WO2021070997A1 - Anti-syntaxin 1a antibody for inhibiting snare complex, and use thereof - Google Patents
Anti-syntaxin 1a antibody for inhibiting snare complex, and use thereof Download PDFInfo
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- WO2021070997A1 WO2021070997A1 PCT/KR2019/013437 KR2019013437W WO2021070997A1 WO 2021070997 A1 WO2021070997 A1 WO 2021070997A1 KR 2019013437 W KR2019013437 W KR 2019013437W WO 2021070997 A1 WO2021070997 A1 WO 2021070997A1
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- syntaxin
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Definitions
- the present invention relates to an anti-Syntaxin 1A antibody that inhibits the SNARE complex and its use.
- SNARE protein refers to a specific group of proteins that are very well conserved across all species, and SNARE complex refers to a complex of these proteins.
- SNARE protein can be divided into target (t-) SNARE and vesicular (v-) SNARE, t-SNARE refers to the SNARE protein present in the presynaptic membrane, and v-SNARE refers to the SNARE protein present in the synaptic vesicle.
- t-SNARE consists of an integral membrane protein called Syntaxin 1A and a soluble NSF attachment protein of 25 kDa (SNAP-25), a peripheral membrane protein, 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).
- VAMP2 vesicle-associated membrane protein 2
- These SNARE proteins have a region of about 60-70 aa called the'SNARE core', and these regions gather together to form a four-helical bundle called the SNARE complex.
- An object of the present invention is to provide an anti-Syntaxin 1A antibody or antigen-binding fragment thereof that inhibits the SNARE complex.
- Another object of the present invention is to provide a fusion anti-Syntaxin 1A antibody or antigen-binding fragment thereof in which a TAT peptide is additionally bound to the anti-Syntaxin 1A 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 comprising 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 Syntaxin 1A antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
- Another object of the present invention is a cosmetic composition for preventing or improving skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient, an antioxidant cosmetic composition, a health functional food composition for preventing or improving skin wrinkles, and for preventing or improving skin wrinkles It is to provide a therapeutic pharmaceutical composition.
- the present invention includes a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3.
- a light chain variable region a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6 anti- A Syntaxin 1A antibody or antigen-binding fragment thereof is provided.
- the present invention provides a fusion anti-Syntaxin 1A 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.
- the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.
- the present invention provides a recombinant expression vector containing the nucleic acid molecule.
- the present invention provides a cell transformed with the recombinant expression vector.
- the present invention provides a composition for detecting Syntaxin 1A antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
- 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.
- the present invention provides an antioxidant cosmetic composition comprising the antibody or antigen-binding fragment thereof as an active ingredient.
- the present invention provides a health functional food composition for preventing or improving skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient.
- 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-Syntaxin 1A antibody that inhibits the SNARE complex and its use, and more particularly, the present invention relates to an anti-Syntaxin 1A antibody or antigen-binding fragment thereof comprising a heavy chain CDR and a light chain CDR of a specific sequence.
- the anti-Syntaxin 1A antibody is expected to be useful for improving or treating skin wrinkles by inhibiting the formation of the SNARE complex.
- Figure 2 shows the size and protein purity results through SDS-PAGE after purification of the cell-permeable anti-Syntaxin 1A scFv protein.
- Figure 3 shows the results of the SNARE complex formation inhibition ability of the cell-permeable anti-Syntaxin 1A scFv protein through native-PAGE analysis.
- Figure 4 shows the results of inhibition of MMP-1 collagenase activity of the cell-permeable anti-Syntaxin 1A scFv protein.
- Figure 5 shows the results of the porcine skin permeability of the cell-permeable anti-Syntaxin 1A scFv protein by DAB staining.
- the present invention provides a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3; And a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6 anti- A Syntaxin 1A antibody or antigen-binding fragment thereof is provided.
- the present invention provides a fusion anti-Syntaxin 1A 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.
- 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).
- the term “antibody” refers to a protein molecule that acts as a receptor for specifically recognizing an antigen, including immunoglobulin molecules that are immunologically reactive with a specific antigen, for example, a monoclonal antibody, c. It may include both clonal antibodies, full-length antibodies, and antibody fragments.
- the term “antibody” may include a bivalent or bispecific molecule (eg, a bispecific antibody), a diabody, a triabbody, or a tetrabody.
- 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 is different from a polyclonal antibody capable of binding to multiple epitopes, It shows a single binding and affinity for the epitope.
- 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 a heavy chain by a disulfide bond.
- the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types, and has subclasses of gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), and gamma 3 ( ⁇ 3). ), gamma4( ⁇ 4), alpha1( ⁇ 1) and alpha2( ⁇ 2).
- the constant region of the light chain has kappa ( ⁇ ) and lambda ( ⁇ ) types.
- IgG is a subtype, and includes IgG1, IgG2, IgG3 and IgG4.
- the term “heavy chain” refers to a full-length heavy chain including a variable region VH and three constant regions CH1, CH2 and CH3 and fragments thereof, including an amino acid sequence having a sufficient variable region sequence to confer antigen specificity. It can contain all.
- the term “light chain” may include both a full-length light chain including a variable region VL and a constant region CL including an amino acid sequence having a sufficient variable region sequence to impart specificity to the antigen, and fragments thereof. have.
- fragment 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, and the like.
- the antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the antibody described herein, but also a biological equivalent thereof within a range capable of exhibiting the ability to specifically bind to Syntaxin 1A.
- 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.
- Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like.
- 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.
- arginine, racine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
- the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.
- nucleic acid molecule has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also sugar or base moieties. Also includes modified analogs.
- the sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified, and the modification includes addition, deletion, or non-conservative or conservative substitution of nucleotides.
- the present invention provides a recombinant expression vector containing the nucleic acid molecule.
- vector refers to a DNA molecule that replicates itself, which is used to carry a clonal gene (or other fragment of clonal DNA).
- expression vector refers to a recombinant DNA molecule comprising a target coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host organism.
- the expression vector may preferably contain one or more selectable markers.
- the marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, and all genes capable of distinguishing a transformed cell from a non-transformed cell are applicable. Examples include antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol, but limited to this. It does not become, and can be appropriately selected by a person skilled in the art.
- any of a wide variety of expression control sequences can be used in the vector.
- useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTR of retroviruses, lac system, trp system, TAC or TRC system, T3 and T7 promoters.
- Promoters and other sequences of constructs and induction known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof may be included.
- the vector expressing the antibody of the present invention may be 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, respectively. In the latter case, both vectors are introduced into host cells through co-transfomation and targeted transformation.
- Simultaneous transformation is a method of simultaneously introducing each vector DNA encoding a light chain and a heavy chain into a host cell, and then selecting cells expressing both the light and heavy chains.
- Target transformation involves selecting cells transformed with a vector containing a light chain (or heavy chain) and transforming the selected cells expressing a light chain with a vector containing a heavy chain (or light chain) to express both light and heavy chains. This is a method of finally selecting cells.
- the present invention provides a cell transformed with a recombinant expression vector.
- Cells capable of stably 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.
- Bacillus strains 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).
- the transformed cells may be cultured according to a suitable medium and culture conditions known in the related art.
- This culture process can be easily adjusted and used by those of ordinary skill in the art according to the selected strain.
- Cell culture is divided into suspension culture and adherent culture according to the cell growth method, and batch, fed-batch, and continuous culture methods according to the culture method.
- the medium used for cultivation should adequately meet the requirements of the specific strain.
- the present invention provides a composition for detecting Syntaxin 1A antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
- 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.
- the composition can inhibit SNARE complex formation.
- the present invention provides an antioxidant cosmetic composition comprising the antibody or antigen-binding fragment thereof as an active ingredient.
- the composition can suppress the generation of active oxygen.
- the cosmetic composition may include a stabilizer, a solubilizing agent, a general auxiliary agent such as vitamins, pigments and fragrances, and a carrier.
- the formulation of the cosmetic composition may be prepared in any formulation conventionally prepared in the art, and external ointment for skin, cream, softening lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment Ment, 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 , Cleansing foam, cleansing lotion, cleansing cream, body lotion, and may have a formulation selected from the group consisting of a body cleanser, but is not limited thereto.
- the composition of each of these formulations may contain various bases and additives necessary and appropriate for formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.
- the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. .
- lactose 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.
- additional chlorofluorohydrocarbon, propane/butane Or a propellant such as dimethyl ether.
- a solvent, a solubilizing agent or an 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, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
- a carrier component such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.
- a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.
- the present invention provides a health functional food composition for preventing or improving skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient.
- the composition can inhibit SNARE complex formation.
- the health functional food composition may be provided in the form of powder, granule, tablet, capsule, syrup, beverage or pill, and the health food composition is used with other foods or food additives in addition to the composition according to the present invention as an active ingredient, and is usually It can be appropriately used according to the phosphorus method.
- the mixing amount of the active ingredient may be appropriately determined according to the 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 can be used in accordance with 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 In the above range, it is clear that the active ingredient can be used in an amount above the above range because there is no problem in terms of safety.
- 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 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 commonly used in formulation, and lactose, dextrose, sucrose, sorbitol, mannitol, starch , Gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear It includes, but is not limited to, magnesium acid and mineral oil.
- 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 and rectal administration, etc. It can be administered as.
- parenteral administration intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and rectal administration, etc. It can be administered as.
