Novel peptides capable of suppressing TGF-β signaling and uses thereof

Abstract

The present invention relates to novel peptides capable of inhibiting TGF-β signaling and uses thereof. The peptides of the present invention can inhibit the TGF-β signaling pathway or inhibit the expression of TGF-β, thereby making it possible to treat or prevent various diseases caused by TGF-β signaling, and thus effectively inhibit cancer processes such as tumor growth and metastasis induced by TGF-β signaling.

Description

Technical Field

[0001] The present invention relates to a novel peptide capable of suppressing TGF-β signal transduction and its use.

Background Art

[0002] The TGF-β cytokine was discovered by its ability to stimulate the formation of cell populations (Roberts AB, et al, Proc Natl Acad Sci USA 78:5339-43, 1981). Since this process is a typical marker of cell transformation, the cytokine has been named transforming growth factor beta, and TGF-β ligands (TGF-β1, TGF-β2, TGF-β3) are recognized as prototypes of multifunctional growth factors. TGF-β is known as an inhibitor of the proliferation of epithelial, endothelial, and hematopoietic cells, and as one of the most potent regulators of the production and deposition of extracellular matrix and tissue repair cascades.

[0003] On the other hand, TGF-β is known to promote tumor growth, invasion, and metastasis during the progression stage of cancer. Specifically, TGF-β activates numerous SMAD-independent signaling pathways including Ras-MAPK and PI3K-AKT that regulate EMT (epithelial-mesenchymal transition) in tumor cells and various other cell activities, and is known to exhibit various activities during the carcinogenesis process.

[0004] Therefore, recently, TGF-β has been considered as a target for preventing tumor growth and metastasis, and various TGF-β inhibitors including monoclonal antibodies against TGF-β, small molecule compounds (kinase inhibitors), antisense oligonucleotides (ASOs), and chimeric proteins are currently being clinically tested. However, the development of therapeutic methods based on the suppression of TGF-β signal has been progressing slowly for a long time.

[0005] In order to solve such problems, it is necessary to develop a therapeutic agent capable of effectively suppressing the TGF-β signal transduction pathway.

Summary of the Invention

Problems to be Solved by the Invention

[0006] One aspect provides Trait a peptide derived from transforming growth factor-β (TGF-β) or an analog thereof.

[0007] Another aspect provides a polynucleotide encoding the peptide of the present invention.

[0008] Still another aspect provides an expression vector containing the polynucleotide.

[0009] Still another aspect provides a pharmaceutical composition for treating or preventing TGF-β-related diseases, comprising the peptide, the polynucleotide, or the expression vector as an active ingredient.

[0010] Still another aspect provides a method for treating or preventing TGF-β-related diseases, comprising administering to an individual a pharmaceutical composition containing the peptide, the polynucleotide, or the expression vector as an active ingredient.

[0011] Still another aspect provides a pharmaceutical composition for treating or preventing cancer, comprising the peptide, the polynucleotide, or the expression vector as an active ingredient.

[0012] Still another aspect provides a method for treating or preventing cancer, comprising administering to an individual a pharmaceutical composition containing the peptide, the polynucleotide, or the expression vector as an active ingredient.

Means for Solving the Problems

[0013] One aspect provides Trait a peptide derived from transforming growth factor-β (TGF-β) or an analog thereof.

[0014] In one embodiment of the present invention, the peptide may inhibit TGF-β signaling.

[0015] As used herein, the term " Trait Transforming growth factor-β (TGF-β)" is a cytokine belonging to the TGF-β superfamily. There are three known forms of TGF-β expressed in mammals: TGF-β1, TGF-β2, and TGF-β3. The signaling by said TGF-β plays a decisive role in various biological processes, and performs various functions such as cell growth inhibition, cell death, differentiation, and epithelial-mesenchymal Trait transition (EMT). The TGF-β signaling system is strictly regulated and plays a decisive role in the maintenance of cell homeostasis as well as in development and organ formation. Therefore, disruption of TGF-β signaling can induce life-threatening diseases such as cancer, fibrosis, and congenital malformations.

[0016] TGF-β is known to exhibit tumor suppressor activity in the early stage of the carcinogenesis process, but promote cancer growth in the later stage of the carcinogenesis process. In particular, TGF-β1 is highly expressed in most cancer tissues, and it is known that cancer patients with high expression of TGF-β1 are often malignant and have a poor prognosis. TGF-β secreted from cells binds to a heteromeric complex of two types of receptors consisting of type I and type II receptors to initiate signaling. When TGF-β binds to the type II receptor, the type I receptor recognizes this and binds to the type II receptor. At this time, when the type II receptor phosphorylates the GS site of the type I receptor, the type I receptor kinase is activated. The phosphorylated TGF-β type I receptor induces the activation of Smad2 and Smad3 by phosphorylating the C-terminal serine residues of the TGF-β signaling mediators Smad2 and Smad3. The activated Smad2 and Smad3 form a complex with Smad4 and translocate to the nucleus to participate in the expression of target genes.

