3 mM, sodium citrate, 0.1 M, pH 4.5) was added and reacted at 37°C for 70 minutes, then 100 μL of stop solution (0.1 M Na ₂ CO ₃ , pH 10) was added to stop the reaction, and absorbance was measured at 405 nm.

si C, et al. Allergy. 2014;69:338-347).The degree of β-hexosaminidase release (%) from RBL-2H3 cells was calculated according to <Equation 2> below as commonly used in previously published papers (Uerm

si C, et al. Allergy. 2014;69:338-347).

<Equation 2>

β-hexosaminidase % release = [absorbance of supernatant / (absorbance of supernatant + absorbance of cell pellet)] × 100

In this example, after DNP-specific IgE sensitization, in RBL-2H3 cells stimulated with DNP-HSA antigen, the secretion of β-hexosaminidase, which is a degranulation indicator of mast cells, of human IgG or oxidized human IgG To verify the effect, the release rate (% release) of β-hexosaminidase calculated through the above formula was comparatively analyzed.

Conditions Stimulants contained β-hexosaminidase release (%) (mean±SEM) *ANOVA p-value
(statistical differences among 4 conditions) One negative control
(DNP-sIgE +, DNP-HSA-) 26.44 ± 0.84 < 0.001
(Tukey's post hoc analysis showed a significant difference between condition 3 and condition 4: p <0.001 )

2 positive control
(DNP-sIgE +, DNP-HSA +) 46.48 ± 0.72 3 hIgG 2 mg
(DNP-sIgE +, DNP-HSA +) 47.81 ± 1.34 4 O ₃ treated hIgG 2 mg
(DNP-sIgE +, DNP-HSA +) 37.81 ± 0.39

human immunoglobulin G (hIgG); Ozone (O ₃ ); dinitropheny-specific IgE (DNP-sIgE); dinitrophenyl-human serum albumin (DNP-HSA); standard error of the mean (SEM); Data are presented as mean±SEM from quadruplicate experiment. *p-value is a one-way ANOVA test, and when the p value is less than 0.05, there is a statistically significant difference in the mean value of β-hexosaminidase release (% release) between the 4 groups. means

Table 12 is the result of comparative analysis of the difference in β-hexosaminidase release (% release) by IgE-antigen-mediated stimulation when human IgG and oxidized human IgG were treated in rat basophilic leukemia cells. indicated.

Referring to Table 12 and FIG. 13, a statistically significant difference was observed in the average values of the % release of β-hexosaminidase under the four experimental conditions (p < 0.001). As a result of Tukey's post-hoc statistical analysis, it was confirmed that the mean value of the release rate of β-hexosaminidase was significantly lower when oxidized human IgG was treated compared to that treated with human IgG (One-way ANOVA test with Tukey'). s post hoc test, p < 0.001). That is, it was confirmed that the oxidized human IgG effectively inhibited the degranulation phenomenon due to IgE-antigen binding from basophil cells. The experimental results of this example confirmed that oxidized IgG exhibits an anti-allergic effect in inhibiting IgE-mediated immediate type allergic reaction more effectively than non-oxidized human IgG. In addition, the above experimental results prove that the oxidized immunoglobulin of the present invention is a substance having an anti-allergic effect useful for preventing or treating allergic diseases.

When the pharmaceutical composition comprising the oxidized immunoglobulin of the present invention is administered to a mammal as an active ingredient, compared with conventional immunoglobulin preparations, it is possible to more effectively prevent or treat diseases related to immunomodulatory dysfunction, and It is possible to prepare a new preventive and therapeutic pharmaceutical composition for dysfunction-related diseases.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (28)

