KR102440312B1 - Thermally stable fgf7 polypeptide and use of the same - Google Patents

Abstract

A polypeptide having FGF7 activity and improved temperature stability is provided. In SEQ ID NO: 1, alanine at position 120 (A) is substituted with cysteine (C), lysine at position 126 (K) is substituted with glutamic acid (E) or aspartic acid (D), or lysine at position 178 (K) is substituted in SEQ ID NO: 1 It contains at least one or more substitutions selected from those substituted with glutamic acid (E) or aspartic acid (D), and has improved temperature stability (thermally stable) polypeptide having an intrinsic activity of FGF7.

Description

FGF7 polypeptide with improved temperature stability and its use

The present disclosure relates to FGF7 polypeptides with improved temperature stability and uses thereof.

FGF (Fibroblast Growth Factor) is a factor that plays an important role in regulating cell growth, proliferation, and differentiation. Various types of FGFs are generated to maintain the function of each tissue in the human body, and they perform unique functions in cell differentiation and proliferation. However, as aging progresses, the concentration of FGFs in each tissue, such as the skin, is gradually lowered. Accordingly, cell regeneration and division functions are weakened, so that wrinkles are formed in the skin and elasticity is reduced.

Among the various FGFs, FGF7 (Fibroblast Growth Factor 7) is also called keratinocyte growth factor, which is known as a strong epithelial cell-specific growth factor in mammalian cells. FGF7 also plays an important role in regulating skin regeneration, cell proliferation and cell differentiation.

FGF7 has already been marketed as a treatment for stomatitis after radiation therapy, and is being developed as a drug that promotes wound healing, and is also widely used as a cosmetic raw material for skin regeneration, wrinkle removal, or increased elasticity.

FGF family, including FGF1, FGF2, FGF7, and FGF10, has very low temperature stability, so when it is left in an aqueous solution for 24 hours, its activity is rapidly lost under temperature conditions above room temperature, and it is reported that the activity loss occurs severely.

Therefore, in order for FGF7 having various functions in the human body to be suitably used for industrial use, it is an essential condition to ensure the thermodynamic stability of FGF7.

The present disclosure intends to provide an FGF7 polypeptide with improved temperature stability.

An object of the present disclosure is to provide a pharmaceutical or cosmetic composition comprising an FGF7 polypeptide with improved temperature stability.

In the FGF7 polypeptide with improved temperature stability according to embodiments, alanine at position 120 in SEQ ID NO: 1 is substituted with cysteine (C), or lysine at position 126 (K) is glutamic acid (E) or aspartic acid (D) It contains at least one or more substitutions selected from the group consisting of substituted with, or the 178th lysine (K) is substituted with glutamic acid (E) or aspartic acid (D), and has improved temperature stability with FGF7 intrinsic activity (thermally stable) is a polypeptide.

Compositions according to embodiments include a polypeptide having improved temperature stability and a pharmaceutically or cosmetically acceptable carrier.

The FGF7 polypeptide according to the Examples exhibits improved temperature stability after manufacturing compared to the wild-type human FGF7 polypeptide.

Polypeptides with improved temperature stability can maintain activity during distribution and storage, unlike existing wild human FGF7 products. Therefore, it can be used as an active ingredient in a pharmaceutical or cosmetic composition.

1 is a polypeptide of wild-type FGF7 (SEQ ID NO: 1).
Figure 2 shows the Δ53N-hFGF7 sequence including the pCold I site with deletion of 2-54 from wild-type FGF7.
3 shows wild-type FGF7 (Δ30N_hFGF7) and Δ53N_hFGF7 (palifermin) and FGF7 mutants (Δ53N_hFGF7(A120C), Δ53N_hFGF7(A120C, K126E), Δ53N_hFGF7(A120C, K178E), Δ53N_hFGF7(A120C, K178E), Δ53N_hFGF7(A120C, K178E), Δ53N_hFGF7. ) shows SDS-PAGE to measure the stability at 37 °C.
4 shows wild-type FGF7 (Δ30N_hFGF7) and Δ53N_hFGF7 (palifermin) and FGF7 variants (Δ53N_hFGF7 (A120C), Δ53N_hFGF7 (A120C, K126E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7. ) is a graph showing the results of measuring the density of the protein bands remaining on the SDS-PAGE over time.
5 shows wild-type FGF7 (Δ30N_hFGF7) and Δ53N_hFGF7 (palifermin) and FGF7 variants (Δ53N_hFGF7(A120C), Δ53N_hFGF7(A120C, K126E), Δ53N_hFGF7(A120C, K178E), Δ53N_hFGF7(A120C, K178E), Δ53N_hFGF7(A120C, K178E), Δ53N_hFGF7. ) shows SDS-PAGE to measure the stability at 45 °C.
Figure 6 shows wild-type FGF7 (Δ30N_hFGF7) and Δ53N_hFGF7 (palifermin) and FGF7 variants (Δ53N_hFGF7(A120C), Δ53N_hFGF7(A120C, K126E), Δ53N_hFGF7(A120C, K178E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7. ) is a graph showing the results of measuring the density of the protein bands remaining on SDS-PAGE over time for measuring the stability at 45°C.
7 shows wild-type FGF7 (Δ30N_hFGF7) and Δ53N_hFGF7 (palifermin) and FGF7 variants (Δ53N_hFGF7 (A120C), Δ53N_hFGF7 (A120C, K126E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7 (A120C, K178E), Δ53N_hFGF7. ) is a graph measuring the change in cell proliferation activity at 45 ℃.