[0017] In addition, TGF-β is known to play an important role in determining immune homeostasis and tolerance, such as regulating immune tolerance and inflammatory responses by inhibiting the functions and expansion of many components of the immune system. In particular, TGF-β is known to play an important role as an arbiter of immunosuppression in the tumor microenvironment. Therefore, it is known that TGF-β plays an important role in promoting cancer growth within the immune environment of tumors, and combination therapies of immune anticancer agents and various TGF-β signaling inhibitors are being researched and developed.

[0018] The peptide may bind to a TGF-β receptor (TGFBR1 and / or TGFBR2) to inhibit TGF-β signaling. Specifically, the peptide may inhibit TGF-β signaling by competing with TGF-β for binding to the TGF-β receptor, thereby preventing the TGF-β cytokine from binding to the TGF-β receptor.

[0019] Alternatively, the peptide may suppress TGF-β signaling by suppressing the expression level of TGF-β itself in cells. Specifically, the peptide may reduce the expression level of intracellular TGF-β through an auto-inhibition pathway, or may suppress TGF-β signaling by a mechanism that reduces the extracellular excretion amount of TGF-β.

[0020] In one embodiment of the present invention, the peptide may contain the amino acid sequence of the following general formula 1 described in SEQ ID NO: 16.

[0021] [General formula 1] F-X 1 -L-G-P-X 2 -P-Y-I-W-X 3 -L-D-T

[0022] In the general formula 1, X 1 , X 2 and X 3may each independently be cysteine (C) or serine (S), and specifically, X 1 -X 2 -X 3 may be C-C-S, C-C-C or S-S-S.

[0023] The peptide may additionally contain any amino acid or peptide. Specifically, the peptide may contain a peptide in which any amino acid or peptide is directly linked and added to one end, preferably the C-terminus, of the amino acid sequence represented by the general formula 1. The added amino acid or peptide may be alanine (A), phenylalanine (F), valine (V), leucine (L), serine (S), or a combination thereof. Non-limiting examples may be alanine (A), phenylalanine (F), valine-leucine-serine-leucine (V-L-S-L) or valine-leucine-serine-phenylalanine (V-L-S-F).

[0024] The peptide may contain one or more of the amino acid sequences of SEQ ID NOs: 1 to 8, and specifically, may consist of one of the amino acid sequences of SEQ ID NOs: 1 to 8.

[0025] In one embodiment of the present invention, the peptide may contain the amino acid sequence of the following general formula 2 described in SEQ ID NO: 17.

[0026] [General formula 2] A-H-C-S-C-D

[0027] The peptide may additionally contain any amino acid or peptide. Specifically, the peptide may be added directly linked to one end, preferably the C-terminus, of the amino acid sequence of the general formula 2. The added amino acid or peptide may be serine (S), arginine (R), aspartic acid (D), asparagine (N), glycine (G), alanine (A), phenylalanine (F), or a combination thereof. As a non-limiting example, it may be serine-arginine-aspartic acid-asparagine (S-R-D-N) or glycine-alanine-phenylalanine-alanine (G-A-F-A).

[0028] The peptide may contain one or more of the amino acid sequences of SEQ ID NOs: 9 and 10. Specifically, it may consist of one of the amino acid sequences of SEQ ID NOs: 9 and 10.

[0029] In one embodiment of the present invention, the peptide may contain the amino acid sequence of the following general formula 3 set forth in SEQ ID NO: 18.

[0030] [General formula 3] T-I-V-Y-Y-V-X 4 -X 5 -K-P-K-V-E-Q

[0031] In the general formula 3, X 4 may be glycine (G), histidine (H), or valine (V), and X 5 may be arginine (R), leucine (L), or isoleucine (I). Specifically, X 4 -X 5 may be G-R, G-L, H-I, or V-I.

[0032] The peptide may contain one or more of the amino acid sequences of SEQ ID NOs: 11 to 14. Specifically, it may consist of one of the amino acid sequences of SEQ ID NOs: 11 to 14.

[0033] In the present invention, the peptide includes, without limitation, not only the amino acid sequences described by each of the above sequence numbers, but also amino acid sequences having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the above sequences, and showing substantially the same or corresponding efficacy as each of the above peptides. Further, as long as it is an amino acid sequence having substantially the same or corresponding biological activity as the peptide of the sequence number substantially described as a sequence having homology with the above sequence, it is obvious that even if it has an amino acid sequence in which some sequences are deleted, modified, substituted, or added, it is also included in the scope of the present invention.

[0034] As used herein, the term "homology" refers to the degree of similarity between a nucleotide sequence encoding a protein or an amino acid sequence. When the homology is sufficiently high, the protein or the expression product of the gene can have the same or similar activities. Also, homology is expressed as a percentage according to the degree of identity with a given amino acid sequence or nucleotide sequence. In this specification, the homology sequence having the same or similar activity as a given amino acid sequence or nucleotide sequence is represented by "% homology". For example, it can be confirmed by calculating parameters such as score, identity, and similarity using standard software, specifically, BLAST 2.0, or by comparing sequences through hybridization experiments used under defined stringent conditions. The appropriate hybridization conditions defined are within the technical scope and can be determined by methods well known to those skilled in the art (for example, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York; Needleman, S.B. and Wunsch, C.D., (1970), Journal of Molecular Biology, 48, 443 - 453).

