KR20230150518A - Anti-SNAP 25 B6 antibodies for preventing and improving skin wrinkle and use thereof - Google Patents

Abstract

The present invention relates to an anti-SNAP 25 B6 antibody having the effect of preventing and improving skin wrinkles and its use. More specifically, the present invention relates to an anti-SNAP 25 B6 antibody comprising a heavy chain CDR and a light chain CDR of a specific sequence, or a use thereof. It is about an antigen-binding fragment. The anti-SNAP 25 B6 antibody is expected to be useful in preventing or treating skin wrinkles by inhibiting SNARE complex formation.

Description

Anti-SNAP 25 B6 antibodies for preventing and improving skin wrinkles and use thereof {Anti-SNAP 25 B6 antibodies for preventing and improving skin wrinkle and use thereof}

The present invention relates to an anti-SNAP 25 B6 antibody having the effect of preventing and improving skin wrinkles and its use.

Membrane fusion that occurs within cells is caused by proteins called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor). SNARE proteins refer to a specific group of proteins that are highly conserved across all species, and SNARE complexes refer to complexes of these proteins. SNARE proteins can be divided into target (t-) SNARE and vesicular (v-) SNARE. t-SNARE refers to a SNARE protein present in the presynaptic membrane, and v-SNARE refers to a SNARE protein present in the synaptic vesicle. t-SNARE is composed of an integral membrane protein called Syntaxin 1A and a peripheral membrane protein, SNAP 25 (soluble NSF attachment protein of 25 kDa), and its functional unit is thought to be its complex (t-SNARE complex). . v-SNARE refers to a membrane protein called vesicle-associated membrane protein 2 (VAMP2 or synaptobrevin). These SNARE proteins have a region of about 60 to 70 aa called the 'SNARE core', and these regions come together to form a four-helical bundle called the SNARE complex.

Among wrinkles, thick wrinkles are created and developed more strongly by frequently used facial muscles, and the exercise of these muscles is achieved through the signal transmission process of the nervous system. In order to control muscle relaxation and contraction, there is a neuromuscular junction in the upper layer of the muscle, and many synaptic vesicles exist at this nerve terminal. Muscles contract by receiving messages from acetylcholine, a neurotransmitter transmitted inside a type of neuroendoplasmic reticulum. In order for these neurotransmitters to be released from the endoplasmic reticulum to the muscle through the nerve terminal, a receptor complex called SNARE must operate normally and fuse the endoplasmic reticulum with the axon membrane of the nerve terminal. In other words, when releasing a neurotransmitter, the synaptic vesicles containing the neurotransmitter must fuse with the presynaptic membrane to release the contents to the outside. In this process, membrane fusion is provided by conjugation and twisting with SNARE proteins that exist as a three-protein complex. In particular, the neurotransmitter release channel is opened by membrane fusion between synaptic vesicles and the presynaptic membrane. The t-SNARE complex, which is a complex of Syntaxin 1a protein and SNAP 25 protein attached to the target membrane, v-SNAREs attached to vesicles are involved, and during the cell membrane fusion process, these SNARE proteins become twisted like a twist. Afterwards, as membrane fusion is completed, acetylcholine, a neurotransmitter within synaptic vesicles, is released. If the SNARE splicing and twisting process is not completely completed, membrane fusion will fail and neurotransmitter release will not occur, resulting in no muscle movement. This process means that it prevents the formation of wrinkles caused by frequently used muscles and can also improve wrinkles that have already formed.

Although research related to the SNARE complex is actively in progress, research on antibodies that inhibit the SNARE complex is still lacking.

Korean Patent Publication No. 10-2014-0137103 (published on December 2, 2014)

An object of the present invention is to provide an anti-SNAP 25 B6 antibody or antigen-binding fragment thereof that inhibits the SNARE complex.

