KR102386477B1 - Novel cell penetrating peptides and use thereof - Google Patents

Abstract

The present invention relates to a novel cell-penetrating peptide and a use thereof. More specifically, provided are: a cell-penetrating peptide consisting of an amino acid represented by general formula I; a polynucleotide encoding the cell-penetrating peptide; a complex comprising the cell-penetrating peptide and a biologically active substance; and a composition for delivering substances comprising the complex.

Description

Novel cell penetrating peptides and use thereof

It relates to a novel cell-penetrating peptide having excellent cell-penetrating ability and uses thereof.

In general, substances that are hydrophilic or have large molecular weights cannot enter the cell by the barrier called the cell membrane. The cell membrane prevents macromolecules such as peptides, proteins, and nucleic acids from entering the cell, and even if it enters the cell through a physiological mechanism called endocytosis by cell membrane receptors, it is fused with the lysosomal compartment of the cell. As it is eventually degraded, there are many restrictions in the treatment and prevention of diseases using the macromolecules. In addition, in the case of anticancer drugs, in order to deliver the drug into cells, obstacles such as multidrug resistance must be overcome. Accordingly, in order to prevent drug degradation, many methods have been proposed to directly deliver various macromolecules and drug carriers into cells without going through endocytosis. These methods include microinjection, electroporation, etc., which have the potential to damage cell membranes. Another method includes a method using a cell-permeable material. However, even if drugs are delivered into cells through this method, there is a problem in that they have to be moved to a specific organ in order to exert the drug effect.

Therefore, various drug delivery systems that can overcome the above limitations and increase stability and mass delivery efficiency have been researched and developed, and representative examples include liposomes and micelles. Liposomes are artificially made phospholipid carriers that can encapsulate both lipophilic and hydrophilic drugs, and since they are biocompatible materials, they are non-toxic and protect the drugs from the external environment. However, absorption is delayed, distribution is limited, metabolic rate is low, and it is captured by cells of the liver or spleen and is rapidly removed from the blood. Although micelles have the ability to increase drug solubility and bioavailability, many studies are still needed on the effects of substances on the movement of substances into cells and their basic medical and clinical applicability. Because of these limitations, there is a need for new agents that can effectively deliver biomaterials into the body, have no cytotoxicity, and do not enter through endocytosis in particular.

In this regard, cell penetrating peptides have been attracting attention as a new alternative. The cell-penetrating peptide is a kind of signal peptide, and is a peptide in which a specific amino acid sequence is combined for the purpose of delivering high molecular substances such as protein, DNA, RNA, etc. into cells. For example, since the 1990s, when the 11 amino acid sequence present in the TAT protein derived from the HIV virus showed that intracellular and intracellular delivery of the beta-galactosidase (120 kDa) protein was possible related research was conducted. Representative first-generation cells include Antennapedia (Penetratin) derived from Drosophila protein, VP22 derived from HSV-1 virus, and Pep-1 derived from Simian Virus 40 large antigen T. It is considered a penetrating peptide and has been widely used in conjunction with TAT. In addition, peptides in which several cationic amino acids, such as arginine and lysine, are repeatedly linked, such as poly arginine and poly lysine, have also been reported to have excellent cell permeability (Korean Patent Publication No. 10-2020- 0104524), being applied to various mass transfers. However, most of these cell-penetrating peptides contain the possibility of immunogenicity and toxicity, and are considered to have poor efficacy when delivered to human cells. Therefore, it is necessary to develop a cell-penetrating peptide that does not cause toxicity, has safety in vivo, and can effectively deliver substances.

As a result of research efforts to develop novel cell-permeable peptides having the above-described excellent effects, the present inventors designed and synthesized cell-permeable peptides in which amino acid sequences were variously modified based on the existing reference peptides, and their excellent cell-permeability was confirmed, thereby completing the present invention.

Accordingly, one aspect is to provide a novel cell-penetrating peptide.

Another aspect is to provide a complex comprising the cell-penetrating peptide and a biologically active substance.

