KR20220159232A - Use of anti-IsaA F10 antibody for preventing or improving atopic dermatitis - Google Patents

Abstract

The present invention relates to a use of an anti-IsaA F10 antibody for preventing or alleviating atopic dermatitis, and more specifically, to a use of an anti-IsaA F10 antibody containing heavy chain CDRs and light chain CDRs of a specific sequence or an antigen-binding fragment thereof, for preventing and improving atopic dermatitis. The anti-IsaA F10 antibody is expected to be useful for alleviating or treating skin inflammation such as atopic disease by inhibiting the growth of Staphylococcus aureus.

Description

Use of anti-IsaA F10 antibody for preventing or improving atopic dermatitis {Use of anti-IsaA F10 antibody for preventing or improving atopic dermatitis}

The present invention relates to the use of an anti-IsaA F10 antibody for preventing and improving atopic dermatitis.

Atopic dermatitis (atopic dermatitis (AD)) is a severe and chronically relapsing inflammatory skin disease accompanied by clinical symptoms such as erythema, edema and lichenification in skin lesions in addition to severe itching and dryness. Although the pathogenesis of atopic dermatitis has not yet been completely identified, it has been known that atopic dermatitis occurs by inducing a chronic inflammatory reaction in the skin due to an overactive immune response (allergic reaction) to allergens in the environment. In addition, atopic dermatitis is a chronic inflammatory disease that occurs before the development of asthma and allergic diseases, and its symptoms differ depending on the concentration, quantitative change or activity level of chemokines, and Th2 type lymphocytes are located in the lesional skin It is also referred to as an intrusion phenomenon.

Currently, 0.5-1% of the world population, especially 5-10% of children, is known to suffer from atopic dermatitis, and it is known that environmental factors are greatly involved in its onset and deterioration. As an example, it is known that the incidence of atopic dermatitis correlates with the amount of exposure to house dust mites and shows a significant correlation with the severity of atopic dermatitis. It is also known that reducing the exposure to house dust mites in the environment can reduce the severity of atopic dermatitis. In addition, atopic dermatitis seems to be caused by a combination of dry skin, characteristics of easily feeling itchy skin compared to normal people, infection by bacteria/viruses/fungi, emotional factors, and environmental factors. In recent decades, the incidence of atopic dermatitis has been increasing, and the number of patients in Korea is also increasing significantly.

Conventionally, the most widely used treatment for atopic dermatitis is a treatment such as a steroid, an antihistamine, or an antibiotic. Among them, although steroids are the most widely used treatment for inflammatory diseases such as bronchial asthma, the mechanism by which steroids react in the body is not yet known.

Therefore, atopic dermatitis treatment agents such as steroids tend to gradually limit their use due to various side effects, and a new concept of atopic dermatitis treatment agent that is effective for atopic dermatitis and has no side effects is required.

US Patent Publication US 2017/0002063 (published on 2017.01.05)

An object of the present invention is a cosmetic composition for preventing or improving atopic dermatitis, a health functional food composition for preventing or improving atopic dermatitis, and a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an anti-IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient. is to provide

Another object of the present invention is a cosmetic composition for preventing or improving atopic dermatitis comprising, as an active ingredient, a fusion anti-IsaA F10 antibody or antigen-binding fragment thereof to which the anti-IsaA F10 antibody or antigen-binding fragment thereof is additionally coupled with a TAT peptide To provide a health functional food composition for preventing or improving atopic dermatitis, and a pharmaceutical composition for preventing or treating atopic dermatitis.

In order to achieve the above object, the present invention comprises a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3. a light chain variable region; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - Provided are a cosmetic composition for preventing or improving atopic dermatitis, a health functional food composition for preventing or improving atopic dermatitis, and a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient.

In addition, the present invention is a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A cosmetic composition for preventing or improving atopic dermatitis comprising, as an active ingredient, a fusion anti-IsaA F10 antibody or an antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is additionally coupled to an IsaA F10 antibody or an antigen-binding fragment thereof, atopic dermatitis It provides a health functional food composition for preventing or improving, and a pharmaceutical composition for preventing or treating atopic dermatitis.