Another object of the present invention is to provide a fusion anti-SNAP 25 B6 antibody or antigen-binding fragment thereof in which a TAT peptide is additionally bound to the anti-SNAP 25 B6 antibody or antigen-binding fragment thereof.

Another object of the present invention is to provide a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, a recombinant expression vector containing the nucleic acid molecule, and a cell transformed with the recombinant expression vector.

Another object of the present invention is to provide a composition for detecting SNAP 25 antigen containing the antibody or antigen-binding fragment thereof as an active ingredient.

Another object of the present invention is to provide a cosmetic composition or health functional food composition for preventing or improving skin wrinkles containing the antibody or antigen-binding fragment thereof as an active ingredient, and a pharmaceutical composition for preventing or treating skin wrinkles.

In order to achieve the above object, the present invention includes a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence shown in 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 shown in SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6. SNAP 25 B6 antibody or antigen-binding fragment thereof is provided.

Additionally, the present invention provides a fusion anti-SNAP 25 B6 antibody or antigen-binding fragment thereof, in which the TAT peptide represented by SEQ ID NO: 7 is additionally bound to the antibody or antigen-binding fragment thereof.

Additionally, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

Additionally, the present invention provides a recombinant expression vector containing the above nucleic acid molecule.

Additionally, the present invention provides cells isolated by transformation with the above recombinant expression vector.

Additionally, the present invention provides a composition for detecting SNAP 25 antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.

Additionally, the present invention provides a cosmetic composition for preventing or improving skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving skin wrinkles containing the antibody or antigen-binding fragment thereof as an active ingredient.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient.

The present invention relates to an anti-SNAP 25 B6 antibody having the effect of preventing and improving skin wrinkles and its use. More specifically, the present invention relates to an anti-SNAP 25 B6 antibody comprising a heavy chain CDR and a light chain CDR of a specific sequence, or a use thereof. It is about an antigen-binding fragment. The anti-SNAP 25 B6 antibody is expected to be useful in preventing or treating skin wrinkles by inhibiting SNARE complex formation.

Figure 1 shows the results of sensitivity analysis of anti-SNAP 25 B6 scFv antibody.
Figure 2 shows the size and protein purity results through SDS-PAGE after purification of the cell-permeable anti-SNAP 25 B6 scFv protein.
Figure 3 shows the cell-penetrating ability of the cell-penetrating anti-SNAP 25 B6 scFv by Western blotting.
Figure 4 shows the results of native-PAGE analysis of the ability of cell-permeable anti-SNAP 25 B6 scFv to inhibit SNARE complex formation.
Figure 5 shows the results of cell-permeable anti-SNAP 25 B6 scFv inhibition of MMP-1 collagenase expression.

The present invention provides a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6. SNAP 25 B6 antibody or antigen-binding fragment thereof is provided.

Additionally, the present invention provides a fusion anti-SNAP 25 B6 antibody or antigen-binding fragment thereof, in which the TAT peptide represented by SEQ ID NO: 7 is additionally bound to the antibody or antigen-binding fragment thereof.

Meanwhile, 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).

In the present invention, 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, for example, a monoclonal antibody, It may include all clone antibodies, full-length antibodies, and antibody fragments. The term “antibody” may also include bivalent or dual-specific molecules (e.g., bispecific antibodies), diabodies, triabodies, or tetrabodies.

As used herein, the term “monoclonal antibody” refers to an antibody molecule of single molecular composition obtained from a population of substantially identical antibodies, and unlike polyclonal antibodies, which can bind to multiple epitopes, these monoclonal antibodies have specific binding properties. It exhibits single binding and affinity for the epitope. In the present invention, the term “full-length antibody” is 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 is subclassed as gamma1 (γ1), gamma2 (γ2), and gamma3 (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 subtypes include IgG1, IgG2, IgG3, and IgG4.