[0035] According to one embodiment of the present invention, in the present invention, a candidate peptide capable of suppressing TGF-β signal transduction is prepared. The peptide binds to the TGF-β receptor or reduces the expression level or extracellular excretion amount of TGF-β to suppress TGF-β signal transduction, and based on this, a TGF-β signal transduction-related disease can be treated or prevented, and thus, TGF-β-expressing cancer can also be effectively treated or prevented.

[0036] Another aspect provides a polynucleotide encoding the peptide. The content regarding "TGF-β" and "TGF-β-derived peptide" is as described above.

[0037] As used herein, the term "polynucleotide" refers to a polymeric substance to which nucleotides are bound and means DNA encoding genetic information.

[0038] In the present invention, the nucleotide sequence encoding the peptide includes not only the nucleotide sequences encoding the amino acids described in each SEQ ID NO, but also nucleotide sequences showing 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the above sequences, and which encode peptides substantially identical or having corresponding efficacy to each of the above peptides without limitation. Further, it is obvious that the present invention also includes, without limitation, nucleotide sequences encoding amino acid sequences having substantially the same or corresponding biological activity as the peptides of the SEQ ID NOs substantially described as sequences having homology with the above sequences, for example, nucleotide sequences encoding amino acid sequences in which some sequences are deleted, modified, substituted, or added.

[0039] Also, in the present invention, the polynucleotide encoding the peptide is varied in the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, taking into account the codons preferred in the organism in which the peptide is to be expressed due to the degeneracy of the codons. Therefore, the polynucleotide includes, without limitation, any polynucleotide sequence encoding each peptide. Further, it includes, without limitation, a probe preparable from a known sequence, for example, a nucleotide sequence that hybridizes under stringent conditions with a complementary sequence to the whole or a part of the polynucleotide sequence and encodes a protein having the activity of the peptide.

[0040] The "stringent conditions" mean conditions that enable specific hybridization between polynucleotides. These conditions are specifically described in the literature (for example, J. Sambrook et al., ibid.). For example, genes with high homology hybridize with each other at a homology of 40% or more, specifically 90% or more, more specifically 95% or more, even more specifically 97% or more, and particularly specifically 99% or more, and do not hybridize with genes having lower homology, or the washing conditions for hybridization that are normally used, such as 60°C, 1XSSC, 0.1% SDS, specifically 60°C, 0.1XSSC, 0.1% SDS, more specifically 68°C, 0.1XSSC, 0.1% SDS. The conditions of washing once, specifically 2 to 3 times, at the corresponding salt concentration and temperature can be enumerated.

[0041] Hybridization requires that two polynucleotides have complementary sequences, even if base mismatches are possible depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, this application can further include not only substantially similar polynucleotide sequences but also isolated polynucleotide fragments complementary to the entire sequence.

[0042] Specifically, polynucleotides having homology can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and the conditions described above. Also, the Tm value may be 60°C, 63°C or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art according to the purpose.

[0043] The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the art (see Sambrook et al., supra, 9.50 - 9.51, 11.7 - 11.8).

[0044] Still other aspects provide an expression vector comprising the polynucleotide. The content regarding "TGF-β" and "polypeptide" is as described above.

[0045] As used herein, the term "expression vector" refers to a recombinant vector that can be introduced into a suitable host cell to express a target protein, and refers to a gene construct containing essential regulatory elements operably linked so that the gene insert is expressed. The term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a general function. The operable linkage with a recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be easily performed using enzymes generally known in the art.

[0046] Preferred expression vectors of the present invention can contain signal sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, start codons, stop codons, polyadenylation signals, and enhancers. The start codon and the stop codon are generally regarded as part of the nucleotide sequence encoding an immunogenic target protein, and when the gene construct is administered, it must necessarily act in an individual and must be in frame with the coding sequence. General promoters can be constitutive or inducible. In the case of prokaryotic cells, there are lac, tac, T3, and T7 promoters. In the case of eukaryotic cells, there are simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), for example, the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Rous sarcoma virus (RSV) promoters, as well as promoters derived from β-actin, human hemoglobin, human muscle creatine, human metallothionein, etc., but are not limited thereto.

[0047] In addition, the expression vector can include a selection marker for selecting a host cell containing the vector. The selection marker is for screening cells transformed with the vector, and markers that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive, so transformed cells can be screened. Furthermore, in the case of an expression vector capable of replication, it can include an origin of replication, which is a specific nucleic acid sequence at which replication is initiated.

[0048] As the recombinant expression vector for inserting a foreign gene, vectors in various forms such as plasmids, viruses, and cosmids can be used. The type of recombinant vector is not particularly limited as long as it functions to express a desired gene and produce a desired protein in various host cells of prokaryotes and eukaryotes. Specifically, a vector that has a promoter showing strong activity and strong expression ability and can mass-produce a foreign protein in a form similar to the natural state is used.

[0049] To express the peptide of the present invention, various combinations of hosts and vectors are used. Examples of expression vectors suitable for eukaryotic hosts include, but are not limited to, expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, adenoassociated virus, cytomegalovirus, and retroviruses. Examples of expression vectors that can be used for bacterial hosts include, but are not limited to, bacterial plasmids obtained from Escherichia coli such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9, or derivatives thereof, plasmids with a broader host range such as RP4, phage DNAs exemplified as phage lambda derivatives such as λgt10, λgt11, or NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. For yeast cells, 2μm plasmids or their derivatives can be used, and for insect cells, pVL941 can be used.

