KR102428940B1 - Thermally stable and protease resistant fgf2 polypeptide and use of the same - Google Patents

Abstract

A polypeptide having FGF2 activity and improved temperature stability and protease resistance is provided. In SEQ ID NO: 1, aspartic acid (D) at position 28 is substituted with glutamic acid (E), cysteine at position 78 (C) is substituted with isoleucine (I) or leucine (L), or cysteine at position 96 (C) in SEQ ID NO: 1 and at least one or more substitutions selected from those substituted with isoleucine (I) or tryptophan (W).

Description

FGF2 polypeptide with improved temperature stability and protease resistance and its use

The present disclosure relates to an FGF2 polypeptide having improved temperature stability and protease resistance, 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, FGF2 (Fibroblast Growth Factor 2) is mainly composed of 155 amino acids and has a molecular weight of about 18 kDa. FGF2 has broad mitogenic and cell survival activity, and plays a role as a potent mediator in wound healing, angiogenesis, and growth of the nervous system.

Therefore, FGF2 is being developed as a pharmaceutical for promoting angiogenesis, promoting wound healing, promoting cartilage or bone formation, and promoting neurogenesis, as well as being widely used as a cosmetic raw material for skin regeneration, wrinkle removal, or elasticity increase. In addition, due to the function of maintaining the cells in a pluripotent state, it is added as a major factor to the culture medium for human pluripotent stem cells (PSC).

As described above, it is reported that FGF2, which has various functions in the human body, is thermodynamically inferior to epithelial growth factor (EGF), insulin-like growth factor (IGF), and vascular endothelial growth factor (VEGF). Also, there is a problem that it is easy to be cleaved by proteolytic enzymes. Therefore, in order for FGF2 to be suitably used for industrial use, it is an essential condition to ensure thermodynamic stability and/or protease resistance of FGF2.

An object of the present disclosure is to provide an FGF2 polypeptide with improved temperature stability and protease resistance.

An object of the present disclosure is to provide a pharmaceutical or cosmetic composition comprising an FGF2 polypeptide having improved temperature stability and protease resistance.

An object of the present disclosure is to provide a culture medium for human pluripotent stem cells comprising an FGF2 polypeptide having improved temperature stability and protease resistance.

In the FGF2 polypeptide with improved temperature stability according to the embodiments, aspartic acid (D) at position 28 in SEQ ID NO: 1 is substituted with glutamic acid (E), or cysteine at position 78 is leucine (L) or isoleucine (I) It contains at least one substitution selected from the group consisting of substituted with or the 96th cysteine (C) is substituted with tryptophan (W) or isoleucine (I), and has improved temperature stability with FGF2 intrinsic activity (thermally stable) poly is a peptide.

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

Human pluripotent stem cell culture medium according to embodiments includes a polypeptide having improved temperature stability and protease resistance as an active ingredient.

The FGF2 polypeptide according to the Examples exhibits improved temperature stability and protease resistance compared to the wild-type human FGF2 polypeptide after product manufacture.

Polypeptides with improved temperature stability and protease resistance can maintain activity during distribution and storage, unlike existing wild human FGF2 products. Therefore, it can be used as an active ingredient in a pharmaceutical or cosmetic composition. In addition, it has the advantage that the activity inducing undifferentiated proliferation can be maintained for a long period of time compared to wild-type FGF2 when used as an active ingredient of human pluripotent stem cell culture medium.