- the oral composition may be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention is an arbitrary device capable of moving the active substance to the target cell. It can be administered by.
- a suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, and is usually As such, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis.
- the pharmaceutical composition of the present invention is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or Alternatively, it may be manufactured by placing it in a multi-capacity container.
- 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 include a dispersant or a stabilizer.
- composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent.
- Bio-panning was performed using an OPAL library having a diversity of 7.6 ⁇ 10 9. 4 ⁇ g of Syntaxin 1A antigen was immobilized on the epoxy magnetic bead and the input phage was reacted. The phage that reacted with the antigen was eluted and the output titer was measured. By measuring the input and output titer every number of times, bio-panning information was obtained and it was confirmed whether it is being performed normally. For each input phage, a phage of 4 ⁇ 10 12 cfu/mL ⁇ was used. The output was 1st-1.2 ⁇ 10 5 , 2nd-2 ⁇ 10 7 , and 3rd-3 ⁇ 10 7 cfu/mL. It can be seen that the amplification increases as bio-panning proceeds. Therefore, it could be confirmed that the bio-panning proceeded normally.
- ELISA analysis was performed to select highly sensitive and highly specific antibodies.
- the obtained phage was infected with E. coli and spread on LB plate to which antibiotics were added. After culturing in a 30° C. incubator for 16 hours, the generated colonies were randomly collected. Each colony was cultured in LB medium, and then treated with IPTG to express scFv, and E. coli was lysed to obtain a soluble fraction, followed by ELISA. First, 1 ⁇ g/mL of Syntaxin 1A recombinant protein was fixed 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.
- 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, it was colored with a TMB substrate, and the scFv antibody bound to the antigen was measured with an ELISA leader. 1 shows the results of ELISA performed using Syntaxin 1A protein. 96 antibodies were analyzed, and 18 species were selected by arbitrarily setting an OD of 0.4 as a positive guideline and 0.1 as a negative guideline (FIG. 1).
- One type of anti-Syntaxin 1A scFv (Table 1) selected above was used as a primer to produce a PCR product containing a restriction enzyme site and TAT. 30 ⁇ L of each PCR product is treated with 4 ⁇ L of buffer 3.1, 1 ⁇ L of Sal I, 1 ⁇ L of Xho I , and 4 ⁇ L of distilled water and reacted at 37°C for an hour, and then the DNA is separated to secure an insert to be inserted into the vector. I did.
- pET28a(+) vector is transformed into DH5 ⁇ competent cells, spread on LB plates with kanamycin added, incubated at 37°C for 16 hours, and colonies are taken the next day and kanamycin It was inoculated on the added LB media and incubated at 37°C for 16 hours.
- the next day after separating the vector using a mini-prep kit, add 30 ⁇ L of vector to 4 ⁇ L of buffer 3.1, 1 ⁇ L of Sal I, 1 ⁇ L of Xho I, 1 ⁇ L of CIP, and 3 ⁇ L of distilled water were treated and reacted at 37° C. for one hour, followed by purification.
- 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 is complete, DH5 ⁇ is transformed, spread on an LB plate containing kanamycin, incubated at 37°C for 16 hours, and colonies are taken the next day, and LB media with kanamycin added. And inoculated at 37° C. for 16 hours. The next day, to check whether the insert was inserted, the plasmid was separated and cut with Sal I and Xho I , and the band was identified by electrophoresis on 1% agarose gel.
- the cloned TAT-anti-Syntaxin 1A scFv antibody clone was transformed into a BL21(DE3) E. coli host, and the transformant was induction of 1 mM IPTG.
- the scFv antibody was suspended in lysis buffer (50 mM Tris-HCl, p H 7.5, 150 mM NaCl), crushed using an ultrasonic grinder, and centrifuged to obtain a supernatant.
- the protein was purified using a resin having an affinity for Ni-NTA. The purified protein was confirmed to have a protein size of about 30 kDa through SDS-PAGE analysis (FIG. 2).
- TAT-anti-Syntaxin 1A scFv antibody In order to investigate the cytotoxicity of the TAT-anti-Syntaxin 1A scFv antibody, PC12 cells, which are mammalian neurons, were cultured in a 37°C, CO 2 incubator. After incubation, the TAT-anti-Syntaxin 1A scFv antibody was treated by concentration, and then further cultured under the same culture conditions. Add MTT ⁇ 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide ⁇ solution to incubate, remove the culture solution, add DMSO (Dimethyl sulfoxide), shake appropriately, and ELISA Reader system at 570 nm The absorbance was measured by, and the values are shown in Table 2.
- MTT ⁇ 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide ⁇ solution to incubate, remove
- TAT-anti-Syntaxin 1A scFv As shown in Table 2, when TAT-anti-Syntaxin 1A scFv was treated at a concentration of 0.01, 0.1, 1, 10, 50, 100 ppm, the change in cell shape and cell viability of mammalian neurons up to 100 ppm concentration was It was confirmed that almost none. Therefore, it was found that the TAT-anti-Syntaxin 1A scFv was a substance without cytotoxicity up to a concentration of 100 ppm.
- Example 4 TAT-anti-Syntaxin 1A Concentration (ppm) Average error 0 100.0 2.39 0.01 89.6 2.05 0.1 81.0 0.71 One 96.3 2.43 10 101.6 7.75 50 123.5 4.85 100 124.3 5.37
- TAT-anti-Syntaxin 1A scFv antibody inhibits SNARE complex formation.
- native-PAGE analysis was performed.
- the SNARE proteins SNAP25, Syntaxin 1A and VAMP2 proteins (LSbio) are mixed at a concentration of 1:1:1 (1 ⁇ g)
- 1 ⁇ g of each protein was added, and each of the TAT-anti-Syntaxin 1A scFv antibodies was treated at different concentrations (0.5, 1, 2, 5, 10, 20 ⁇ g), then quickly mixed, and then reacted at 4° C. for 1 hour.
- TAT-anti-Syntaxin 1A scFv the inhibitory effect of TAT-anti-Syntaxin 1A scFv on MMP-1 activity was tested using a Real Time PCR method.
- PC12 cells which are mammalian neurons, were cultured in a 37°C, CO 2 incubator. After incubation, UVA was irradiated using a UV irradiator system. Thereafter, 10 ppm of TAT-anti-Syntaxin 1A scFv antibody and 100 ppm of adenosine as a positive control were treated, and then further cultured under the same culture conditions.
- RT-PCR was performed using 1 ⁇ g of total RNA and TOPscript RT dry mix. MMP-1 and GAPDH used in Real Time PCR were synthesized and used in Macrogen, and the nucleotide sequences are shown in Table 3 below.
- Real Time PCR was performed using TOPreal TM Qpcr 2X PreMIX. As shown in FIG. 4, TAT-anti-Syntaxin 1A scFv inhibited MMP-1 collagen-degrading enzyme, which is the cause of increased wrinkle formation by ultraviolet rays. It was confirmed that the wrinkle improvement effect was far superior to that of the control when 10 ppm was treated.
- TAT-anti-Syntaxin 1A scFv In order to confirm the skin penetration of TAT-anti-Syntaxin 1A scFv, it was confirmed by tissue staining using pig skin.
- Pig skin was treated with 10 ⁇ g of TAT-anti-Syntaxin 1A scFv antibody and incubated for 16 hours in a 37°C, CO 2 incubator. After fixing the pig skin with 4% formaldehyde, a paraffin block was prepared, and the tissue was excised at 5 ⁇ m using a microtome. Thereafter, the tissue was mounted on the slide, paraffin was removed, and the tissue section was blocked for 30 minutes after undergoing a hydration process. Anti-His HRP antibody was reacted for 1 hour at room temperature to the blocked tissue.
- 5 is a result of comparing the skin permeability of anti-Syntaxin 1A scFv and TAT-anti-Syntaxin 1A scFv. As shown in FIG. 5, it was confirmed that the anti-Syntaxin 1A scFv was not delivered into the pig skin tissue at all, whereas the TAT-anti-Syntaxin 1A scFv had an efficient skin delivery ability.
- PC12 cells which are mammalian neurons, were treated with TAT-anti-Syntaxin 1A scFv antibody and then the intracellular active oxygen content was fluorescently stained It was measured using.
- PC12 cells inoculated in a 96-well plate were cultured in a 37° C., CO 2 incubator and treated with TAT-anti-Syntaxin 1A scFv antibody (10, 20 ppm). After incubation for 2 hours, 0.2 mM hydrogen peroxide was added to each well, and then further cultured under the same culture conditions.
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Abstract
The present invention relates to an anti-syntaxin 1A antibody for inhibiting a SNARE complex, and a use thereof and, more specifically, to an anti-syntaxin 1A antibody comprising heavy chain CDRs and light chain CDRs of specific sequences, or an antigen-binding fragment thereof. The anti-syntaxin 1A antibody inhibits the formation of a SNARE complex, and thus is expected to be effectively used in the reduction or treatment of skin wrinkles.
Description
본 발명은 SNARE 복합체를 억제하는 항-Syntaxin 1A 항체 및 이의 용도에 대한 것이다.The present invention relates to an anti-Syntaxin 1A antibody that inhibits the SNARE complex and its use.
