Conjugates with enhanced half-life, stability, cell penetration and skin penetration and uses thereof

Abstract

The present invention relates to a conjugate comprising a biologically active molecule and a cell-penetrating peptide, and a use thereof. The biologically active molecule has an excellent half-life and / or stability, and the conjugate is capable of penetrating cells, tissues, and / or skin. According to the present invention, provided is a conjugate obtained by applying anti-ubiquitination technology (AUT) and / or binding a cell-penetrating peptide to biologically active molecules, and thus having an increased half-life and / or stability and exhibiting effects on cell, tissue, and / or skin penetration. Therefore, the conjugate according to the present invention can be useful as a raw material for topical cosmetics and / or skin-penetrating cosmetics.

Description

Conjugates with improved cell and skin permeability and increased half-life and stability, and uses thereof

[0001] The present invention relates to a conjugate comprising a biologically active molecule and a cell-penetrating peptide, and uses thereof. The biologically active molecule has excellent half-life and / or stability, and the conjugate is characterized by being capable of penetrating cells, tissues, and / or skin.

[0002] Many biologically active molecules, such as Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), and Superoxide Dismutase (SOD), are widely used as raw materials for specific functions in products that help whiten the skin, improve wrinkles, give a smooth skin tone, or protect the skin from UV rays.

[0003] Meanwhile, macromolecules such as unspecified peptides including proteins cannot penetrate the lipid bilayer of the plasma membrane without the help of specific receptors or transporters. Therefore, the protein transduction domain (PTD) helps transport the substances (peptides, proteins, nucleic acids, etc.) to be transported to deep tissues within the body. In this case, the Basic PTD is an auxiliary domain that does not freely penetrate the biological membrane but induces endocytosis from the outside to the inside, and has the limitation that continuous transport between cells is impossible.

[0004] The ubiquitination process is one of the phenomena that has the greatest impact on homeostasis. It refers to the phenomenon in which ubiquitin binds to target proteins in the protein degradation control mechanism, and the biggest role of this process is known to induce protein degradation. When ubiquitination of a target protein occurs, the protein is degraded by the proteasome in the cytoplasm, which is called the ubiquitin-proteasome pathway. Most proteins expressed in the body undergo ubiquitination, and this process is sequentially promoted by ubiquitin E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin-ligase) enzymes (Kim et al., 2021).

[0005] There is a need for the development of technologies that can inhibit the degradation of biologically active molecules through ubiquitination and increase cell penetration and / or skin penetration.

[0006] The present inventors have completed the present invention by developing a conjugate having improved cell, tissue and / or skin penetration by conjugating a cell-penetrating peptide to biologically active molecules and increasing the half-life and / or stability by avoiding ubiquitination of biologically active molecules.

[0007] An example of the present application provides a conjugate comprising a biologically active molecule; and a cell-penetrating peptide having an amino acid sequence of SEQ ID NO: 1 linked to the N-terminus, the C-terminus, or both of the biologically active molecule.

[0008] Another example provides a use for the above conjugate.

[0009] The present inventors have confirmed through previous research that protein degradation can be inhibited by blocking the ubiquitin-proteasome pathway.

[0010] In addition, the present inventors renamed the hydrophobic MTS (Macromolecule Transduction Sequences) (AAVLLPVLLAAP, SEQ ID NO: 1) derived from the signal peptide of Fibroblast Growth Factor (FGF) among the Macromolecule Transduction Domains (MTDs) as BMTS (Biological materials transdermal system).

[0011] Accordingly, the present inventors applied anti-ubiquitination technology (AUT) to develop biologically active molecules with increased half-life and / or stability, thereby substituting specific lysine residues within the amino acid sequence of biologically active molecules with conservative amino acids, such as arginine. In addition, the present invention was completed by combining the cell-penetrating peptide BMTS to improve cell and / or skin penetration.

[0012] According to the present invention, a conjugate is provided that exhibits increased half-life and / or stability and also exhibits cell penetration and / or skin penetration effects by applying anti-ubiquitination technology (AUT) to biologically active molecules and / or binding a cell-penetrating peptide. Therefore, the conjugate of the present invention can be usefully used as a raw material for topical cosmetics and / or skin-penetrating cosmetics.

[0013] Figures 1a and 1b are diagrams showing vector maps for the production of conjugates of biologically active molecules and cell-penetrating peptides (BMTS). Figure 1a shows a map of a conjugate produced using the pET24a vector, and Figure 1b shows a vector map of a conjugate produced using the EE-tag vector.

[0014] Figure 2 shows the results of inducing expression of biologically active molecules and cell-penetrating peptides (BMTS). The results are before expression (U: Un-induction), after expression (W: Whole cell lysate), lysate supernatant (S: Soluble), and pellet (IB: Inclusion body), respectively.

[0015] Figure 3 is a schematic diagram of the purification process by classifying the expression-induced conjugate into those expressed in a soluble and in-soluble state.

[0016] Figure 4a shows the results of treating fibroblasts with BMTS-AUT-FGF-1 and measuring the degree of cell proliferation.

[0017] Figure 4b shows the results of treating fibroblasts with BMTS-AUT-FGF-2 and measuring the degree of cell proliferation.

[0018] Figure 4c shows the results of measuring the intracellular activity of BMTS-AUT-SOD-1.

[0019] Figure 5 shows the results of comparing the skin penetration ability of BMTS-BT-LC and AUT-EGF-5L-BMTS with that of the case where BMTS is not bound.

[0020] Figure 6 is a graph showing the change and improvement rate of the Wrinkles small value when using the cosmetic composition of Manufacturing Example 1.

[0021] Figure 7 is an image showing wrinkles at the corners of the eyes before and after using the cosmetic composition of Manufacturing Example 1.

[0022] Figure 8 is a graph showing the change and improvement rate of the affected area value when using the cosmetic composition of Manufacturing Example 1.

[0023] Figure 9 is an image showing the pore area before and after using the cosmetic composition of Manufacturing Example 1.

[0024] Figure 10 is a graph showing the change in a* value and improvement rate of the application area and non-application area of ​​the cosmetic composition of Manufacturing Example 2.

[0025] Figure 11 is an image showing the application area and non-application area of ​​the cosmetic composition of Manufacturing Example 2.

[0026] Figure 12 shows the change in R2 value and improvement rate when using the cosmetic composition of Manufacturing Example 2.

[0027] Figure 13 is a graph showing the change in the Wrinkles large value and the improvement rate when using the cosmetic composition of Manufacturing Example 3.

[0028] Figure 14 is an image showing neck wrinkles before and after using the cosmetic composition of Manufacturing Example 3.

[0029] Figure 15 shows the change in maximum a* value and improvement rate when using the cosmetic composition of Manufacturing Example 4.

[0030] Figure 16 shows photographs of the scalp condition before and after use of the cosmetic composition of Manufacturing Example 4.

[0031] Hereinafter, the present invention will be described in more detail.

[0032] An example of this application is:

[0033] biologically active molecules; and

[0034] A cell-penetrating peptide having an amino acid sequence of SEQ ID NO: 1, linked to the N-terminus, C-terminus or both of the above biologically active molecule.

[0035] A conjugate comprising:

[0036] As used herein, the term “cell penetrating peptide (CPP)” refers to a peptide capable of transporting molecules linked across cell membranes. The present inventors have renamed and used a cell penetrating peptide having the amino acid sequence of “AAVLLPVLLAAP (SEQ ID NO: 1)” as BMTS (Biological Materials Transdermal System). The cell penetrating peptide is a hydrophobic membrane-translocating sequence (MTS) derived from the signal peptide of fibroblast growth factor (FGF). The biologically active molecule in the conjugate may have increased cell penetration and / or skin penetration ability compared to a molecule not bound to the cell penetrating peptide.

[0037] As used herein, the term “skin penetration ability” means the ability or property of a peptide to penetrate the skin and into the interior of the skin.

[0038] The above-mentioned cell-penetrating peptide may exhibit excellent skin retention. As used herein, the term "skin retention" refers to the ability of a peptide that has penetrated the skin to remain bound to tissue within the skin without passing through the skin tissue and being transferred to the circulation. In the case of pharmaceutical preparations or cosmetics targeting skin tissue, it is preferable to use a carrier with excellent skin tissue retention properties so that the component bound to the peptide can act on the skin tissue or skin cells for a long period of time. The skin penetration-promoting peptide of the present invention exhibits not only excellent skin permeability but also excellent skin retention, and thus can be used as a carrier for pharmaceutical preparations or cosmetics.

[0039] The conjugate may comprise a biologically active molecule and a cell-penetrating peptide in any order. In one example, the cell-penetrating peptide is bound to the N-terminus and / or C-terminus of the biologically active molecule, but is not limited to this orientation.

[0040] In this specification, the term “conjugate” may be used with the same meaning as fusion protein or conjugate.

[0041] As used herein, the term “biologically active molecule” refers to any substance, including a drug, that can perform, participate in, or initiate various biochemical changes in the body of a human or animal, and that can exert a desired effect in the body. More specifically, the biologically active molecule may be one or more selected from the group consisting of peptides, proteins, glycoproteins, nucleic acids, carbohydrates, lipids, glycolipids, compounds, natural products, semi-synthetic drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorochromes, but is not limited thereto.

[0042] In one embodiment, the biologically active molecule may be a growth factor.

[0043] In this specification, “growth factor” refers to an important protein or peptide that regulates cell growth, division, survival, differentiation, etc. Growth factors are secreted from outside the cell and activate intracellular signaling pathways by binding to specific receptors on the cell surface, which plays an important role in changing cell behavior. More specifically, the above-mentioned growth factor may be at least one selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), stem cell factor (SCF), keratinocyte growth factor (KGF), and transforming growth factor (TGF). More specifically, the fibroblast growth factor may be FGF-1, FGF-2 or FGF-10, the insulin-like growth factor may be IGF-1, the transforming growth factor may be TGF-α, TGF-β (TGF-β1, TGF-β2 and TGF-β3), the platelet-derived growth factor may be PDGF-A or PDGF-B, the vascular endothelial growth factor may be VEGF-A, and the keratinocyte growth factor may be KGF-2, but is not limited thereto. In addition, the platelet-derived growth factor may be a dimeric form of PDGF-A or PDGF-B, and may be expressed herein as PDGF-AA and PDGF-BB, respectively. In one embodiment, PDGF-BB, which is a dimeric form of PDGF-B, may exhibit an increased skin improvement effect compared to PDGF-B, which is a monomer.

[0044] Epidermal growth factor (EGF) is known to play a role in promoting wound healing, and is a substance that is being marketed as a cosmetic material with the 'skin regeneration' effect as its main point.

[0045] Fibroblast growth factor (FGF) is known as a peptide that promotes the proliferation of vascular cells or the growth of nerve cells, and is a substance that is used in marketing as a cosmetic material with the main point of 'wrinkle improvement effect'.

[0046] Platelet-derived growth factor (PDGF) is an important growth factor in the cell division process. It is known to be involved in blood vessel formation along with vascular endothelial growth factor (VEGF), making aged skin healthy and mediating the regeneration of damaged wounds.

[0047] Insulin-like growth factor-1 (IGF-1) acts on various cellular physiological functions and, together with EGF and other growth factors, helps the growth and division of skin cells and helps produce ECM substances such as collagen, fibronectin, elastin, and keratin in the dermis, preventing aging and wrinkles and hair damage and hair loss.

[0048] Stem cell factor (SCF) exists in both membrane-bound and soluble protein forms, and is involved in blood formation, sperm formation, and melanin formation. It is an essential element that activates stem cells present in almost all tissues, including the skin, and is known to prevent aging in the skin and promote the formation of new hair follicles in the hair.

[0049] Keratinocyte growth factor (KGF), also known as FGF-7, is a growth factor. KGF promotes the growth and division of epithelial cells, particularly keratinocytes. It promotes keratinocyte migration and proliferation, thereby aiding wound healing. It protects epithelial tissue from damage caused by various environmental factors, and is known to be involved in hair follicle development and maintenance.

[0050] Transforming growth factor beta (TGF-β) was first discovered as a substance that induces fibroblast transformation, and is known as a factor that regulates development, cell differentiation and proliferation, migration, survival, extracellular matrix production, and the immune system. TGF-β plays a particularly important role in the development of blood vessels and the production of blood cells, and unusually, rather than promoting growth, it has been reported to have a growth inhibitory effect in most cells. Its most important role is reported to be suppressing scar and fibrosis formation by being expressed at high concentrations during wound repair.

[0051] In one embodiment, the biologically active molecule described above may be selected from the group consisting of a botulinum toxin serotype selected from the group consisting of A, B, C, D, E, F, G and H, a recombinant botulinum toxin, a modified botulinum toxin, a fragment of a botulinum toxin and a combination thereof.

[0052] As used herein, “botulinum toxin” refers to a toxin secreted by the anaerobic bacterium Clostridium botulinum. Botulinum toxin exists in serotypes A to H, and types A and B can cause disease in humans and are used commercially and medically. Botulinum toxin is known to be effective in improving wrinkles by paralyzing muscles by inhibiting the secretion of the neurotransmitter acetylcholine at nerve endings. In one specific example, a peptide (BT-LC) was used in which only the light chain portion of type A botulinum toxin was recombined into a single chain, but is not limited thereto.

[0053] In one embodiment, the biologically active molecule may be at least one selected from the group consisting of noggin, superoxide dismutase (SOD), acetyl hexapeptide-8 (AH), acetyl pentapeptide-4 (AP), tissue inhibitor of metalloproteinase (TIMP), and aldehyde dehydrogenase (ALDH), but is not limited thereto.