A pharmaceutical composition for preventing or treating diseases related to immune dysregulation, comprising oxidized immunoglobulin as an active ingredient. The method according to claim 1, wherein the oxidized immunoglobulin is ozone (ozone, O ₃ ), hydrogen peroxide (H ₂ O ₂ ), sodium hypochlorite (Sodium hypochlorite, NaClO) and at least one oxidizing agent from the group consisting of an oxidizing agent; A pharmaceutical composition characterized in that the reaction is mixed with immunoglobulin. The pharmaceutical composition according to claim 2, wherein the oxidized immunoglobulin is reacted by mixing 0.1 to 4 μg of ozone with respect to 1 mg of immunoglobulin. The pharmaceutical composition according to claim 1, wherein the immunoglobulin is any one from the group consisting of IgG, IgA, IgM, IgD and IgE. The pharmaceutical composition according to claim 4, wherein the immunoglobulin is IgG. The pharmaceutical composition according to claim 5, wherein the IgG is immunoglobulin G isolated from mammalian blood. The pharmaceutical composition according to claim 6, wherein the IgG is an IgG isolated from the mammal's own blood or an IgG isolated from the blood of other mammals. The pharmaceutical composition according to claim 1, wherein the immune dysregulation-related disease is any one from the group consisting of an allergic disease, a chronic inflammatory disease, an autoimmune disease, and a malignant tumor disease. The pharmaceutical composition according to claim 8, wherein the allergic disease is any one from the group consisting of bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, urticaria, food allergy, anaphylaxis, and drug allergy. The method according to claim 8, wherein the chronic inflammatory disease or autoimmune disease is degenerative arthritis, chronic inflammatory gastroenteritis, ankylosing spondylitis, chronic pustular dermatitis, rheumatoid arthritis, systemic lupus erythematosus, pemphigus, autoimmune thyroiditis, autoimmune hepatitis, chronic inflammatory Intestinal disease, autoimmune nephritis, chronic inflammatory gastroenteritis, Sjogren's syndrome, scleroderma, and psoriasis pharmaceutical composition, characterized in that any one of the group consisting of. The method according to claim 8, wherein the malignant tumor is a solid cancer selected from the group consisting of lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, thyroid cancer, breast cancer, liver cancer, kidney cancer, prostate cancer and mesothelioma, leukemia, lymphoma and multiple A pharmaceutical composition, characterized in that it is any one of hematologic cancers selected from the group consisting of myeloma. The pharmaceutical composition according to claim 11, wherein the oxidized immunoglobulin has an anticancer immunotherapeutic effect by immunomodulation through activation of cytotoxic T cells. The pharmaceutical composition according to claim 1, further comprising an adjuvant in addition to the oxidized immunoglobulin. The method of claim 13, wherein the adjuvant is aluminum hydroxide (aluminum hydroxide), calcium phosphate (calcium phosphate), tyrosine (tyrosine), monophosphoryl lipid A (monophosphoryl lipid A, MPL) or histamine (histamine) from the group consisting of A pharmaceutical composition, characterized in that at least one adjuvant. isolating the immunoglobulin from the mammal's own blood or isolating the immunoglobulin from the blood of other mammals (first step);
preparing oxidized immunoglobulin by mixing and reacting the isolated immunoglobulin with an oxidizing agent (second step); and
A method for preventing or treating an immune dysregulation-related disease, comprising administering the oxidized immunoglobulin to an individual suffering from an immune dysregulation-related disease (step 3). The method of claim 15, wherein the oxidizing agent is ozone (ozone, O ₃ ), hydrogen peroxide (H ₂ O ₂ ), sodium hypochlorite (NaClO) method characterized in that at least one oxidizing agent from the group consisting of. The method according to claim 15, wherein in the second step, 0.1 to 4 μg of ozone is mixed and reacted with 1 mg of the isolated immunoglobulin. The method according to claim 15, wherein the immune dysregulation-related disease is any one from the group consisting of an allergic disease, a chronic inflammatory disease, an autoimmune disease, and a malignant tumor disease. The method according to claim 18, wherein the allergic disease is any one from the group consisting of bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, urticaria, food allergy, anaphylaxis, and drug allergy. The method of claim 18, wherein the chronic inflammatory disease or autoimmune disease is degenerative arthritis, chronic inflammatory gastroenteritis, ankylosing spondylitis, chronic pustular dermatitis, rheumatoid arthritis, systemic lupus erythematosus, pemphigus, autoimmune thyroiditis, autoimmune hepatitis, chronic inflammatory Intestinal disease, autoimmune nephritis, chronic inflammatory gastroenteritis, Sjogren's syndrome, scleroderma and psoriasis, characterized in that any one of the group. The method according to claim 18, wherein the malignant tumor is a solid cancer selected from the group consisting of lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, thyroid cancer, breast cancer, liver cancer, kidney cancer, prostate cancer and mesothelioma, leukemia, lymphoma and multiple A method characterized in that it is any one of hematologic cancers selected from the group consisting of myeloma. isolating the immunoglobulin from the mammal's own blood or isolating the immunoglobulin from the blood of other mammals (first step);
A method of preparing a pharmaceutical composition for preventing or treating a disease related to immune dysregulation, comprising the step (second step) of preparing oxidized immunoglobulin by mixing the isolated immunoglobulin with an oxidizing agent. The method of claim 22, wherein the oxidizing agent ozone (ozone, O ₃ ), hydrogen peroxide (hydrogen peroxide, H ₂ O ₂ ), sodium hypochlorite (sodium hypochlorite, NaClO), characterized in that one or more oxidizing agents from the group consisting of pharmaceutical A method for preparing the composition. 23. The method of claim 22, wherein in the second step, 0.1 to 4 μg of ozone is mixed and reacted with 1 mg of the isolated immunoglobulin. The method of claim 22, wherein the immune dysregulation-related disease is any one of an allergic disease, a chronic inflammatory disease, an autoimmune disease, and a malignant tumor disease. The method of claim 25, wherein the allergic disease is any one from the group consisting of bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, urticaria, food allergy, anaphylaxis, and drug allergy. The method of claim 25, wherein the chronic inflammatory disease or autoimmune disease is degenerative arthritis, chronic inflammatory gastroenteritis, ankylosing spondylitis, chronic pustular dermatitis, rheumatoid arthritis, systemic lupus erythematosus, pemphigus, autoimmune thyroiditis, autoimmune hepatitis, chronic inflammatory Intestinal disease, autoimmune nephritis, chronic inflammatory gastroenteritis, Sjogren's syndrome, scleroderma, and psoriasis, characterized in that any one of the group consisting of a method for producing a pharmaceutical composition. The method according to claim 25, wherein the malignant tumor is a solid cancer selected from the group consisting of lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, uterine cancer, thyroid cancer, breast cancer, liver cancer, kidney cancer, prostate cancer and mesothelioma, leukemia, lymphoma and multiple A method for preparing a pharmaceutical composition, characterized in that it is any one of hematologic cancers selected from the group consisting of myeloma.

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