Hereinafter, embodiments will be described in detail so that those of ordinary skill in the art can easily implement it. The embodiment may be implemented in various different forms, and is not limited to the specific embodiment described herein.

Unless the definitions of some terms used in this disclosure are otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The techniques and processes described in this disclosure are generally performed according to conventional methods, which are presented throughout this application. In general, the nomenclature and laboratory procedures in molecular biology, biochemistry, analytical chemistry and cell culture used in this disclosure are well known in the art and are the same as commonly used.

variant

The present disclosure provides FGF7 polypeptides that are thermally stabilized by site-directed mutagenesis. In the present disclosure, mutations were prepared by site-specific mutagenesis after rationally predicting the most optimal amino acid at a new position that was not previously known through bioinformation analysis and protein design using a computer.

1 shows the wild-type human FGF7 polypeptide sequence.

In the present disclosure, the term 'wild type' refers to a native FGF7 having the most common amino acid sequence among members of the species. In the present disclosure, wild-type FGF7 is human FGF7, a protein of 194 amino acids in length (SEQ ID NO: 1, FIG. 1).

In the present disclosure, a 'fragment' refers to a functional fragment of a FGF7 polypeptide having FGF7 activity. In addition, it refers to a functional fragment of the FGF7 polypeptide having at least 85% sequence identity with the sequence of SEQ ID NO: 1. Fragments of the FGF7 polypeptide also have at least one or more substitutions according to the invention. At least 96%, 97%, 98%, 99% or 100% sequence identity is preferred. Fragments are intended to be polypeptides that consist only of part of the intact polypeptide sequence and structure, and variants may have C-terminal deletions or N-terminal deletions. Such a functional fragment may have a cell-binding region and a heparin-binding segment of the subject FGF7 protein according to the present invention.

In the present disclosure, 'sequence identity' means that identical amino acid residues are found in the FGF7 polypeptide according to the present invention as described above. When the specified contiguous segments of the amino acid sequence of the FGF7 polypeptide are aligned and compared to the specific amino acid sequence corresponding to the reference molecule, the wild-type human FGF7 polypeptide is used as a reference. The % sequence identity is calculated by determining the number of positions in which the same amino acid residue is present in both sequences, dividing it by the total number of positions in the segment compared to the reference molecule, and multiplying this by 100. It is calculated by calculating the % identity. Sequence alignment methods are well known in the art. Reference sequence as used herein refers to the specific corresponding human wild-type FGF7 protein according to the present invention. For example, in mammalian species such as mice, rats, rabbits, primates, pigs, dogs, cattle, horses and humans, FGF7 is highly conserved and exhibits greater than 85% sequence identity across a wide range of species. Preferably, the sequence identity is at least 96%, 97%, 98%, or 99% or greater or 100%. A person skilled in the art will know that the remaining 15% or less amino acids in the full length of the FGF7 protein according to the present invention may be prepared using, for example, other sources of FGF7 species or suitable non-FGF7 peptide sequences generally known in the art or It will be appreciated that, due to the addition of tags, this may be variable. FGF7 protein according to an embodiment of the present invention having 85% or more identity to wild-type FGF7, other members of the FGF family generally have very low sequence identity, so it is unlikely to contain similar proteins other than FGF7.

The present inventors confirmed that the 120th, 126th and 178th positions in wild-type human FGF7 are positions associated with the thermal stability of the FGF7 polypeptide, respectively.

Changing to the most appropriate amino acid at a position associated with thermal stability requires an inventive step of the inventor.

The present inventors confirmed that thermal stability can be improved by inducing an intramolecular disulfide bond by substituting cysteine (C) for alanine (A) at the 120th position.

In addition, it was confirmed that thermal stability could be further improved by inducing ionic bonding with arginine (R) at position 175 by substituting lysine (K) at position 126 with glutamic acid (E) or aspartic acid (D). .

In addition, it was confirmed that thermal stability could be further improved by inducing ionic bonding with arginine (R) at position 175 by substituting lysine (K) at position 178 with glutamic acid (E) or aspartic acid (D). .

Variants possible in the present disclosure may be any one of various variants disclosed in Table 1 below.