The present invention relates to a use of an anti-IsaA F10 antibody for preventing and improving atopic dermatitis, and more specifically, the present invention relates to atopic dermatitis of an anti-IsaA F10 antibody or an antigen-binding fragment thereof comprising heavy chain CDRs and light chain CDRs of a specific sequence. It is for preventive and remedial use. The anti-IsaA F10 antibody is expected to be useful for improving or treating skin inflammation such as atopic disease by inhibiting the growth of Staphylococcus aureus.

Figure 1 shows the intracellular permeability of the cell-permeable anti-IsaA F10 scFv antibody protein by confocal microscopy.
Figure 2 shows the antibacterial activity of the cell-permeable anti-IsaA F10 scFv antibody against Staphylococcus aureus.
Figure 3 shows the degree of inhibition of cytokine TNF-α, IL-1β, IL-6 mRNA expression by cell-permeable anti-IsaA F10 scFv antibody using Staphylococcus aureus.
Figure 4 shows the degree of suppression of the top cytokine TSLP and chemokine TARC mRNA expression by the cell-permeable anti-IsaA F10 scFv antibody using Staphylococcus aureus.
Figure 5 shows the skin cell healing promoting effect of the cell-permeable anti-IsaA F10 scFv antibody.

The present invention comprises a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - Provided is a cosmetic composition for preventing or improving atopic dermatitis comprising an IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient.

In addition, the present invention is a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - Provided is a cosmetic composition for preventing or improving atopic dermatitis comprising, as an active ingredient, a fusion anti-IsaA F10 antibody or an antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is additionally coupled to an IsaA F10 antibody or an antigen-binding fragment thereof. .

The cosmetic composition may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and flavors, and carriers in addition to active ingredients.

The formulation of the cosmetic composition may be prepared in any formulation conventionally prepared in the art, and external skin ointments, creams, softening lotions, nutrient lotions, packs, essences, hair tonics, shampoos, rinses, hair conditioners, and hair treatments Treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, foundation, nutrition essence, sunscreen, soap , It may have a formulation selected from the group consisting of cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto. The composition of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. .

When the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/butane or a propellant such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the formulation is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose as a carrier component , aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

In addition, the present invention is a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - Provided is a health functional food composition for preventing or improving atopic dermatitis comprising an IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient.

In addition, the present invention is a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A functional food composition for preventing or improving atopic dermatitis comprising, as an active ingredient, a fusion anti-IsaA F10 antibody or an antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is additionally coupled to an IsaA F10 antibody or an antigen-binding fragment thereof, to provide.

The health functional food composition may be provided in the form of powder, granule, tablet, capsule, syrup, drink or pill, and the health food composition is used together with other foods or food additives in addition to the active ingredient, the composition according to the present invention, and is usually It can be used appropriately depending on the method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use thereof, for example, prevention, health or therapeutic treatment.

The effective dose of the antibody or antigen-binding fragment thereof contained in the health functional food composition may be used according to the effective dose of the pharmaceutical composition, but in the case of long-term intake for the purpose of health and hygiene or health control It may be less than the above range, and since the active ingredient has no problem in terms of safety, it is certain that it can be used in an amount greater than the above range.

There is no particular limitation on the type of health food, and examples include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, vitamin complexes, and the like are exemplified.

In addition, the present invention is a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - Provided is a pharmaceutical composition for preventing or treating atopic dermatitis comprising an IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient.

In addition, the present invention is a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - Provided is a pharmaceutical composition for preventing or treating atopic dermatitis comprising, as an active ingredient, an IsaA F10 antibody or an antigen-binding fragment thereof and a fusion anti-IsaA F10 antibody or an antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is additionally coupled. .

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is one commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, and starch. , acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear including, but not limited to, acid magnesium and mineral oil, and the like. The composition for preventing or treating cancer metastasis of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. can be administered with When administered orally, since proteins or peptides are digested, oral compositions can be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention can be used in any device through which the active agent can move to target cells. can be administered by

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

On the other hand, CDRs consisting of amino acids represented by SEQ ID NO: 1 to SEQ ID NO: 6 are listed in Table 1.

In addition, the amino acid sequence of the TAT peptide used in the present invention is “YGRKKRRQRRR” (SEQ ID NO: 7), and the base sequence of the TAT peptide is “TAT GGC CGC AAA AAA CGC CGC CAG CGC CGC CGC” (SEQ ID NO: 8).