[0054] More specifically, the superoxide dismutase may be SOD-1, the TIMP may be TIMP-1, or the aldehyde dehydrogenase may be ALDH-2, but is not limited thereto.

[0055] Noggin is reported to be a key protein that antagonizes BMP-2 / -4 signaling, which inhibits hair follicle differentiation in early developmental stages. Its overexpression is known to potently promote hair formation and growth. The interplay of various factors is essential for early hair follicle formation, with laminin, noggin, and PDGF playing key roles.

[0056] TIMP-1 (Tissue inhibitor of metalloproteinase 1) is an in vivo inhibitory protein for various matrix metalloproteinases (MMPs) expressed in the skin. It is a cytoprotective factor that inhibits the breakdown of intercellular substances found in aging and cell damage caused by external stimuli, promotes cell differentiation, and has anti-apoptotic effects. It is known as a biological anti-aging factor that can prevent wrinkles by inhibiting proteases involved in aging and wrinkle formation.

[0057] Superoxide dismutase (SOD) is one of the most powerful antioxidant proteins in the body, decomposing superoxide into oxygen and hydrogen peroxide, thereby stabilizing it. Among these, SOD-1 functions within human cells and acts as an anti-aging factor.

[0058] Acetyl pentapeptide-4 (AP) is a matripeptide composed of five amino acids linked to a 16-carbon aliphatic chain (palmitoyl). It is known to activate specific genes involved in extracellular matrix regeneration and cell proliferation processes, and to have a wrinkle-improving effect by activating the synthesis of extracellular matrix polymers.

[0059] Acetyl hexapeptide-8 (AH) acts to reduce muscle contraction by inhibiting the release of neurotransmitters in neurons instead of SNAP-25 (Synaptosomal Associated Protein 25).

[0060] Aldehyde dehydrogenase (ALDH) is an enzyme involved in aldehyde metabolism, playing a crucial role in the detoxification of both exogenous and endogenous aldehydes. ALDH catalyzes the conversion of acetaldehyde, the primary metabolite of ethanol, to acetate, playing a crucial role in alcohol metabolism. It is also known to protect cells from oxidative stress.

[0061] As used herein, “biologically active molecule” may include a wild-type biologically active molecule, a variant of a biologically active molecule, or both.

[0062] In one embodiment, the biologically active molecule may be a variant that has been modified to increase half-life and / or stability in the body.

[0063] In one embodiment, one or more (for example, one or more, two or more, three or more, and the upper limit is not particularly limited and may be, but is not limited to, 10 or less, 20 or less, or 30 or less) lysine residues in the amino acid sequence of the biologically active molecule may be substituted with a conservative amino acid. In the present invention, a "conservative amino acid substitution" means that the amino acid residue is substituted by another amino acid residue having a side chain with similar chemical properties, for example, having a charge or hydrophobicity. Generally, the functional properties of the protein are not substantially changed by conservative amino acid substitutions. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate; and 7) sulfur-containing side chains: cysteine ​​and methionine.

[0064] In the present invention, one or more lysine residues in the amino acid sequence of the biologically active molecule may be substituted with arginine or histidine containing a basic side chain, for example, may be substituted with an arginine residue.

[0065] To inhibit ubiquitination of the biologically active molecules, the present inventors applied anti-ubiquitination technology (AUT), which involves substituting specific lysine residues within the amino acid sequence of the biologically active molecules with conservative amino acids, such as arginine. This anti-ubiquitination technology can increase the half-life and / or in vivo stability of the biologically active molecules.

[0066] In one embodiment, the conjugate described above may or may not include a linker between the biologically active molecule and the cell-penetrating peptide.

[0067] As used herein, the term "linker" refers to a molecule or group of atoms that connects, couples, or binds two or more components together. Each component of the conjugate herein, for example, a biologically active molecule and a cell-penetrating peptide, may be connected or bound together by any suitable means. The linker may have additional functions, such as increasing or decreasing water solubility, increasing the distance between the two components being connected to provide flexibility, or enhancing stability. However, it is preferred that the linker not affect the activity of the biologically active molecule.

[0068] The linker may be a peptide linker and may be, but is not limited to, 1 to 10, or 2 to 10 amino acids in length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., up to 20) amino acids in length, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 amino acids in length. For example, the peptide linker may be comprised of neutral amino acids (more specifically, Gly, Ser, Ala, Thr or a combination of these four amino acids). For example, [GGGGS]n, (GS)n, (G2S)n, (G3S)n, (G4S)n, Gn, LE, SSGG or GGGGSGGGGG (wherein G is Gly, S is Ser, L is Leu, E is Glu, and n is an integer from 1 to 10, such as but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), and for example, GS, GGGGS, LE, SSGG, GG, GGGGG, GGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, GGGGSGGGGSGGGGSGGGGS, or GGGGSGGGGSGGGGSGGGGSGGGGS.

[0069] As used herein, the term "connected" means that components are directly or indirectly connected together. The individual components may be connected covalently or non-covalently.

[0070] In one embodiment, the conjugate described above may comprise one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 33.

[0071] Another example of the present application provides a nucleic acid molecule encoding the biologically active molecule or conjugate described above.

[0072] As used herein, the terms "nucleic acid molecule," "nucleic acid," or "nucleic acid sequence" refer to a polymer of deoxyribonucleotides or ribonucleotides, either single-stranded or double-stranded. The nucleic acid molecule encompasses RNA genomic sequences, cDNA, and RNA sequences transcribed therefrom, and, unless otherwise specified, also includes analogs of natural nucleic acids.

[0073] The nucleic acid molecule comprises not only a nucleic acid sequence encoding the amino acid sequence of the conjugate, but also a complementary sequence to that sequence. The complementary sequence includes not only a perfectly complementary sequence but also a substantially complementary sequence, meaning a sequence that can hybridize with the nucleic acid sequence encoding the amino acid sequence of the conjugate, for example, under stringent conditions known in the art.

[0074] The above nucleic acid molecule may be an isolated nucleic acid molecule.

[0075] Another example of the present application provides a recombinant vector (expression vector) comprising a nucleic acid molecule encoding the biologically active molecule or conjugate described above and / or a recombinant cell comprising the nucleic acid molecule and / or the recombinant vector.

[0076] As used herein, the term "recombinant vector (expression vector)" refers to a nucleic acid construct operably linked to a gene insert encoding a protein of interest so as to be expressed. In one embodiment, the expression vector may be linear or circular, or single-stranded or double-stranded DNA, cDNA, RNA, etc. encoding two or more proteins of interest. The expression vector may be part of a vector that can be used to transform, transfect, or transfect a host, but is not limited thereto, and may itself be transcribed and / or translated in vitro.

[0077] As used herein, the term "operably linked" means that the linkage between nucleic acid sequences is functionally related. For example, a coding sequence (e.g., a sequence encoding a protein of interest) may be operably linked to appropriate regulatory elements to allow its replication, transcription, and / or translation. For example, a coding sequence is operably linked to a promoter if the promoter is capable of driving transcription of the coding sequence. Regulatory elements need not be adjacent to the coding sequence so long as they function properly. For example, an intervening sequence that is not translated but is transcribed may be present between a promoter sequence and a coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

[0078] Each component within the recombinant vector must be operably linked to one another, and the linkage of these component sequences can be accomplished by ligation at convenient restriction enzyme sites, or, if such sites do not exist, by using synthetic oligonucleotide adaptors or linkers according to conventional methods.

[0079] A recombinant vector may contain transcription and translation control sequences that enable the gene of interest to be expressed in a selected host. Expression control sequences may include a promoter for initiating transcription, an optional operator sequence for regulating such transcription, and / or sequences for regulating the termination of transcription and translation. The initiation and termination codons are generally considered to be part of the nucleic acid sequence encoding the protein of interest, and must be functional in a subject when the genetic construct is administered, and must be in frame with the coding sequence.

[0080] For example, a promoter refers to a DNA base sequence region to which transcriptional regulatory factors bind, and for the purpose of the present invention, a promoter capable of inducing strong and stable gene expression can be used to increase the gene expression rate.

[0081] Promoters can be constitutive or inducible. Examples of promoters include, but are not limited to, the early and late promoters of adenovirus, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV) promoter, Epstein virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, RNA polymerase ± promoter, T3 and T7 promoters, and the major operator and promoter region of phage lambda.

[0082] In addition, the recombinant vector may suitably contain adapters or linkers, enhancers, selectable markers (e.g., antibiotic resistance markers), replicative units, polyA sequences, purification tags or other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0083] Recombinant vectors can use various types of vectors, such as plasmids, viral vectors, bacteriophage vectors, and cosmid vectors.

[0084] In one embodiment, the recombinant cell may be an isolated recombinant cell.

[0085] In the present invention, the recombinant cell is a host cell capable of stably and continuously cloning or expressing the recombinant vector, and any host cell known in the art can be used, and as a prokaryotic cell, E. coli, for example, E. coliJM109, E. coliBL21, E. coliRR1, E. coliLE392, E. coliB, E. coliX 1776, E. Examples of host cells that can be used include, but are not limited to, strains of the genus Bacillus, such as coliW3110, Bacillus subtilis, and Bacillus thuringiensis, and strains of enterobacteria, such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. In the case of transforming eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, such as CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, and MDCK cell lines, can be used.

[0086] The introduction of a recombinant vector into a cell can be accomplished using any suitable standard technique known in the art, such as electroporation, electroinjection, microinjection, calcium phosphate co-precipitation, calcium chloride / rubidium chloride, retroviral infection, DEAE-dextran, cationic liposome, polyethylene glycol-mediated uptake, gene gun, etc., but is not limited thereto. At this time, the circular structure can be introduced in a linear form by cutting it with an appropriate restriction enzyme.

[0087] The method for selecting the above recombinant cells can be easily performed using a method widely known in the art, utilizing a phenotype expressed by a selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformants can be easily selected by culturing the transformants in a medium containing the antibiotic.

[0088] Culturing recombinant cells can be accomplished using various methods known in the art. For example, recombinant cells can be inoculated into a culture medium and cultured. Once the cell density reaches a certain level, IPTG can be added to the medium to induce protein expression. This culturing can then yield proteins secreted into the cells or into the medium.

[0089] Proteins secreted into cells or the medium can be purified using various purification methods known in the art, but are preferably purified using affinity chromatography using an affinity tag. For example, if the conjugate is fused to GST, the desired protein can be easily obtained using a glutathione-bound resin column, and if fused to His, the desired protein can be easily obtained using IMAC (immobilized metal affinity chromatography).

[0090] Another example of the present application provides a method for producing a conjugate, comprising the step of expressing the nucleic acid molecule or the recombinant vector in a cell.

[0091] The conjugate can be prepared by expressing a nucleic acid molecule encoding the conjugate provided herein in a suitable host cell as described above.

[0092] In one embodiment, the method for producing the conjugate may include a step of culturing a cell comprising the nucleic acid molecule or the recombinant vector under conditions suitable for expression. The culturing step may be performed under conventional culture conditions as described above. In addition, the method may further include a step of isolating and / or purifying the conjugate from the culture after the culturing step.

[0093] Another example of the present application provides a method for preparing a conjugate comprising the step of linking a cell-penetrating peptide having an amino acid sequence of SEQ ID NO: 1 to a biologically active molecule.

[0094] In one embodiment, the connecting step may be performed ex vivo or in vitro.

[0095] In one embodiment, the linking may be performed chemically or recombinantly, but is not limited thereto.

[0096] In one embodiment, the method for preparing the conjugate may additionally comprise, prior to the step of linking the cell penetrating peptide to the N-terminus, C-terminus, or both of the biologically active molecule, a step of preparing a variant of the biologically active molecule in which one or more lysine residues in the amino acid sequence of the biologically active molecule are substituted with histidine residues or arginine residues.

[0097] Another example of the present application provides a cosmetic composition comprising the above-described conjugate as an active ingredient.

[0098] The above-described cosmetic composition may be a cosmetic composition for improving skin condition. The improvement in skin condition may include, but is not limited to, skin moisturizing, skin whitening, skin soothing, skin irritation relief, skin elasticity improvement, skin inflammation improvement, UV-induced skin damage suppression, pore reduction, or wrinkle improvement.

[0099] In one embodiment, the improvement in skin condition may include, but is not limited to, skin soothing, skin irritation relief, skin elasticity improvement, and wrinkle improvement.

[0100] In this specification, “skin moisturizing” refers to increasing moisture in the skin and maintaining it in a moist state.

[0101] In this specification, “skin whitening” means not only brightening the skin tone by inhibiting the synthesis of melanin pigment, but also improving skin hyperpigmentation such as freckles or blemishes caused by ultraviolet rays, hormones, or genetics.

[0102] In this specification, “skin soothing” means relieving the heat or pain of irritated skin, and may include soothing skin irritated by skin inflammation.

[0103] In this specification, “skin irritation relief” means alleviating irritation to which the skin is sensitive and returning the skin to a healthy and stable state.

[0104] In this specification, “improvement in skin elasticity” means alleviating the degree to which the skin sags or stretches, and refers to the maintenance of skin elasticity in a state where elastic fibers composed of elastin exist together with collagen fibers, and where elastin and collagen are sufficiently present.