[0050] Still another aspect provides a pharmaceutical composition for the treatment or prevention of TGF-β-related diseases, comprising as an active ingredient the peptide of the present invention, the polynucleotide of the present invention, or the expression vector of the present invention. The content regarding "TGF-β" and "peptide" is as described above.

[0051] As used herein, the term "TGF-β-related disease" or "TGF-β disease" means any disorder, disease, or illness that benefits from treatment with a peptide that suppresses the TGF-β signaling pathway of the present invention, including chronic and acute diseases or illnesses, including pathological conditions in which an individual is susceptible to the disease.

[0052] The TGF-β related diseases may include diseases characterized by the accumulation of extracellular matrix, diseases induced by circulating TGF-β or TGF-β activated at local sites, diseases induced by the suppression of the immune system due to the production of endogenous TGF-β, severe injuries, burns and serious illnesses, such as acute immunodeficiency starting from viral or bacterial infections, multi-organ systemic diseases due to the production or overproduction of TGF-β, and TGF-β-producing tumors.

[0053] Examples of the TGF-β related diseases include, but are not limited to, cancer, neurodegenerative diseases (such as Alzheimer's dementia and Huntington's disease), fibrotic skin diseases (such as scleroderma, CNS pathological scar tissue, skin scarring, keloid scarring and nerve scarring), fibrotic diseases of the lung, liver and kidney (such as chronic hepatic fibrosis, acute liver injury, epileptic lung and renal fibrosis, and cirrhosis), cystic fibrosis, cardiac fibrosis, atherosclerosis and arteriosclerosis, systemic sclerosis, and ocular fibrosis.

[0054] As used herein, the term "treatment" means any act by which the symptoms of the disease are improved or favorably changed by administration of the composition of the present invention.

[0055] As used herein, the term "prevention" means any act by which the disease or the likelihood of the onset of the disease is suppressed or delayed by administration of the composition of the present invention.

[0056] The pharmaceutical composition can include a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" can mean a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the injected compound. Here, the meaning of "pharmaceutically acceptable" is that it has no more toxicity than can be tolerated by the subject to which it is applied (formulated) without suppressing the activity of the active ingredient.

[0057] Any type of carrier that can be used in the present invention can be used as long as it is a pharmaceutically acceptable carrier commonly used in the art. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These can be used alone or in combination of two or more. The pharmaceutical composition contains a pharmaceutically acceptable carrier in addition to the active ingredient and can be manufactured into an oral dosage form or a parenteral dosage form depending on the administration route by a conventional method known in the art.

[0058] The pharmaceutical composition can be formulated and used in the form of an oral dosage form such as powder, granule, tablet, pill, sugar-coated tablet, capsule, liquid, gel, syrup, suspension, aerosol, etc., external preparation, suppository or sterilized injection solution by a conventional method. When formulating the pharmaceutical composition, diluents or disintegrants such as fillers, extenders, binders, wetting agents, disintegrants, or surfactants generally used can be added for preparation.

[0059] When the pharmaceutical composition is manufactured into an oral dosage form, it can be manufactured into dosage forms such as powder, granule, tablet, pill, sugar-coated tablet, capsule, liquid, gel, syrup, suspension, wafer, etc. by a method known in the art together with a suitable carrier. At this time, examples of suitable pharmaceutically acceptable carriers include saccharides such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starches such as corn starch, potato starch, wheat starch, celluloses such as cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, and the like. In the case of formulation, it can be formulated containing diluents and / or disintegrants such as fillers, extenders, binders, wetting agents, disintegrants, surfactants as required.

[0060] When the pharmaceutical composition is manufactured into a parenteral dosage form, it can be formulated into the form of injections, transdermal administration agents, nasal inhalants and suppositories by known methods in the art together with suitable carriers. When formulating into an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol and propylene glycol, or mixtures thereof. Preferably, Ringer's solution, PBS (phosphate buffered saline) containing triethanolamine, sterile water for injection, isotonic solutions such as 5% dextrose can be used. When formulating into a transdermal administration agent, it can be formulated into the form of ointments, creams, lotions, gels, topical solutions, pastes, liniments, aerosol agents, etc. In the case of nasal inhalants, it may be formulated into an aerosol spray form using suitable propellants such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, etc. When formulating into a suppository, as its base, witepsol, tween 61, polyethylene glycols, cacao butter, laurin fat, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, etc. can be used.

[0061] The pharmaceutical composition is administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dosage level can be determined by factors such as the severity of the disease, the activity of the drug, the age, weight, health, gender of the patient, the sensitivity of the patient to the drug, the administration time of the composition of the present invention used, the administration route and excretion ratio, the treatment period, elements including drugs formulated or used concomitantly with the composition of the present invention, and other elements well known in the medical field. The pharmaceutical composition is administered alone or in combination with components known to exhibit a therapeutic effect on the known disease. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all the above elements.