# variant One (A120C) 2 (K126E) 3 (K126D) 4 (K178E) 5 (K178D) 6 (A120C, K126E) 7 (A120C, K126D) 8 (A120C, K178E) 9 (A120C, K178D) 10 (K126E, K178E) 11 (K126E, K178D) 12 (K126D, K178E) 13 (K126D, K178D) 14 (A120C, K126E, K178E) 15 (A120C, K126D, K178E) 16 (A120C, K126E, K178D) 17 (A120C, K126D, K178D)

Among the above various variants, a mutation at a single position may also improve thermal stability, but two or more mutations may be desirable for improving thermal stability. Furthermore, three mutations may be preferred for further improved thermal stability.

In general, the coding gene for FGF7 is cloned and then expressed in a transformed organism, preferably a microorganism. The host organism expresses a foreign gene to produce FGF7 under expression conditions. In addition, synthetic recombinant FGF7 can be made in eukaryotes such as yeast or human cells. According to a recombinant production method, FGF7 may be in the form of 194 amino acids, 141 amino acids in which 2 to 54 are deleted, 164 amino acids in which 1 to 30 are deleted, or a mixture thereof. Figure 2 exemplifies the Δ53N-hFGF7 sequence with deletion of 2-54 and a pCold I site. The amino acids synthesized at this time may be in the form of 141 amino acids in which 2-54 are deleted or 142 amino acids in which methionine (M), the first sequence of the pCold I site, remains.

The description presented herein demonstrates for the first time that some changes in wild-type FGF7 construct FGF7 mutants with higher temperature stability and longer half-life than wild-type protein.

The FGF7 protein according to the invention used to insert the substitutions described herein can be, for example, mouse , rats, rabbits, primates, pigs, dogs, cattle, horses, whales and humans. Preferably, the FGF7 protein of interest is from a human source. However, the mammal having 85% or more, most preferably about 96% or more, 97% or more, 98% or more or 99% or more sequence identity to the amino acid sequence of the human FGF7 protein of SEQ ID NO: 1 used as a basis of comparison. All biologically active variants for FGF7 can be used in the present invention.

In some embodiments, the stable FGF7 polypeptides according to the invention described herein can be used to facilitate detection, purification, tagging to specific tissues or cells, improved stability, extended activity, improved expression, etc. It may further comprise a tag or sequence other than any additional FGF peptide known in the art.

Pharmaceutical and cosmetic compositions

The various variants disclosed in Table 1 may be provided as pharmaceutical and/or cosmetic compositions together with a pharmaceutically or cosmetically acceptable carrier.

Various variants disclosed in Table 1 promote angiogenesis, promote wound healing, promote chondrogenesis or bone formation, or subjects in need of promoting neurogenesis or wrinkle improvement, skin elasticity improvement, skin aging prevention, hair loss prevention or hair growth It can be administered to a subject in need of improvement of a skin condition, such as promotion, improvement of skin moisture, removal of age spots or treatment of acne. In addition, after radiation treatment, it may be administered to a subject as a therapeutic agent for stomatitis, a therapeutic agent for head and neck cancer, a therapeutic agent for graft-versus-host disease, and the like. The various variants disclosed in Table 1 may be administered in "native" form or, if desired, in the form of salts, esters, amides, prodrugs, derivatives, etc., provided that the salts, esters, amides, prodrugs or derivatives are used. can be selected from substances that are pharmacologically compatible, ie, effective for the method(s). Salts, esters, amides, prodrugs and other derivatives of peptides are known to those skilled in the art of synthetic organic chemistry and can be prepared using, for example, standard known procedures.

The various variants disclosed in Table 1 are transdermally administered as products for subcutaneous, parenteral, topical, oral, nasal (or otherwise inhaled), rectal, or topical administration, such as in the form of aerosols, creams, serums and patches. can be formulated. The composition may be administered in various unit dosage forms depending on the method of administration. Suitable unit dosage forms may include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained release formulations, lipid complexes, and the like.

When the various variants disclosed in Table 1 are combined with a cosmetically acceptable carrier to form a cosmetic composition, fillers (e.g., hyaluronic fillers, polymethylmethacrylate (PMMA) microspheres and collagen fillers), etc. may additionally include. The composition may preferably be for topical, subcutaneous, or transdermal administration.

The composition may be an injectable composition.

The composition may further comprise collagen (eg bovine, porcine, or human collagen) hyaluronic acid. The collagen may be synthetic collagen, and the hyaluronic acid may be a chicken meal or a fermentation product of a microorganism.

The composition may further comprise an anesthetic (eg, lidocaine).

The composition may be a skin cream (eg, a face cream, a neck cream, a body cream).

The composition may be a liquid formulation in the form of a serum or toner.

The composition may be a semi-solid preparation in a gel state.