In the present invention, the term “heavy chain” refers to a full-length heavy chain and fragments thereof including a variable region VH and three constant regions CH1, CH2, and CH3, including an amino acid sequence with sufficient variable region sequence to confer specificity to the antigen. All can be included. Additionally, in the present invention, the term “light chain” may include both a full-length light chain including a variable region VL and a constant region CL containing an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, as well as fragments thereof. there is.

In the present invention, the terms “fragment,” “antibody fragment,” and “antigen-binding fragment” are used interchangeably to refer to any fragment of the 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.

The antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the antibody described herein, but also its biological equivalent, to the extent that it can exhibit the ability to specifically bind to SNAP 25. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. So, based on the benefits, arginine, lacine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.

Additionally, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.

As used herein, the term “nucleic acid molecule” is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also sugar or base sites. Also includes modified analogues. The sequences of nucleic acid molecules encoding the heavy and light chain variable regions of the invention may be modified, including additions, deletions, or non-conservative or conservative substitutions of nucleotides.

Additionally, the present invention provides a recombinant expression vector containing the above nucleic acid molecule.

In the present invention, “vector” refers to a self-replicating DNA molecule used to transport a clonal gene (or other fragment of clonal DNA).

In the present invention, “expression vector” refers to a recombinant DNA molecule containing a desired coding sequence and an appropriate nucleic acid sequence essential for expressing the operably linked coding sequence in a specific host organism. The expression vector may preferably contain one or more selectable markers. The marker is a nucleic acid sequence that has characteristics that can be generally selected by chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples include, but are limited to, antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol. This does not mean that it is possible, and can be appropriately selected by a person skilled in the art.

To express the DNA sequence of the present invention, any of a wide variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include, for example, SV40 or adenovirus early and late promoters, CMV promoters and enhancers, retroviral LTR, lac system, trp system, TAC or TRC system, T3 and T7 promoters. , the main operator and promoter regions of phage lambda, the regulatory region of the fd code protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of the above phosphatases, such as Pho5, of the yeast alpha-mating system. Promoters and other sequences of composition and induction known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof, may be included.

The vector expressing the antibody of the present invention can be either a vector system in which the light and heavy chains are simultaneously expressed in one vector, or a system in which the light and heavy chains are expressed in separate vectors. In the latter case, the two vectors are introduced into the host cell through co-transformation and targeted transformation. Simultaneous transformation is a method of simultaneously introducing vector DNA encoding light and heavy chains into a host cell and then selecting cells that express both light and heavy chains. Targeted transformation selects cells transformed with a vector containing a light chain (or heavy chain) and transforms the selected cells expressing the light chain again with a vector containing a heavy chain (or light chain) to express both light and heavy chains. This is the final method of selecting cells.

Additionally, the present invention provides cells transformed with a recombinant expression vector.

Cells capable of stably and continuously cloning and expressing the vector of the present invention may be any host cell known in the art, such as Escherichia coli, Bacillus subtilis, and Bacillus thuringen. Strains of the genus Bacillus, such as cis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g. Examples include, but are not limited to, prokaryotic host cells such as Staphylocus carnosus.

In the method for producing the antibody or antigen-binding fragment thereof, the transformed cells can be cultured according to appropriate media and culture conditions known in the related art. This culture process can be easily adjusted and used by a person skilled in the art according to the selected strain. Cell culture is divided into suspension culture and adherent culture depending on the growth method of the cells, and batch, fed-batch, and continuous culture depending on the culture method. The medium used for culture must appropriately meet the requirements of the specific strain.

Additionally, the present invention provides a composition for detecting SNAP 25 antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.

Additionally, the present invention provides a cosmetic composition for preventing or improving skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient. Specifically, the composition can inhibit SNARE complex formation.

In addition to the active ingredients, the cosmetic composition may include conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.

The formulation of the cosmetic composition can be prepared in any formulation commonly manufactured in the art, including skin ointment, cream, softening lotion, nourishing lotion, pack, essence, hair tonic, shampoo, rinse, hair conditioner, and hair treatment. Cream, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, eye cream, moisture cream, hand cream, foundation, nutritional essence, sunscreen, soap , 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, tracant, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide can be used as the carrier ingredient. .