1 is a polypeptide of wild-type FGF2 (SEQ ID NO: 1).
2 shows SDS-PAGE of wild-type FGF2 and FGF2 mutants (pQE80_hFGF2(S137P), pQE80_hFGF2 (D28E, S137P)).
3 shows wild-type FGF2 (Δ9N-hFGF2) and FGF2 variants (Δ9N-hFGF2 (D28E, S137P), Δ9N-hFGF2 (D28E, C78L, C96I, S137P), Δ9N-hFGF2 (D28E, C78I, C96I, S137P), Δ9N-hFGF2 (D28E, C78L, C96W, S137P), Δ9N-hFGF2 (D28E, C78I, C96W, S137P)) at 37 ° C. SDS-PAGE for determining the stability is shown.
4 shows wild-type FGF2 (Δ9N-hFGF2) and FGF2 variants (Δ9N-hFGF2 (D28E, S137P), Δ9N-hFGF2 (D28E, C78L, C96I, S137P), Δ9N-hFGF2 (D28E, C78I, C96I, S137P), Δ9N-hFGF2 (D28E, C78L, C96W, S137P), and Δ9N-hFGF2 (D28E, C78I, C96W, S137P)) at 45° C. are shown SDS-PAGE to determine the stability.
5 is a graph measuring changes in cell proliferation activity at 37° C. of wild-type FGF2 and FGF2 variants (pQE80_hFGF2(S137P), pQE80_hFGF2 (D28E, S137P)).
6 shows wild-type FGF2 (Δ9N-hFGF2) and FGF2 variants (Δ9N-hFGF2 (D28E, S137P), Δ9N-hFGF2 (D28E, C78L, C96I, S137P), Δ9N-hFGF2 (D28E, C78I, C96I, S137P)) is a graph measuring the change in cell proliferation activity at 37 ℃.
7 shows wild-type FGF2 (Δ9N-hFGF2) and FGF2 variants (Δ9N-hFGF2 (D28E, S137P), Δ9N-hFGF2 (D28E, C78L, C96I, S137P), Δ9N-hFGF2 (D28E, C78I, C96I, S137P)) is a graph measuring the change in cell proliferation activity at 42 ℃.
8 shows SDS-PAGE for measuring the resistance of wild-type FGF2 (Δ9N-hFGF2) to proteolytic enzymes.
FIG. 9 shows SDS-PAGE for measuring the resistance of FGF2 variants (Δ9_hFGF2 D28E + C78L + C96I + S137P) to proteolytic enzymes.

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 FGF2 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 FGF2 polypeptide sequence.

In the present disclosure, the term 'wild type' refers to a native FGF2 having the most common amino acid sequence among members of the species. In the present disclosure, wild-type FGF2 is human FGF2, an 18 kDa protein 155 amino acids long (SEQ ID NO: 1, FIG. 1).

In the present disclosure, a 'fragment' refers to a functional fragment of a FGF2 polypeptide having FGF2 activity. In addition, it refers to a functional fragment of the FGF2 polypeptide having 85% or more sequence identity with the sequence of SEQ ID NO: 1. Fragments of the FGF2 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 FGF2 protein according to the present invention.

In the present disclosure, 'sequence identity' means that identical amino acid residues are found in the FGF2 polypeptide according to the present invention as described above. When the specified contiguous segments of the amino acid sequence of the FGF2 polypeptide are aligned and compared to the specific amino acid sequence corresponding to the reference molecule, the wild-type human FGF2 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 FGF2 protein according to the present invention. For example, in mammalian species such as mouse, rat, rabbit, primate, pig, dog, bovine, horse and human, FGF2 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%. The person skilled in the art will know that the remaining 15% or less amino acids in the full length of the FGF2 protein according to the present invention can be prepared using, for example, other sources of FGF2 species or suitable non-FGF2 peptide sequences generally known in the art or It will be appreciated that, due to the addition of tags, this may be variable. The FGF2 protein according to an embodiment of the present invention having at least 85% identity to wild-type FGF2 is unlikely to include similar proteins other than FGF2, since other members of the FGF family generally have very low sequence identity.

The present inventors confirmed that position 28 or position 137 in wild-type human FGF2 is a position associated with thermal stability and/or protease resistance of the FGF2 polypeptide. In addition, it was confirmed that the cysteine at the 78th or 96th position among the cysteines exposed on the surface of FGF2 is a position associated with thermal stability and/or protease resistance.