[0062] The dosage of the pharmaceutical composition can be determined by those skilled in the art in consideration of the purpose of use, the severity of the disease, the age, weight, gender, medical history of the patient, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng to about 1,000 mg / kg, preferably 1 ng to about 100 mg / kg per adult. The dosing frequency of the composition of the present invention is not particularly limited thereto, but it can be administered once a day or the dose can be divided and administered several times. The dosage or dosing frequency does not limit the scope of the present application in any way.

[0063] Still another aspect provides a method for treating or preventing a TGF-β related disease, comprising the step of administering to an individual a pharmaceutical composition for treating or preventing the TGF-β related disease. The descriptions of the "TGF-β related disease" and the "pharmaceutical composition" are as described above.

[0064] As used herein, the term "individual" can include mammals such as mice, livestock, humans, etc. in which a TGF-β related disease has occurred or is at risk of occurring, birds, reptiles, farmed fish, etc. without limitation.

[0065] The pharmaceutical composition can be administered in a single or multiple doses in a pharmaceutically effective amount. At this time, the composition can be formulated into a dosage form such as a solution, powder, aerosol, injection, infusion (Ringer's), capsule, pill, tablet, suppository or patch for administration. The administration route of the pharmaceutical composition for preventing or treating cancer can be through any general route that can reach the target tissue.

[0066] The pharmaceutical composition is not particularly limited thereto, and depending on the purpose, it can be administered through routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, pulmonary administration, rectal administration, etc. However, during oral administration, it can also be administered in a form that is not formulated, and since the active ingredient of the pharmaceutical composition can be denatured or decomposed by gastric acid, the oral composition may be coated with an active drug or administered orally in a form formulated to be protected from degradation in the stomach or in the form of an oral patch. Further, the composition can be administered by any device through which the active substance can move to the target cells.

[0067] According to one embodiment of the present invention, the peptide contained in the composition can suppress TGF-β signal transduction by binding to the TGF-β receptor or reducing the expression level or extracellular excretion amount of TGF-β, whereby TGF-β-related diseases can be effectively treated or prevented.

[0068] Still another aspect provides a pharmaceutical composition for treating or preventing cancer, which contains the peptide of the present invention, the polynucleotide of the present invention, or the expression vector of the present invention as an active ingredient. The contents regarding "TGF-β", "peptide", and "pharmaceutical composition" are as described above.

[0069] As used herein, the term "cancer" refers to a tumor that has abnormally grown due to autonomous overgrowth of body tissues, or a disease that forms a tumor. The cancer may be a cancer that expresses TGF-β, more preferably, a cancer characterized by excessive activation of TGF-β. Specifically, it may be liver cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, cancer near the anus, colon cancer, breast cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, but is not limited thereto.

[0070] Yet another aspect provides a method for treating or preventing cancer, comprising the step of administering to an individual a pharmaceutical composition for treating or preventing the cancer. The descriptions of the "cancer" and "pharmaceutical composition" are as described above.

[0071] As used herein, the term "individual" can include mammals such as mice, livestock, and humans in which a cancer disease has occurred or is at risk of occurring, birds, reptiles, cultured fish, etc. without limitation.

[0072] According to one embodiment of the present invention, the peptide contained in the composition can suppress TGF-β signaling, such as by binding to the TGF-β receptor or reducing the expression level or extracellular excretion amount of TGF-β. Therefore, since the composition can effectively suppress the TGF-β-related signaling pathway that plays an important role in the carcinogenesis process or the regulation of the cancer microenvironment, it can be used to treat or prevent cancer.

Advantages of the Invention

[0073] Since the novel peptide of the present invention can suppress the signal transduction pathway by TGF-β, it can effectively inhibit the carcinogenesis process such as TGF-β-related diseases, tumor growth and metastasis induced by TGF-β. Therefore, the composition containing the peptide has the merit of being able to effectively treat or prevent TGF-β-related diseases and thus cancer.

Brief Description of the Drawings

[0074]

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Modes for Carrying Out the Invention

[0075] One aspect provides the following novel peptide; a polynucleotide encoding the same; or an expression vector containing the polynucleotide.

[0076] In one embodiment of the present invention, the peptide may include the amino acid sequence of the following general formula 1 described in SEQ ID NO: 16.

[0077] [General formula 1] F-X 1 -L-G-P-X 2 -P-Y-I-W-X 3 -L-D-T

[0078] In the general formula 1, X 1 , X 2 and X 3 may each independently be cysteine (C) or serine (S), and specifically, X 1 -X 2 -X 3 may be C-C-S, C-C-C or S-S-S.

[0079] The peptide may additionally contain any amino acid or peptide. Specifically, the peptide may contain a peptide in which any amino acid or peptide is directly linked and added to one end, preferably the C-terminus, of the amino acid sequence represented by the general formula 1. The added amino acid or peptide may be alanine (A), phenylalanine (F), valine (V), leucine (L), serine (S), or a combination thereof. Non-limiting examples include alanine (A), phenylalanine (F), valine-leucine-serine-leucine (V-L-S-L) or valine-leucine-serine-phenylalanine (V-L-S-F).

[0080] The peptide may include one or more of the amino acid sequences of SEQ ID NOs: 1-8, and specifically, may consist of one of the amino acid sequences of SEQ ID NOs: 1-8.