[0105] As used herein, "improving skin inflammation" refers to suppressing skin inflammation. Inflammation is a defense response of living tissue to a certain stimulus, and refers to a complex lesion that causes three things: tissue degeneration, circulatory disturbance, exudation, and tissue proliferation. The inflammatory response is a mechanism for regenerating damage caused by physical actions, harmful substances, chemical stimuli, bacterial infections, etc. as part of the defense response of living tissue to external stimuli. However, a persistent inflammatory response can actually cause mucosal damage and promote skin tissue damage, inflammation, and skin troubles.

[0106] In this specification, “pore reduction” refers to a reduction in pore area (reduction in pore size).

[0107] In this specification, “wrinkle improvement” means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already formed.

[0108] In one embodiment, the wrinkles may be frown lines, crow's feet wrinkles, mouth wrinkles, neck wrinkles, nose wrinkles, crow's feet wrinkles, or neck wrinkles, and may be crow's feet wrinkles or neck wrinkles, but are not limited thereto.

[0109] As demonstrated in the examples of the present invention described below, a cosmetic composition including the above-described conjugate has effects of improving wrinkles, reducing pores, soothing skin, improving skin elasticity, and soothing the scalp, and can be usefully used as a cosmetic composition for improving skin condition.

[0110] The above-described cosmetic composition may also be a cosmetic composition for improving hair loss.

[0111] In this specification, “hair loss” means a phenomenon in which hair falls out from the skin or a condition in which hair becomes thick or thin, and specifically, it may be at least one selected from the group consisting of nutritional alopecia, endocrine alopecia, vascular alopecia, premature alopecia, alopecia areata, neural alopecia, alopecia pityriasis, trichotillomania, alopecia malignant, female pattern alopecia, male pattern alopecia, androgenetic alopecia, telogen effluvium, tinea capitis, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex, drug-induced alopecia universalis, mechanical alopecia, traumatic alopecia, compression alopecia, anagen alopecia, alopecia pityriasis, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, cicatricial alopecia, and congenital alopecia.

[0112] In this specification, “hair loss improvement” means any action that at least reduces the severity of symptoms, such as parameters related to the alleviation or treatment of hair loss.

[0113] As demonstrated in the examples of the present invention described below, a cosmetic composition comprising a conjugate of a biologically active molecule of the present invention and a cell-penetrating peptide has a scalp soothing effect and can be used as a cosmetic composition for improving hair loss.

[0114] In one embodiment, the cosmetic composition may include not only the above-described conjugate as an active ingredient, but also ingredients commonly used in cosmetic compositions, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances, and carriers.

[0115] As the above carrier, purified water, monohydric alcohols (ethanol or propyl alcohol), polyhydric alcohols (glycerol, 1,3-butylene glycol or propylene glycol), higher fatty acids (palmitic acid or linolenic acid), oils (wheat germ oil, camellia oil, jojoba oil, olive oil, squalene, sunflower oil, macadamia peanut oil, avogar oil, soybean hydrolyzed lecithin or fatty acid glycerides), etc. can be used, but are not limited thereto. In addition, surfactants, bactericides, antioxidants, ultraviolet absorbers, anti-inflammatory agents and refreshing agents can be added as necessary.

[0116] Examples of surfactants that can be used include polyoxyethylene, hydrogenated castor oil, polyoxyethylene, oleyl ether, polyoxyethylene monooleate, polyoxyethylene, glyceryl monostearate, sorbitan monostearate, polyoxyethylene monooleate, sorbitan, sucrose fatty acid ester, monolaurate hexaglycerin, polyoxyethylene reduced lanolin, POE, glyceryl pyroglutamic acid, isostearic acid, diester, N-acetylglutamine, and isostearyl ester.

[0117] Antiseptics that can be used include hinoctiol, triclosan, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, and zinc pyritaone.

[0118] Any of butylhydroxyanisole, gallic acid, propyl gallic acid, and erythorbic acid can be used as antioxidants.

[0119] Examples of UV absorbers that can be used include benzophenones such as dihydroxybenzophenone, melanin, ethyl para-aminobenzoate, para-dimethylaminobenzoic acid 2-ethylhexyl ester, cinoxite, para-methoxycinnamic acid 2-ethylhexyl ester, 2-(2-hydroxy-5-methylphenyl) benzotriazole, urocanic acid, and metal oxide fine particles.

[0120] Anti-inflammatory agents include dipotassium glycyrrhizinate or allantoin, and refreshing agents include red pepper tincture or 1-menthol.

[0121] In one embodiment, the cosmetic composition described above can be prepared in a formulation selected from the group consisting of a solution, an external ointment, a gel, a cream, a foam, a nourishing toner, an emollient toner, a pack, an emollient, a milky lotion, a makeup base, an essence, an ampoule, a hair ampoule, a scalp treatment, a hair tonic, a hair conditioner, a hair treatment, a hair lotion, a hair shampoo, a hair rinse, a shampoo with rinse function, a hair nourishing toner, a hair gel, a hair wax, a hair spray, a dye, a soap, a liquid cleanser, a bath agent, a sunscreen cream, a sun oil, a suspension, an emulsion, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, a patch, and a spray, but is not limited thereto. In addition, the composition of the present invention can be formulated and used in the form of a liquid, an aerosol, or a sterile injection solution, and when formulating the composition, diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants can be used.

[0122] In one embodiment, the cosmetic composition comprises, in addition to the above-described conjugate, water, butylene glycol, dipropylene glycol, niacinamide, glycerin, 1,2-hexanediol, betaine, hydroxyacetophenone, tromethamine, carbomer, octyldodeceth-16, xanthan gum, adenosine, sodium hyaluronate, disodium EDTA, fragrance, hydrolyzed sodium hyaluronate, It may further include at least one selected from the group consisting of Decapeptide-40, Pentylene Glycol, C10-30 Alkyl Acrylate Crosspolymer, Hydroxyethylcellulose, Ethanol, Propanediol, Sodium Citrate, Menthol, Salicylic Acid, and Panthenol, but is not limited thereto.

[0123] In one embodiment, the cosmetic composition may include one or more conjugates.

[0124] In one embodiment, the above-described conjugate may be included in an amount of 0.00001 to 5 wt% relative to the weight of the entire cosmetic composition, more specifically 0.00001 to 5 wt%, 0.00001 to 1 wt%, 0.00001 to 0.1 wt%, 0.00001 to 0.01 wt%, 0.00005 to 5 wt%, more specifically 0.00005 to 5 wt%, 0.00005 to 1 wt%, 0.00005 to 0.1 wt%, 0.00005 to 0.01 wt%, 0.0001 to 5 wt%, more specifically 0.0001 to 5 wt%, 0.0001 to 1 wt%, 0.0001 to 0.1 wt%, or It may be included in an amount of 0.0001 to 0.01 wt%, but is not limited thereto. If the content of the above-mentioned conjugate is less than 0.00001 wt% of the total weight of the cosmetic composition, it is difficult to expect a substantial skin improvement effect, and if it is more than 5 wt%, problems such as the formulation becoming unstable may occur.

[0125] Another example of the present application provides a pharmaceutical composition for preventing or treating skin diseases, comprising the above-described conjugate as an active ingredient.

[0126] In one embodiment, the skin disease may be a skin wrinkle-related disease, and the skin wrinkle-related disease may be a skin wrinkle-related disease caused by skin photodamage. "Skin photodamage" refers to skin damage caused by exposure of the skin to sunlight or UV light (particularly, UV-B). In addition, the skin wrinkle-related disease may be any one or more selected from the group consisting of, but not limited to, elastosis, thinning of the skin, skin atrophy, reduction of collagen fibers and elastic fibers, loss of skin elasticity, dryness, wrinkle formation, and premature skin aging.

[0127] In one embodiment, the skin disease may be a skin pigmentation disease. For example, the skin disease may be at least one selected from the group consisting of, but not limited to, lentigines, freckles, hypopigmentation, and hyperpigmentation.

[0128] In one embodiment, the skin disease may be selected from the group consisting of keratosis pilaris, erythema nodosum, prickly heat, pityriasis versicolor, strophulosis, calluses and dermatitis.

[0129] Another example of the present application provides a preventive pharmaceutical composition for preventing or treating hair loss comprising the above-described conjugate as an active ingredient.

[0130] As demonstrated in the examples of the present invention described below, a cosmetic composition comprising a conjugate of a biologically active molecule of the present invention and a cell-penetrating peptide has a scalp soothing effect and can be used as a pharmaceutical composition for preventing or treating hair loss.

[0131] In this specification, prevention of hair loss refers to all acts of suppressing or delaying the occurrence of hair loss by using a composition containing the above-described conjugate as an active ingredient, and treatment of hair loss refers to all acts of managing or improving the symptoms of hair loss by using a composition containing the above-described conjugate as an active ingredient.

[0132] The pharmaceutical composition of the present invention described above may further include appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods, and carriers, excipients, and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0133] The pharmaceutical composition of the present invention can be delivered to the body by various routes including oral, transcutaneous, subcutaneous, intravenous or intramuscular administration, and can be administered as an injectable preparation. In addition, the pharmaceutical composition of the present invention can be formulated according to methods well known to those skilled in the art so as to achieve rapid release, delayed release or slow release after administration according to the above method. The formulations include tablets, pills, powders, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders, etc.Suitable carriers, excipients and diluents include lactose, dextrose, sucrose, mannitol, xylitol, erythritol, maltitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearate and mineral oil. In addition, the formulation may further include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, etc. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the extent of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, the pharmaceutical composition of the present invention may be administered once a day or divided into several doses. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0134] In one embodiment, the pharmaceutical composition comprises, in addition to the above-described conjugate, water, butylene glycol, dipropylene glycol, niacinamide, glycerin, 1,2-hexanediol, betaine, hydroxyacetophenone, tromethamine, carbomer, octyldodeceth-16, xanthan gum, adenosine, sodium hyaluronate, disodium EDTA, fragrance, hydrolyzed sodium hyaluronate, It may further include at least one selected from the group consisting of Decapeptide-40, Pentylene Glycol, C10-30 Alkyl Acrylate Crosspolymer, Hydroxyethylcellulose, Ethanol, Propanediol, Sodium Citrate, Menthol, Salicylic Acid, and Panthenol, but is not limited thereto.

[0135] Another example of the present application provides a pharmaceutical composition for preventing and / or improving skin diseases, comprising the above-described conjugate as an active ingredient.

[0136] The above skin diseases are as described above.

[0137] Another example of the present application provides a pharmaceutical composition for preventing and / or improving hair loss, comprising the above-described conjugate as an active ingredient.

[0138] The above hair loss, hair loss prevention, and hair loss improvement are as described above.

[0139] In this specification, “quasi-drug” means a product that has a milder effect than a pharmaceutical product among products used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals. For example, according to the Pharmaceutical Affairs Act, a quasi-drug is a product excluding products used for the purpose of pharmaceutical products, and includes products used for treating or preventing diseases of humans or animals, products that have a mild effect on the human body or do not act directly, etc.

[0140] The quasi-drug composition of the present invention is not particularly limited in its formulation, and can be formulated in various forms of quasi-drugs known in the art. The formulated quasi-drugs include, but are not limited to, disinfectant cleansers, shower foams, ointments, wet tissues, coating agents, hair tonics, hair lotions, hair creams, hair sprays, hair mousses, hair gels, hair conditioners, hair shampoos, hair rinses, hair packs, hair ampoules, hair essences, hair treatments, eyebrow hair growth agents, eyelash hair growth agents, eyelash nutrients, pet shampoos, pet rinses, soaps, ointments, creams, lotions, oils, waxes, aerosols, or patches, and the like. The composition includes all quasi-drugs in the conventional sense, and the formulation method, dosage, method of use, components, etc. of the quasi-drugs can be appropriately selected from conventional techniques known in the art.

[0141] In addition to the above-mentioned ingredients, the quasi-drug composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent, as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effects of the present invention, and may include, for example, fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc.

[0142] Representative examples of pharmaceutically acceptable carriers, excipients or diluents of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia gum, alginate, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil, injectable esters, withepsol, macrogol, Tween 61, cacao butter, lauric acid, etc.

[0143] In one embodiment, the above-described pharmaceutical composition comprises, in addition to the above-described conjugate, water, butylene glycol, dipropylene glycol, niacinamide, glycerin, 1,2-hexanediol, betaine, hydroxyacetophenone, tromethamine, carbomer, octyldodeceth-16, xanthan gum, adenosine, sodium hyaluronate, disodium EDTA, fragrance, hydrolyzed sodium hyaluronate, It may further include at least one selected from the group consisting of Decapeptide-40, Pentylene Glycol, C10-30 Alkyl Acrylate Crosspolymer, Hydroxyethylcellulose, Ethanol, Propanediol, Sodium Citrate, Menthol, Salicylic Acid, and Panthenol, but is not limited thereto.

[0144] Another example of the present application provides a food composition comprising the conjugate described above.

[0145] In one embodiment, the food composition may have a skin disease prevention and / or improvement effect.

[0146] In one embodiment, the food composition may have a hair loss prevention and / or improvement effect.

[0147] The above skin diseases are as described above.

[0148] The above hair loss, hair loss prevention, and hair loss improvement are as described above.

[0149] The food composition of the present invention may include not only the conjugate as an active ingredient, but also ingredients commonly added during food manufacturing. The added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the carbohydrates mentioned above include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents [thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is manufactured as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, Eucommia extract, jujube extract, licorice extract, etc. may be additionally included in addition to the extract of the present invention.

[0150] In the present invention, the food composition may be used as various food additives or health functional foods. The food may be manufactured in the form of powder, granules, tablets, capsules, or beverages, and may specifically be beverages, meat, chocolate, food, confectionery, pizza, ramen, other noodles, gum, ice cream, alcoholic beverages, vitamin complexes, or health supplements.