세포 내에서 일어나는 막융합은 SNARE(soluble N-ethylmaleimide sensitive factor attachment protein receptor)라고 하는 단백질들에 의해 일어난다. SNARE 단백질은 모든 종에 걸쳐 매우 잘 보존되어 있는 특정 단백질 군을 말하며, SNARE 복합체(complex)는 이 단백질들의 복합체를 의미한다. SNARE 단백질은 target (t-) SNARE와 vesicular (v-) SNARE로 나눌 수 있으며, t-SNARE는 presynaptic membrane에 존재하는 SNARE 단백질을 말하며, v-SNARE는 synaptic vesicle에 존재하는 SNARE 단백질을 일컫는다. t-SNARE는 Syntaxin 1A로 불리우는 막단백질(integral membrane protein)과 peripheral membrane protein인 SNAP-25(soluble NSF attachment protein of 25 kDa)으로 이루어지며 그 기능적 단위는 이의 복합체 (t-SNARE complex)로 생각되고 있다. v-SNARE는 vesicle-associated membrane protein 2 (VAMP2 또는 synaptobrevin)라는 막 단백질을 말한다. 이러한 SNARE 단백질들은 'SNARE core'라고 불리는 60~70 aa 정도의 부위를 가지고 있는데, 이 부위들이 서로 모여 SNARE 복합체라고 불리는 four-helical bundle을 형성하게 된다.Membrane fusion within cells is caused by proteins called soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE). SNARE protein refers to a specific group of proteins that are very well conserved across all species, and SNARE complex refers to a complex of these proteins. SNARE protein can be divided into target (t-) SNARE and vesicular (v-) SNARE, t-SNARE refers to the SNARE protein present in the presynaptic membrane, and v-SNARE refers to the SNARE protein present in the synaptic vesicle. t-SNARE consists of an integral membrane protein called Syntaxin 1A and a soluble NSF attachment protein of 25 kDa (SNAP-25), a peripheral membrane protein, and its functional unit is thought to be its complex (t-SNARE complex). have. v-SNARE refers to a membrane protein called vesicle-associated membrane protein 2 (VAMP2 or synaptobrevin). These SNARE proteins have a region of about 60-70 aa called the'SNARE core', and these regions gather together to form a four-helical bundle called the SNARE complex.
상기 SNARE 복합체와 관련된 연구가 활발하게 진행 중이지만, SNARE 복합체를 억제하는 항체에 대한 연구는 여전히 부족한 실정이다.Although studies related to the SNARE complex are actively in progress, studies on antibodies that inhibit the SNARE complex are still insufficient.
본 발명의 목적은 SNARE 복합체를 억제하는 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편을 제공하는 데에 있다.An object of the present invention is to provide an anti-Syntaxin 1A antibody or antigen-binding fragment thereof that inhibits the SNARE complex.
본 발명의 다른 목적은 상기 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편에 추가적으로 TAT 펩타이드가 결합된 융합 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편을 제공하는 데에 있다.Another object of the present invention is to provide a fusion anti-Syntaxin 1A antibody or antigen-binding fragment thereof in which a TAT peptide is additionally bound to the anti-Syntaxin 1A antibody or antigen-binding fragment thereof.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자, 상기 핵산 분자를 포함하는 재조합 발현벡터 및 상기 재조합 발현벡터로 형질전환된 세포를 제공하는 데에 있다. 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 comprising the nucleic acid molecule, and a cell transformed with the recombinant expression vector.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 Syntaxin 1A 항원 검출용 조성물을 제공하는 데에 있다. Another object of the present invention is to provide a composition for detecting Syntaxin 1A antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
본 발명의 또 다른 목적은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 개선용 화장료 조성물, 항산화용 화장료 조성물, 피부 주름 예방 또는 개선용 건강기능식품 조성물과, 피부 주름 예방 또는 치료용 약학 조성물을 제공하는 데에 있다.Another object of the present invention is a cosmetic composition for preventing or improving skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient, an antioxidant cosmetic composition, a health functional food composition for preventing or improving skin wrinkles, and for preventing or improving skin wrinkles It is to provide a therapeutic pharmaceutical composition.
상기 목적을 달성하기 위하여, 본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2 및 서열번호 3으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3을 포함하는 경쇄 가변 영역; 및 서열번호 4로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2 및 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3을 포함하는 중쇄 가변 영역을 포함하는 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편을 제공한다.In order to achieve the above object, the present invention includes a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3. A light chain variable region; And a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6 anti- A Syntaxin 1A antibody or antigen-binding fragment thereof is provided.
또한, 본 발명은 항체 또는 그의 항원 결합 단편에 추가적으로 서열번호 7로 표시되는 TAT 펩타이드가 결합된 융합 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편을 제공한다.In addition, the present invention provides a fusion anti-Syntaxin 1A 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.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자를 제공한다.In addition, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.
또한, 본 발명은 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공한다.In addition, the present invention provides a recombinant expression vector containing the nucleic acid molecule.
또한, 본 발명은 상기 재조합 발현벡터로 형질전환된 세포를 제공한다.In addition, the present invention provides a cell transformed with the recombinant expression vector.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 Syntaxin 1A 항원 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting Syntaxin 1A antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 개선용 화장료 조성물을 제공한다.In addition, 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.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 항산화용 화장료 조성물을 제공한다.In addition, the present invention provides an antioxidant cosmetic composition comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving skin wrinkles comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 치료용 약학조성물을 제공한다.In addition, 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.
본 발명은 SNARE 복합체를 억제하는 항-Syntaxin 1A 항체 및 이의 용도에 관한 것으로서, 더욱 상세하게, 본 발명은 특정 서열의 중쇄 CDR 및 경쇄 CDR을 포함하는 항-Syntaxin 1A 항체 또는 이의 항원 결합 단편에 대한 것이다. 상기 항-Syntaxin 1A 항체는 SNARE 복합체 형성을 억제하여 피부 주름 개선 또는 치료에 유용하게 활용될 수 있을 것으로 예상된다.The present invention relates to an anti-Syntaxin 1A antibody that inhibits the SNARE complex and its use, and more particularly, the present invention relates to an anti-Syntaxin 1A antibody or antigen-binding fragment thereof comprising a heavy chain CDR and a light chain CDR of a specific sequence. will be. The anti-Syntaxin 1A antibody is expected to be useful for improving or treating skin wrinkles by inhibiting the formation of the SNARE complex.
도 1은 항-Syntaxin 1A scFv 항체 선별을 위한 ELISA 분석 결과를 나타낸 것이다.1 shows the results of ELISA analysis for screening anti-Syntaxin 1A scFv antibodies.
도 2은 세포투과성 항-Syntaxin 1A scFv 단백질의 정제 후 SDS-PAGE를 통해 크기 및 단백질 순도 결과를 나타낸 것이다.Figure 2 shows the size and protein purity results through SDS-PAGE after purification of the cell-permeable anti-Syntaxin 1A scFv protein.
도 3은 세포투과성 항-Syntaxin 1A scFv 단백질의 SNARE 복합체 형성 저해능을 native-PAGE 분석을 통해 결과를 나타낸 것이다.Figure 3 shows the results of the SNARE complex formation inhibition ability of the cell-permeable anti-Syntaxin 1A scFv protein through native-PAGE analysis.
도 4은 세포투과성 항-Syntaxin 1A scFv 단백질의 MMP-1 콜라겐 분해효소 활성 억제 결과를 나타낸 것이다.Figure 4 shows the results of inhibition of MMP-1 collagenase activity of the cell-permeable anti-Syntaxin 1A scFv protein.
도 5는 세포투과성 항-Syntaxin 1A scFv 단백질의 돼지피부 투과능을 DAB 염색법으로 결과를 나타낸 것이다.Figure 5 shows the results of the porcine skin permeability of the cell-permeable anti-Syntaxin 1A scFv protein by DAB staining.
본 발명은 서열번호 1로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2 및 서열번호 3으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3을 포함하는 경쇄 가변 영역; 및 서열번호 4로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2 및 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3을 포함하는 중쇄 가변 영역을 포함하는 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편을 제공한다.The present invention provides a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3; And a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6 anti- A Syntaxin 1A antibody or antigen-binding fragment thereof is provided.
또한, 본 발명은 항체 또는 그의 항원 결합 단편에 추가적으로 서열번호 7로 표시되는 TAT 펩타이드가 결합된 융합 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편을 제공한다.In addition, the present invention provides a fusion anti-Syntaxin 1A 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.
한편, 서열번호 1 내지 서열번호 6로 표시되는 아미노산으로 이루어진 CDR들은 표 1에 기재하였다.Meanwhile, the CDRs consisting of amino acids represented by SEQ ID NO: 1 to SEQ ID NO: 6 are listed in Table 1.
또한, 본 발명에 사용된 TAT 펩타이드의 아미노산 서열은 "YGRKKRRQRRR" (서열번호 7)이고, TAT 펩타이드의 염기서열은 "TAT GGC CGC AAA AAA CGC CGC CAG CGC CGC CGC" (서열번호 8)이다. 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).
본 발명에서 용어, “항체”는 면역학적으로 특정 항원과 반응성을 갖는 면역글로불린 분자를 포함하는, 항원을 특이적으로 인식하는 수용체 역할을 하는 단백질 분자를 의미하며, 그 예로, 단일 클론 항체, 다클론 항체, 전장항체(full-length antibody) 및 항체 단편을 모두 포함할 수 있다. 또한 상기 용어, “항체”는 이가(bivalent) 또는 이중 특이성 분자(예컨대, 이중특이성 항체), 디아바디, 트리아바디 또는 테트라바디를 포함할 수 있다.In the present invention, the term "antibody" refers to a protein molecule that acts as a receptor for specifically recognizing an antigen, including immunoglobulin molecules that are immunologically reactive with a specific antigen, for example, a monoclonal antibody, c. It may include both clonal antibodies, full-length antibodies, and antibody fragments. In addition, the term “antibody” may include a bivalent or bispecific molecule (eg, a bispecific antibody), a diabody, a triabbody, or a tetrabody.