If the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as the carrier ingredient. In particular, in the case of a spray, chlorofluorohydrocarbon, propane/butane may be used as the carrier ingredient. Alternatively, it may include 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 esters of glycerol, fatty acid esters of polyethylene glycol or sorbitan.

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

In addition, the present invention provides a health functional food composition for preventing or improving skin wrinkles containing the antibody or antigen-binding fragment thereof as an active ingredient. Specifically, the composition can inhibit SNARE complex formation.

The health functional food composition may be provided in the form of powder, granules, tablets, capsules, syrup, beverage, or pills, and the health food composition is used with other foods or food additives in addition to the composition according to the present invention, which is an active ingredient, and is commonly used It can be used appropriately according to the method. The mixing amount of the active ingredient can be appropriately determined depending on its purpose of use, 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 in accordance with the effective dose of the pharmaceutical composition, but in case of long-term intake for the purpose of health and hygiene or health control It may be below the above range, and since the active ingredient has no safety issues, it is certain that it can be used in amounts above the above range.

There are no particular restrictions on the types of health foods, and examples include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Examples include drinks, alcoholic beverages, and vitamin complexes.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient. Specifically, the composition can inhibit SNARE complex formation.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is one commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch. , acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear. Includes, but is not limited to, magnesium acid and mineral oil. The composition for preventing or treating cancer metastasis of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. 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. It can be administered. When administered orally, proteins or peptides are digested, so 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. It can be administered by.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and is usually A skilled doctor can easily determine and prescribe an effective dosage for desired treatment or prevention.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. Alternatively, it can be manufactured by placing it in a multi-capacity 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 dispersant 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.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating 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-SNAP 25 scFv Antibody screening

1. Implementation of bio-panning

Bio-panning was performed using the OPAL library with a diversity of 7.6 × ¹⁰⁹ . 4 μg of SNAP 25 antigen was immobilized on epoxy magnetic beads and input phage was reacted. The output titer was measured by eluting the phage that reacted with the antigen. The input and output titers were measured every time to obtain bio-panning information and confirm whether it was being implemented normally. For each cycle, 4 × 10 ¹² cfu/mL ≥ phage was used as input phage. The output was 1st - 5 × 10 ⁶ , 2nd - 1.5 × 10 ⁸ , and 3rd - 1.7 × 10 ⁷ cfu/mL, showing that bio-panning was in progress.

2. ELISA analysis

ELISA analysis was performed to select antibodies with high sensitivity and specificity. The obtained phage was infected with E. coli and plated 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 cultured in LB medium, treated with IPTG to express scFv, E. coli was lysed, the soluble fraction was taken, and ELISA was performed. First, 1 μg/mL of SNAP 25 recombinant protein was immobilized on a 96 well ELISA plate and blocked with PBS containing 1% BSA. After 1 hour, the cell lysate obtained above was treated and reacted at 4°C for 16 hours. After washing the plate three times with PBS containing 0.1% Tween20, HRP-conjugated anti-HA antibody was diluted 1:1000 in blocking solution and reacted at room temperature for 1 hour. The plate was washed five times with PBS containing 0.1% Tween20, developed with TMB substrate, and scFv antibodies bound to the antigen were measured using an ELISA reader. 288 antibodies were analyzed, and OD 0.4 was arbitrarily set as a positive guideline and 0.1 as a negative guideline, and 9 types were selected.

3. Sequence analysis

Individual clones were selected through sequencing for the nine positive clones of anti-SNAP 25 selected through ELISA analysis. Sequence analysis was necessary because there was a possibility that overlapping clones existed among the positive clones selected previously. As a result of sequence analysis, it was confirmed that 6 out of 9 positive clones for anti-SNAP 25 were individual clones.