Changing to the most appropriate amino acid at a position associated with thermal stability and/or protease resistance requires an inventor's inventive step.

The present inventors confirmed that thermal stability can be improved by substituting glutamic acid (E) for aspartic acid (D) at the 28th position.

In addition, it was confirmed that thermal stability could be further improved by substituting proline (P) for serine (S) at the 137th position.

In addition, it was confirmed that thermal stability can be further improved by substituting both the 28th position and the 137th position.

In the case of position 78 and position 96, stabilization comparison after 19 mutations was performed through SDM ((http://mari.bioc.cam.ac.uk, University of Cambridge) and Discovery studio 2019 (BIOVIA) and confirmed. did.

In the case of the 78th position, the SDM analysis result showed the highest predicted value (Predicted pseudo ΔG) of 0.33 when mutated to leucine (L), and the mutation energy change value ( kcal/mol) was predicted to be the most stable as -1.20.

In the case of the 96th position, in the case of SDM analysis, the predicted value (Predicted pseudo ΔG) of 0.19 was the highest when mutated to isoleucine (L), and the mutation energy change value ( kcal/mol) was -0.39, which was predicted to be the most stable.

In US9169309, US2017-0291931, EP3380508, US20180319857, etc., cysteine at position 78 is substituted with serine (S) or tyrosine (Y), or cysteine at position 96 is replaced with serine (S), tyrosine (Y), threonine (T) or asparagine (N) and the like are disclosed. In the case of these substituted amino acids, the majority are hydrophilic and uncharged amino acids, whereas in the present disclosure, substituted amino acids are hydrophobic amino acids and can be viewed as a category that cannot be easily predicted from the prior art.

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

# variant One (D28E) 2 (C78I) 3 (C78L) 4 (C96I) 5 (C96W) 6 (S137P) 7 (D28E, C78I) 8 (D28E, C78L) 9 (D28E, C96I) 10 (D28E, C96W) 11 (D28E, C137P) 12 (C78I, C96I) 13 (C78I, C96W) 14 (C78L, C96I) 15 (C78L, C96W) 16 (C78I, S137P) 17 (C78L, S137P) 18 (C96I, S137P) 19 (C96W, S137P) 20 (D28E, C78I, C96I) 21 (D28E, C78I, C96W) 22 (D28E, C78L, C96I) 23 (D28E, C78L, C96W) 24 (D28E, C78I, S137P) 25 (D28E, C78L, S137P) 26 (D28E, C96I, S137P) 27 (D28E, C96W, S137P) 28 (C78I, C96I, S137P) 29 (C78I, C96W, S137P) 30 (C78L, C96I, S137P) 31 (C78L, C96W, S137P) 32 (D28E, C78I, C96I, S137P) 33 (D28E, C78I, C96W, S137P) 34 (D28E, C78L, C96I, S137P) 35 (D28E, C78L, C96W, S137P)

Among the above various variants, a single position mutation may also improve thermal stability and/or protease resistance, but two or more mutations may be desirable for improving thermal stability and/or protease resistance. Furthermore, three to four mutations may be desirable for more thermal stability and/or improved protease resistance. In general, the coding gene for FGF2 is cloned and then expressed in a transformed organism, preferably a microorganism. The host organism expresses a foreign gene to produce FGF2 under expression conditions. In addition, synthetic recombinant FGF2 can be made in eukaryotes such as yeast or human cells. FGF2 may be in the form of 146 amino acids, 153-155 amino acids, or a mixture thereof depending on the recombinant production method.

The description presented herein demonstrates for the first time that some changes in wild-type FGF2 construct FGF2 mutants with higher temperature stability and longer half-life than wild-type protein.