본 발명에서 용어, “단일 클론 항체”는 실질적으로 동일한 항체 집단에서 수득한 단일 분자 조성의 항체 분자를 지칭하고, 이러한 단일 클론 항체는 다클론 항체가 여러 개의 에피토프에 결합할 수 있는 것과 달리, 특정 에피토프에 대해 단일 결합성 및 친화도를 나타낸다. 본 발명에서 용어, “전장항체”는 2 개의 전체 길이의 경쇄 및 2 개의 전체 길이의 중쇄를 가지는 구조이며, 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다. IgG는 서브타입(subtype)으로, IgG1, IgG2, IgG3 및 IgG4를 포함한다.In the present invention, 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 is different from a polyclonal antibody capable of binding to multiple epitopes, It shows a 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 a 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 has subclasses of gamma 1 (γ1), gamma 2 (γ2), and gamma 3 (γ3). ), gamma4(γ4), alpha1(α1) and alpha2(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.
본 발명에서 용어, “중쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 VH 및 3 개의 불변 영역 CH1, CH2 및 CH3를 포함하는 전체길이 중쇄 및 이의 단편을 모두 포함할 수 있다. 또한, 본 발명에서 용어, “경쇄”는 항원에 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 VL 및 불변 영역 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 포함할 수 있다.In the present invention, the term “heavy chain” refers to a full-length heavy chain including a variable region VH and three constant regions CH1, CH2 and CH3 and fragments thereof, including an amino acid sequence having a sufficient variable region sequence to confer antigen specificity. It can contain all. In addition, 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 including an amino acid sequence having a sufficient variable region sequence to impart specificity to the antigen, and fragments thereof. have.
본 발명에서 용어, “단편”, “항체 단편” 및 “항원 결합 단편”은 항체의 항원결합 기능을 보유하는 본 발명의 항체의 임의의 단편을 지칭하는 것으로 호환적으로 사용된다. 예시적인 항원 결합 단편은 Fab, Fab', F(ab')2 및 Fv 등을 포함하나, 이에 제한되지 않는다.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, and the like.
본 발명의 항체 또는 그의 항원 결합 단편은 Syntaxin 1A에 특이적으로 결합하는 능력을 나타낼 수 있는 범위 내에서, 본 명세서에 기재된 항체의 서열뿐만 아니라, 이의 생물학적 균등물도 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은 예를 들어, 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 리신과 히스티딘은 모두 양전하를 띈 잔기이고; 알라닌, 글리신과 세린은 유사한 크기를 가지며; 페닐알라닌, 트립토판과 티로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이점에 기초하여, 아르기닌, 라신과 히스티딘; 알라닌, 글리신과 세린; 그리고 페닐알라닌, 트립토판과 티로신은 생물학적으로 기능 균등물이라 할 수 있다.The antibody or antigen-binding fragment thereof of the present invention may include not only the sequence of the antibody described herein, but also a biological equivalent thereof within a range capable of exhibiting the ability to specifically bind to Syntaxin 1A. 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. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, 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. Thus, on the basis of this, arginine, racine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자를 제공한다.In addition, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof.
본 명세서에서 사용되는 용어, “핵산 분자”는 DNA(gDNA 및 cDNA) 및 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산 분자에서 기본 구성단위인 뉴클레오티드는 자연의 뉴클레오티드뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다. 본 발명의 중쇄 및 경쇄 가변 영역을 코딩하는 핵산 분자의 서열은 변형될 수 있으며, 상기 변형은 뉴클레오티드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다.As used herein, the term "nucleic acid molecule" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also sugar or base moieties. Also includes modified analogs. The sequence of the nucleic acid molecule encoding the heavy and light chain variable regions of the present invention may be modified, and the modification includes addition, deletion, or non-conservative or conservative substitution of nucleotides.
또한, 본 발명은 상기 핵산 분자를 포함하는 재조합 발현벡터를 제공한다.In addition, the present invention provides a recombinant expression vector containing the nucleic acid molecule.
본 발명에 있어서, "벡터"는 클론유전자(또는 클론 DNA의 다른 조각)를 운반하는데 사용되는 스스로 복제되는 DNA 분자를 의미한다.In the present invention, "vector" refers to a DNA molecule that replicates itself, which is used to carry a clonal gene (or other fragment of clonal DNA).
본 발명에서 있어서, “발현 벡터”는 목적한 코딩 서열과, 특정 숙주 생물에서 작동 가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다. 발현 벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질 전환된 세포를 비 형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 앰피실린(Ampicillin), 카나마이신(Kanamycin), 제네티신(Geneticin; G418), 블레오마이신(Bleomycin), 하이그로마이신(Hygromycin), 클로람페니콜(Chloramphenicol) 과 같은 항생제 내성 유전자가 있으나, 이에 한정되는 것은 아니며, 당업자에 의해 적절히 선택 가능하다.In the present invention, "expression vector" refers to a recombinant DNA molecule comprising a target coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host organism. The expression vector may preferably contain one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, and all genes capable of distinguishing a transformed cell from a non-transformed cell are applicable. Examples include antibiotic resistance genes such as Ampicillin, Kanamycin, Geneticin (G418), Bleomycin, Hygromycin, and Chloramphenicol, but limited to this. It does not become, and can be appropriately selected by a person skilled in the art.
본 발명의 DNA 서열을 발현시키기 위하여, 매우 다양한 발현 조절 서열 중 어느 것이라도 벡터에 사용될 수 있다. 유용한 발현 조절서열의 예에는, 예를 들어, SV40 또는 아데노바이러스의 초기 및 후기 프로모터들, CMV의 프로모터와 인핸서, 레트로바이러스의 LTR, lac 시스템, trp 시스템, TAC 또는 TRC 시스템, T3 및 T7 프로모터들, 파지 람다의 주요 오퍼레이터 및 프로모터 영역, fd 코드 단백질의 조절 영역, 3-포스포글리세레이트 키나제 또는 다른 글리콜분해 효소에 대한 프로모터, 상기 포스파타제의 프로모터들, 예를 들어 Pho5, 효모 알파-교배 시스템의 프로모터 및 원핵세포 또는 진핵 세포 또는 이들의 바이러스의 유전자의 발현을 조절하는 것으로 알려진 구성과 유도의 기타 다른 서열 및 이들의 여러 조합이 포함될 수 있다.In order 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, early and late promoters of SV40 or adenovirus, promoters and enhancers of CMV, LTR of retroviruses, lac system, trp system, TAC or TRC system, T3 and T7 promoters. , The main operator and promoter region of phage lambda, the regulatory region of the fd coding protein, the promoter for 3-phosphoglycerate kinase or other glycolase, the promoters of the phosphatase, e.g. Pho5, of the yeast alpha-crossing system Promoters and other sequences of constructs and induction known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof may be included.
본 발명의 항체를 발현하는 벡터는, 경쇄와 중쇄가 하나의 벡터에서 동시에 발현되는 벡터 시스템이거나 또는 경쇄와 중쇄를 각각 별도의 벡터에서 발현시키는 시스템 모두 가능하다. 후자의 경우, 두 벡터는 동시 형질전환(co-transfomation) 및 표적 형질전환(targeted transformation)을 통하여 숙주세포로 도입된다. 동시 형질전환은 경쇄 및 중쇄를 코딩하는 각각의 벡터 DNA를 동시에 숙주세포로 도입한 뒤 경쇄와 중쇄를 모두 발현하는 세포를 선별하는 방법이다. 표적 형질전환은 경쇄(또는 중쇄)를 포함하는 벡터로 형질전환 된 세포를 선별하고 경쇄를 발현하는 선별된 세포를 중쇄(또는 경쇄)를 포함하는 벡터로 다시 형질전환 하여 경쇄 및 중쇄 모두를 발현하는 세포를 최종적으로 선별하는 방법이다.The vector expressing the antibody of the present invention may be 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, respectively. In the latter case, both vectors are introduced into host cells through co-transfomation and targeted transformation. Simultaneous transformation is a method of simultaneously introducing each vector DNA encoding a light chain and a heavy chain into a host cell, and then selecting cells expressing both the light and heavy chains. Target transformation involves selecting cells transformed with a vector containing a light chain (or heavy chain) and transforming the selected cells expressing a light chain with a vector containing a heavy chain (or light chain) to express both light and heavy chains. This is a method of finally selecting cells.
또한, 본 발명은 재조합 발현벡터로 형질전환된 세포를 제공한다. In addition, the present invention provides a cell transformed with a recombinant expression vector.
본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 세포는 관련 기술 분야에 공지된 임의의 숙주 세포일 수 있으며, 예컨대, 에스케리치아 콜라이(Escherichia coli), 바실러스 서브틸리스 및 바실러스 츄린겐시스와 같은 바실러스 속 균주, 스트렙토마이세스(Streptomyces), 슈도모나스(Pseudomonas)(예를 들면, 슈도모나스 푸티다(Pseudomonas putida)), 프로테우스 미라빌리스(Proteus mirabilis) 또는 스타필로코쿠스(Staphylococcus)(예를 들면, 스타필로코쿠스 카르노수스(Staphylocus carnosus))와 같은 원핵 숙주 세포를 포함하나, 이에 제한되는 것은 아니다.Cells capable of stably 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. Bacillus strains 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).