Another aspect is to provide a composition for mass transfer comprising the complex, and a mass transfer method comprising the step of treating cells with the composition.

Another aspect is to provide a polynucleotide encoding the cell penetrating peptide.

Other objects and advantages of the present application will become more apparent from the following detailed description in conjunction with the appended claims and drawings. Content not described in this specification will be omitted because it can be sufficiently recognized and inferred by those skilled in the technical field or similar technical field of the present application.

One aspect provides a cell-penetrating peptide consisting of an amino acid sequence represented by the following [General Formula I].

[General Formula I]

X ₁ -[X _a -X _b -[EX _c -L]-X _d -X _e -DE-X _f -SV-X _g -X _h ]-X ₂

In Formula I, X _a is GH, AH, SH, or absent;

X _b is His(H) or Ala(A);

X _c is Arg(R) or Ala(A);

X _d is Lys(K) or Arg(R);

X _e is Ser(S), Cys(C), Ala(A), Leu(L), or Gin(Q);

X _f is Trp(W) or Ile(I);

X _g is Thr(T), Asn(N), Lys(K), or Arg(R);

X _h is SG, SV, SA, QG, or absent; and

X ₁ and X ₂ are absent or at least one or more is present, wherein X ₁ is ESKAVKWSAL and X ₂ can be G, GL, GLIES, GLIESESAET, or GLIECESAET.

One aspect relates to a cell-permeable peptide that can be usefully used in the field of basic research, diagnosis and treatment of various diseases, and the like, and relates to a basic platform peptide structure composed of various amino acids that can be expanded with an unlimited number of designs.

The term “amino acid” as used herein includes the 22 standard amino acids that are naturally incorporated into peptides, as well as D-isomeric and modified amino acids. Accordingly, the peptide may be a peptide containing D-amino acids. Meanwhile, in another aspect of the present invention, the peptide may include post-translational modified non-standard amino acids and the like. Examples of post-translational modifications include phosphorylation, glycosylation, acylation (including, e.g., acetylation, myristoylation, and palmitoylation), alkylation ), carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, change in chemical properties (eg, beta-elimination deimidation) , deamidation) and structural changes (eg, formation of disulfide bridges). In addition, changes in amino acids caused by chemical reactions occurring in the course of binding with crosslinkers to form a peptide conjugate, for example, changes in amino acids such as changes in amino groups, carboxy groups or side chains are included. Meanwhile, amino acid sequences used in the present specification are abbreviated according to the IUPAC-IUB nomenclature.

As used herein, the term "cell permeability" refers to the ability or property of a peptide to penetrate a cell (membrane) and penetrate into a cell.

As used herein, the term "peptide" refers to a polymer of amino acids, and a form in which a small number of amino acids are linked is usually called a peptide, and when many amino acids are linked, it is called a protein. In these peptide and protein structures, the linkage between amino acids consists of an amide bond or a peptide bond. A peptide bond is a bond between a carboxyl group (-COOH) and an amino group (-NH ₂ ) through which water (H ₂ O) escapes and forms -CO-NH-.

Therefore, as used herein, the term "cell penetrating peptide (CPP)" refers to a peptide capable of moving a cargo into a cell in vitro and/or in vivo . . As used herein, "cargo" includes all substances that can move into cells by binding to cell-penetrating peptides, for example, any substances that want to increase cell permeation efficiency, for example, through a general route. It may refer to a biologically active material that does not easily move into cells.

In one embodiment, the peptide may be composed of an amino acid sequence represented by the following [General Formula II].

[General Formula Ⅱ]

X _1a -[X _aa -X _ba -ERLK-X _ca -DEWSV-X _da -X _ea ]-X _2a

In Formula II, X _aa is AH or absent; X _ba is His(H) or Ala(A); X _ca is Ala(A), Ser(S), or Cys(C); X _da is Thr(T) or Arg(R); X _ea is SG, QG, or absent; and X _1a and X _2a are absent or at least one or more is present, wherein X _1a is ESKAVKWSAL and X _2a can be G, GL, GLIES, GLIESESAET, or GLIECESAET.