As used herein, the term “antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes, for example, monoclonal antibodies, Clonal antibodies, full-length antibodies and antibody fragments may all be included. Also, the term “antibody” may include bivalent or bispecific molecules (eg, bispecific antibodies), diabodies, triabodies or tetrabodies.

As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such a monoclonal antibody, unlike polyclonal antibodies that can bind to several epitopes, is a specific antibody molecule. Shows single binding and affinity for the epitope. In the present invention, the term "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses include gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types. IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.

As used herein, the term "heavy chain" refers to a full-length heavy chain and fragments thereof comprising a variable region VH and three constant regions CH1, CH2 and CH3 comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen. can include all In addition, as used herein, the term “light chain” may include both a full-length light chain and fragments thereof including a variable region VL and a constant region CL, which include an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen. have.

In the present invention, the terms "fragment", "antibody fragment" and "antigen-binding fragment" are used interchangeably to refer to any fragment of an antibody of the present invention that retains the antigen-binding function of the antibody. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv, and the like.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

< Example 1> Anti- IsaA scFv antibody selection

1. Implementation of bio-panning

Bio-panning was performed using an OPAL library having a diversity of 7.6 × 10 ⁹ . 10 μg of IsaA antigen was immobilized on the epoxy magnetic bead and the input phage reacted. The output titer was measured by eluting the phage reacted with the antigen. The input and output titers were measured every time to obtain bio-panning information and to confirm whether it was normally performed. For each input phage, a phage of 1 × 10 ¹² cfu/mL ≥ was used. The output was measured as 1st - 6.5 × 10 ⁶ , 2nd - 1.13 × 10 ⁴ , 3rd - 4.21 × 10 ⁷ cfu/mL. Therefore, it was confirmed that bio-panning proceeded normally.

2. ELISA assay

An ELISA assay was performed to select highly sensitive and highly specific antibodies. The obtained phage was infected with Escherichia coli and spread on an LB plate containing antibiotics. After culturing in an incubator at 30° C. for 16 hours, the resulting colonies were randomly collected. Each colony was inoculated and cultured in LB medium, treated with IPTG to express scFv antibody, and E. coli was lysed to obtain a soluble fraction to perform ELISA. First, 50 ng of IsaA antigen was immobilized on a 96-well ELISA plate and blocked with PBS containing 3% BSA. After 1 hour, the cell lysate obtained above was treated and reacted at 37°C for 2 hours. After washing the plate three times with PBS containing 0.1% Tween20, the HRP-conjugated anti-HA antibody was diluted 1:1000 in a blocking solution and reacted at room temperature for 1 hour. After washing the plate 5 times with PBS containing 0.1% Tween20, the plate was developed with TMB substrate and the scFv antibody bound to the antigen was measured with an ELISA reader. 96 antibodies were analyzed, and 10 types were selected by arbitrarily setting an OD of 0.4 as a positive guideline and 0.1 as a negative guideline.

< Example 2> Anti- IsaA scFv Antibody sequencing

Individual clones were selected through sequencing for the positive clones of 10 types of IsaA scFv selected through ELISA analysis. Sequence analysis was necessary because there is a possibility that overlapping clones may exist among the previously selected positive clones. As a result of sequence analysis, it was confirmed that 7 out of 10 positive clones were individual clones.

< Example 3> Selected anti- IsaA F10 scFv Antibody purification and sensitivity analysis

1. Anti- IsaA F10 scFv antibody purification

Anti-IsaA F10 scFv antibodies (Table 1) with the highest antigen-binding ability among the selected 7 species were purified. ER2738 E.coli cells transformed with the anti-IsaA scFv clone (F10) were cultured in 500 mL of SB media, and the cells were lysed using 1 × TES buffer. The cell lysate was reacted with 0.5 mL Ni beads and eluted using imidazole.