[0081] In one embodiment of the present invention, the peptide may include the amino acid sequence of the following general formula 2 set forth in SEQ ID NO: 17.

[0082] [General formula 2] A-H-C-S-C-D

[0083] The peptide may additionally include any amino acid or peptide. Specifically, the peptide may include a peptide in which any amino acid or peptide is directly linked and added to one end, preferably the C-terminus, of the amino acid sequence represented by the general formula 1. The added amino acid or peptide may be serine (S), arginine (R), aspartic acid (D), asparagine (N), glycine (G), alanine (A), phenylalanine (F), or a combination thereof. Non-limiting examples may be serine-arginine-aspartic acid-asparagine (S-R-D-N) or glycine-alanine-phenylalanine-alanine (G-A-F-A).

[0084] The peptide may include one or more amino acid sequences of SEQ ID NOs: 9 and 10. Specifically, it may consist of one amino acid sequence of SEQ ID NOs: 9 and 10.

[0085] In one embodiment of the present invention, the peptide may include the amino acid sequence of the following general formula 3 set forth in SEQ ID NO: 18.

[0086] [General formula 3] T-I-V-Y-Y-V-X 4 -X 5 -K-P-K-V-E-Q

[0087] In the general formula 3, X 4 may be glycine (G), histidine (H), or valine (V), and X 5may be arginine (R), leucine (L), or isoleucine (I), and specifically, X 4 -X 5 may be G-R, G-L, H-I or V-I.

[0088] The peptide may contain one or more amino acid sequences among SEQ ID NOs: 11 to 14, and specifically, may consist of one amino acid sequence among SEQ ID NOs: 11 to 14.

[0089] Another aspect provides the use of the above peptide; the above polynucleotide; or the above expression vector; for the treatment or prevention of TGF-β related diseases, particularly cancers in which TGF-β is expressed.

[0090] Examples of the TGF-β related diseases include, but are not limited to, cancers, brain nerve diseases (e.g., Alzheimer's dementia and Huntington's disease), fibrotic skin diseases (e.g., scleroderma, CNS pathological scar tissue, skin scar formation, keloid scar formation and nerve scar formation), fibrotic diseases of the lung, liver and kidney (e.g., chronic hepatic fibrosis, acute liver injury, epileptic lung and renal fibrosis, and cirrhosis), cystic fibrosis, cardiac fibrosis, atherosclerosis and arteriosclerosis, systemic sclerosis, and ocular fibrosis.

[0091] The cancer may be, but is not limited to, liver cancer, lung cancer, pancreatic cancer, non-small cell lung cancer, colon cancer, bone cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, cancer near the anus, colon cancer, breast cancer, fallopian tube cancer, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma.

Examples

[0092] The following will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0093] Example 1: Design of TGF-β-signaling inhibitory peptide In order to develop the TGF-β-signaling inhibitory peptide of the present invention, 14 candidate amino acid sequences derived from TGF-β targeting the TGF-β receptor were designed and prepared. Specific information on the candidate sequences is described in Table 1 and FIG. 1 below.

[0094] [Table 1]

[0095] For the preparation of the above candidate peptides, the crystal structure of the protein (PDB: 3KFD) registered in the Protein Data Bank (http: / / www.rcsb.org) was used to predict the site that can bind to the TGF-β receptor 1 (TGFBR1). Based on this, specific peptides were designed, and after confirming the binding possibility by Autodock, a molecular modeling program, the predicted structure was generated. As a result, it was confirmed that in the case of the representative P3, P5, P3 / 5 hybrid, and P6 peptides, they can bind to the TGF-β receptor 1 (FIG. 2).

[0096] Based on the above results, it can be seen that the candidate peptides prepared in the present invention can suppress the signal transduction pathway induced by TGF-β by binding to the TGF-β receptor.

[0097] Example 2: Establishment of conditions for confirming the TGF-β-signaling inhibitory effect In order to establish the conditions for confirming the TGF-β-signaling inhibitory effect of the candidate peptides prepared in Example 1 above, the following experiments were conducted.

[0098] The TGF-β signaling pathway proceeds from the TGF-β receptor. When the TGF-β receptor is activated by TGF-β, the TGF-β signaling pathway is known to proceed through the process of phosphorylation of Smad2 / 3 proteins. Therefore, since the activation or inhibition of the TGF-β signaling pathway can be confirmed by changes in the expression level of phosphorylated-Smad2 / 3 (p-Smad2 / 3), the induction conditions of p-Smad2 / 3 by TGF-β treatment were confirmed.

[0099] Specifically, in order to analyze the expression of phosphorylated-Smad2 / 3 expressed from cells at each time point by Western blotting experiments, after treating pancreatic cancer cell lines Bxpc3 and Aspc1 with the TGF-β cytokine at 50 ng / ml, a cell lysis buffer containing a protease inhibitor (Thermo Scientific) was added to the cells obtained at each time point and lysed, and then sampled to prepare samples for Western blotting. The samples were electrophoresed on SDS-PAGE, transferred to a nitrocellulose membrane, and blocked using skim milk powder. Next, the membrane was incubated with a primary antibody against Smad2 / 3 or pSmad2 / 3 at 4°C for 1 day and washed, and then incubated with a horseradish peroxidase-conjugated secondary antibody that can bind to the primary antibody at room temperature for 2 hours. After that, it was visualized by chemiluminescence using Western ECL substrate (Thermo scientific), and the obtained luminescence image was analyzed with Chemidoc (biorad).