상기 항체 또는 그의 항원 결합 단편의 제조 방법에서 형질전환 세포의 배양은 관련 기술 분야에 공지된 적당한 배지와 배양조건에 따라 이루어질 수 있다. 이러한 배양과정은 통상의 기술자라면 선택되는 균주에 따라 용이하게 조정하여 사용할 수 있다. 세포 배양은, 세포의 성장 방식에 따라 현탁배양과 부착배양, 배양방법에 따라 회분식, 유가식 및 연속배양식의 방법으로 구분된다. 배양에 사용되는 배지는 특정한 균주의 요구조건을 적절하게 만족시켜야 한다.In the method for preparing the antibody or antigen-binding fragment thereof, the transformed cells may be cultured according to a suitable medium and culture conditions known in the related art. This culture process can be easily adjusted and used by those of ordinary skill in the art according to the selected strain. Cell culture is divided into suspension culture and adherent culture according to the cell growth method, and batch, fed-batch, and continuous culture methods according to the culture method. The medium used for cultivation should adequately meet the requirements of the specific strain.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 Syntaxin 1A 항원 검출용 조성물을 제공한다.In addition, the present invention provides a composition for detecting Syntaxin 1A antigen comprising the antibody or antigen-binding fragment thereof as an active ingredient.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 개선용 화장료 조성물을 제공한다. 상세하게는, 상기 조성물은 SNARE 복합체 형성을 억제할 수 있다.In addition, 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.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 항산화용 화장료 조성물을 제공한다. 상세하게는, 상기 조성물은 활성산소 생성을 억제할 수 있다.In addition, the present invention provides an antioxidant cosmetic composition comprising the antibody or antigen-binding fragment thereof as an active ingredient. Specifically, the composition can suppress the generation of active oxygen.
상기 화장료 조성물은 유효성분 외에 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함할 수 있다.In addition to the active ingredient, the cosmetic composition may include a stabilizer, a solubilizing agent, a general auxiliary agent such as vitamins, pigments and fragrances, and a carrier.
상기 화장료 조성물의 제형은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 피부외용연고, 크림, 유연화장수, 영양화장수, 팩, 에센스, 헤어토닉, 샴푸, 린스, 헤어 컨디셔너, 헤어 트리트먼트, 젤, 스킨 로션, 스킨소프너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스처 로션, 영양로션, 마사지 크림, 영양크림, 아이크림, 모이스처 크림, 핸드 크림, 파운데이션, 영양에센스, 선스크린, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디 로션 및 바디 클렌저로 이루어지는 군으로부터 선택된 제형을 가질 수 있으나, 이에 제한되지 않는다. 이들 각 제형의 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다.The formulation of the cosmetic composition may be prepared in any formulation conventionally prepared in the art, and external ointment for skin, cream, softening lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment Ment, 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 , Cleansing foam, cleansing lotion, cleansing cream, body lotion, and may have a formulation selected from the group consisting of a body cleanser, but is not limited thereto. The composition of each of these formulations may contain various bases and additives necessary and appropriate for formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.
상기 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. .
상기 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.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. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane/butane Or a propellant such as dimethyl ether.
상기 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation is a solution or emulsion, a solvent, a solubilizing agent or an 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, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.
상기 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the above formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose , Aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 개선용 건강기능식품 조성물을 제공한다. 상세하게는, 상기 조성물은 SNARE 복합체 형성을 억제할 수 있다.In addition, the present invention provides a health functional food 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.
상기 건강기능식품 조성물은 분말, 과립, 정제, 캡슐, 시럽, 음료 또는 환의 형태로 제공될 수 있으며, 상기 건강식품조성물은 유효성분인 본 발명에 따른 조성물 이외에 다른 식품 또는 식품 첨가물과 함께 사용되고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 그의 사용 목적 예를 들어 예방, 건강 또는 치료적 처치에 따라 적합하게 결정될 수 있다.The health functional food composition may be provided in the form of powder, granule, tablet, capsule, syrup, beverage or pill, and the health food composition is used with other foods or food additives in addition to the composition according to the present invention as an active ingredient, and is usually It can be appropriately used according to the phosphorus method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use, for example, prevention, health or therapeutic treatment.
상기 건강기능식품 조성물에 함유된 항체 또는 그의 항원 결합 단편의 유효용량은 상기 약학조성물의 유효용량에 준해서 사용할 수 있으나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로도 사용될 수 있음은 확실하다.The effective dose of the antibody or antigen-binding fragment thereof contained in the health functional food composition can be used in accordance with 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 In the above range, it is clear that the active ingredient can be used in an amount above the above range because there is no problem in terms of safety.
상기 건강식품의 종류에는 특별한 제한이 없고, 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등을 들 수 있다.There is no particular limitation on the kind of health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, Drinks, alcoholic beverages, and vitamin complexes.
또한, 본 발명은 상기 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 치료용 약학조성물을 제공한다. 상세하게는, 상기 조성물은 SNARE 복합체 형성을 억제할 수 있다.In addition, 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.
본 발명의 약학 조성물은 약제학적으로 허용되는 담체를 추가로 포함할 수 있으며, 상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 암 전이 예방 또는 치료용 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is commonly used in formulation, and lactose, dextrose, sucrose, sorbitol, mannitol, starch , Gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear It 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 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.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장 내 투여 등으로 투여할 수 있다. 경구 투여시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 될 수 있으며, 본 발명의 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.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 and rectal administration, etc. It can be administered as. When administered orally, since the protein or peptide is digested, the oral composition may be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention is an arbitrary device capable of moving the active substance to the target cell. It can be administered by.
본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, and is usually As such, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis.
본 발명의 약학 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화하여 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or Alternatively, it may be manufactured by placing it in a multi-capacity container. In this case, 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 include a dispersant or a stabilizer.
본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
<실시예 1> 항-Syntaxin 1A scFv 항체 선별<Example 1> Anti-Syntaxin 1A scFv antibody selection
1. bio-panning 시행1. Implement bio-panning
7.6 × 109의 다양성을 가지는 OPAL library를 이용하여 bio-panning을 수행하였다. Epoxy magnetic bead에 4 μg의 Syntaxin 1A 항원을 고정시키고 input phage를 반응시켰다. 항원과 반응한 phage를 용출(elution)하여 output titer를 측정하였다. 매 횟수마다 input과 output titer를 측정하여 bio-panning의 정보를 획득하고 정상적으로 시행되고 있는지를 확인하였다. 매 횟수마다 input phage는 4 × 1012 cfu/mL ≥의 phage를 사용하였다. output은 1차 - 1.2 × 105, 2차 - 2 × 107, 3차 - 3 × 107 cfu/mL으로 bio-panning이 진행될수록 증폭되는 것을 알 수 있었다. 따라서 bio-panning이 정상적으로 진행되었다는 것을 확인할 수 있었다.Bio-panning was performed using an OPAL library having a diversity of 7.6 × 10 9. 4 μg of Syntaxin 1A antigen was immobilized on the epoxy magnetic bead and the input phage was reacted. The phage that reacted with the antigen was eluted and the output titer was measured. By measuring the input and output titer every number of times, bio-panning information was obtained and it was confirmed whether it is being performed normally. For each input phage, a phage of 4 × 10 12 cfu/mL ≥ was used. The output was 1st-1.2 × 10 5 , 2nd-2 × 10 7 , and 3rd-3 × 10 7 cfu/mL. It can be seen that the amplification increases as bio-panning proceeds. Therefore, it could be confirmed that the bio-panning proceeded normally.
2. ELISA 분석2. ELISA analysis
고민감도, 고특이성 항체의 선별을 위해 ELISA 분석법을 수행하였다. 획득한 phage를 대장균에 감염시켜 항생제가 첨가된 LB plate에 도말하였다. 16 시간 동안 30℃ 인큐베이터에서 배양한 후 생성된 콜로니(colony)를 랜덤하게 채집하였다. 각 콜로니(colony)를 LB 배지에 넣어 배양한 후 IPTG를 처리하여 scFv를 발현시키고 대장균을 용해(lysis)하여 수용성 분획(soluble fraction)을 취하여 ELISA를 수행하였다. 먼저, 1 μg/mL의 Syntaxin 1A 재조합 단백질을 96 well ELISA plate에 고정시키고, 1% BSA가 함유된 PBS로 블락킹(blocking)하였다. 1 시간 후, 위에서 얻은 세포 용해물(cell lysate)을 처리하여 4℃에서 16 시간 동안 반응시켰다. 0.1% Tween20가 함유된 PBS로 plate를 3회 씻어준 후 블락킹 용액(blocking solution)에 HRP-conjugated anti-HA 항체를 1:1000으로 희석하여 상온에서 1 시간 동안 반응시켰다. 0.1% Tween20가 함유된 PBS로 plate를 5회 씻어준 후 TMB substrate로 발색시켜 ELISA leader로 항원에 결합된 scFv 항체를 측정하였다. 도 1은 Syntaxin 1A 단백질을 이용하여 진행한 ELISA의 결과를 나타낸다. 96개의 항체를 분석하였고 OD 0.4를 양성 가이드라인(positive guideline), 0.1을 음성 가이드라인(negative guideline)으로 임의로 정하여 18종을 선별하였다(도 1). ELISA analysis was performed to select highly sensitive and highly specific antibodies. The obtained phage was infected with E. coli and spread on LB plate to which antibiotics were added. After culturing in a 30° C. incubator for 16 hours, the generated colonies were randomly collected. Each colony was cultured in LB medium, and then treated with IPTG to express scFv, and E. coli was lysed to obtain a soluble fraction, followed by ELISA. First, 1 μg/mL of Syntaxin 1A recombinant protein was fixed 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 a blocking solution and reacted at room temperature for 1 hour. After washing the plate 5 times with PBS containing 0.1% Tween20, it was colored with a TMB substrate, and the scFv antibody bound to the antigen was measured with an ELISA leader. 1 shows the results of ELISA performed using Syntaxin 1A protein. 96 antibodies were analyzed, and 18 species were selected by arbitrarily setting an OD of 0.4 as a positive guideline and 0.1 as a negative guideline (FIG. 1).