[0151] Another example of the present application provides a biologically active molecule, wherein at least one lysine residue in the amino acid sequence of the biologically active molecule is substituted with an arginine residue or a histidine residue. More specifically, the lysine residue may be substituted with an arginine residue.

[0152] The biologically active molecules are as described above.

[0153] To inhibit ubiquitination of the biologically active molecules, the present inventors applied anti-ubiquitination technology (AUT), which involves substituting specific lysine residues in the amino acid sequence of the biologically active molecules with arginine. This anti-ubiquitination technology can increase the half-life and / or in vivo stability of the biologically active molecules.

[0154] In one embodiment, the biologically active molecule is selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factor (TGF), a botulinum toxin serotype selected from the group consisting of A, B, C, D, E, F, G and H, a recombinant botulinum toxin, a modified botulinum toxin, a fragment of a botulinum toxin, noggin, superoxide dismutase (SOD), acetyl hexapeptide-8 (acetyl hexapeptide-8). AH), acetyl pentapeptide-4 (AP), tissue inhibitor of metalloproteinase (TIMP), aldehyde dehydrogenase (ALDH), and combinations thereof, but is not limited thereto.

[0155] The above variant may have characteristics such as advantages in recombinant production, increased half-life (e.g., in vivo half-life), and / or enhanced stability (in vivo stability; prevention of degradation by degrading enzymes, etc.) compared to a wild-type biologically active molecule in which no mutation has been introduced.

[0156] In one embodiment, the variant of the biologically active molecule may comprise, but is not limited to, an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 33.

[0157] Other examples of the present application provide a nucleic acid sequence encoding the biologically active molecule, a recombinant vector (expression vector) comprising the nucleic acid sequence, and / or a recombinant cell comprising the nucleic acid molecule and / or the recombinant vector. The contents of the nucleic acid sequence, the recombinant vector, and the recombinant cell are as described above.

[0158] Another example of the present application provides a use for the preparation of a pharmaceutical composition for preventing or treating a skin disease of the biologically active molecule and / or the conjugate.

[0159] Another example of the present application provides a method for preventing or treating a skin disease, comprising administering to a patient in need of prevention or treatment of a skin disease a composition comprising the biologically active molecule; the conjugate; and / or the biologically active molecule and / or the conjugate.

[0160] Another example of the present application provides a use of the biologically active molecule; the conjugate; and / or a composition comprising the biologically active molecule and / or the conjugate for preventing, treating, and / or improving a skin disease.

[0161] Another example of the present application provides a use of the biologically active molecule; the conjugate; and / or a composition comprising the biologically active molecule and / or the conjugate for improving a skin condition.

[0162] Another example of the present application provides a use for the preparation of a pharmaceutical composition for preventing or treating hair loss of the biologically active molecule and / or the conjugate.

[0163] Another example of the present application provides a method for preventing or treating hair loss, comprising administering to a patient in need of prevention or treatment of hair loss a composition comprising the biologically active molecule; the conjugate; and / or the biologically active molecule and / or the conjugate.

[0164] Another example of the present application provides a use of the biologically active molecule; the conjugate; and / or a composition comprising the biologically active molecule and / or the conjugate for preventing, treating, and / or ameliorating hair loss.

[0165] In this specification, the phrase “a polynucleotide (which may be used interchangeably with “gene” or “nucleic acid molecule”) or “a polypeptide (which may be used interchangeably with “protein”)” “comprises a specific nucleic acid sequence or amino acid sequence” or “consists of a specific nucleic acid sequence or amino acid sequence” or “is represented by a specific nucleic acid sequence or amino acid sequence” may mean that the polynucleotide or polypeptide consists of or essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a “substantially equivalent sequence” in which a mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or the desired function of the polynucleotide or polypeptide is maintained.

[0166] In one example, a protein or nucleic acid molecule "comprises or has a particular amino acid sequence or nucleic acid sequence" or "consists of or is represented by a particular amino acid sequence or nucleic acid sequence" can mean that the polynucleotide or polypeptide (i) consists of or essentially comprises the particular nucleic acid sequence or amino acid sequence, or (ii) consists of or essentially comprises an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% homologous to the particular nucleic acid sequence or amino acid sequence and retains its original function and / or desired function.

[0167] As used herein, the term "homology (identity or similarity)" refers to the degree of identity with a given nucleic acid sequence or amino acid sequence, and can be expressed as a percentage (%). For nucleic acid sequence homology, for example, it can be determined using the algorithm BLAST by the literature (see: Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see: Methods Enzymol., 183, 63, 1990). Based on the algorithm BLAST, programs called BLASTN and BLASTX have been developed (see: http: / www.ncbi.nlm.nih.gov).

[0168] Hereinafter, the present invention will be described in more detail based on examples. The following examples are intended only to illustrate the present invention and are not intended to limit the present invention.

[0169] Example 1: Preparation of a conjugate of a biologically active molecule and a cell-penetrating peptide (BMTS)

[0170] 1.1. Synthesis of a conjugate of a biologically active molecule expressible in E. coli and a cell-penetrating peptide (BMTS)

[0171] We commissioned GeneScript (China) to synthesize a conjugate of a biologically active molecule and a cell-penetrating peptide (BMTS). The cell-penetrating peptide (BMTS) has the amino acid sequence of SEQ ID NO: 1, and the sequence identified by Blast search was used.

[0172] [Sequence number 1]

[0173] AAVLLPVLLAAP

[0174] Biologically active molecules were used, including biologically active molecules that had been mutated by substituting specific lysine residues with arginine residues in the amino acid sequence to avoid ubiquitination (hereinafter, AUT (Anti-Ubiquitination Technology) applied) and wild-type biologically active molecules. The biologically active molecules, sequences, and the positions of mutations when AUT was applied are shown in Table 1 below. Mutated residues are underlined.

[0175] AUT applied biologically active molecule name biologically active molecule sequence (mutation position) sequence number AUT-BT-LC Botulinum toxin type A light chain recombined into a single chain PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGRFATDPAVTLAH ELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDK LKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNK (K223R)2AUT-FGF-1fibroblast growth factor-1; FGF-1)FNLPPGNYKKPKLLYCSNGGHFLRILPDGTVDGTRDRSDQHIQLQLSAESVGEVYIKSTETGQYLAMDTDGLLYGSQTPNEECLFLERLEENHYNTYISKKHAERNWFVGLKKNGSCKRGPRTHYGQKAILFLPLPVSSD (K117R)3AUT-FGF-10Fibroblast Growth Factor-10 (fibroblast growth factor-10;FGF-10)LGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQRTRRKNTSAHFLPMVVHS (K164R)4AUT-PDGF-A혈소판유래 성장인자 subunit A(platelet-derived growth factor subunit A; PDGF-A)SIEEAVPAVCKTRTVIYEIPRSQVDPTSANFLIWPPCVEVKRCTGCCNTSSVKCQPSRVHHRSVKVAKVEYVRKKPKLREVQVRLEEHLECACATTSLNPDYREEDTGRPRESGKKRKRKRLKPT (K91R)5AUT-PDGF-B혈소판유래 성장인자 subunit B(platelet-derived growth factor subunit B; PDGF-B)SLGSLTIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNNRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACRCETVAAARPVT (K120R)6AUT-FGF-2섬유아세포 성장인자-2 (fibroblast growth factor-2; FGF-2)AAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRRYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS (K130R)7AUT-SOD-1슈퍼옥사이드 디스뮤테이스-1 (Superoxide dismutase-1;SOD-1)ATKAVCVLRGDGPVQGIINFEQKESNGPVRVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHERADDLGKGGNEESTKTGNAGSRLACGVIGIAQ (K21R, K42R, K134R)8AUT-VEGFA혈관내피 성장인자 A (Vascular endothelial growth factor A; VEGF-A)APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCRCSCKNTDSRCKARQLELNERTCRCDKPRR (K148R)9AUT-IGF-1인슐린유사 성장인자-1 (insulin-like growth factor-1; IGF-1)GPETLCGAELVDALQFVCGDRGFYFNRPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA (K64R)10AUT-TGF-β2전환 성장인자-β2 (Transforming growth factor-β2; TGF-β2MALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVRSCKCS (K107R)11AUT-EGF상피세포 성장인자(epidermal growth factor;EGF)MNSDSECPLSHDGYCLHDGVCMYIEALDRYACNCVVGYIGERCQYRDLKWWELR (K29R)12Aut-NOG노긴(noggin)QHYLHIRPAPSDNLPLVDLIEHPDPIFDPKEKDLNETLLRSLLGGHYDPGFMATSPPEDRPGGGGGAAGGAEDLAELDQLLRQRPSGAMPSEIRGLEFSEGLAQGKKQRLSKKLRRKLQMWLWSQTFCPVLYAWNDLGSRFWPRYVKVGSCFSKRSCSVPEGMVCKPSKSVHLTVLRWRCQRRGGQRCGWIPIQYPIISECKCSC (K94R)13Aut-TIMP1Tissue inhibitor of metalloproteinase-1CTCVPPHPQTAFCNSDLVIRAKFVGTPEVNQTTLYQRYEIKMTKMYKGFQALGDAADIRFVYTPAMESVCGYFHRSHNRSEEFLIAGKLQDGLLHITTCSFVAPWNSLSLAQRRGFTRTYTVGCEECTVFPCLSIPCKLQSGTHCLWTDQLLQGSEKGFQSRHLACLPREPGLCTWQSLRSQIA (K118R)14Aut-SCF줄기세포 인자 (stem cell factor; SCF)EGICRNRVTNNVRDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA (K13R)15;

[0176] For two of these biologically active molecules, a biological linker (linker; 5x GGGGSGGGGSGGGGSGGGGSGGGGS) was used to link them to BMTS, which is indicated as '5L'. The synthesized base sequences were each cloned into the pET24a vector, and in the case of the growth factor SCF, they were transferred to the EE tag vector and cloned. Information on the synthesized sequences is shown in Table 2 below, and the respective DNA maps are shown in Figures 1a and 1b.

[0177]

[0178] 1.2. Peptide synthesis of conjugates of biologically active molecules and BMTS

[0179] Additionally, conjugates of biologically active molecules and BMTS were synthesized through peptide synthesis. Two wild-type biologically active molecules were used (see Table 3).

[0180] NameBiologically active molecule (wild type)SequenceSequence numberAHacetyl hexapeptide-8 (AH)EEMQRR30APacetyl pentapeptide-4 (AP)KTTKS31

[0181] For peptide synthesis, it was produced without the start codon methionine (M). The biologically active molecules used, the positions of the mutant residues, and the BMTS conjugation direction are shown in Table 4 below. The peptide synthesis method is described in Example 3.

[0182] NameBMTS Junction DirectionAmino Acid SequenceSequence NumberAH-BMTSC-TerminalEEMQRRAAVLLPVLLAAP32AP-BMTSC-TerminalKTTKSAAVLLPVLLAAP33

[0183] Example 2: Establishment of conditions for expression and purification of a conjugate expressible in E. coli

[0184] 2.1. Establishment of expression conditions for conjugates of biologically active molecules and BMTS

[0185] (1) Presentation of expression induction conditions for each conjugate

[0186] The conjugates synthesized in Example 1.1 were inoculated at 0.1% stock in LB broth containing 100 ug / ml kanamycin or ampicillin on the first day, and then cultured in a shaking incubator at 37°C. The following day, they were inoculated at 1% stock in LB broth containing 100 ug / ml kanamycin or ampicillin, and cultured at 37°C until OD600nm reached approximately 0.6. Upon reaching this, 0.5 to 1 mM IPTG was added, and each conjugate was cultured at 18°C ​​for 18 hours or at 37°C for 6 hours.

[0187] (2) Confirmation of expression by conjugate

[0188] Some of the expression-induced conjugates were collected, dissolved in PBS, and then sonicated to disrupt the cells. Then, centrifuged at 12,000 rpm for 15 minutes, and divided into whole cells after expression induction, supernatant (soluble form), and pellet (insoluble form) after expression induction. These were subjected to PAGE using a 4-12% gradient SDS gel, and then stained and destained to confirm whether the expression status was soluble or insoluble. At this time, some cells collected before expression induction were also disrupted through the same process and used as a control group (see Fig. 2).

[0189] 2.2. Purification process of the conjugate expressed in a soluble form

[0190] Among the expression-induced conjugates, proteins expressed in a soluble form were purified using the following method (Fig. 3);

[0191] 2.2.1. Purification of BMTS-AUT-BT-LC

[0192] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0193] 2) Supernatant recovery: The obtained cell pellet was dissolved in lysis buffer (20 mM MES (pH 6.5), 1 mM EDTA) at a ratio of 1:50, and the cells were disrupted with a homogenizer on ice. Centrifugation was performed at 17,000 xg for 30 minutes to obtain only the supernatant containing the expressed protein.

[0194] 3) Primary purification: The obtained supernatant was filtered using a 0.4 um polyethersulfone (PES) syringe and filtered using a SP Sepharose FF column equilibrated with the corresponding A buffer (20 mM MES pH 6.5), and then washed with A buffer (20 mM MES pH 6.5). The proteins bound to the column were eluted using A and buffer (20 mM MES pH 6.5, 1 M NaCl), using a linear gradient so that the concentration of B buffer was 0 to 40%.