[0100] As a result, in the case of the Bxpc3 and Aspc1 cell lines, since it was confirmed that the expression level of pSmad2 / 3 was high 12 to 24 hours after treatment with TGF-β (Figure 3), in the following experiments, the expression level of pSmad2 / 3 24 hours after treatment with TGF-β was confirmed to check for the presence or absence of inhibition of the TGF-β signaling pathway.

[0101] Next, to confirm the expression level of TGF-β in pancreatic cancer cell lines, pancreatic cancer cell lines Bxpc3, Aspc1, Panc1, Capan2, and miapaca2, and the cancer cell line SNU213 derived from a patient were cultured in a 6-well plate at 1×10 6 / well for 48 hours, and then the concentration of the TGF-β cytokine in the culture medium was analyzed by the ELISA method.

[0102] As a result, it was confirmed that the TGF-β cytokine levels in BxPC3, AsPC1, Panc1, Capan2, Miapaca2, and SNU213 cells were shown (Figure 4). From this, it can be seen that the above cell lines can be used in the experiment for confirming the TGF-β signal transduction inhibitory effect by the mechanism of suppressing the expression level of TGF-β.

[0103] Next, to confirm the expression of the TGF-β receptor (TGFBR) to which TGF-β binds in pancreatic cancer cell lines, the expressions of TGFBR1, TGFBR2, and TGFBR3 receptors were analyzed by Western blotting experiments in pancreatic cancer cell lines BxPC3, AsPC1, and Miapaca2 cells. Specifically, after adding cell lysis buffer to each cell to lyse it, sampling was performed to prepare Western blotting samples, and the expressions of each receptor were compared using primary antibodies capable of detecting TGFBR1, TGFBR2, and TGFBR3 by the Western blotting method described in Example 2.

[0104] As a result, it was confirmed that the AsPC1 pancreatic cancer cell line generally showed high expression levels of TGFBR1, TGFBR2, and TGFBR3, and it was also confirmed that other cell lines expressed TGFBR (Figure 5). From this, it can be seen that the above cell lines can be used in the experiment for confirming the TGF-β signal transduction inhibitory effect by the mechanism of inhibiting the binding of TGF-β and the TGF-β receptor.

[0105] Example 3: Confirmation of the effect of the candidate peptide on suppressing the expression level of TGF-β The following experiment was conducted to confirm whether the candidate peptide prepared in Example 1 above could suppress the expression level of extracellular TGF-β.

[0106] Specifically, Aspc1, a pancreatic cancer cell line, was seeded in a 96-well plate at a concentration of 2×10 4 / well and cultured for 1 day. After that, TGF-β at 50 ng / ml alone or TGF-β + inhibitor (candidate peptide and control group peptide) was treated for 48 hours, and then the concentration of TGF-β cytokine in the cancer cell culture medium was analyzed by ELISA. Using a TGF-β detection ELISA kit, the concentration of extracellularly secreted TGF-β cytokine was measured with a multiplate reader and graphed by measuring the absorbance.

[0107] As a result, in the case of Aspc1 cells, it was confirmed that among the candidate peptides, peptides P5, P6, P7, and P14 directly inhibited the excretion amount of TGF-β cytokine itself (Figure 6). Based on the above results, it can be seen that the TGF-β inhibitory peptide of the present invention can inhibit the TGF-β signaling pathway by suppressing the expression level of TGF-β, specifically, the excretion amount of TGF-β itself.

[0108] Example 4: Confirmation of the TGF-β-signaling inhibitory effect of candidate peptides In order to confirm the inhibitory effect of the candidate peptides prepared in Example 1 on TGF-β signaling, the following experiment was conducted.

[0109] Specifically, Aspc1, Bxpc3, and Panc1 cell lines, which are pancreatic cancer cell lines, were seeded in a 6-well plate at a concentration of 1×10 6 / well and cultured for 1 day. The next day, TGF-β or TGF-β + candidate peptide was treated for 24 hours (control group: 15% ACN - candidate peptide solvent, DMSO - P144 peptide solvent, P144), and the expression levels of pSmad2 / 3 and total Smad2 / 3 were analyzed by the Western blotting method described in Example 2.

[0110] As a result, in the case of Aspc1 cells, it was confirmed that the peptide according to the present invention suppresses the expression level of pSmad2 / 3, and among them, it was particularly confirmed that the P6 peptide is the most excellent in the ability to suppress the expression level of pSmad2 / 3 (Figure 7). Also, in the case of Bxpc3 and Panc1 cell lines, it was confirmed that the peptide according to the present invention significantly suppresses the expression level of pSmad2 / 3 (Figures 8 and 9). Based on the above results, it can be seen that the TGF-β signal transduction inhibitory peptide prepared in the present invention can actually effectively suppress TGF-β signal transduction.