Pharmaceutically acceptable carriers may be approved by a federal or state regulatory agency or may be approved by the U.S. include those listed in the pharmacopoeia or other generally recognized pharmacopeias for use in animals, more particularly in humans or animals, more particularly humans. "Carrier" means, for example, a diluent, adjuvant, excipient, adjuvant or vehicle with which one or more peptides described herein are administered.

A pharmaceutically acceptable carrier may contain, for example, one or more physiologically acceptable compounds that act to stabilize the composition or increase or decrease absorption of the various variants disclosed in Table 1. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, lactose, maltose, trehalose, mannitol, levan or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protective and absorption enhancers such as lipids, compounds that reduce the clearance or hydrolysis of peptides, or other excipients, stabilizers and/or pH adjusting buffers.

Other physiologically acceptable compounds, particularly used in the manufacture of tablets, capsules, gel caps, and the like, may include, but are not limited to, binders, diluents/fillers, disintegrants, lubricants, and suspending agents.

To prepare an oral dosage form (eg, a tablet) excipients, optional disintegrants, binders and optional lubricants, etc. are added to the various variants disclosed in Table 1 and the resulting composition can be compressed. If desired, the compressed product may be coated using known methods for taste masking or enteric dissolution or sustained release.

Other physiologically acceptable compounds that may be formulated with the various variants disclosed in Table 1 may include wetting agents, emulsifying agents, dispersing agents or preservatives that are particularly useful for preventing the growth or action of microorganisms. Excipients can be used in a sterile, contaminant-free state.

The various variants disclosed in Table 1 can be incorporated into formulations for cosmetic use and applied topically and can be applied to skin creams (eg, face creams, neck creams, body creams) or body lotions, wrinkle-removing creams, moisturizing creams. , can be formulated as an eye cream, a whitening cream, or incorporated into a cosmetic, sunscreen, or moisturizer.

The various variants disclosed in Table 1 may also be incorporated into formulations optionally further comprising fillers, moisturizers, vitamins (eg, vitamin E, vitamin C), and/or colorants/dyes.

Suitable injectable cosmetic formulations may include, but are not limited to, formulations incorporating the various variants disclosed in Table 1 together with one or more filler substances. Exemplary materials usable as injectable cosmetic wrinkle fillers include, but are not limited to, temporary (absorbable) fillers such as collagen (eg, synthetic collagen, bovine collagen, porcine collagen, human collagen, etc.), hyaluronic acid gel, calcium hydride. lyxylapatite (typically implanted in the form of a gel), or poly-L-lactic acid (PLLA), and the like. The peptides may also be incorporated into injectable cosmetic formulations containing permanent (non-absorbable) fillers. Exemplary “permanent” fillers may include, but are not limited to, polymethylmethacrylate beads (PMMA microspheres).

The various variants disclosed in Table 1 may be incorporated into or administered with dermal fillers, injectable formulations, etc. Such injectable formulations may further comprise an anesthetic (e.g., lidocaine or an analog thereof). . The injectable formulation is substantially sterile or sterile and/or conforms to institutional guidelines for subcutaneous injectable fillers.

The various variants disclosed in Table 1 may be administered to a subject using any route known in the art, which route may be, for example, by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular). intradermal, or intradermal), inhalation, transdermal application, rectal administration, vaginal administration, or oral administration. Preferred routes of administration include subcutaneous, transdermal, or topical application.

Effective amounts of the various variants disclosed in Table 1 can be administered via topical (ie, non-systemic) administration, including, but not limited to, peripheral intramuscular, intravascular, and subcutaneous administration.

Administration of the various variants disclosed in Table 1 may be in any convenient manner, for example, by injection, intravenous and arterial stents (including eluting stents), catheter, oral administration, inhalation, transdermal application, rectal administration, and the like. .

The various variants disclosed in Table 1 may be formulated with a pharmaceutically acceptable carrier prior to administration, eg, as described above. A pharmaceutically acceptable carrier is determined in part by the particular composition to be administered, as well as by the particular method used to administer the composition.

The dose administered in a subject should, in the context of the methods described herein, be sufficient to affect a beneficial therapeutic response (eg, increased subcutaneous adipogenesis) in the subject over time. The dose will be determined by the efficacy of the particular vehicle/delivery method employed, the site of administration, the route of administration, and the condition of the subject, as well as the body weight or surface area of the subject being treated. The size of the dose will also be determined by the presence, sex, and extent of any adverse side effects that accompany administration of a particular peptide in a particular subject.

The various variants disclosed in Table 1 can be administered systemically (eg, orally, or as an injection) according to standard methods well known to those skilled in the art. The peptides may be administered to the oral cavity in various forms, such as lozenges, aerosol sprays, mouthwashes, coated swabs, and the like. A variety of buccal, and sublingual formulations are also contemplated. The various variants disclosed in Table 1 can be administered as a depot formulation when formulated as an injectable to provide treatment over a period of time.