<<
실시예Example
2> 항- 2> anti-
Syntaxin Syntaxin
1A 1A
scFvscFv
항체 서열 분석 Antibody sequence analysis
ELISA 분석을 통하여 선별된 항-Syntaxin 1A 18종의 양성 클론(positive clone)에 대하여 서열분석(sequencing)을 통해 개별적 클론(clone)을 선별하였다. 앞서 선별한 양성 클론(positive clone) 중에 중복되는 클론(clone)이 존재할 가능성이 있기 때문에 서열분석이 필요하였다. 서열 분석 결과, 항-Syntaxin 1A의 경우 18 종의 양성 클론(positive clone) 중에 7종이 개별적인 클론(clone)임을 확인하였다.Individual clones were selected through sequencing for 18 positive clones of anti-Syntaxin 1A selected through ELISA analysis. Sequencing was required because there is a possibility that duplicate clones exist among the positive clones selected previously. As a result of sequence analysis, in the case of anti-Syntaxin 1A, it was confirmed that 7 of the 18 positive clones were individual clones.
<<
실시예Example
3> 세포투과성 항- 3> Cell permeable anti-
Syntaxin Syntaxin
1A 1A
scFvscFv
제작 making
1. TAT-항-1.TAT-anti-
Syntaxin Syntaxin
1A 1A
scFvscFv
construct 제작 construct creation
앞서 선별된 1종의 항-Syntaxin 1A scFv (표 1)를 primer를 이용하여 제한효소 사이트(restriction enzyme site)와 TAT을 삽입한 PCR 산물을 생산하였다. 각 PCR 산물 30 μL에 완충액(buffer) 3.1 4 μL, Sal
I 1 μL, Xho
I 1 μL, 증류수 4 μL를 처리하여 37℃에서 한 시간 동안 반응시킨 후 DNA를 분리하여 vector에 삽입할 insert를 확보하였다. pET28a(+) vector는 DH5α competent cell에 형질전환(transformation) 한 후, 카나마이신(kanamycin)이 첨가된 LB plate에 도말하여 37℃에서 16 시간 동안 배양하고 다음 날 콜로니(colony)를 취하여 카나마이신(kanamycin)이 첨가된 LB media에 접종하여 37℃에서 16 시간 동안 배양하였다. 다음날 mini-prep kit를 이용하여 vector를 분리한 후 vector 30 μL 에 완충액(buffer) 3.1 4 μL, Sal
I 1 μL,
Xho
I 1 μL, CIP 1 μL, 증류수 3 μL를 처리하여 37℃에서 한 시간 동안 반응시킨 후 정제하였다. 정제된 vector와 insert의 농도를 nanodrop으로 측정하고 vector와 insert의 비율별로 T4 ligase를 이용하여 상온에서 16 시간 동안 ligation을 진행하였다. Ligation이 끝난 후 DH5α에 형질전환(transformation) 한 후 카나마이신(kanamycin)이 첨가된 LB plate에 도말하여 37℃에서 16 시간 동안 배양하고 다음 날 콜로니(colony)를 취하여 카나마이신(kanamycin)이 첨가된 LB media에 접종하여 37℃에서 16 시간 동안 배양하였다. 다음날 insert가 삽입되었는지 확인하기 위해 플라스미드(plasmid)를 분리하여 Sal
I과 Xho
I으로 절단(restriction)하고, 1% 아가로스 젤(agarose gel)에 전기영동하여 밴드를 확인하였다. One type of anti-Syntaxin 1A scFv (Table 1) selected above was used as a primer to produce a PCR product containing a restriction enzyme site and TAT. 30 μL of each PCR product is treated with 4 μL of buffer 3.1, 1 μL of Sal I, 1 μL of Xho I , and 4 μL of distilled water and reacted at 37°C for an hour, and then the DNA is separated to secure an insert to be inserted into the vector. I did. pET28a(+) vector is transformed into DH5α competent cells, spread on LB plates with kanamycin added, incubated at 37°C for 16 hours, and colonies are taken the next day and kanamycin It was inoculated on the added LB media and incubated at 37°C for 16 hours. The next day, after separating the vector using a mini-prep kit, add 30 μL of vector to 4 μL of buffer 3.1, 1 μL of Sal I, 1 μL of Xho I, 1 μL of CIP, and 3 μL of distilled water were treated and reacted at 37° C. for one hour, followed by purification. 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 is complete, DH5α is transformed, spread on an LB plate containing kanamycin, incubated at 37°C for 16 hours, and colonies are taken the next day, and LB media with kanamycin added. And inoculated at 37° C. for 16 hours. The next day, to check whether the insert was inserted, the plasmid was separated and cut with Sal I and Xho I , and the band was identified by electrophoresis on 1% agarose gel.
TargetTarget | CloneClone | Light chain Light chain CDRCDR sequence sequence | Heavy chain Heavy chain CDRCDR sequence sequence | ||||
CDR1CDR1 | CDR2CDR2 | CDR3CDR3 | CDR1CDR1 |
CDR2 | CDR3CDR3 | ||
SyntaxinSyntaxin 1A 1A | 77 | TGSSSNIGNNSVTTGSSSNIGNNSVT | DNNQDNNQ | ATWDSSLNGATWDSSLNG | NYDMSNYDMS | SISSNDGSKSISSNDGSK | RDLRTCRLMRCSSDDGMDVRDLRTCRLMRCSSDDGMDV |
2. TAT-항-2. TAT-anti-
Syntaxin Syntaxin
1A 1A
scFvscFv
항체 정제 Antibody purification
클로닝 된 TAT-항-Syntaxin 1A scFv 항체 클론을 BL21(DE3) 대장균 숙주에 형질전환(transformation) 하고, 형질전환체를 1 mM의 IPTG 유도(induction) 하였다. scFv 항체를 lysis buffer (50 mM Tris-HCl, p H 7.5, 150 mM NaCl)에 현탁하고 초음파 분쇄기를 이용하여 파쇄한 후, 원심분리하여 상층액을 얻었다. 단백질의 정제는 Ni-NTA에 친화력을 갖는 레진을 사용해서 단백질 정제하였다. 정제된 단백질은 SDS-PAGE 분석을 통하여 단백질 크기는 약 30 kDa 으로 확인하였다(도 2). The cloned TAT-anti-Syntaxin 1A scFv antibody clone was transformed into a BL21(DE3) E. coli host, and the transformant was induction of 1 mM IPTG. The scFv antibody was suspended in lysis buffer (50 mM Tris-HCl, p H 7.5, 150 mM NaCl), crushed using an ultrasonic grinder, and centrifuged to obtain a supernatant. The protein was purified using a resin having an affinity for Ni-NTA. The purified protein was confirmed to have a protein size of about 30 kDa through SDS-PAGE analysis (FIG. 2).
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실시예Example
4> 세포투과성 항- 4> Cell permeable anti-
Syntaxin Syntaxin
1A 1A
scFv의scFv
세포독성 시험 Cytotoxicity test
TAT-항-Syntaxin 1A scFv 항체에 대한 세포독성을 알아보기 위하여, 포유류 신경 세포인 PC12 세포를 37℃, CO2 인큐베이터에서 배양하였다. 배양 후 TAT-항-Syntaxin 1A scFv 항체를 농도별로 처리한 다음 같은 배양 조건에서 추가 배양하였다. MTT{3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide} 용액을 넣어 배양한 다음 배양액을 제거하고 DMSO(Dimethyl sulfoxide)를 넣고 적당히 흔들어 준 뒤 570 nm에서 ELISA Reader system으로 흡광도를 측정하였으며, 그 값은 표 2에 나타내었다. 표 2에 나타낸 바와 같이, TAT-항-Syntaxin 1A scFv를 0.01, 0.1, 1, 10, 50, 100 ppm 농도로 처리했을 때, 100 ppm 농도까지 포유류 신경 세포의 세포모양 변형 및 세포생존률의 변화가 거의 없음을 확인하였다. 따라서, TAT-항-Syntaxin 1A scFv의 100 ppm 농도까지는 세포 독성이 없는 물질임을 알 수 있었다.In order to investigate the cytotoxicity of the TAT-anti-Syntaxin 1A scFv antibody, PC12 cells, which are mammalian neurons, were cultured in a 37°C, CO 2 incubator. After incubation, the TAT-anti-Syntaxin 1A scFv antibody was treated by concentration, and then further cultured under the same culture conditions. Add MTT{3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide} solution to incubate, remove the culture solution, add DMSO (Dimethyl sulfoxide), shake appropriately, and ELISA Reader system at 570 nm The absorbance was measured by, and the values are shown in Table 2. As shown in Table 2, when TAT-anti-Syntaxin 1A scFv was treated at a concentration of 0.01, 0.1, 1, 10, 50, 100 ppm, the change in cell shape and cell viability of mammalian neurons up to 100 ppm concentration was It was confirmed that almost none. Therefore, it was found that the TAT-anti-Syntaxin 1A scFv was a substance without cytotoxicity up to a concentration of 100 ppm.