[0111] Example 5: Confirmation of cancer cell growth inhibition by candidate peptides In order to confirm the cancer cell growth inhibitory effect by suppressing TGF-β signal transduction of the candidate peptide prepared in Example 1 above, the following experiment was conducted.

[0112] Specifically, Aspc1, a pancreatic cancer cell line, was seeded in a 96-well plate at a concentration of 2×10 4 / well and cultured for 1 day. After that, after treating with TGF-β 50 ng / ml or TGF-β + inhibitor (candidate peptide, galunisertib or LY2109761) for 24 hours, the ratio of viable cells was measured with a multi-plate reader using the CCK-8 assay reagent (sigma) and graphed by measuring the absorbance. Galunisertib and LY2109761 were used at a concentration of 100 μm as well-known TGF-β inhibitors.

[0113] As a result, it was confirmed that when the peptide according to the present invention, which was confirmed to suppress TGF-β signal transduction prior to the cancer cell line, was treated, the growth ability of the cancer cells was suppressed (Figure 10).

[0114] Based on the above results, it can be seen that the TGF-β signal transduction inhibitory peptide prepared in the present invention can suppress the growth of cancer cells by the mechanism of suppressing TGF-β signal transduction.

[0115] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily transformed into other specific forms without changing the technical idea and essential features of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive.

Industrial Applicability

[0116] The present invention relates to a peptide capable of suppressing TGF-β signal transduction and its uses, and to a method for treating or preventing TGF-β signal transduction-related diseases, and thus cancers in which TGF-β is expressed.

[0117] [Sequence Listing Free Text] <110> Korea Institute of Science and Technology <120> Novel peptides capable of inhibiting TGF-beta signaling and uses thereof <130> OP21-013PCT <150> KR 10-2020-0182441 <151> 2020-12-23 <160> 18 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> P1 <400> 1 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Ala 1 5 10 15 <210> 2 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> P2 <400> 2 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Cys Leu Asp Thr 1 5 10 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> P3 <400> 3 Phe Ser Leu Gly Pro Ser Pro Tyr Ile Trp Ser Leu Asp Thr 1 5 10 <210> 4 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> P4 <400> 4 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Phe 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> P5 <400> 5 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Cys Leu Asp Thr Phe 1 5 10 15 <210> 6 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> P6 <400> 6 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Val Leu 1 5 10 15 Ser Leu <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> P7 <400> 7 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Cys Leu Asp Thr Val Leu 1 5 10 15 Ser Leu <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> P8 <400> 8 Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Cys Leu Asp Thr Val Leu 1 5 10 15 Ser Phe <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> P9 <400> 9 Ala His Cys Ser Cys Asp Ser Arg Asp Asn 1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> P10 <400> 10 Ala His Cys Ser Cys Asp Gly Ala Phe Ala 1 5 10 <210> 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> P11 <400> 11 Thr Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln 1 5 10 <210> 12 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> P12 <400> 12 Thr Ile Val Tyr Tyr Val Gly Leu Lys Pro Lys Val Glu Gln 1 5 10 <210> 13 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> P13 <400> 13 Thr Ile Val Tyr Tyr Val His Ile Lys Pro Lys Val Glu Gln 1 5 10 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> P14 <400> 14 Thr Ile Val Tyr Tyr Val Val Ile Lys Pro Lys Val Glu Gln 1 5 10 <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> P144 <400> 15 Thr Ser Leu Asp Ala Ser Ile Trp Ala Met Met Gln Asn Ala 1 5 10 <210> 16 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 <220> <221> MISC_FEATURE <222> (2) <223> Xaa is cysteine or serine <220> <221> MISC_FEATURE <222> (6) <223> Xaa is cysteine or serine <220> <221> MISC_FEATURE <222> (11) <223> Xaa is cysteine or serine <400> 16 Phe Xaa Leu Gly Pro Xaa Pro Tyr Ile Trp Xaa Leu Asp Thr 1 5 10 <210> 17 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 <400> 17 Ala His Cys Ser Cys Asp 1 5 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 <220> <221> MISC_FEATURE <222> (7) <223> Xaa is glycine, histidine or valine <220> <221> MISC_FEATURE <222> (8) <223> Xaa is arginine, leucine or isoleucine <400> 18 Thr Ile Val Tyr Tyr Val Xaa Xaa Lys Pro Lys Val Glu Gln 1 5 10

Claims

Claim 1 A peptide derived from transforming growth factor-beta (TGF-β) or an analog thereof, comprising the amino acid sequence of SEQ ID NO: 5, 6 or 7. Claim 2 The peptide according to claim 1, wherein the peptide suppresses TGF-β signal transduction. Claim 3 A polynucleotide encoding the peptide according to claim 1 or 2. Claim 4 An expression vector containing the polynucleotide according to claim 3. Claim 5 A pharmaceutical composition for treating or preventing TGF-β-related diseases, wherein the pharmaceutical composition comprises a pharmaceutically effective amount of the peptide according to claim 1 or 2, wherein the TGF-β-related diseases are cancer, neurological diseases, fibrotic skin diseases, pulmonary fibrosis, liver fibrosis, renal fibrosis, cystic fibrosis, cardiac fibrosis, atherosclerosis, arteriosclerosis, systemic sclerosis, or ocular fibrosis, the said pharmaceutical composition.

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