< Example 2> Selected anti-SNAP 25 scFv Antibody purification and sensitivity analysis

1. Anti-SNAP 25 scFv antibody purification

Among the selected positive clones, anti-SNAP 25 scFv B6 antibody (Table 1) with high antigen binding ability was purified. ER2738 E. coli cells transformed with anti-SNAP 25 scFv clone (B6) were cultured in 500 mL of SB media, and the cells were lysed using 1 × TES buffer. Cell lysates were reacted with 0.5 mL Ni beads and eluted using imidazole.

Target Clone Light chain CDR sequence Heavy chain CDR sequence CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 SNAP 25 B6 SGSSSNIGNNYVS ANSH AAWDASLSG SYYMS AISPGSSNK RNSHMFDY

2. Purified anti-SNAP 25 B6 scFv Sensitivity analysis of antibodies

ELISA analysis was used to analyze the sensitivity of the purified anti-SNAP 25 B6 scFv antibody. First, various concentrations of SNAP 25 antigen were immobilized on the bottom of the ELISA plate, blocked using PBS containing 3% BSA, and each well was treated with 10 μg/mL of anti-SNAP 25 B6 scFv antibody for reaction. Unreacted antibodies were removed by washing with PBS containing 0.05% Tween 20, and then analyzed using HRP-conjugated anti-HA antibody diluted 1:1000 in blocking solution. As a result, the Kd of the anti-SNAP 25 B6 scFv antibody was analyzed to be 25.792 nM, and the LOD was analyzed to be 5.07 nM (Figure 1).

< Example 3> Cell permeable anti-SNAP 25 B6 scFv produce

1. TAT-ANTI-SNAP 25 B6 scFv construct production

A PCR product was produced in which a restriction enzyme site and TAT were inserted using primers for the previously selected anti-SNAP 25 B6 scFv antibody gene. The PCR product (insert) and pET28a(+) vector were treated with 1 μL of EcoR I and 1 μL of Xho I , respectively, reacted at 37°C for one hour, and then purified. The concentration of the purified vector and insert was measured with nanodrop, and ligation was performed at room temperature for 16 hours using T4 ligase for each ratio of vector and insert. After ligation, DH5α competent cells were transformed, plated on an LB plate with kanamycin added, and cultured at 37°C for 16 hours. The next day, colonies were taken and inoculated into LB medium with kanamycin added, and incubated at 37°C for 16 hours. It was cultured for some time. The next day, to check whether the insert was inserted, the plasmid was isolated, restricted with EcoR I and Xho I , and the band was confirmed by electrophoresis on a 1% agarose gel.

2. TAT-anti-SNAP 25 scFv refine

The cloned TAT-anti-SNAP 25 scFv clone (B6) was transformed into a BL21 (DE3) E. coli host. The transformants were cultured in LB medium containing kanamycin until the OD ₆₀₀ value reached 0.6, and expression was induced by treatment with 0.5 mM IPTG at 16°C. Cells obtained by centrifuging the culture medium were suspended in lysis buffer (50mM Tris-HCl, pH 7.5, 150mM NaCl), disrupted using an ultrasonicator, and then centrifuged to obtain the supernatant. Resin with affinity to Ni-NTA was used to separate and purify only the inducibly expressed protein, and the purity and size of the purified protein were confirmed through SDS-PAGE analysis (Figure 2).

< Example 4> Cell permeable anti-SNAP 25 B6 scFv Cytotoxicity test

To examine the cytotoxicity of TAT-anti-SNAP 25 B6 scFv, PC12 cells, which are mammalian neural cells, were cultured in a CO ₂ incubator at 37°C. Cultured cells were treated with TAT-anti-SNAP 25 B6 scFv at different concentrations and then 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 TAT-anti-SNAP 25 B6 scFv was treated at concentrations of 0.01, 0.1, 1, 10, 50, and 100 ppm, there was little change in cell shape deformation and cell viability up to a concentration of 100 ppm. Confirmed. Therefore, it was found that TAT-anti-SNAP 25 B6 scFv was not cytotoxic up to a concentration of 100 ppm.