The various variants disclosed in Table 1 can be administered topically, for example, to the skin surface, to a local lesion or wound, to a surgical site, or the like.

The various variants disclosed in Table 1 can be delivered through the skin using a conventional transdermal drug delivery system, i.e., a transdermal "patch", and the various variants disclosed in Table 1 are typically for drug delivery that will adhere to the skin. It may be contained within a layered structure provided as a device.

Other formulations for topical delivery include, but are not limited to, ointments, gels, sprays, fluids, and creams. Ointments may be semi-solid preparations, typically based on petrolatum or other petroleum derivatives. As with other carriers or vehicles, the ointment base must be inert, stable, non-irritating and non-sensitizing. Creams containing the various variants disclosed in selected Table 1 may typically be viscous liquid or semi-solid emulsions, often oil-in-water or water-in-oil. Cream bases are typically water washable and contain an oil phase, an emulsifier and an aqueous phase. The particular ointment or cream base to be used is one that will provide for optimal drug delivery, as will be appreciated by those skilled in the art.

The various variants disclosed in Table 1 are in a storage container ready for dilution (e.g., in a pre-measured volume), or in a soluble capsule ready for addition to a large amount of water, alcohol, hydrogen peroxide, or other diluent, " as a "concentrate". For example, the peptide can be lyophilized for later reconstitution.

The various variants disclosed in Table 1 may have various uses. The various variants disclosed in Table 1 may find use in many applications. For example, since subcutaneous fat provides fullness and firmness to the skin, enhancing the formation of subcutaneous fat has use in cosmetic surgery procedures. Aging skin contains less subcutaneous fat. Therefore, administration of one or more of the various variants disclosed in Table 1 described in the present disclosure to a desired site to promote subcutaneous fat formation can result in fuller and younger looking skin. This approach can replace current methods of transplanting fat cells from other parts of the body (eg, thighs or buttocks), a process often with low success rates.

The various variants disclosed in Table 1 can be administered to selectively enhance subcutaneous adipose tissue (e.g., to enhance subcutaneous adipose tissue without substantially increasing visceral fat and/or other adipose tissue). . In response to administration of the various variants disclosed in Table 1, adipocyte formation occurs in dermal fibroblasts, and volume can be added within a selected subcutaneous site in a subject.

The various variants disclosed in Table 1 can be used to reduce scarring. This can be achieved by administering one or more of the various variants disclosed in Table 1 in an amount sufficient to reduce the scar area and/or improve the appearance of the scar area. A scar may be, for example, a scar produced by a burn, a scar produced by surgery, a scar produced by acne, a scar produced by a biopsy, or a scar produced by an injury.

The various variants disclosed in Table 1 can be used in various cosmetic procedures, for example, to improve the appearance of the skin. This may be accomplished by administering one or more peptides to the site of the subject in an amount sufficient to improve the appearance of the skin. Such administration may include subcutaneous administration to areas such as the lips, eyelids, cheeks, forehead, chin, neck, and the like. The peptides are used in these methods to reduce wrinkles, reduce sagging skin, improve the surface texture of the skin, reduce, remove or fill in wrinkles, remove or reduce age spots, and/or remove dark circles under the eyes etc. can be used. These cosmetic applications are exemplary and not intended to be limiting.

The various variants disclosed in Table 1 can be used to improve tissue volume at the site of a subject. This may be accomplished by administering one or more of the peptides described herein in an amount sufficient to increase tissue volume at the site of the subject. For example, increasing tissue volume may include firming or augmenting breast tissue and/or firming or augmenting hip tissue or other parts of the body or face.

The FGF7 used at this time may be used in an amount of 0.01 to 10 ppm. Above 10 ppm, there is a possibility of side effects that may induce adverse reactions in excessive amounts. Therefore, the practical use range is 0.01 to 10 ppm, preferably 0.01 to 2 ppm.

The various variants disclosed in Table 1 may also be used to soften the skin in the area of a subject. This may be accomplished by administering one or more of the peptides described herein in an amount sufficient to soften the skin at the desired site. The smoothing may include smoothing skin scarred by acne, smoothing out areas of cellulite, smoothing or reducing stretch marks, and/or smoothing wrinkles.

The various variants disclosed in Table 1 can be used to recruit stem cells to the formation of subcutaneous fat in a subject. This can be achieved by administering the various variants disclosed in Table 1 in an amount sufficient to recruit stem cells to the formation of subcutaneous fat. This has utility, for example, in various reconstructive surgical procedures and the like.

The various variants disclosed in Table 1 can be used to reconstruct tissue in a subject. Such reconstruction may include, for example, breast reconstruction (eg, after surgery to remove a tumor), or face or limb reconstruction (eg, after an automobile accident or burn). This can be achieved by administering the various variants disclosed in Table 1 in an amount that increases the volume of the tissue during or after the tissue reconstruction process. The various variants disclosed in Table 1 may optionally be used in combination with tissue graft materials or other procedures that enhance the healing of skin or injured tissue.