[0195] 4) Secondary purification: The eluted fraction was filtered using an anion exchange column (Q FF), and only the flow-through fraction was mixed with a 50 mM Tris (pH 7.2) solution containing 1 M NaCl. Afterwards, to remove salt, it was filtered again using a desalting column 26 / 10 using PBS to obtain the final protein.

[0196] 2.2.2. Purification of BMTS-AUT-FGF-1, BMTS-AUT-FGF-2, and BMTS-AUT-FGF-10

[0197] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0198] 2) Supernatant recovery: The obtained cell pellet was dissolved in lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 5 mM 2-mercaptoethanol) at a ratio of 1:10, and the cells were disrupted with a homogenizer on ice. Centrifugation was performed at 11,000 xg for 30 minutes to obtain only the supernatant containing the expressed protein.

[0199] 3) 1st purification: The obtained supernatant was filtered using a 0.4 um polyethersulfone (PES) syringe and filtered using a Heparin (Hiprep 16 / 10) column equilibrated with the corresponding A buffer (50 mM Tris pH 8.0, 500 mM NaCl), and then washed with a solution of 5 times the volume of the column with a mixture of A and B buffers (50 mM Tris pH 8.0, 2 M NaCl), and the proteins bound to the column were extracted with 5 times the volume of the column while adjusting the relative ratios of A and B buffers to 50-100%, and then extracted once more with 4 times the volume of the column with only B buffer at 100%.

[0200] 4) Secondary purification: The eluted fraction was subjected to size exclusion chromatography using a Superdex 75 column to obtain the final protein.

[0201] 2.2.3. BMTS-AUT-SOD-1 tablets

[0202] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0203] 2) Cell disruption: The obtained cell pellet was disrupted three times with a homogenizer at 10,000 bar at 10 ℃, centrifuged at 17,000 xg at 4 ℃ for 30 minutes, the pellet was removed, and the supernatant was obtained.

[0204] 3) Removal of impurities: To remove impurities, the cell lysate was placed in a beaker and heated using a stirrer while mixing until the stirring machine temperature reached 200℃. Afterwards, the stirring was continued while the temperature was lowered for 5 minutes so that the temperature of the cell lysate reached 70℃, and then the temperature was immediately lowered by placing it in ice water. The washed cell lysate was centrifuged at 4℃ and 17,000 xg for 30 minutes to obtain the supernatant.

[0205] 4) Primary and secondary purification: The obtained supernatant was filtered using an anion exchange column (Q FF), and only the flow-through fraction was mixed with a 50 mM Tris (pH 8.0) solution containing 2 M AmSO4 and filtered using a hydrophobic interaction column (phenyl). Afterwards, only the eluted fraction was collected separately and filtered again using a desalting column 26 / 10 using PBS to remove salt, thereby obtaining the final protein.

[0206] 2.3. Purification process of the conjugate expressed in an in-soluble form

[0207] 2.3.1. Purification of AUT-EGF-1-5L-BMTS

[0208] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0209] 2) Cell disruption and inclusion body acquisition: The obtained cell pellet was dissolved in lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5% Triton X-100), disrupted twice using a high-pressure homogenizer (800 to 1,200 bar), and centrifuged at 13,000 xg at 4 ℃ for 30 minutes to obtain a pellet (inclusion body).

[0210] 3) Solubilization process: The obtained inclusion bodies are washed twice with IB washing buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% Triton X-100) and then washed once more with 50 mM Tris-HCl (pH 8.0). Then, they are solubilized overnight at RT using solubilization buffer (50 mM Tris-HCl (pH 8.0), 8 M urea), and centrifuged at 13,000 xg for 30 minutes at 4°C to obtain only the solubilized supernatant.

[0211] 4) Refolding process: The obtained solubilized supernatant is mixed with 40 times the volume of 50 mM Tris-HCl (pH 8.0) and dialysis is performed overnight at RT using a 3 kDa MWCO membrane. The supernatant is centrifuged at 13,000 xg for 30 minutes at 4°C to separate only the supernatant and filtered through a 0.2 micrometer membrane.

[0212] 5) Primary purification: The anion exchange purification (DEAE) column was equilibrated with 10 times the column volume of equilibration buffer (50 mM Tris-HCl (pH 8.0)), and the supernatant was applied to the anion exchange purification (DEAE) column. The column was then eluted with elution buffer (50 mM Tris-HCl (pH 8.0), 0.5 M NaCl), and the fractions containing AUT-EGF-5L-BMTS were collected.

[0213] 6) Secondary purification: The fractions collected for further purification were pooled and subjected to gel filtration chromatography, and the fraction containing AUT-EGF-5L-BMTS was collected. The eluted AUT-EGF-5L-BMTS was dialyzed against 100 x elution volume dialysis buffer (phosphate-buffered saline) at 4°C for 12 h, and the purified AUT-EGF-5L-BMTS was concentrated to 1 mg / ml using a 10 kDa cutoff Centricon and stored at -80°C.

[0214] 2.3.2. Purification of BMTS-5L-AUT-IGF-1

[0215] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0216] 2) Cell disruption and inclusion body acquisition: The obtained cell pellet was dissolved in lysis buffer (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5% Triton X-100), disrupted twice using a high-pressure homogenizer (800 to 1,200 bar), and centrifuged at 17,000 xg at 4 ℃ for 30 minutes to obtain a pellet (inclusion body).

[0217] 3) Solubilization process: The obtained inclusion bodies were washed twice with IB washing buffer (50 mM Tris-HCl (pH 8.0), 1 M Urea, 1 mM EDTA) and centrifuged at 17,000 xg at 4°C for 30 minutes to obtain only the solubilized supernatant.

[0218] 4) Refolding process: The obtained 50 mg inclusion bodies were solubilized with 3 ml of 50 mM Tris-HCl (pH 8.0), 1 M urea, and 1 mM EDTA at RT for 12 h, and then centrifuged at 17,000 x g for 20 min at room temperature to isolate only the supernatant. Refolding was performed by gradually adding refolding buffer (50 mM Tris-HCl pH 5.0, 2 mM GSSG, 2 mM GSH, 3 M urea) until the protein concentration reached 5 mg / ml at room temperature. After refolding, the pH of the IBs solution was adjusted to 5 with 2 mM acetic acid, and after 2 h, the suspension was centrifuged at 17,000 x g, 4°C for 30 min.

[0219] 5) Purification process: After equilibrating a Hitrap Capto S column with 5 column volumes (CV) of buffer A (50 mM sodium acetate pH 5.4), the sample containing BMST-5L-AUT-IGF-1 was loaded onto the column, washed with 5 CV of buffer A, eluted with a 20 CV linear gradient of 1 M NaCl, and the fractions were analyzed by SDS-PAGE. The fractions containing mainly BMTS-5L-AUT-IGF-1 were merged and concentrated to 3 ml by ultrafiltration (3K, Amicon Ultra, Millipore, Germany). Afterwards, the concentrated IGF-1 solution was applied to a HiPrep 16 / 60 Sephacryl S-100 high-resolution column (Cytiva, USA) and further purified using buffer A. The eluted fraction was analyzed by SDS-PAGE, and the fraction containing BMST-5L-AUT-IGF-1 was dialyzed in 50 mM Sodium Acetate + 100 mM NaCl (pH 5.4) for 24 hours, concentrated, and stored at -80 °C.

[0220] 2.3.3. Purification of BMTS-AUT-VEGFA

[0221] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0222] 2) Cell disruption and inclusion body acquisition: The obtained cell pellet was resuspended in lysis buffer (20 mM Tris-HCl, 5 mM EDTA, pH 8.0), homogenized 4 times at 10°C and 10,000 bar, and centrifuged at 10,000 x g for 30 min at 10°C. The supernatant was then discarded, and the pellet was washed twice with washing buffer (2 M Urea, 20 mM Tris-HCl, 0.5 M NaCl, 1% Triton X-100, 2 mM Mercaptoethanol, pH 8.0) at room temperature for 20 min, and centrifuged at 11,000 g for 40 min at 8°C with increased temperature. Finally, the inclusion body was washed twice with 20 mM Tris-HCl buffer (pH 8.0).

[0223] 3) Solubilization process: 1 g of inclusion body was completely dissolved in guanidine hydrochloride buffer (7 M Guanidine hydrochloride, 20 mM Tris-HCl, 1 mM EDTA, 100 mM Mercaptoethanol pH 8.0) to dissolve the protein, and the dissolved inclusion body was dialyzed against 20 mM Tris-HCl buffer (pH 8.0) to precipitate the protein. After centrifugation, the precipitate was dissolved in 8 M urea buffer (8 M Urea, 20 mM Tris-HCl, 1 mM EDTA, 100 mM Mercaptoethanol, pH 8.0) at room temperature and the protein concentration was adjusted to 0.2 mg / ml.

[0224] 4) Refolding process: Afterwards, the dissolved protein was refolded by dialyzing against 2 L refolding buffer (20 mM Tris-HCl, 1 mM EDTA, 0.1 mM GSSG, 1 mM GSH, pH 8.0) at 10°C for 18 hours.

[0225] 5) Purification process: First, the refolded protein was purified using a DEAE-Sepharose Fast Flow column, and then analyzed by SDS-PAGE. The fractions mainly containing BMTS-AUT-VEGFA were pooled and concentrated to 3 ml via ultrafiltration (3K, Amicon Ultra, Millipore, Germany). Then, the concentrated BMTS-AUT-VEGFA was applied to a Sephacryl S-100 column for further purification, and the eluted fractions were analyzed by SDS-PAGE. The fractions containing BMTS-AUT-VEGFA were concentrated and stored at -80 °C.

[0226] 2.3.4. Purification of BMTS-AUT-PDGF-AA

[0227] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0228] 2) Cell disruption and inclusion body acquisition: The obtained pellet was resuspended 1:10 (w / v) in lysis buffer (20 mM MES (pH 6.0), 1 mM EDTA, 0.1 mM PMSF) and disrupted using a homogenizer on ice. The suspension was centrifuged at 11,000 xg for 30 min, and the supernatant containing BMTS-AUT-PDGF-A was obtained.

[0229] 3) Acquisition of BMTS-AUT-PDGF-AA: To obtain BMTS-AUT-PDGF-AA inclusion bodies, the pH was adjusted to 4.0 by adding 1 M sodium acetate (pH 3.5), centrifuged at 11,000 rpm for 45 minutes, and the pellet was dissolved with 8 M urea, 20 mM CHES pH 10.0, and 10 mM DTT. The denatured PDGF-AA was adjusted to pH 6.0 using 1 M sodium acetate pH 3.5.

[0230] 4) Primary purification of denatured BMTS-AUT-PDGF-AA: The solution was filtered using a 0.4 μm polyethersulfone (PES) syringe filter, and the filtrate was applied to a SP FF column equilibrated with 5 column volumes (CV) A buffer (20 mM MES pH 6.0, 8 M urea, 10 mM DTT), and the column was washed with 5 CV A buffer. The bound BMTS-AUT-PDGF-AA protein was then eluted with 100% B buffer (20 mM MES pH 6.0, 8 M urea, 10 mM DTT, 1 M NaCl), and proteins larger than 30 kDa were removed using a 30,000 Da amicon.

[0231] 5) BMTS-AUT-PDGF-AA refolding: A 20-fold volume of the denatured BMTS-AUT-PDGF-A solution was added to a refolding buffer (100 mM Tris-HCl pH 7.6, 200 mM L-arginine, 500 mM NaCl, 5 mM GSH, 0.5 mM GSSG) and dialyzed for 3 days. The refolded BMTS-AUT-PDGF-A solution was desalted with a 50 mM Tris-HCl pH 7.6 and 200 mM arginine solution.

[0232] 6) Secondary purification of refolded BMTS-AUT-PDGF-AA: The refolded BMTS-AUT-PDGF-AA solution was filtered using a 0.4 μm polyethersulfone (PES) syringe filter, and the filtrate was applied to a SP FF column equilibrated with 5 column volumes (CV) A buffer (50 mM Tris-HCl pH 7.6), and the column was washed with 5 CV A buffer. The bound BMTS-AUT-PDGF-AA protein was eluted with 5 CV A and B buffers (50 mM Tris pH 8.0, 2 M NaCl) using a linear gradient of 20–100% B buffer and 4 CV 100% B buffer, and the eluted fraction was purified by size exclusion chromatography (Superdex 75) using a column equilibrated with 1X PBS and stored at -80 °C.

[0233] 2.3.5. Purification of BMTS-AUT-PDGF-BB

[0234] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0235] 2) Cell disruption and inclusion body acquisition: The obtained pellet was disrupted three times using a homogenizer at 10℃, 10,000 bar, centrifuged at 17,000 xg, 4℃ for 30 minutes, the supernatant was discarded, the pellet was washed twice with washing buffer (50 mM Tris-HCl pH6.0, 2 M urea, 5 mM EDTA) for 40 minutes, centrifuged at 17,000 xg, 4℃ for 30 minutes, and 1 g of inclusion body was dissolved in buffer (50 mM Tris-HCl pH6.0, 5 mM EDTA, 6 M urea, 10 mM dithiothreitol) and stirred at 250 rpm at 4-8℃ overnight. The dissolved IB was centrifuged at 17,000 xg and room temperature for 20 minutes.