TAT-α-SNAP 25 B6 scFv Concentration (ppm) average error 0 100.00 7.456 0.01 100.04 0.710 0.1 106.26 2.861 One 102.02 2.904 10 101.42 2.534 50 107.56 4.319 100 105.50 0.637

< Example 5> Cell permeable anti-SNAP 25 B6 scFv Cell permeation assay

To confirm cell penetration of TAT-anti-SNAP 25 B6 scFv, PC12 cells were cultured in 6-well plates, replaced with 1.5 mL of fresh culture medium without FBS, and incubated with various concentrations of TAT-anti-SNAP 25 B6. scFv was processed. One hour after treatment, the cells were thoroughly washed with PBS, recovered, and the degree of penetration of TAT-anti-SNAP 25 B6 scFv into the cells was confirmed by Western blot analysis. In order to determine whether the TAT-anti-SNAP 25 B6 scFv permeated into the cells was identified at the correct location (size), purified TAT-anti-SNAP 25 B6 scFv (Control) was also electrophoresed. As shown in Figure 3, it was confirmed that TAT-anti-SNAP 25 B6 scFv effectively penetrated cells in a concentration-dependent manner.

< Example 6> Cell permeable anti-SNAP 25 B6 scFv SNARE complex formation inhibitory ability test

To confirm whether the TAT-anti-SNAP 25 B6 scFv antibody inhibits SNARE complex formation, native-PAGE analysis was performed. Whether the complex formed when SNARE proteins Snap 25, Syntaxin 1A, and Vamp2 protein (LSbio) were mixed at a concentration (1 μg) of 1:1:1, respectively, is inhibited by the addition of TAT-anti-SNAP 25 B6 scFv. was confirmed. 1 μg of each protein was added, treated with TAT-anti-SNAP 25 B6 scFv at various concentrations (0.01, 0.1, 1, 2.5, 5, 10, 20 μg), thoroughly mixed, and reacted at 4°C for 1 hour. When the reaction was completed, sample buffer was added to stop the reaction, and then SNARE complex formation was confirmed on 10% native-PAGE (Figure 4). As a result, it was found that TAT-anti-SNAP 25 B6 scFv effectively inhibits SNARE complex formation.

< Example 7> Cell permeable anti-SNAP 25 B6 scFv MMP -1 Expression suppression evaluation

To investigate the effect of TAT-anti-SNAP 25 B6 scFv on collagen production, changes in expression of MMP-1 (Matrix metalloproteinase-1), known as a collagen degrading enzyme, were measured using Real-Time PCR method. PC12 cells, which are mammalian nerve cells, were maintained at 37°C and cultured in an incubator containing 5% carbon dioxide and then irradiated with UVA using a UV irradiator system. Afterwards, the cells were treated with 1 ppm of TAT-anti-SNAP 25 B6 scFv and 100 ppm of Adenosine as a positive control, and then further cultured under the same culture conditions. After incubation, cells were collected with 300 μL of TRIzol, transferred to a 1.5 mL tube, 50 μL of chloroform was added, mixed, and left at room temperature for 5 minutes. Afterwards, it was centrifuged at 4°C and 15,000 rpm for 15 minutes, and the supernatant was transferred to a new tube, mixed with an equal amount of 2-propanol, left at room temperature for 5 minutes, and then centrifuged at 4°C and 12,000 rpm for 20 minutes. After centrifugation, 2-propanol was discarded, 300 μL of 75% ethanol was added, centrifuged at 4°C and 10,000 rpm for 10 minutes, 75% ethanol was discarded, and the RNA pellet was dried at room temperature to remove remaining ethanol. After dissolving 30 μL of DEPC-treated purified water in the pellet, quantification was performed at 260 nm, and RT (reverse transcription)-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 by Macrogen, Inc., and their base sequences are shown in Table 3. Real-time PCR was performed using TOPreal ^TM Qpcr 2X PreMIX, and the results are shown in Figure 5. TAT-anti-SNAP 25 B6 scFv inhibited MMP-1 collagenase, which causes increased wrinkle formation by ultraviolet rays, and showed a similar effect when treated at 1 ppm as when treated at 100 ppm in the control group.