The various variants disclosed in Table 1 can be used to reduce heel pain in a subject by administering in an amount sufficient to reduce heel pain experienced by the subject when walking.

The various variants disclosed in Table 1 may be administered for augmentation of subcutaneous fat to increase thermoregulation and/or improve immune function. Subjects can be treated with the various variants disclosed in Table 1 to prevent disease or to treat ongoing disease associated with increased organ fat, including but not limited to cardiovascular disease, and other obesity associated diseases.

Administration in any of these methods may be topical or systemic, and may be by any route described herein, such as topical, subcutaneous, transdermal, oral, nasal, vaginal, and/or rectal administration. Preferably, the various variants disclosed in Table 1 may be administered by subcutaneous injection. Alternatively, the various variants disclosed in Table 1 above may be administered topically in the form of a skin cream such as a face cream, or may be administered transdermally through a transdermal patch.

Although the above uses and methods are described with respect to use in humans, they are also suitable for use in animals, eg, veterinary use. Accordingly, certain preferred organisms include, but are not limited to, humans, non-human primates, canines, horses, cats, pigs, ungulates, rabbits, and the like.

badge

The various variants disclosed in Table 1 may be included in a 'medially effective amount' corresponding to the amount necessary to maintain the pluripotent stem cells in an undifferentiated state for at least 5 passages, and may be provided as a human pluripotent stem cell medium. .

In the present disclosure, including both human embryonic stem cells and induced pluripotent stem cells, the term 'human pluripotent stem cells' refers to pluripotent stem cells that are capable of generating virtually all cell types in the human body with the same progeny. It is characterized by the ability to form - self-renewal capacity.

In the present disclosure, the term 'maintaining stem cells in a pluripotent state' means maintaining the cells in an undifferentiated state having the ability to differentiate into virtually any cell type. This pluripotent state depends on a stemness-supporting cocktail of growth factors, of which FGF7 is the most important growth factor. FGF7 supports self-renewal in several ways: directly activating the mitogen-activated protein kinase pathway and indirectly catalyzing transforming growth factor β1 and activin signaling (Greber, et al. 2008, Stem Cells25). , 455-464). FGF7, through cell adhesion and survival functions, contributes to the pluripotency of human PSCs complexly (Eisellova, et al. 2009, Stem Cells 27, 1847-1857).

The present disclosure relates to the characterization of an engineered subject FGF7, demonstrating the effect of substitution in the protein, methods of using the protein in human PSC culture, and comprising one or more thermostable FGF7 proteins described herein suitable for culturing human PSCs in an undifferentiated state. provide badges. The human embryonic stem cells (ESCs) used in the examples provided herein were derived from blastocyst embryos obtained with the informed consent of the physician. A well-characterized human ESC cell line (Adewumi, et al. 2007, Nat Biotechnol 25, 803-816) CCTL14 (Center of Cell Therapy Line) was used for passages 29-41. As in human induced pluripotent stem cells (iPSCs), the AM13 cell line, derived using Yamanaka's cocktail and reprogramming of dermal fibroblasts by Sendai virus transfection, was used as the 34-41 cell line. Accounting status was used (Kruta et al. 2014, Stem Cells and Development 23, 2443-2454).

Hereinafter, preferred experimental examples are presented to help the understanding of the present invention, but the following experimental examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

Experimental Example 1: Construction, purification and thermal stability analysis of mutants using pCold I vector

The mutation at position 1 (A120C), mutation at position 2 (A120C, K126E), mutation at position 2 (A120C, K178E), mutation at position 3 (A120C, K126E, K178E) of FGF7 were synthesized and His It was subcloned into pCold I vector with -Tag. The recombinant vector into which FGF7 was inserted was transformed into BL21 (DE3) cells and expressed.

10ml LB media (Ambrothia) (0.25g used) was inoculated and 10ul of ampicillin (Ampicillin, 50mg/ml) was added and then pre-cultured at 37°C.

10ml of the pre-culture solution and 1ml of ampicillin (Ampicillin, 50mg/ml) were inoculated into 1L LB media (ambrothia) (25g used) and cultured at 37°C. When the value of OD ₆₀₀ is 0.6, the culture medium is cooled in a refrigerator at 4°C for 10 minutes, and 5 mM of beta-di-1-thiogalactopyranoside (β-Dl-thiogalactopyranoside; IPTG) is added thereto for 24 hours at 17°C. E. coli cells induced for expression were obtained.