[0236] 3) Purification of monomeric BMTS-AUT-PDGF-B: A Hitrap Capto S column was equilibrated with 5 column volumes (CV) of buffer A (50 mM Tris-HCl pH 6.0, 5 mM EDTA, 6 M urea, 10 mM dithiothreitol), and the sample containing BMTS-AUT-PDGF B was loaded onto the column. The column was washed with 5 CV of buffer A and eluted with a 20 CV linear gradient of buffer B (50 mM Tris-HCl pH 6.0, 5 mM EDTA, 6 M urea, 1 M NaCl). Fractions were analyzed by SDS-PAGE, and the fractions containing mainly BMTS-AUT-PDGF B were pooled and concentrated to 3 ml by ultrafiltration (3K, Amicon Ultra, Millipore, Germany).

[0237] 4) Refolding and dimerization of dimeric BMTS-AUT-PDGF-BB: The protein concentration was adjusted to 20 mg / ml, diluted with Tris-EDTA buffer (50 mM Tris-HCl pH 7.8, 5 mM EDTA), and incubated at 4°C for 8 h to allow dimerization. The dimerized protein was concentrated to 2.0–2.5 mg / ml using an ultrafiltration membrane with a cutoff of 10 kDa, and then filtered through 0.1 M sodium acetate (pH 5.0).

[0238] 2.3.6. Purification of BMTS-AUT-TGFβ2

[0239] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0240] 2) Cell lysis and inclusion body acquisition: The cell pellet was suspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM EDTA, 1.0% Triton X-100) and lysed twice using a high-pressure homogenizer (800 to 1,200 bar). The lysate was centrifuged at 13,000 g and 4 °C for 30 minutes to obtain inclusion bodies. The obtained inclusion bodies were washed twice with IB washing buffer (20 mM Tris-HCl (pH 8.0), 1 M NaCl, 1 mM EDTA, 0.5% Triton X-100, 2 M urea) and then once with 20 mM Tris-HCl (pH 8.0).

[0241] 3) Solubilization process: The separated inclusion bodies were solubilized overnight at room temperature using a solubilization buffer (20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 mM EDTA, 8 M urea, 1% DTT (w / v)). The supernatant was recovered by centrifugation (13,000 g, 30 min, 4 °C) and filtered (0.2 μm). Then, 20 mM sodium acetate solid was added to the supernatant, the pH was adjusted to 4.0 using acetic acid, and the supernatant was recovered by centrifugation (13,000 g, 30 min, 4 °C) and filtered (0.2 μm).

[0242] 4) Purification of BMTS-AUT-TGFβ2: A cation exchange purification (SP) column was equilibrated with 10 column volumes of equilibration buffer (20 mM sodium acetate, 8 M urea, 0.1% DTT (w / v), pH 4.2), and the supernatant was applied to the SP column for cation exchange purification. The column was eluted with elution buffer (20 mM sodium acetate, 8 M urea, 0.1% DTT (w / v), 1 M NaCl, pH 4.2), and the collected fractions for the AUT-TGFβ2-BMTS refolding process were pooled and a 1-volume volume of 100 mM Tris-HCl buffer (pH 9.5) containing 8 M urea was added, and the half-diluted pool solution was diluted with a 10-volume volume of refolding buffer (50 mM Tris-HCl (pH 9.5), 0.9 M urea, 0.78 M CHES, 2.2 mM GSH, 1.1 mM GSSG) at 4°C for 24 h. The solution diluted with refolding buffer was reacted at 4°C for 4 days for protein refolding, and then the supernatant was obtained through centrifugation (13,000 g, 40 min, 4°C), filtered (0.2 micrometer), and the refolded TGF-β2 was concentrated to 0.2 mg / mL using a 3 kDa MWCO centricon and stored at -80°C.

[0243] 2.3.7. Refinement of BMTS-AUT-NOG

[0244] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0245] 2) Cell disruption and inclusion body acquisition: The cell pellet was suspended in lysis buffer (50 mM Tris-HCl (pH 9.0), 1 mM EDTA, 1 mM PMSF), disrupted twice using a high-pressure homogenizer (800 to 1,200 bar), centrifuged at 13,000 g, 4°C for 30 minutes to obtain inclusion bodies, and washed with washing buffer (50 mM Tris-HCl (pH 8.5), 1 M NaCl, 1% Triton X-100, 5 mM DTT).

[0246] 3) Solubilization process: The separated inclusion bodies were solubilized using a solubilization buffer (8 M urea, 40 mM CHES (pH 10.0), 10 mM DTT, 1 mM EDTA), and the solution containing denatured BMTS-AUT-NOG was adjusted to pH 4.5 using 3 M sodium acetate (pH 3.6) and filtered using a 0.4 μm polyethersulfone (PES) syringe filter.

[0247] 4) BMTS-AUT-NOG refolding process: The filtrate was applied to a SP FF column equilibrated with 5 column volumes (CV) A buffer (50 mM sodium acetate pH 4.5, 6 M urea, 10 mM DTT), the column was washed with 5 CV A buffer, and the bound BMTS-AUT-NOG protein was eluted with 100% B buffer (50 mM sodium acetate (pH 4.5), 6 M urea, 1 mM DTT, 1 M NaCl), and then placed in a 30,000 Da Amicon and centrifuged at 3,500 rpm for 30 minutes to obtain a refolding solution with proteins larger than 30 kDa removed. The eluted BMTS-AUT-NOG was subjected to secondary refolding at 4°C for 3 days in a refolding buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM GSH, 0.2 mM GSSH, 150 mM Gdn-HCl), and then the refolded BMTS-AUT-NOG was desalted with a 50 mM Tris-HCl pH 7.0 solution.

[0248] 5) Purification of BMTS-AUT-NOG: After equilibrating the SP FF column with 5 column volumes (CV) A buffer (50 mM Tris-HCl (pH 7.0)), the refolded BMTS-AUT-NOG was applied to the SP FF column and the column was washed with 5 CV A buffer. The bound BMTS-AUT-NOG protein was eluted with 100% B buffer (50 mM Tris-HCl (pH 7.0), 2 M NaCl), and the final purified BMTS-AUT-NOG elution was stored by changing the buffer to 1X PBS (pH 7.4).

[0249] 2.3.8. Refinement of BMTS-AUT-SCF

[0250] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0251] 2) Cell lysis and inclusion body acquisition: The cell pellet was suspended in lysis buffer (50 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, 1.0% Triton X-100) and lysed twice using a high-pressure homogenizer (800 to 1,200 bar). The lysate was centrifuged at 13,000 g and 4 °C for 30 minutes to obtain inclusion bodies. The obtained inclusion bodies were washed twice with IB washing buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 2 M urea) and then once with 20 mM Tris-HCl (pH 7.5).

[0252] 3) Solubilization process: The separated BMTS-AUT-SCF inclusion body was solubilized overnight at room temperature using solubilization buffer (20 mM Tris-HCl (pH 8.0), 10 mM NaCl, 1 mM EDTA, 8 M urea, 2 mM DTT), and the supernatant was recovered by centrifugation (13,000 g, 30 min, 4 °C). The supernatant was dialyzed against 40 times the volume of refolding buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 mM GSH, 0.2 mM GSSG) at 4 °C for 24 h by dilution method. The supernatant was obtained by centrifugation (13,000 g, 40 min, 4 °C) and filtered through a 0.2 μm filter.

[0253] 4) BMTS-AUT-SCF 1st purification process: After equilibrating the anion exchange purification (Q) column with 10 column volumes of equilibration buffer (50 mM Tris-HCl (pH 7.0)), the supernatant was applied to the anion exchange purification (Q) column, and the column was eluted using elution buffer (50 mM Tris-HCl (pH 7.0), 1 M NaCl), and the fraction containing BMTS-AUT-SCF was collected. The collected fractions were pooled for further purification, and the NaCl concentration was adjusted to 2 M using a 4 M NaCl stock solution, and then filtered through a 0.2 micrometer filter after adjusting the NaCl concentration.

[0254] 5) BMTS-AUT-SCF secondary purification process: The hydrophobic interaction chromatography column (butyl) was equilibrated with 10 column volumes of equilibration buffer (50 mM Tris-HCl (pH 7.0), 2 M NaCl), and then the supernatant was applied to the hydrophobic interaction chromatography column (butyl), and the BMTS-AUT-SCF protein was eluted using elution buffer (50 mM Tris-HCl (pH 7.0)), and dialyzed against 100 x elution volume dialysis buffer (Phosphate buffer saline pH 7.4) at 4°C for 12 hours. The purified BMTS-AUT-SCF was concentrated to 1 mg / ml using a 3 kDa cutoff centricon and stored at -80°C.

[0255] 2.3.9. Purification of AUT-TIMP-1-BMTS

[0256] 1) Cell pellet recovery: After large-scale expression induction was performed again under the expression conditions confirmed above, the culture medium was centrifuged at 4°C, 5,000 rpm for 15 minutes to obtain a cell pellet.

[0257] 2) Cell disruption and inclusion body acquisition: The cell pellet was suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 100 μM PMSF) and disrupted twice using a high-pressure homogenizer (800 to 1,200 bar). The pellet was centrifuged at 13,000 g and 4°C for 30 minutes to obtain inclusion bodies, which were then washed with washing buffer (50 mM Tris-HCl (pH 8.0), 1 M NaCl, 2 M urea, 0.1% Triton X-100).

[0258] 3) Solubilization process: The separated inclusion bodies were solubilized overnight at room temperature using solubilization buffer (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 6 M Gdn-HCl), and the supernatant was recovered through centrifugation (13,000 g, 30 min, 4 °C).

[0259] 4) AUT-TIMP-1-BMTS 1st purification process: The affinity (Ni-NTA) column was equilibrated with 10 column volumes of equilibration buffer (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 6 M Gdn-HCl), and the recovered supernatant was applied to the Affinity (Ni-NTA) column, and the target protein was eluted using elution buffer (50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 6 M Gdn-HCl, 500 mM imidazole), and the fraction containing his-TEVcs-BMTS-AUT-TIMP1 was collected.

[0260] 5) AUT-TIMP-1-BMTS refolding process: The eluted his-TEVcs-BMTS-AUT-TIMP1 was subjected to 1ST refolding using the direct dilution method in refolding buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 2 mM GSH, 0.2 mM GSSH) at 4 °C at a rate of >0.2 ml / min, and was added very slowly to protein (2 mg / ml X 20 ml), and then refolding buffer (50 mM Tris-HCl (pH 7.5), 300 mM Gdn-HCl, 2 mM GSH, 0.2 mM GSSH) was added very slowly dropwise (>0.5 ml / min) to protein (2 mg / ml X 200 ml) at 4 °C.

[0261] 6) AUT-TIMP-1-BMTS secondary purification process and TEV cleavage process: Then, the SP FF column was equilibrated with 5 column volumes (CV) A buffer (50 mM Tris-HCl pH 7.5), and the bound protein was eluted with 5 CV A and B buffers (50 mM Tris pH 7.5, 1 M NaCl), and the eluted protein was treated in TEV at 30°C for 1 hour.

[0262] 7) His-tag removal and AUT-TIMP-1-BMTS purification completed: TEV-treated BMTS-AUT-TIMP1 was equilibrated on an affinity (Ni-NTA) column with 10 column volumes of equilibration buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl), and then the supernatant was applied to an Affinity (Ni-NTA) column to remove the His-tag and only the eluted solution was obtained to complete the purification of AUT-TIMP-1-BMTS.

[0263] Example 3: Peptide synthesis of biologically active molecules and BMTS conjugates

[0264] AH-BMTS and AP-BMTS, the conjugates of Example 1.2, were synthesized through peptide synthesis as follows.

[0265] 1) Preparation of Resins: Place the resin in a flask, add 20% (v / v) piperidine in DMF (N,N-Dimethylformamide) (approximately 10% mL / gm resin), and react while shaking at room temperature for 30 minutes. Then, filter the resin and wash it several times with DMF.

[0266] 2) Coupling process: The N-terminal protecting group was removed according to the standard deprotection protocol, and 1 g of resin was mixed in 10 mL of dichloromethane (DCM), and then 5 times the amount of DMF was added to completely dissolve the amino acid derivative.

[0267] 3) Capping process: The resins were filtered and washed several times with DMF, mixed with a DMF solution containing acetic anhydride and pyridine, and gently shaken at room temperature for 30 minutes. The resins were filtered and washed again with DMF. If the Kaiser test result was not negative, the capping process was repeated.

[0268] 4) Resin cleavage process: Before starting the cleavage process, the N-terminal Fmoc group was removed, the resin was slurried in a trifluoroacetic acid (TFA) cocktail buffer, filtered through a glass funnel filter, and washed three times with small portions of TFA. The filtrates were then combined, and 8–10 times the volume of cold ether was added to precipitate the peptide overnight at 4°C. The peptide was then filtered using a glass funnel filter, and the crude peptide was further washed with cold ether.

[0269] 5) Deprotection process for Fmoc peptide synthesis: The peptide was dissolved in 5% (w / w) phenol / TFA (approximately 10 μmol / mL), and cleavage of the Mtr group was monitored by HPLC. After cleavage was complete, the solution was evaporated to dryness, the residue was partitioned between water and dichloromethane, and the aqueous layer was washed approximately four times with dichloromethane and lyophilized to obtain the peptide.

[0270] Example 4: Confirmation of the cell-mediated efficacy of a conjugate of a biologically active molecule and BMTS.

[0271] 4.1. Confirmation of cell growth in fibroblasts

[0272] In order to confirm the cell growth activity of the conjugate of biologically active molecules and BMTS, BMTS-AUT-FGF-1 (SEQ ID NO: 17) and BMTS-AUT-FGF-2 (SEQ ID NO: 21) prepared in Example 2.2.2 were treated to NIH3T3 cells, which are mouse fibroblasts, and the degree of cell proliferation was measured.