primer order MMP -One forward 5'-ACGCAGATTTAGCCTCCGAA-3' reverse 5'-TGACTTGGTAATGGGTTGCC-3' GAPDH forward 5'-GACATGCGCGCTGGAGAAAAC-3' reverse 5'-AGCCCAGGATGCCCTTTAGT-3'

< Example 8> Cell permeable anti-SNAP 25 B6 scFv Active oxygen inhibition effect test

To confirm the intracellular reactive oxygen species (ROS) inhibitory effect of TAT-anti-SNAP 25 B6 scFv, PC12 cells, which are mammalian neural cells, were treated with TAT-anti-SNAP 25 B6 scFv and the intracellular reactive oxygen species content was measured using fluorescent staining. It was measured using PC12 cells inoculated in a 96-well plate were maintained at 37°C and cultured in an incubator containing 5% carbon dioxide, and then treated with TAT-anti-SNAP 25 B6 scFv at various concentrations. After incubation for 2 hours, 0.2mM hydrogen peroxide was added to each well and further cultured under the same culture conditions. 20 μM carboxy-H ₂ DCF-DA was added and reacted at 37°C for 30 minutes. After washing thoroughly with PBS, 100 μL of PBS was added again, and fluorescence was measured at 485/525 nm using an ELISA Reader system. As shown in Table 4, it was found that TAT-anti-SNAP 25 B6 scFv effectively inhibits ROS generation.

sample density
(ppm) ROS (fold of control) non treat control 0.2 mm H ₂ O ₂ average error average error TAT-α-SNAP 25 B6 scFv 0 1.000 0.099 1.913 0.215 0.1 0.911 0.035 1.844 0.195 One 0.954 0.043 1.399 0.176 10 0.969 0.051 1.478 0.123 25 1.054 0.048 1.346 0.221 50 1.054 0.074 1.286 0.062 100 1.094 0.095 1.236 0.052

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred implementation examples and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> HAUUL BIO <120> Anti-SNAP 25 antibodies for preventing and improving skin wrinkles and use it <130> DP-2022-0068 <160> 8 <170>CopatentIn 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 <400> 1 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser 1 5 10 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 <400> 2 Ala Asn Ser His One <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 <400> 3 Ala Ala Trp Asp Ala Ser Leu Ser Gly 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 4 Ser Tyr Tyr Met Ser 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 5 Ala Ile Ser Pro Gly Ser Ser Asn Lys 1 5 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 <400> 6 Arg Asn Ser His Met 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 (10)

A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6. SNAP 25 B6 antibody or antigen-binding fragment thereof. A fusion anti-SNAP 25 B6 antibody or antigen-binding fragment thereof, in which the TAT peptide represented by SEQ ID NO: 7 is additionally bound to the antibody or antigen-binding fragment thereof of claim 1. A nucleic acid molecule encoding the antibody of claim 1 or 2 or an antigen-binding fragment thereof. A recombinant expression vector containing the nucleic acid molecule of claim 3. Cells isolated after being transformed with the recombinant expression vector of claim 4. A composition for detecting SNAP 25 antigen comprising the antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient. A cosmetic composition for preventing or improving skin wrinkles, comprising the antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient. The cosmetic composition for preventing or improving skin wrinkles according to claim 7, wherein the composition inhibits the formation of SNARE complexes. A health functional food composition for preventing or improving skin wrinkles, comprising the antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient. A pharmaceutical composition for preventing or treating skin wrinkles, comprising the antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.