After centrifugation, the optimally dissolved supernatant was injected into a column with Ni-NTA beads. The first wash buffer (20 mM Tris pH 8.0, 200 mM NaCl) and the second wash buffer (20 mM Tris pH 8.0, 200 mM NaCl, 30 mM imidazole) equal to three times the volume of pCold I_FGF7 protein injected into the column. ), and using 100ml elution buffer (elution buffer, 20mM Tris pH 8.0, 200mM NaCl, 1M imidazole) sequentially increasing the imidazole concentration and eluting the FGF7 protein for primary purification.

The supernatant purified through the first affinity chromatography (Ni-NTA) experiment was secondarily injected into a column with heparin beads. The column was washed with the first wash buffer (20 mM Tris 8.0, 200 mM NaCl) three times the volume of the pCold I_hFGF7 protein injected into the column, and eluted with 60 ml elution buffer (20 mM Tris pH 8.0, 1500 mM NaCl) for secondary purification.

Finally, the pCold I_hFGF7 protein fraction was prepared using a HiLoad ^TM 16/60 Superdex 75 (Amersham Biosciences) column with 1X PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4), (WELGENE) was purified by gel filtration using

37℃ stability test

Purified FGF7 proteins were reacted for 0, 4, 8, 12, and 15 days at 37°C at a concentration of 0.5 mg/ml using 1X PBS buffer. carried out. Protein bands remaining in SDS-PAGE GEL were quantitatively measured for density using ImageJ program (Wayne Rasband, NIH).

As illustrated in FIG. 3 , it can be seen that the thermal stability of the variants was improved from the FGF7 polypeptide bands identified through 15% SDS-PAGE.

For density measurement, the density of SDS-PAGE GEL was measured using the imageJ program (Wanyne Rasband). The results are shown in Table 2 below, and are graphically represented in FIG. 4 .

0 day 4 days 8 days 12 days 15 days Δ30_hFGF7 (wild type) 100 40 14 4 4 Δ53_hFGF7 (palifermin) 100 60 46 23 18 Δ53_hFGF7 (A120C) 100 87 77 73 66 Δ53_hFGF7 (A120C, K126E) 100 97 89 103 96 Δ53_hFGF7 (A120C, K178E) 100 107 94 107 100 Δ53_hFGF7 (A120C, K126E, K178E) 100 116 120 112 118

unit %

From the results of Table 2 and Figure 4, Δ53N_hFGF7 (palifermin) showed a decrease in the protein band to 18% on the 15th day, whereas Δ53N_hFGF7 (A120C) induced disulfide bonds had 66% remaining on the 15th day. It can be seen that Δ53N_hFGF7 (A120C, K126E) and Δ53N_hFGF7 (A120C, K178E), which induced disulfide bonds and ionic bonds, had 96% and 100% protein remaining on the 15th day, respectively. △53N_hFGF7 (A120C, K126E, K178E), which introduced mutations at all three positions, showed an error that increased to 118% on the 15th day than on the 0th day, but it can be seen that the bands near 100% were stably maintained for 15 days. .

As a result of comparing the stability of △30N_hFGF7(wild) and △53N_hFGF7(palifermin) to confirm the stability along the length of the N-terminal, △30N_hFGF7 (wild type) and N-terminus with the signal peptide removed were more It can be seen that the short Δ53N_hFGF7(palifermin) has a density of 4% and 18% on the 15th day, respectively, indicating that Δ53N_hFGF7(palifermin), which has a shorter N-terminal length, is more stable at 37°C.

45℃ Stability Test

Purified FGF7 proteins were reacted for 0, 1, 2, 3, 4, 5, 6, and 7 days at 45°C at a concentration of 0.5 mg/ml using 1X PBS buffer as a standard, and were stained with Coomassie blue dye to 15% SDS-PAGE electrophoresis was performed. The result is illustrated in FIG. 5 .

As illustrated in FIG. 5 , it can be seen that the thermal stability of the variants was improved from the FGF7 polypeptide bands identified through 15% SDS-PAGE.

For density measurement, the density of the SDS-PAGE gel was measured using the imageJ program (Wanyne Rasband). The results are shown in Table 3 and Figure 6 below.

0 day 1 day 2 days 3 days 4 days 5 days 6 days 7 days Δ30_hFGF7 (wild type) 100 0 0 0 0 0 0 0 Δ53_hFGF7 (palifermin) 100 0 0 0 0 0 0 0 Δ53_hFGF7 (A120C) 100 16 6 0 0 0 0 0 Δ53_hFGF7 (A120C, K126E) 100 57 51 42 30 29 19 22 Δ53_hFGF7 (A120C, K178E) 100 66 51 43 22 21 10 9 Δ53_hFGF7 (A120C, K126E, K178E) 100 84 86 81 74 80 76 79

unit %

From the results of Table 3 and Figure 6, it can be seen that all mutants have improved thermal stability than wild-type hFGF7.