The FGF2 protein according to the invention used to insert the substitutions described herein can be, for example, a mouse, as long as it meets the criteria specified herein, i.e. is heat-stabilized while retaining the desired biological activity of wild-type FGF2. , rats, rabbits, primates, pigs, dogs, cattle, horses and humans. Preferably, the FGF2 protein of interest is from a human source. However, 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 FGF2 protein of SEQ ID NO: 2 used as a basis of comparison, mammalian All biologically active variants for FGF2 can be used in the present invention.

In some embodiments, the stable FGF2 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. 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).

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, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, protective and absorption enhancers such as lipids , a compound that reduces the clearance or hydrolysis of the peptide, 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 may be incorporated into formulations for cosmetic use and applied topically and may be formulated as skin creams (eg, face creams) or body lotions, wrinkle-removing creams, or as cosmetic, sunscreen agents. , or may be incorporated into a moisturizer.

The various variants disclosed in Table 1 may also be incorporated into formulations optionally further comprising fillers, humectants, vitamins (eg, vitamin E), 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.

In this case, FGF2 used 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 are 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 to be provided as a human pluripotent stem cell medium. can

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, with FGF2 being the most important growth factor. FGF2 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). FGF2, 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 provides a method for characterizing an engineered subject FGF2, demonstrating the effect of substitution in a protein, methods of using the protein in human PSC culture, and comprising one or more thermostable FGF2 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 the pQE80 vector

Mutations at one position (S137P) and two positions (D28E, S137P) of FGF2 were synthesized and subcloned into pQE80L vector with His-Tag. The recombinant vector into which FGF2 was inserted was transformed into Rosetta(DE3)pLysS 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 OD600 is 0.6, the culture medium is cooled in a refrigerator at 4°C for 10 minutes, and then 0.5 mM of beta-di-1-thiogalactopyranoside (β-D-1-thiogalactopyranoside; IPTG) is added at 20°C for 20 hours. E. coli cells induced for expression were obtained.

The expressed pQE80_FGF2 was dissolved in a lysis buffer solution (20mM Tris pH 8.0, 200mM NaCl, 3mM DTT), sonicated, and centrifuged at 13000 r.p.m for 30 minutes, followed by purification.

After centrifugation, the optimally dissolved supernatant was injected into a column with Heparin beads. The first wash buffer (20 mM Tris pH 8.0, 200 mM NaCl, 3 mM DTT) and the second wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 3 mM) equal to three times the volume of pQE80_FGF2 protein injected into the column. DTT), and eluted with 60ml elution buffer, 20mM Tris pH 8.0, 1800mM NaCl, 3mM DTT) for primary purification.

Finally, the pQE80_FGF2 protein fraction was purified by gel filtration using a HiLoad™ 16/60 Superdex 75 (Amersham Biosciences) column and 1X PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), (WELGENE). became

Purified FGF2 proteins were reacted for 0, 2, 4, and 6 days at 37°C at a concentration of 0.5 mg/ml using 1X PBS buffer. . The result is illustrated in FIG. 2 .

As illustrated in Figure 2, both mutated polypeptides (pQE80_hFGF2 (S137P), pQE80_hFGF2 (D28E, S137P)) from the FGF2 polypeptide bands identified through 15% SDS-PAGE) hFGF2 (wild-type) poly It can be seen that the stability on SDS-PAGE was improved than that of the peptide.

Experimental Example 2: Construction, purification and thermal stability analysis of mutants using the pET17b vector

Mutation at position 1 (S137P), mutation at position 2 (D28E, S137P), mutation at position 4 ((D28E, C78L, C96I, S137P), (D28E, C78L, C96W, S137P), (D28E) , C78I, C96I, S137P), (D28E, C78I, C96W, S137P)) were synthesized and subcloned into pET17b vector with His. The recombinant vector into which FGF2 was inserted was transformed into Rosetta(DE3)pLysS 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 OD600 is 0.6, the culture medium is cooled in a refrigerator at 4°C for 10 minutes, and then 0.5 mM of beta-di-1-thiogalactopyranoside (β-D-1-thiogalactopyranoside; IPTG) is added at 20°C for 20 hours. E. coli cells induced for expression were obtained.