Target Clone Light chain CDR sequences Heavy chain CDR sequences CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 IsaA F10 TGSSSNIGNNNVN ANSQ GAWDSSLSAYV GYAMG VISSGGGST KVRRVFDY

< Example 4> cell permeable anti- IsaA F10 scFv antibody production

1. TAT-anti- IsaA F10 scFv DNA construct construction

A PCR product was produced in which a restriction enzyme site and TAT were inserted using a primer for the previously selected anti-IsaA scFv. The PCR product (insert) and the pET28a(+) vector were treated with 1 μL of EcoR I and 1 μL of Xho I , reacted at 37° C. for one hour, and then purified. The concentration of the purified vector and insert was measured by nanodrop, and ligation was performed at room temperature for 16 hours using T4 ligase for each ratio of vector and insert. After ligation, DH5α was transformed, smeared on LB plates supplemented with kanamycin, and cultured at 37°C for 16 hours. Colonies were picked the next day and inoculated into LB media supplemented with kanamycin, and cultured at 37°C for 16 hours. The next day, in order to confirm whether the insert was inserted, the plasmid was isolated, restricted with Xho I and EcoR I , and subjected to electrophoresis on a 1% agarose gel to confirm the band.

2. TAT-anti- IsaA F10 scFv antibody purification

The cloned TAT-anti-IsaA F10 scFv antibody clone was transformed into a BL21(DE3) E. coli host. Transformants were cultured in LB medium containing kanamycin until an OD ₆₀₀ value of 0.6, and expression was induced by treatment with 0.5 mM IPTG at 16°C. Cells obtained by centrifugation of the culture medium were suspended in lysis buffer (50 mM Tris-Hcl, pH 7.5, 150 mM NaCl), disrupted using a sonicator, and then centrifuged to obtain a supernatant. In order to separate and purify only the induced protein, a resin having affinity for Ni-NTA was used, and the purified protein was confirmed through SDS-PAGE analysis.

< Example 5> cell permeable anti- IsaA F10 scFv Antibody cell permeation assay

Immunofluorescence was used to confirm cell permeation of the TAT-anti-IsaA F10 scFv antibody. After dispensing 1 × 10 ⁵ human skin cells on a 10 mm coverslip coated with 0.1% gelatin for 1 hour, 10% FBS (fetal bovine serum), penicillin (100 U/mL), and streptomycin (100 μg/mL) were added. Put the RPMI medium containing the cultured in an incubator containing 5% carbon dioxide by maintaining 37 ℃. After culturing, it was replaced with 1.5 mL of fresh culture medium without FBS and treated with 50 ppm of TAT-anti-IsaA F10 scFv. After 12 hours of treatment, the cells were fixed in a fixative, blocked with 5% FBS, and immunized with an anti-His-FITC antibody. After staining the TAT-anti-IsaA F10 scFv (green), it was confirmed with a confocal microscope. As shown in Figure 1, it was confirmed that the TAT-anti-IsaA F10 scFv was penetrated into cells.

< Example 6> cell permeable anti- IsaA F10 scFv Cytotoxicity test

In order to examine the cytotoxicity of the TAT-anti-IsaA F10 scFv antibody, human skin cells were dispensed in 1 × 10 ⁴ per well in a 96-well plate, maintained at 37 ° C, and cultured in an incubator containing 5% carbon dioxide. did After 24 hours, the TAT-anti-IsaA F10 scFv was treated at different concentrations and further cultured under the same culture conditions. After incubation, add MTT{3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide} solution and react for 3 hours, then remove the culture medium and add 100 μL of DMSO (Dimethyl sulfoxide) gave. After shaking for 15 minutes, absorbance was measured at 570 nm using an ELISA reader. As shown in Table 2, when the TAT-anti-IsaA F10 scFv was treated at concentrations of 0.01, 0.1, 1, 10, 50, and 100 ppm, it was confirmed that there was little change in cell shape transformation and cell viability up to 100 ppm concentration. did Accordingly, it was found that the TAT-anti-IsaA F10 scFv antibody was non-cytotoxic up to a concentration of 100 ppm.

TAT-anti-IsaA F10 scFv Concentration (ppm) Average error 0 100.00 0.577 0.01 105.66 0.208 0.1 110.05 0.532 One 109.78 0.653 10 109.73 1.020 50 108.44 0.231 100 106.25 0.508

< Example 7> Cell permeable anti- IsaA F10 scFv's Staphylococcus aureus antimicrobial activity test

1. Confirmation of the inhibitory effect of TAT-anti-IsaA F10 scFv on ceramidase

The effect of the TAT-anti-IsaA F10 scFv antibody on the production of Ceramidase by Staphylococcus aureus was investigated.