[0273] NIH3T3 cells used in the experiment were purchased from American Type Culture Collection (ATCC, USA) and cultured in DMEM (GenDEPOT, USA) medium containing 10% (v / v) fetal bovine serum (Gibco, USA), 100 units / mL penicillin, and 100 μg / mL streptomycin (Gibco, USA) at 37°C in a 5% CO2 incubator. NIH3T3 cells were seeded at 5 × 10 in a 96-well plate. 3Cells were seeded at 1 cell / well and cultured for 24 hours in 10% FBS / DMEM medium. The culture medium was removed and cultured for another 24 hours in serum-free DMEM medium. After that, BMTS-AUT-FGF-1 and BMTS-AUT-FGF-2 were treated at the test concentration range and cultured for 48 hours. 10 μl of Cell Proliferation Reagent WST-1 (Roche) solution was added to each well and incubated at 37°C for 3 hours. The absorbance was measured at 450 nm and the degree of proliferation of NIH3T3 cells was evaluated compared to the control group. The control group was treated with the same concentration of PBS instead of BMTS-AUT-FGF-1 and BMTS-AUT-FGF-2.

[0274] As a result, as shown in Figures 4a and 4b, both BMTS-AUT-FGF-1 and BMTS-AUT-FGF-2 increased the proliferation of NIH3T3 cells within the test concentration range. In the case of BMTS-AUT-FGF-1, cell proliferation increased 1.9-fold compared to the control group, and ED 50 was 6.37 ng / ml (Fig. 4a), and in the case of BMTS-AUT-FGF-2, cell proliferation increased 3.3-fold compared to the control group, and ED 50 was 0.84 ng / ml (Fig. 4b).

[0275] 4.2. Measurement of SOD-1 activity

[0276] The activity of BMTS-AUT-SOD-1 (SEQ ID NO: 22) manufactured in Example 2.2.3 was measured using an SOD assay kit (Dojindo, USA) according to the method described in the manual. 20 μl of diluted samples by concentration were dispensed into a 96-well plate, 200 μl of WST working solution was added, mixed, 20 μl of enzyme working solution was added, and the mixture was incubated at 37°C for 20 minutes. The absorbance was measured at 450 nm using a microplate reader. SOD activity was expressed as a percentage (%) as the difference in absorbance between the sample-added and -unadded groups (Fig. 4c).

[0277] As a result, as shown in Fig. 4c, the SOD unit of BMTS-AUT-SOD-1 was 1075 units / ml, confirming that BMTS-AUT-SOD-1 has excellent activity.

[0278] Example 5: Confirmation of skin penetration ability of conjugates of biologically active molecules and BMTS

[0279] In order to confirm the skin permeability of the conjugate of biologically active molecules and BMTS, the skin permeability of BMTS-AUT-BT-LC (SEQ ID NO: 16) and AUT-EGF-5L-BMTS (SEQ ID NO: 29) was confirmed by the Korea Biotechnology Research Institute (KBI), and the results are shown in Fig. 5. As shown in Fig. 5, it was confirmed using a fluorescent substance that the skin permeability was significantly superior in the groups treated with BMTS-AUT-BT-LC and AUT-EGF-5L-BMTS than in the groups treated with only AUT-BT-LC and AUT-EGF.

[0280] Example 6: Confirmation of the effect of improving wrinkles at the corners of the eyes and the area of ​​pores of a cosmetic composition containing a conjugate of a biologically active molecule and BMTS.

[0281] 6.1. Manufacturing Example 1: Manufacturing of a cosmetic composition for improving wrinkles around the corners of the eyes and / or improving pore area

[0282] According to the composition shown in Table 5 below, a cosmetic composition for improving wrinkles around the corners of the eyes and / or improving pore area was prepared (Preparation Example 1).

[0283] Ingredients (% by weight) Manufacturing Example 1 Water 78.5 Butylene Glycol 6 Dipropylene Glycol 5 Niacinamide 4.8 Glycerin 3.24 6 1,2-Hexanediol 0.8 Betaine 0.5 Hydroxyacetophenone 0.5 Tromethamine 0.18 Carbomer 0.22 Octyldodeceth-16 0.1 Xanthan Gum 0.04 Adenosine 0.04 Sodium Hyaluronate 0.03 Disodium EDTA EDTA) 0.02 Fragrance 0.01 Hydrolyzed Sodium Hyaluronate 0.005 Decapeptide-40 0.005 AH-BMTS 0.002 5 AUT-EGF-5L-BMTS 0.0002 BMTS-AUT-SOD-10.0001 BMTS-AUT-BT-LC 0.00007 5 BMTS-AUT-FGF-10.00005 AUT-FGF-100.00005 BMTS-AUT-PDGF-BB 0.00005 BMTS-AUT-IGF-10.00003

[0284] 6.2. Evaluation of the effect of improving wrinkles around the corners of the eyes

[0285] 6.2.1 Evaluation Method

[0286] In this study, ANTERA 3D (Miravex, Ireland) was used to evaluate the improvement of crow's feet wrinkles by the test substance (cosmetic composition of Manufacturing Example 1 above). The same tester measured the wrinkle area around the left eye of all subjects, and to ensure reproducibility of the measurement, the same area was measured by overlapping the image measured before using the test substance. The captured images were matched using ANTERA CS software, a dedicated software for ANTERA 3D, and the matched measurement area was used for analysis. The measured value was analyzed as the 'Wrinkles small value', which represents wrinkles on the skin, using the Indentation index. A decrease in the measured value compared to before using the test substance means an improvement in crow's feet wrinkles. The device measurements were taken before using the test substance and immediately after the first use.

[0287] 6.2.2. Evaluation Results

[0288] The changes in Wrinkles small values, the improvement rate of Wrinkles small values, and the results of statistical analysis of Wrinkles small values ​​are shown in Tables 6 to 8 below. In the statistical analysis, p-values ​​were analyzed using the Wilcoxon signed-rank test (*p<0.05, *p<0.01, ***p<0.001).

[0289] Wrinkles small value change (N=23) - Before use, immediately after use, average 9.278.30, standard deviation 2.762.40

[0290] Wrinkles small value improvement rate (%) - Improvement rate immediately after 1 use (%) 10.42

[0291] Improvement rate (%) = (measurement after use - measurement before use) / measurement before use × 100

[0292] Wrinkles small value statistical analysis - p-value 0.000*** immediately after 1 use

[0293] In addition, a graph showing the change and improvement rate of the Wrinkles small value is shown in Fig. 6, and an image showing wrinkles at the corners of the eyes before and after using the cosmetic composition of Manufacturing Example 1 is shown in Fig. 7.

[0294] As shown in Tables 6 to 8 and Figures 6 and 7, it was confirmed that the Wrinkles small value was statistically significantly reduced when the cosmetic composition of Manufacturing Example 1 was used.

[0295] In this way, it was confirmed that the cosmetic composition of Preparation Example 1 including AH, AUT-EGF, AUT-SOD-1, AUT-BT-LC, AUT-FGF-1, AUT-FGF-10, AUT-PDGF-B and AUT-IGF-1 exhibited an excellent effect of improving wrinkles at the corners of the eyes.

[0296] 6.3. Evaluation of pore area improvement effect

[0297] 6.3.1. Evaluation Method

[0298] In this study, ANTERA 3D was applied to evaluate the improvement (reduction) of pore area by the test substance (cosmetic composition of Manufacturing Example 1 above). The same tester measured the left cheek area of ​​all test subjects, and to ensure reproducibility of the measurement, the same area was measured by overlapping the image measured before using the test substance. The captured images were matched using ANTERA CS software, a dedicated software for ANTERA 3D, and the matched measurement area was used for analysis. The measured value was analyzed as the Affected area value, a measurement variable representing the pore area of ​​the skin, using a Pores small filter, and the unit of measurement is ㎟. A decrease in the measured value compared to before using the test substance means that the pore area has improved. The device measurements were performed before using the test substance and immediately after the first use.

[0299] 6.3.2. Evaluation Results

[0300] The changes in affected area values, the rate of improvement in affected area values, and the results of statistical analysis of affected area values ​​are shown in Tables 9 to 11 below. During statistical analysis, p-values ​​were analyzed using the Wilcoxon signed-rank test (*p<0.05, *p<0.01, ***p<0.001).

[0301] Changes in affected area values ​​(N=23) - Before use, immediately after use, average 65.0346.41, standard deviation 33.8624.30

[0302] Affected area value improvement rate (%) - Improvement rate immediately after 1 use (%) 28.64

[0303] Improvement rate (%) = (measurement after use - measurement before use) / measurement before use × 100

[0304] Affected area value statistical analysis - p-value 0.000*** immediately after 1 use

[0305] In addition, a graph showing the change in the affected area value and the rate of improvement in the affected area value is shown in Fig. 8, and an image showing the pore area before and after using the cosmetic composition of Manufacturing Example 1 is shown in Fig. 9.

[0306] As shown in Tables 9 to 11 and Figures 8 and 9, it was confirmed that the pore area was statistically significantly reduced when the cosmetic composition of Manufacturing Example 1 was used.

[0307] In this way, it was confirmed that the cosmetic composition of Preparation Example 1 including AH, AUT-EGF, AUT-SOD-1, AUT-BT-LC, AUT-FGF-1, AUT-FGF-10, AUT-PDGF-B, and AUT-IGF-1 exhibited an excellent pore area improvement effect.

[0308] Example 7: Confirmation of the skin soothing effect and skin elasticity improvement effect of a cosmetic composition containing a conjugate of a biologically active molecule and BMTS.

[0309] 7.1. Manufacturing Example 2: Manufacturing of a cosmetic composition for skin soothing and / or skin elasticity improvement

[0310] According to the composition shown in Table 12 below, a cosmetic composition for skin soothing and / or skin elasticity improvement was prepared (Preparation Example 2).

[0311] Ingredients (% by weight) Manufacturing Example 2 Water 78.5 Butylene Glycol 6 Dipropylene Glycol 5 Niacinamide 4.8 Glycerin 3.24 6 1,2-Hexanediol 0.8 Betaine 0.5 Hydroxyacetophenone 0.5 Tromethamine 0.18 Carbomer 0.22 Octyldodeceth-16 0.1 Xanthan Gum 0.04 Adenosine 0.04 Sodium Hyaluronate 0.03 Disodium EDTA EDTA) 0.02 Fragrance 0.01 Hydrolyzed Sodium Hyaluronate 0.005 Decapeptide-40 0.005 AH-BMTS 0.005 0 AUT-EGF-5L-BMTS 0.0002 BMTS-AUT-SOD-10.0001 BMTS-AUT-FGF-10.00005 AUT-FGF-100.00005 BMTS-AUT-PDGF-B 0.00005 BMTS-AUT-IGF-10.00003

[0312] 7.2. Evaluation of skin soothing effect after external stimulation

[0313] 7.2.1. Evaluation Method

[0314] 1) Induction of skin irritation by tape stripping

[0315] In this test, skin irritation was induced by tape stripping on both forearms of the test subject prior to instrumental measurement. The same tester attached 18 mm tape (Scotch® Transparent Tape, 3M Company, USA) to the designated test area, applied equal pressure for 2 seconds, and then stripped in the same direction. Stripping was repeated 40 times using a new tape each time, and instrumental measurement was performed on areas where the test substance (the cosmetic composition of Manufacturing Example 2 above) was applied and areas where it was not applied.

[0316] 2) Evaluation of skin soothing effect after external stimulation by ANTERA 3D

[0317] In this test, ANTERA 3D (Miravex, Ireland) was applied to evaluate the skin soothing effect after external stimulation of the test substance (cosmetic composition of Manufacturing Example 2 above). The same tester measured both forearms of all subjects (test substance application area, non-application area), and to ensure reproducibility of the measurement, the same area was measured by overlapping the image measured before using the test substance. The captured images were analyzed using ANTERA CS software, a dedicated software for ANTERA 3D, to obtain Average CIE L*, a*, b* values. The L* value represents brightness, the a* value represents redness, and the b* value represents yellowness.

[0318] The a* value, which represents the redness of the skin, was analyzed as a value measuring the skin soothing effect after external stimulation of the test substance. The more the change in the measured value of the applied area is significantly (p<.05) reduced compared to the area without the test substance application, the more it means that there is a skin soothing effect after external stimulation. The device measurement was performed before using the test substance, after stimulation, and immediately after the first use.

[0319] 7.2.2. Evaluation Results

[0320] The results of statistical analysis of Δa* value change, Δa* value improvement rate, and a* value between the test substance application site and the non-application site are shown in Tables 13 to 15. In the statistical analysis, p-values ​​were analyzed through Repeated measures ANOVA (*p<0.05, *p<0.01, ***p<0.001).

[0321] Changes in Δa* value (N=23) - Application area Non-application area Δa*1 Δa*2 Δa*1 Δa*2 Mean 5.5 3 3.1 1 5.4 1 4.99 Standard deviation 1.1 4 1.1 8 1.7 2 1.55

[0322] Δa* value improvement rate - application area, non-application area, immediately after 1 use, immediately after 1 use, standard deviation 43.847.84

[0323] Improvement rate (%) = (measurement value after use - measurement value before use (after stimulation)) / measurement value before use (after stimulation) × 100

[0324] a* value statistical analysis between the test substance application site and the non-application site - time - time - group p-value 0.000*** 0.000***

[0325] In addition, the change in a* value and improvement rate of the applied and unapplied areas are shown in Fig. 10, and the images of the applied and unapplied areas are shown in Fig. 11.