The expressed pET17b_FGF2 was dissolved in a lysis buffer solution (20 mM Tris pH 8.0, 200 mM NaCl, 3 mM DTT), sonicated, centrifuged at 13000 r.p.m for 30 minutes, and then purified.

After centrifugation, the optimally dissolved supernatant was injected into a column with Heparin beads. The first wash buffer (20 mM Tris pH 8.0, 200 mM NaCl, 3 mM DTT) and the second wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 3 mM) equal to three times the volume of pET17b_FGF2 protein injected into the column. DTT), and eluted with 60ml elution buffer, 20mM Tris pH 8.0, 1800mM NaCl, 3mM DTT) for primary purification.

The 4-position mutants were purified by affinity chromatography using a solution prepared not to contain 3 mM DTT in both buffer and elution buffer.

Finally, the pET17b_FGF2 protein fraction was purified by gel filtration using a HiLoad™ 16/60 Superdex 75 (Amersham Biosciences) column and 1X PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), (WELGENE). became

37℃ stability test

Purified FGF2 proteins were reacted for 0, 3, 6, and 9 days at 37°C at a concentration of 0.5 mg/ml using 1X PBS buffer. . The result is illustrated in FIG. 3 .

As illustrated in FIG. 3 , it can be seen that the thermal stability of the variants was improved from the FGF2 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 2 below.

variant 0 days 3 days 6 days 9 days △9N_hFGF2 100 84 69 65 △9N_hFGF2 (D28E, S137P) 100 100 65 17 △9N_hFGF2 (D28E, C78L, C96I, S137P) 100 86 102 89 △9N_hFGF2 (D28E, C78I, C96I, S137P) 100 98 102 103 △9N_hFGF2 (D28E, C78L, C96W, S137P) 100 99 96 94 △9N_hFGF2 (D28E, C78I, C96W, S137P) 100 112 92 65

It can be seen from the results in Table 2 of the unit % that the mutants at the four positions have improved thermal stability than wild-type hFGF2. In particular, in the case of Δ9N-hFGF2 (D28E, C78L, C96I, S137P), Δ9N-hFGF2 (D28E, C78L, C96W, S137P), Δ9N-hFGF2 (D28E, C78I, C96I, S137P) compared to other variants It can be seen that the thermal stability is relatively better.

45℃ Stability Test

Purified FGF2 proteins were reacted for 0, 1, 2, 3, 4, 5, and 6 days at 45°C at a concentration of 0.5 mg/ml using 1X PBS buffer as a basis. PAGE electrophoresis was performed. The result is illustrated in FIG. 4 .

As illustrated in FIG. 4 , it can be seen that the thermal stability of the variants was improved from the FGF2 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 below.

0 days 1 day 2 days 3 days 4 days 5 days 6 days 7 days △9N_hFGF2 100 0 0 0 0 0 0 0 △9N_hFGF2 (D28E, S137P) 100 73 54 37 17 9 3 2 △9N_hFGF2 (D28E, C78L, C96I, S137P) 100 93 62 41 38 16 22 15 △9N_hFGF2 (D28E, C78L, C96W, S137P) 100 76 20 5 0 0 0 0 △9N_hFGF2 (D28E, C78I, C96I, S137P) 100 84 72 54 49 38 35 31 △9N_hFGF2 (D28E, C78I, C96W, S137P) 100 58 21 15 0 0 0 0

From the results of Table 3, it can be seen that all mutants have improved thermal stability than wild-type hFGF2. In particular, it can be seen that Δ9N-hFGF2 (D28E, C78L, C96I, S137P) and Δ9N-hFGF2 (D28E, C78I, C96I, S137P) have relatively better thermal stability compared to other variants.