실시예4Example 4 | ||
TAT-항-Syntaxin 1A TAT- |
||
농도 (ppm)Concentration (ppm) | 평균Average | 오차error |
00 | 100.0 100.0 | 2.39 2.39 |
0.010.01 | 89.6 89.6 | 2.05 2.05 |
0.10.1 | 81.0 81.0 | 0.71 0.71 |
1One | 96.3 96.3 | 2.43 2.43 |
1010 | 101.6 101.6 | 7.75 7.75 |
5050 | 123.5 123.5 | 4.85 4.85 |
100100 | 124.3 124.3 | 5.37 5.37 |
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실시예Example
5> 세포투과성 항- 5> Cell permeable anti-
Syntaxin Syntaxin
1A 1A
scFv의scFv
SNARE 복합체 형성 SNARE complex formation
억제능Inhibitory ability
실험 Experiment
TAT-항-Syntaxin 1A scFv 항체의 SNARE 복합체 형성 억제 여부를 확인하기 위해, native-PAGE 분석을 수행하였다. SNARE 단백질인 SNAP25, Syntaxin 1A와 VAMP2 단백질 (LSbio사)을 각각 1:1:1의 농도 (1 μg)로 혼합했을 경우 형성되는 복합체가 TAT-항-Syntaxin 1A scFv 항체의 첨가에 의해 억제되는지를 확인하였다. 각각의 단백질 1 μg 넣고 TAT-항-Syntaxin 1A scFv 항체 각각을 농도별 (0.5, 1, 2, 5, 10, 20 μg)로 처리한 후 재빨리 혼합한 다음 4℃에서 1시간 반응시켰다. 반응이 종료되면 샘플 완충액을 넣어 반응을 정지시킨 후 10% native-PAGE 상에서 SNARE 복합체 형성여부를 확인하였다(도 3). 도 3에 나타낸 바와 같이, TAT-항-Syntaxin 1A scFv는 농도 의존적 (5, 10, 20 μg)으로 SNARE 복합체를 효과적으로 저해하였으며, TAT-항-Syntaxin 1A scFv의 10 μg 농도에서 SNARE 복합체 형성을 50% 이상 억제하였다.To determine whether the TAT-anti-Syntaxin 1A scFv antibody inhibits SNARE complex formation, native-PAGE analysis was performed. When the SNARE proteins SNAP25, Syntaxin 1A and VAMP2 proteins (LSbio) are mixed at a concentration of 1:1:1 (1 μg), it is determined whether the complex formed is inhibited by the addition of the TAT-anti-Syntaxin 1A scFv antibody. Confirmed. 1 μg of each protein was added, and each of the TAT-anti-Syntaxin 1A scFv antibodies was treated at different concentrations (0.5, 1, 2, 5, 10, 20 μg), then quickly mixed, and then 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 (FIG. 3). As shown in Figure 3, TAT-anti-Syntaxin 1A scFv effectively inhibited the SNARE complex in a concentration-dependent manner (5, 10, 20 μg), and SNARE complex formation at a concentration of 10 μg of TAT-anti-Syntaxin 1A scFv was 50 % Or more was suppressed.
<실시예 6> 세포투과성 항-Syntaxin 1A scFv의 MMP-1 발현억제 평가<Example 6> Evaluation of cell-permeable anti-Syntaxin 1A scFv inhibition of MMP-1 expression
TAT-항-Syntaxin 1A scFv에 대하여, 콜라겐 생성에 미치는 영향을 알아보고자 Real Time PCR 방법을 사용하여 TAT-항-Syntaxin 1A scFv에 의한 MMP-1 활성 억제효능을 실험하였다. 포유류 신경 세포인 PC12 세포를 37℃, CO2인큐베이터에서 배양하였다. 배양 후 자외선조사기기(UV irradiator system)를 이용하여 UVA를 조사하였다. 그 후, TAT-항-Syntaxin 1A scFv 항체 10 ppm과 양성대조구로서 adenosine 100 ppm을 처리한 다음 같은 배양 조건에서 추가 배양하였다. 배양 후 세포를 TRIzol 300 μL로 모아 1.5 mL tube에 옮기고, chloroform 50 μL를 첨가한 다음 혼합하여 상온에 5분간 방치한다. 이후 4℃, 15,000 rpm으로 15분간 원심분리하여 상층액을 새로운 tube로 옮기고 동량의 2-propanol과 섞어 상온에서 5분간 방치한 다음 4℃, 12,000 rpm에서 20분간 원심분리한다. 원심분리 후 2-propanol은 버리고 75% 에탄올 300 μL를 첨가하여 4℃, 10,000 rpm에서 10분간 원심분리한다. 75% 에탄올은 버리고 RNA pellet은 상온에서 말려 남아있는 에탄올을 제거한다. Pellet에 DEPC가 처리된 정제수 30 μL를 넣어 녹인 후 260 nm에서 정량한다. RT-PCR은 1 μg의 total RNA와 TOPscript RT dry mix를 이용하여 수행하였다. Real Time PCR에 사용된 MMP-1과 GAPDH는 마크로젠에서 합성하여 사용하였으며 염기서열은 아래 표 3에 나타내었다. TOPrealTM Qpcr 2X PreMIX를 이용하여 Real Time PCR을 실시하였다. 도 4에 나타낸 바와 같이, TAT-항-Syntaxin 1A scFv는 자외선에 의해 증가된 주름 생성의 원인이 되는 MMP-1 콜라겐 분해효소를 억제하였다. 10 ppm 처리 시 대조구 보다 주름개선 효과가 월등히 우수함을 확인하였다.In order to investigate the effect of TAT-anti-Syntaxin 1A scFv on collagen production, the inhibitory effect of TAT-anti-Syntaxin 1A scFv on MMP-1 activity was tested using a Real Time PCR method. PC12 cells, which are mammalian neurons, were cultured in a 37°C, CO 2 incubator. After incubation, UVA was irradiated using a UV irradiator system. Thereafter, 10 ppm of TAT-anti-Syntaxin 1A scFv antibody and 100 ppm of adenosine as a positive control were treated, and then further cultured under the same culture conditions. After incubation, collect the cells with 300 μL of TRIzol, transfer to a 1.5 mL tube, add 50 μL of chloroform, mix, and leave for 5 minutes at room temperature. Then, centrifuge at 4°C and 15,000 rpm for 15 minutes, transfer the supernatant to a new tube, mix with the same amount of 2-propanol, stand for 5 minutes at room temperature, and then centrifuge at 4°C and 12,000 rpm for 20 minutes. After centrifugation, discard 2-propanol, add 300 μL of 75% ethanol, and centrifuge at 4°C and 10,000 rpm for 10 minutes. Discard the 75% ethanol and dry the RNA pellet at room temperature to remove the remaining ethanol. After dissolving 30 μL of DEPC-treated purified water into the pellet, it is quantified 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 in Macrogen, and the nucleotide sequences are shown in Table 3 below. Real Time PCR was performed using TOPreal TM Qpcr 2X PreMIX. As shown in FIG. 4, TAT-anti-Syntaxin 1A scFv inhibited MMP-1 collagen-degrading enzyme, which is the cause of increased wrinkle formation by ultraviolet rays. It was confirmed that the wrinkle improvement effect was far superior to that of the control when 10 ppm was treated.
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실시예Example
7> 세포투과성 항- 7> Cell permeable anti-
Syntaxin Syntaxin
1A 1A
scFv의scFv
피부 투과 분석 Skin penetration analysis
TAT-항-Syntaxin 1A scFv의 피부 투과를 확인하기 위하여, 돼지 피부를 사용하여 조직염색으로 확인하였다. 돼지 피부에 TAT-항-Syntaxin 1A scFv 항체 10 μg을 처리하고 37℃, CO2 인큐베이터에서 16시간 배양하였다. 돼지 피부를 4% 포름알데하이드로 고정시킨 뒤 파라핀 블록을 제조하였으며, 마이크로톰을 이용하여 5 μm로 조직을 절제하였다. 그 후, 슬라이드 위에 조직을 마운팅한 다음 파라핀을 제거하고, 수화과정을 거친 후 조직 절편을 30분간 블락킹하였다. 블락킹이 끝난 조직에 anti-His HRP antibody를 상온에서 1시간 반응시켰다. PBST로 씻어준 다음 DAB-chromogen 용액에 5분간 반응시킨 후 H&E 염색기법으로 염색하여 광학 현미경으로 관찰하였다. 도 5는 항-Syntaxin 1A scFv과 TAT-항-Syntaxin 1A scFv의 피부 투과력을 비교한 결과이다. 도 5에 나타낸 바와 같이, 항-Syntaxin 1A scFv는 돼지 피부 조직 내로 전혀 전달되지 않음에 반해, TAT-항-Syntaxin 1A scFv는 효율적인 피부 전달 능력을 가짐을 확인할 수 있었다. In order to confirm the skin penetration of TAT-anti-Syntaxin 1A scFv, it was confirmed by tissue staining using pig skin. Pig skin was treated with 10 μg of TAT-anti-Syntaxin 1A scFv antibody and incubated for 16 hours in a 37°C, CO 2 incubator. After fixing the pig skin with 4% formaldehyde, a paraffin block was prepared, and the tissue was excised at 5 μm using a microtome. Thereafter, the tissue was mounted on the slide, paraffin was removed, and the tissue section was blocked for 30 minutes after undergoing a hydration process. Anti-His HRP antibody was reacted for 1 hour at room temperature to the blocked tissue. After washing with PBST, reacting with DAB-chromogen solution for 5 minutes, stained with H&E staining technique, and observed with an optical microscope. 5 is a result of comparing the skin permeability of anti-Syntaxin 1A scFv and TAT-anti-Syntaxin 1A scFv. As shown in FIG. 5, it was confirmed that the anti-Syntaxin 1A scFv was not delivered into the pig skin tissue at all, whereas the TAT-anti-Syntaxin 1A scFv had an efficient skin delivery ability.