0.1 ml of Staphylococcus aureus strain was spread on TSB solid medium and then dried. After diluting TAT-anti-IsaA F10 scFv to 10 ppm and 100 ppm, 50 ul each was dropped on a paper disc having a diameter of 8 mm, placed on a solid medium, and then incubated at 37° C. for 24 hours. As a result of observing and measuring the size of the bacterial growth inhibition region generated around the paper disc, the TAT-anti-IsaA F10 scFv inhibits the growth of Staphylococcus aureus, and thus the possibility of inhibiting the activity of ceramide was confirmed (Table 3).

sample name Size of fungal growth arrest ring PBS 0mm penicillin 15 mm TAT-anti-IsaA F10 scFv 10 ppm 4 mm TAT-anti-IsaA F10 scFv 100 ppm 8 mm

2. TAT-anti- IsaA F10 scFv's Staphylococcus aureus growth inhibitory effect confirmed

The effect of TAT-anti-IsaA F10 scFv antibody on the growth of Staphylococcus aureus was investigated.

The strain was inoculated into TSB medium (Tryptic soy broth) and cultured until an OD of 0.1, and then treated with TAT-anti-IsaA F10 scFv antibody. After incubation at 37° C. for 24 hours, absorbance was measured at 600 nm to investigate the growth ability of Staphylococcus aureus. Lactobacillus rhamnosus was used as a negative control. It was confirmed that the TAT-anti-IsaA F10 scFv inhibited the growth of Staphylococcus aureus (FIG. 2).

< Example 8> Cell permeable anti- IsaA F10 scFv's Inhibitory effect on inflammatory cytokine expression

In order to determine whether the TAT-anti-IsaA F10 scFv antibody inhibits the expression of cytokines TNF-α, IL-1β, and IL-6 mRNA, which are mainly found in atopic dermatitis skin, human skin cells were plated in wells in a 6-well plate. After dispensing per 1 × 10 ⁵ , maintained at 37 ℃ and cultured in an incubator containing 5% carbon dioxide. After 24 hours, 10 ppm of TAT-anti-IsaA F10 scFv and 1 × 10 ⁴ Staphylococcus aureus were aliquoted in the serum-free RPMI culture medium. After culturing, total RNA was extracted from the cells using TRIzol. After adding 30 μL of DEPC-treated purified water to the pellet, dissolve it and quantify at 260 nm. RT-PCR was performed using 1 μg of total RNA and TOPscript RT dry mix. MMP-1 and GAPDH used in Real Time PCR were synthesized and used by Macrogen, and the base sequences are shown in Table 4 below. Real-time PCR was performed using TOPreal ^TM Qpcr 2X PreMIX. As shown in Figure 3, the TAT-anti-IsaA F10 scFv suppressed the cytokines TNF-α, IL-1β, and IL-6, which were increased by Staphylococcus aureus, and showed effects similar to those treated with control penicillin.

primer order TNF-α forward 5'-AACCTGAACCTTCCAAAGATGG-3' reverse 5′-TCTGGCTTGTTCCTCACTACT-3′ IL-1β forward 5'-TTACAGTGGCAATGAGGATGAC-3' reverse 5′-GTCGGAGATTCGTAGCTGGAT-3′ IL-6 forward 5′-GACAGCCACTCACCTCTTCA-3′ reverse 5′-AGTGCCTCTTTGCTGCTTTC-3′ GAPDH forward 5′-GACATGCCGCCTGGAGAAAC-3′ reverse 5′-AGCCCAGGATGCCCTTTAGT-3′

< Example 9> Cell permeable anti- IsaA F10 scFv's anti-atopy activity inhibitory effect

In order to examine whether the TAT-anti-IsaA F10 scFv antibody suppresses the mRNA expression of the top cytokine TSLP (thymic stromal lymphopoietin) and chemokine TARC (thymus and activationregulated chemokine/CCL17), which are increased in the serum of patients with atopic dermatitis, human skin cells After dispensing 1 × 10 ⁵ per well in a 6-well plate, it was maintained at 37 ° C and cultured in an incubator containing 5% carbon dioxide. After 24 hours, 10 ppm of TAT-anti-IsaA F10 scFv and 1 × 10 ⁴ Staphylococcus aureus were aliquoted in the serum-free RPMI culture medium. After culturing, total RNA was extracted from the cells using TRIzol. After adding 30 μL of DEPC-treated purified water to the pellet, dissolve it and quantify at 260 nm. RT-PCR was performed using 1 μg of total RNA and TOPscript RT dry mix. TSLP, TARC and GAPDH used in Real Time PCR were synthesized and used by Macrogen, and the base sequences are shown in Table 5 below. Real-time PCR was performed using TOPreal ^TM Qpcr 2X PreMIX. As shown in Figure 4, the TAT-anti-IsaA F10 scFv inhibited the top cytokine TSLP and chemokine TARC, which were increased by Staphylococcus aureus, and showed an effect similar to that of the control penicillin treatment.