Experimental Example 3: Confirmation of cell proliferative ability of mutants using pQE80 vector

For the mutants prepared in the same manner as in Experimental Example 1, BALB3T3 cells were used, and cultured and maintained in DMEM medium containing 10% bovine serum. In order to confirm the cell proliferation activity by FGF2, the cells were insulin 10 ug/ml, dexamethasone 1 uM, transferrin 10 ug/ml, sodium selenite 10 ng/ml, It was cultured in F12/DMEM medium containing 100 ug/ml of ovalbumin and 5 ug/ml of fibronectin.

Cells were cultured in a 96-well plate at 0.5 x 10 ⁴ /well, and treated with FGF2 (0.3 ng/ml) together with heparin (10 ug/ml) for 42 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 3 times, and expressed as 'mean ± standard deviation'. FGF2 proteins were stored at 37°C for 0, 2, 4, and 6 days, respectively, and cell proliferation activity changes were confirmed.

The results are illustrated in FIG. 5 . Referring to FIG. 5 , a decrease in activity of hFGF2 was observed under the condition of 2 days after storage at 37°C, and only 60% of the activity of the protein was observed on day 0 of storage at 37°C after 4 days of storage at 37°C. On the other hand, the hFGF2 S137P mutant was observed to have lower activity than hFGF2 under the condition of storage for 2 days at 37°C, and only 60% of the activity of the protein at 37°C for storage on day 0 was observed under the condition of storage for 4 days at 37°C. On the other hand, it can be seen that the hFGF2 D28E+S137P mutant shows a decrease in activity as the storage date at 37° C. is longer, but shows a much less decrease in activity than the hFGF2 and hFGF2 S137P mutants.

Experimental Example 4: Confirmation of cell proliferative ability of mutants using pET17b vector

For the mutants prepared in the same manner as in Experimental Example 2, BALB3T3 cells were used, and cultured and maintained in DMEM medium containing 10% bovine serum. In order to confirm the cell proliferation activity by FGF2, the cells were insulin 10 ug/ml, dexamethasone 1 uM, transferrin 10 ug/ml, sodium selenite 10 ng/ml, It was cultured in F12/DMEM medium containing 100 ug/ml of ovalbumin and 5 ug/ml of fibronectin.

Cells were cultured in a 96-well plate at 0.5 x 10 ⁴ /well, and treated with FGF2 (0.3 ng/ml) together with heparin (10 ug/ml) for 42 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 3 times, and expressed as 'mean ± standard deviation'. FGF2 proteins were stored at 37°C for 0, 3, 6, 9, and 12 days, respectively, and cell proliferation activity changes were confirmed.

The results are illustrated in FIG. 6 . Referring to FIG. 6 , it was confirmed that Δ9_hFGF2 (wild type) had reduced activity under the condition of storage at 37°C for 3 days, and almost lost activity after 6 days of storage at 37°C. Δ9_hFGF2 D28E+S137P mutant showed a longer activity than the wild-type protein, and showed a decrease in activity after 9 days of storage at 37°C. Unlike these, Δ9_hFGF2 D28E + C78L + C96I + S137P mutant and Δ9_hFGF2 D28E + C78I + C96I + S137P mutant showed no decrease in activity even under the storage condition of 37°C for 12 days. Therefore, it was confirmed that the protein activity was stably maintained at 37° C. in the variants according to the Examples.

FGF2 proteins were stored at 45°C for 0, 1, 2, 3, 4, 5, 6, and 7 days, respectively, and cell proliferation activity changes were confirmed. The results are illustrated in FIG. 7 . Referring to FIG. 7 , it was confirmed that Δ9_hFGF2 (wild type) had a sharp decrease in activity from 45° C. storage for 1 day, and almost lost activity from 45° C. storage for 2 days. The Δ9_hFGF2 D28E+S137P mutant protein showed a result that the activity was observed longer than that of the wild-type protein, but showed a decrease in activity after storage for 2 days at 45°C. Unlike these, the activity of △9_hFGF2 D28E + C78L + C96I + S137P mutant protein and △9_hFGF2 D28E + C78I + C96I + S137P mutant protein decreases slightly depending on the storage date at 45℃, but the activity is maintained longer than the wild type. , it could be confirmed that the proliferative activity of more than 2 times was still maintained even after storage at 45°C for 6 days.