[0326] As shown in Tables 13 to 15, and FIGS. 10 and 11, it was confirmed that the a* value indicating redness of the skin was statistically significantly reduced when the cosmetic composition of Manufacturing Example 2 was used.

[0327] In this way, it was confirmed that the cosmetic composition of Preparation Example 2 including AH, AUT-EGF, AUT-SOD-1, AUT-BT-LC, AUT-FGF-1, AUT-FGF-10, AUT-PDGF-B, and AUT-IGF-1 exhibited an excellent skin soothing effect.

[0328] 7.3. Elasticity improvement evaluation using Cutometer

[0329] 7.3.1. Evaluation Method

[0330] In this study, a Cutometer (Cutometer dual MPA580, Courage+Khazaka Electronic GmbH, Cologne, Germany) was used to evaluate the improvement in elasticity of the test substance (the cosmetic composition of Manufacturing Example 2). The same tester fixed the probe to the right cheek of all subjects with adhesive tape, and then suctioned and released the probe for 2 seconds three times at a pressure of 450 mbar. The average value was calculated and used for analysis. This analysis was performed using the software MPA CTplus, a Cutometer-specific analysis program. The Cutometer measures skin elasticity using the pressure inside the probe aperture through the principle of skin suction and relaxation. The probe apertures are 2, 4, 6, and 8 mm to limit the area of ​​skin to which suction is applied. A larger aperture is suitable for use on thicker skin. To measure the improvement in elasticity of the test substance, a 2 mm probe that delivers suction to the skin was used. The R2 value, which represents skin elasticity, was used as a value to measure the improvement in elasticity, and the unit of measurement is %. An increase in the measured value compared to before use of the test substance indicates improved elasticity. Instrument measurements were conducted before and immediately after the first use of the test substance.

[0331] 7.3.2. Evaluation Results

[0332] The changes in R2 value, improvement rate in R2 value, and statistical analysis results for R2 value are shown in Tables 16 to 18 below. During statistical analysis, p-values ​​were analyzed using the Wilcoxon signed-rank test (*p<0.05, *p<0.01, ***p<0.001).

[0333] Changes in R2 values ​​(N=23) - Before use, immediately after use, average 66.10, 68.11, standard deviation 2.95, 3.06

[0334] R2 value improvement rate (%) - Improvement rate immediately after 1 use (%) 3.04

[0335] Improvement rate (%) = (measurement after use - measurement before use) / measurement before use × 100

[0336] R2 value statistical analysis - p-value 0.000*** immediately after 1 use

[0337] Additionally, the change in R2 value and improvement rate are shown in Figure 12.

[0338] As shown in Tables 16 to 18 and Fig. 12, it was confirmed that the R2 value indicating skin elasticity was statistically significantly reduced when the cosmetic composition of Manufacturing Example 2 was used.

[0339] In this way, it was confirmed that the cosmetic composition of Preparation Example 2 including AH, AUT-EGF, AUT-SOD-1, AUT-BT-LC, AUT-FGF-1, AUT-FGF-10, AUT-PDGF-B and AUT-IGF-1 exhibited an excellent skin elasticity improvement effect.

[0340] Example 8: Confirmation of the neck wrinkle improvement effect of a cosmetic composition containing a conjugate of a biologically active molecule and BMTS.

[0341] 8.1. Manufacturing Example 3: Manufacturing of a cosmetic composition for improving neck wrinkles

[0342] According to the composition shown in Table 19 below, a cosmetic composition for improving neck wrinkles was prepared (Preparation Example 3).

[0343] Ingredients (% by weight) Manufacturing Example 3 Water 82.561 Dipropylene Glycol 4 Niacinamide 4.8 Glycerin 4.246 1,2-Hexanediol 0.8 Betaine 0.5 Hydroxyacetophenone 0.5 Tromethamine 0.16 Carbomer 0.1 Octyldodeceth-16 0.1 Adenosine 0.04 Sodium Hyaluronate 0.02 Disodium EDTA 0.02 Fragrance 0.01 Pentylene Glycol Glycol)2C10-30 Alkyl Acrylate Crosspolymer)0.1Hydroxyethylcellulose)0.04AP-BMTS0.0015AH-BMTS0.001AUT-EGF-5L-BMTS0.0001BMTS-AUT-SOD-10.000025AUT-FGF-20.0001AUT-TGF-β2-BMTS0.00005BMTS-AUT-PDGF-AA0.00005AUT-TIMP1-BMTS0.00003

[0344] 8.2. Evaluation of neck wrinkle improvement effect

[0345] 8.2.1. Evaluation Method

[0346] In this study, ANTERA 3D (Miravex, Ireland) was used to evaluate the effect of the test substance (cosmetic composition of Manufacturing Example 3 above) on improving neck wrinkles. The same tester measured the neck wrinkle area of ​​all subjects, and to ensure reproducibility of the measurement, the same area was measured by overlapping the image measured before using the test substance. The captured images were matched using ANTERA CS software, a dedicated software for ANTERA 3D, and the matched measurement area was used for analysis. The measured values ​​were analyzed as the Wrinkles large value, which represents skin wrinkles, using the Indentation Index. A decrease in the measured value compared to before using the test substance means that neck wrinkles have improved. The device measurements were performed before using the test substance and after 2 weeks of use.

[0347] 8.2.2. Evaluation Results

[0348] The changes in the Wrinkles large value, the improvement rate of the Wrinkles large value, and the results of statistical analysis of the Wrinkles large value are shown in Tables 20 to 22 below. In the statistical analysis, the p-value was analyzed using the Wilcoxon signed-rank test (*p<0.05, *p<0.01, ***p<0.001).

[0349] Changes in Wrinkles large values ​​(N=21) - Before use, After 2 weeks of use, Average 38.55, 33.80, Standard deviation 16.50, 13.43

[0350] Wrinkles large value improvement rate (%) - After 2 weeks of use, improvement rate (%) 12.33

[0351] Improvement rate (%) = (measurement after use - measurement before use) / measurement before use × 100

[0352] Wrinkles large value statistical analysis - after 2 weeks of use p-value 0.000***

[0353] In addition, a graph showing the change and improvement rate of the Wrinkles large value is shown in Fig. 13, and an image showing neck wrinkles before and after using the cosmetic composition of Manufacturing Example 3 is shown in Fig. 14.

[0354] As shown in Tables 20 to 22, and FIGS. 13 and 14, it was confirmed that when the cosmetic composition of Manufacturing Example 3 was used, the Wrinkles large value indicating wrinkles on the skin was statistically significantly reduced.

[0355] In this way, it was confirmed that the cosmetic composition of Preparation Example 3 containing AP, AH, AUT-EGF, AUT-SOD-1, AUT-FGF-2, AUT-TGF-β2, AUT-PDGF AA, and AUT-TIMP1 exhibited an excellent neck wrinkle improvement effect.

[0356] Example 9: Confirmation of the scalp soothing effect of a cosmetic composition containing a conjugate of a biologically active molecule and BMTS.

[0357] 9.1. Manufacturing Example 4: Manufacturing of a cosmetic composition for scalp soothing

[0358] According to the composition shown in Table 23 below, a cosmetic composition for skin soothing and / or skin elasticity improvement was prepared (Preparation Example 3).

[0359] Ingredients (% by weight) Manufacturing Example 4 Water 64.909 Niacinamide 0.1 Octyldodeceth-16 0.5 Sodium Hyaluronate 0.4 Disodium EDTA 0.03 Fragrance 0.01 Ethanol 30 Propanediol 3 Sodium Citrate 0.3 Menthol 0.3 Salicylic Acid 0.25 Panthenol (Panthenol)0.2BMTS-AUT-SOD-10.00015AUT-FGF-100.00015BMTS-AUT-PDGF-B0.00005BMTS-AUT-IGF- 10.00005BMTS-AUT-VEGFA0.0002BMTS-AUT-SCF0.00015BMTS-AUT-FGF-20.00005BMTS-AUT-NOG0.00001

[0360] 9.2. Evaluation of scalp soothing effect

[0361] 9.2.1. Evaluation Method

[0362] In this study, a video microscope (KONG PC Camera, Bomtech, Korea) and an image analysis program (Image J, National Institutes of Health, USA) were used to evaluate the scalp soothing effect of the test substance (cosmetic composition of Manufacturing Example 4 above). The video microscope was used to measure the crown area of ​​all subjects at 80x magnification by the same tester in the same position and with uniform lighting to ensure consistent filming.

[0363] The measured image can be extracted into three values, L*, a*, and b*, using an image analysis program to analyze the RGB color skin image using the ICA (independent component analysis) algorithm. The L* value represents brightness, the a* value represents redness as it detects hemoglobin, and the b* value represents yellowness as it detects melanin. The maximum a* value, which represents the redness of the skin, was used in the analysis to measure the scalp soothing effect of the test substance, and a decrease in the measured value compared to before using the test substance means that there is a scalp soothing effect. The device measurements were performed before using the test substance and after 2 weeks of use.

[0364] 9.2.2. Evaluation Results

[0365] The results of statistical analysis of maximum a* value change, Δmaximum a* value change, maximum a* value improvement rate, and maximum a* value are shown in Tables 24 to 27 below. In the statistical analysis, p-values ​​were analyzed using paired t-test (*p<0.05, *p<0.01, ***p<0.001).

[0366] Maximum a* value change (N=21) - Before use, After 2 weeks of use, Average 7.714.17, Standard deviation 3.822.06

[0367] Δmaximum a* value change-ΔMa*1 average 7.71 standard deviation 3.82

[0368] ΔMa*1= After 2 weeks of use - Before use

[0369] Maximum a* value improvement rate - Improvement rate after 2 weeks of use (%) 45.97

[0370] Maximum a* value statistical analysis - p-value 0.000*** after 2 weeks of use

[0371] Additionally, the maximum a* value change and improvement rate are shown in Figure 15, and the scalp condition before and after use is shown in Figure 16.

[0372] As shown in Tables 24 to 27, and FIGS. 15 and 16, it was confirmed that when the cosmetic composition of Manufacturing Example 4 was used, the maximum a* value indicating redness of the skin was statistically significantly reduced.

[0373] In this way, it was confirmed that the cosmetic composition of Preparation Example 4 containing AUT-SOD-1, AUT-FGF-10, AUT-PDGF-B, AUT-IGF-1, AUT-VEGFA, SCF, AUT-FGF2 and NOG exhibited an excellent scalp soothing effect.

[0374] According to the present invention, a conjugate is provided that exhibits increased half-life and / or stability and also exhibits cell penetration and / or skin penetration effects by applying anti-ubiquitination technology (AUT) to biologically active molecules and / or binding a cell-penetrating peptide. Therefore, the conjugate of the present invention can be usefully used as a raw material for topical cosmetics and / or skin-penetrating cosmetics.

Claims

biologically active molecules; and A cell-penetrating peptide having an amino acid sequence of SEQ ID NO: 1, linked to the N-terminus, C-terminus or both of the above biologically active molecule. A conjugate comprising: A conjugate in claim 1, wherein at least one lysine residue in the amino acid sequence of the biologically active molecule is substituted with an arginine residue or a histidine residue. A conjugate according to claim 1 or 2, wherein the biologically active molecule is a growth factor. In claim 3, the growth factor is at least one selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), stem cell factor (SCF), keratinocyte growth factor (KGF), and transforming growth factor (TGF). A conjugate according to claim 1 or 2, wherein the biologically active molecule is selected from the group consisting of botulinum toxin serotypes selected from the group consisting of A, B, C, D, E, F, G and H, recombinant botulinum toxin, modified botulinum toxin, fragments of botulinum toxin and combinations thereof. A conjugate according to claim 1 or 2, wherein the biologically active molecule is at least one selected from the group consisting of noggin, superoxide dismutase (SOD), acetyl hexapeptide-8 (AH), acetyl pentapeptide-4 (AP), TIMP (Tissue inhibitor of metalloproteinase), and aldehyde dehydrogenase (ALDH). A conjugate according to claims 1 to 6, comprising a linker between the biologically active molecule and the cell-penetrating peptide. A conjugate according to claims 1 to 7, comprising one amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO:

33. A nucleic acid molecule encoding a conjugate of any one of claims 1 to 8. A recombinant vector comprising the nucleic acid molecule of claim 9. A recombinant cell comprising a conjugate of any one of claims 1 to 8, a nucleic acid molecule encoding the conjugate, or a recombinant vector comprising the nucleic acid molecule. A cosmetic composition comprising a conjugate of any one of claims 1 to 8 as an active ingredient. A method for producing a conjugate, comprising the step of expressing the nucleic acid molecule of claim 9 in a cell. A method for preparing a conjugate, comprising the step of linking a cell-penetrating peptide having an amino acid sequence of SEQ ID NO: 1 to a biologically active molecule. As a biologically active molecule, The biologically active molecule is selected from the group consisting of epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factor (TGF), botulinum toxin serotype A, B, C, D, E, F, G and H, recombinant botulinum toxin, modified botulinum toxin, fragment of botulinum toxin, noggin, superoxide dismutase (SOD), acetyl hexapeptide-8 (AH), It is selected from the group consisting of acetyl pentapeptide-4 (AP), tissue inhibitor of metalloproteinase (TIMP), aldehyde dehydrogenase (ALDH), and combinations thereof, A biologically active molecule, wherein at least one lysine residue in the amino acid sequence of the biologically active molecule is substituted with an arginine residue or a histidine residue. A biologically active molecule comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 33, in claim 15.

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