primer order TSLP forward 5′-TCCTCTGAAGACCTGACC-3′ reverse 5′-TCTCCTTTTCTCCCTAATCCTC-3′ TARC forward 5′-CGCAGCTCGAGGGACCAATGTG-3′ reverse 5'-CAAGACCTCTCAAGGCTTTGCAGG-3' GAPDH forward 5′-GACATGCCGCCTGGAGAAAC-3′ reverse 5′-AGCCCAGGATGCCCTTTAGT-3′

< Example 10> cell permeable anti- IsaA F10 scFv's skin cells healing promoting effect

In order to examine the promotion of skin cell healing for the TAT-anti-IsaA F10 scFv antibody, human skin cells were dispensed in 1 × 10 ⁵ per well in a 6-well plate, maintained at 37 ° C, and incubated with 5% carbon dioxide. cultured in. After 24 hours, TAT-anti-IsaA F10 scFv was treated with 50 ppm in serum-free RPMI culture medium, and scratch wound was generated using a 1,000p tip after 1 hour. After culturing for 48 hours, cells in the scratch margin area grew, and the degree of wound closure and wound area were measured under a microscope (FIG. 5). As shown in Figure 5, the TAT-anti-IsaA F10 scFv was observed to have a wound healing effect.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

<110> HAUUL BIO <120> Use of anti-IsaA F10 antibody for preventing or improving atopic dermatitis <130> DP-2021-0132 <160> 8 <170> KopatentIn 2.0 <210> 1 <211> 13 <212> PRT <213> artificial sequence <220> <223> light chain CDR1 <400> 1 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Asn Val Asn 1 5 10 <210> 2 <211> 4 <212> PRT <213> artificial sequence <220> <223> light chain CDR2 <400> 2 Ala Asn Ser Gln One <210> 3 <211> 11 <212> PRT <213> artificial sequence <220> <223> light chain CDR3 <400> 3 Gly Ala Trp Asp Ser Ser Leu Ser Ala Tyr Val 1 5 10 <210> 4 <211> 5 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR1 <400> 4 Gly Tyr Ala Met Gly 1 5 <210> 5 <211> 9 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR2 <400> 5 Val Ile Ser Ser Gly Gly Gly Ser Thr 1 5 <210> 6 <211> 8 <212> PRT <213> artificial sequence <220> <223> heavy chain CDR3 <400> 6 Lys Val Arg Arg Val Phe Asp Tyr 1 5 <210> 7 <211> 11 <212> PRT <213> artificial sequence <220> <223> TAT peptide <400> 7 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 8 <211> 33 <212> DNA <213> artificial sequence <220> <223> TAT peptide <400> 8 tatggccgca aaaaacgccg ccagcgccgc cgc 33

Claims (6)

A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A cosmetic composition for preventing or improving atopic dermatitis comprising an IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient. A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A cosmetic composition for preventing or improving atopic dermatitis comprising, as an active ingredient, an IsaA F10 antibody or an antigen-binding fragment thereof, additionally a fusion anti-IsaA F10 antibody or an antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is coupled. A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A functional food composition for preventing or improving atopic dermatitis comprising an IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient. A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A functional food composition for preventing or improving atopic dermatitis comprising, as an active ingredient, an IsaA F10 antibody or an antigen-binding fragment thereof, additionally a fusion anti-IsaA F10 antibody or an antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is coupled. A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A pharmaceutical composition for preventing or treating atopic dermatitis comprising an IsaA F10 antibody or an antigen-binding fragment thereof as an active ingredient. A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 6 - A pharmaceutical composition for preventing or treating atopic dermatitis comprising, as an active ingredient, a fusion anti-IsaA F10 antibody or an antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is additionally coupled to an IsaA F10 antibody or an antigen-binding fragment thereof.

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