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실시예Example
8> 세포투과성 항- 8> Cell permeable anti-
Syntaxin Syntaxin
1A 1A
scFv의scFv
활성 산소 억제 효과 실험 Free radical suppression effect experiment
TAT-항-Syntaxin 1A scFv 항체의 세포 내에서 활성산소(ROS) 저해효과를 확인하기 위해, 포유류 신경 세포인 PC12 세포에 TAT-항-Syntaxin 1A scFv 항체를 처리 후 세포내 활성산소 함량을 형광염색법을 이용하여 측정하였다. 96-well 플레이트에 접종된 PC12 세포를 37℃, CO2 인큐베이터에서 배양한 후 TAT-항-Syntaxin 1A scFv 항체 (10, 20 ppm)를 처리하였다. 2시간 동안 배양한 후 각 well에 0.2 mM 과산화수소를 첨가한 다음 같은 배양 조건에서 추가 배양하였다. 20 μM carboxy-H2DCF-DA를 첨가하고 37℃, 30분간 반응시켰다. PBS로 세척한 다음 PBS를 재 첨가하여 485/525 nm에서 ELISA Reader system으로 형광을 측정하였다. 표 4에 나타낸 바와 같이, TAT-항-Syntaxin 1A scFv는 ROS 생성을 효과적으로 억제함을 알 수 있었다.In order to confirm the inhibitory effect of TAT-anti-Syntaxin 1A scFv antibody on active oxygen (ROS) in cells, PC12 cells, which are mammalian neurons, were treated with TAT-anti-Syntaxin 1A scFv antibody and then the intracellular active oxygen content was fluorescently stained It was measured using. PC12 cells inoculated in a 96-well plate were cultured in a 37° C., CO 2 incubator and treated with TAT-anti-Syntaxin 1A scFv antibody (10, 20 ppm). After incubation for 2 hours, 0.2 mM hydrogen peroxide was added to each well, and then 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 with PBS, PBS was added again, and fluorescence was measured at 485/525 nm with an ELISA Reader system. As shown in Table 4, it was found that TAT-anti-Syntaxin 1A scFv effectively inhibited ROS production.
시료sample | 농도(ppm)Concentration (ppm) | ROS (fold of control)ROS (fold of control) | |||
non treat controlnon treat control | 0.2 mM H2O2 0.2 mM H 2 O 2 | ||||
평균Average | 오차error | 평균Average | 오차error | ||
TAT-항-Syntaxin 1A scFvTAT- |
00 | 1.0001.000 | 0.0290.029 | 1.4741.474 | 0.0770.077 |
1010 | 0.8620.862 | 0.0830.083 | 1.1331.133 | 0.0370.037 | |
2020 | 0.8170.817 | 0.0500.050 | 1.0481.048 | 0.0380.038 |
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다. As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.
Claims (12)
- 서열번호 1로 표시되는 아미노산 서열로 이루어진 경쇄 CDR1, 서열번호 2로 표시되는 아미노산 서열로 이루어진 경쇄 CDR2 및 서열번호 3으로 표시되는 아미노산 서열로 이루어진 경쇄 CDR3을 포함하는 경쇄 가변 영역; 및 서열번호 4로 표시되는 아미노산 서열로 이루어진 중쇄 CDR1, 서열번호 5로 표시되는 아미노산 서열로 이루어진 중쇄 CDR2 및 서열번호 6으로 표시되는 아미노산 서열로 이루어진 중쇄 CDR3을 포함하는 중쇄 가변 영역을 포함하는 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편.A light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 3; And a heavy chain CDR1 consisting of an amino acid sequence represented by SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence represented by SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence represented by SEQ ID NO: 6 anti- Syntaxin 1A antibody or antigen-binding fragment thereof.
- 제1항의 항체 또는 그의 항원 결합 단편에 추가적으로 서열번호 7로 표시되는 TAT 펩타이드가 결합된 융합 항-Syntaxin 1A 항체 또는 그의 항원 결합 단편.A fusion anti-Syntaxin 1A antibody or antigen-binding fragment thereof in which the TAT peptide represented by SEQ ID NO: 7 is additionally bound to the antibody of claim 1 or antigen-binding fragment thereof.
- 제1항 또는 제2항의 항체 또는 그의 항원 결합 단편을 코딩하는 핵산 분자.A nucleic acid molecule encoding the antibody of claim 1 or 2 or an antigen-binding fragment thereof.
- 제3항의 핵산 분자를 포함하는 재조합 발현벡터.A recombinant expression vector comprising the nucleic acid molecule of claim 3.
- 제4항의 재조합 발현벡터로 형질전환된 세포.Cells transformed with the recombinant expression vector of claim 4.
- 제1항 또는 제2항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 Syntaxin 1A 항원 검출용 조성물.A composition for detecting Syntaxin 1A antigen comprising the antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.
- 제1항 또는 제2항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 개선용 화장료 조성물.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.
- 제7항에 있어서, 상기 조성물은 SNARE 복합체 형성을 억제하는 것을 특징으로 하는 피부 주름 예방 또는 개선용 화장료 조성물.The cosmetic composition for preventing or improving skin wrinkles according to claim 7, wherein the composition inhibits the formation of a SNARE complex.
- 제1항 또는 제2항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 항산화용 화장료 조성물.An antioxidant cosmetic composition comprising the antibody of claim 1 or 2 or an antigen-binding fragment thereof as an active ingredient.
- 제9항에 있어서, 상기 조성물은 활성산소 생성을 억제하는 것을 특징으로 하는 항산화용 화장료 조성물.The cosmetic composition for antioxidants according to claim 9, wherein the composition inhibits the production of active oxygen.
- 제1항 또는 제2항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 개선용 건강기능식품 조성물.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.
- 제1항 또는 제2항의 항체 또는 그의 항원 결합 단편을 유효성분으로 포함하는 피부 주름 예방 또는 치료용 약학 조성물.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.
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US20050228169A1 (en) * | 2002-03-19 | 2005-10-13 | Hiroshi Yokota | Polypeptide binding to human syntaxin 1a |
KR100883757B1 (en) * | 2007-03-12 | 2009-02-12 | 성균관대학교산학협력단 | Polyphenol compounds with modulating neurotransmitter release |
CN103641888A (en) * | 2013-12-03 | 2014-03-19 | 广州健坤生物科技有限公司 | Multifunctional novel small peptide, composition including same and application of small peptide |
KR101640694B1 (en) * | 2011-09-29 | 2016-07-18 | 셀스냅, 엘엘씨 | Compositions and methods for toxigenicity testing |
KR102017240B1 (en) * | 2018-08-23 | 2019-09-02 | 주식회사 하울바이오 | Anti-VAMP2 antibodies inhibiting SNARE complex and use thereof |
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WO2011002179A2 (en) | 2009-06-30 | 2011-01-06 | (주)아모레퍼시픽 | Cosmetic composition containing a rubus coreanus extract for diminishing skin wrinkles |
KR101803201B1 (en) | 2015-07-30 | 2017-11-30 | 성균관대학교산학협력단 | SNARE complex formation inhibiting composition comprising myricetin derivatives |
KR101997474B1 (en) | 2016-06-13 | 2019-07-09 | 기초과학연구원 | method for controlling neurotransmitter using the cucurbit[n]uril, and use thereof |
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US20050228169A1 (en) * | 2002-03-19 | 2005-10-13 | Hiroshi Yokota | Polypeptide binding to human syntaxin 1a |
KR100883757B1 (en) * | 2007-03-12 | 2009-02-12 | 성균관대학교산학협력단 | Polyphenol compounds with modulating neurotransmitter release |
KR101640694B1 (en) * | 2011-09-29 | 2016-07-18 | 셀스냅, 엘엘씨 | Compositions and methods for toxigenicity testing |
CN103641888A (en) * | 2013-12-03 | 2014-03-19 | 广州健坤生物科技有限公司 | Multifunctional novel small peptide, composition including same and application of small peptide |
KR102017240B1 (en) * | 2018-08-23 | 2019-09-02 | 주식회사 하울바이오 | Anti-VAMP2 antibodies inhibiting SNARE complex and use thereof |
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