It can be seen that Δ53N_hFGF7 had a protein band of 0% within one day, while Δ53N_hFGF7 (A120C), which induced disulfide bonds, became 0% on the 3rd day. It was confirmed that Δ53N_hFGF7 (A120C, K126E) and Δ53N_hFGF7 (A120C, K178E) induced disulfide bond and ionic bond remained 22% and 9% protein on the 7th day, respectively. Δ53N_hFGF7 (A120C, K126E, K178E), which introduced all of the previously identified mutations, was able to confirm 79% of the protein band on the 7th day.

In addition, it was confirmed that there was no significant difference according to the length of the N-terminal, such as Δ30_hFGF7 (wild type) and Δ53_hFGF7 (palifermin) at 45°C.

Experimental Example 3: Confirmation of cell proliferative capacity of mutants

For the mutants prepared in the same manner as in Experimental Example 1, HaCaT cells were used, and cultured and maintained in DMEM medium containing 10% fetal bovine serum. used a serum-free DMEM medium containing 0.03% BSA.

Cells were cultured in a 96-well plate at 0.6x10 ⁴ /well, and treated with FGF7 (300 ng/ml) together with heparin (10 ug/ml) for 40 hours. Cell number increase was performed using WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt], It was confirmed by measuring the production level of WST-8 formazan formed by an electron mediator and intracellular dehydrogenases. The degree of WST-8 formazan production can be confirmed through absorbance (450 nm). The experiment was repeated 4 times, and expressed as 'mean ± standard deviation'.

FGF7 proteins were stored at 45° C. for 0, 0.5, 1, 2, 4, 6, and 8 days, respectively, and cell proliferation activity changes were confirmed.

The results are illustrated in Table 4 and FIG. 7 .

division 50% active (ED50) Δ30_hFGF7 (wild type) 0.8 days Δ53_hFGF7 (palifermin) 1.7 days Δ53_hFGF7 (A120C) 8.0 days Δ53_hFGF7 (A120C, K126E) 1107 days Δ53_hFGF7 (A120C, K178E) 33.9 days Δ53_hFGF7 (A120C, K126E, K178E)

Referring to Table 4 and Figure 7, in the case of Δ30N_hFGF7 (wild type), the cell proliferation activity decreased as the time to store the protein at 45 degrees increased, and the time point with 50% activity (ED50) was 0.8 days, whereas In the case of Δ53N_hFGF7 (palifemin), it was confirmed that the ED50 was increased up to 1.7 days. On the other hand, it was confirmed that the ED50 of Δ53N_hFGF7 (A120C) was increased up to 8 days, the ED50 of Δ53N_hFGF7 (A120C, K178E) was increased by 33.9 days, and that of Δ53N_hFGF7 (A120C, K126E) was increased by 1107 days. In addition, Δ53N_hFGF7 (A120C, K126E, K178E) showed no decrease in cell proliferation activity even after storage at 45°C for 8 days. Confirmed.

Although various embodiments have been described above, the scope of the rights is not limited thereto. The implemented form can be implemented with various modifications within the scope of the detailed description and accompanying drawings of the invention, and it is natural that this also falls within the scope of rights.

<110> Korea Institute of Ocean Science and Technology <120> THERMALLY STABLE FGF7 POLYPEPTIDE AND USE OF THE SAME <130> DPP20202668KR <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> FGF7 of Homo Sapiens <400> 1 Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 20 25 30 Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 70 75 80 Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95 Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala 180 185 190 Ile Thr <210> 2 <211> 157 <212> PRT <213> Artificial Sequence <220> <223> FGF7 variant of Homo Sapiens <400> 2 Met Asn His Lys Val His His His His His His Ile Glu Gly Arg His 1 5 10 15 Met Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile Arg Val Arg Arg Leu 20 25 30 Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp Lys Arg Gly Lys Val 35 40 45 Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn Ile Met Glu Ile Arg 50 55 60 Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly Val Glu Ser Glu Phe 65 70 75 80 Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr Ala Lys Lys Glu Cys 85 90 95 Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu Glu Asn His Tyr Asn 100 105 110 Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly Gly Glu Met Phe Val 115 120 125 Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly Lys Lys Thr Lys Lys 130 135 140 Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala Ile Thr 145 150 155

Claims (8)

Amino acids 2 to 54 are deleted from the N-terminus of SEQ ID NO: 1 (Δ53N),
Alanine at position 120 (A) is substituted with cysteine (C),
lysine at position 126 (K) is substituted with glutamic acid (E),
Lysine at position 178 (K) is substituted with glutamic acid (E),
A polypeptide having FGF7 activity and having improved temperature stability compared to a wild-type human FGF7 polypeptide. delete delete delete delete The method of claim 1,
The polypeptide having improved temperature stability is a polypeptide in which methionine (M) is bound to the deletion site. delete a polypeptide according to claim 1 or 6; and
A composition comprising a cosmetically acceptable carrier.

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