Experimental Example 5: Confirmation of resistance to protease cleavage of mutants using pET17b vector

Resistance to protease cleavage of Δ9_hFGF2 (wild type) and Δ9_hFGF2 D28E + C78L + C96I + S137P mutant proteins prepared in the same manner as in Experimental Example 2 was measured.

8 and 9 show the results of SDS-PAGE measurement of Δ9_hFGF2 (wild type) and Δ9_hFGF2 D28E + C78L + C96I + S137P mutant proteins, respectively. In FIGS. 8 and 9, No. 1 is a case measured immediately after protease treatment without incubation, No. 2 is a case measured after protease treatment and incubation at 37° C. for 3 hours, and No. 3 to 14 FGF2 0.25 mg/mL , shows the results measured after treatment with 0.0025 mg/mL of each of 12 different types of proteolytic enzymes and incubation at 37° C. for 3 hours.

To measure the density of each band, the density of the SDS-PAGE gel was measured using the imageJ program (Wanyne Rasband). The results are shown in Table 4 below.

number proteolytic enzyme △9_hFGF2 (wild type) △9_hFGF2 D28E + C78L + C96I + S137P One X 100 100 2 X 85 89 3 α-Chymotrypsin (α-C) 33 83 4 Trypsin (TR) 21 68 5 Elastase (EL) 70 82 6 Papain (PA) 84 95 7 Subtilisin (SU) 20 79 8 Endoproteinase Glu-C (EG-C) 82 93 9 Proteinase K (P-K) 35 79 10 Clostripain (Endoproteinase-Arg-C) (CL) 46 85 11 Pepsin (PE) 90 84 12 Thermolysin (TH) 47 81 13 Bromelain (BR) 85 90 14 Actinase (A-E) 11 66

From the results of FIGS. 8 and 9 and the contents of Table 4, it can be seen that Δ9_hFGF2 D28E + C78L + C96I + S137P mutant protein has overall improved resistance to protease than Δ9_hFGF2 (wild type).

In particular, for α-Chymotrypsin (α-C), Trypsin (TR), Subtilisin (SU), Proteinase K (P-K), Clostrripain (Endoproteinase-Arg-C) (CL), Thermolysin (TH), Actinase (A-E) It can be seen that the resistance is relatively significantly improved.

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 AND PROTEASE RESISTANT FGF2 POLYPEPTIDE AND USE OF THE SAME <130> DPP20204187KR <150> KR 10-2019-0152362 <151> 2019-11-25 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 155 <212> PRT <213> Artificial Sequence <220> <223> FGF2 of Homo Sapines <400> 1 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser 145 150 155

Claims (8)

Aspartic acid (D) at position 28 in SEQ ID NO: 1 is substituted with glutamic acid (E),
cysteine at position 78 (C) is substituted with isoleucine (I) or leucine (L),
the 96th cysteine (C) is substituted with isoleucine (I),
Serine (S) at position 137 is substituted with proline (P),
A polypeptide having FGF2 activity and having improved temperature stability as compared to a wild-type human FGF2 polypeptide. delete delete delete The method of claim 1,
The 28th aspartic acid (D) is substituted with glutamic acid (E),
A polypeptide in which the 78th cysteine (C) is substituted with isoleucine (I). delete A polypeptide according to claim 1 or 5; and
A composition comprising a cosmetically acceptable carrier. A human pluripotent stem cell culture medium comprising the polypeptide according to claim 1 or 5 as an active ingredient.

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