In one embodiment, the peptide may be composed of an amino acid sequence represented by the following [General Formula Ⅲ].

[General Formula Ⅲ]

X _1b -[GH-X _ab -[EX _bb -L]-X _cb -DE-X _db -SV-X _eb -X _fb ]-X _2b

In Formula III, X _ab is His(H) or Ala(A); X _bb is Arg(R) or Ala(A); X _cb is KS, KA, KQ, KL, KC, RS, or RC; X _db is Trp(W) or Ile(I); X _eb is Thr(T), Lys(K), or Arg(R); X _fb is SG, QG, or absent; and X _1b and X _2b are absent or at least one or more is present, wherein X _1b is ESKAVKWSAL, and X _2b may be G, GL, GLIES, GLIESESAET, or GLIECESAET.

In one embodiment, the peptide may be composed of an amino acid sequence represented by the following [General Formula IV].

[General Formula IV]

X _1c -[SHHERLK-X _ac -DEWSV-X _bc -X _cc ]-X _2c

In the general formula IV, X _ac is Ser(S) or Cys(C); X _bc is Asn(N) or Thr(T); X _cc is SG, SV, SA or absent; and X _1c and X _2c are absent or at least one or more is present, wherein X _1c is ESKAVKWSAL and X _2c can be G, GL, GLIES, GLIESESAET, or GLIECESAET.

In one embodiment, the novel peptide of [General Formula I] may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 162, for example, belonging to [General Formula II] It may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 23, for example, any one selected from the group consisting of SEQ ID NO: 24 to SEQ ID NO: 110 as belonging to [General Formula III] It may be an amino acid sequence of, or may be any one amino acid sequence selected from the group consisting of SEQ ID NO: 111 to SEQ ID NO: 162 as belonging to [General Formula IV], but is not limited thereto. In this case, the cell-penetrating peptide is 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 91%, 92%, 93 of the amino acid sequence shown in SEQ ID NOs: 1 to 162, respectively. %, 94%, 95%, 96%, 97%, 98%, 99% or more of an amino acid sequence having sequence homology.

In the present specification, the amino acid of the peptide can be changed, and such change refers to changing the physicochemical properties of the peptide. For example, amino acid changes can be performed to improve the thermostability of the peptide, alter substrate specificity, change the optimum pH, and the like.

Various amino acids capable of enhancing the effect as the cell-penetrating peptide may be additionally added or deleted to the N-terminus and C-terminus of the cell-penetrating peptide represented by the [General Formula I].

In addition, the peptide may have chemical stability, enhanced pharmacological properties (half-life, absorption, potency, potency, etc.), altered specificity (eg, broad spectrum of biological activity), reduced antigenicity, N- or C of the peptide -A protecting group may be attached to the terminal. In one embodiment, the N-terminus of the peptide is an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group group), a stearyl group, a butoxycarbonyl group, an aryloxycarbonyl group, and a polyethylene glycol (PEG) It may be bonded to any one protecting group selected from the group consisting of, ; And / or the C-terminus of the peptide is an amino group (amino group, -NH ₂ ), a tertiary alkyl group (tertiary alkyl group) and azide (azide, -NHNH ₂ ) to be bonded to any one protecting group selected from the group consisting of can In addition, the peptide may optionally further include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability.

As used herein, the term "stability" may refer to storage stability (eg, room temperature storage stability) as well as in vivo stability to protect the peptide from attack by proteolytic enzymes in vivo.

In one embodiment, the types of cells permeable to the cell-penetrating peptide include, but are not limited to, for example, brain-vascular barrier endothelial cells, cancer cells, blood cells, lymphocytes, and lymphocytes. ), immune cells, stem cells, induced pluripotent stem cells (iPSCs), neural stem cells (NSCs), T cells, B cells, natural killer cells (NK cells), vs. It may be any one selected from the group consisting of macrophages, neurons, glial cells, microglia, astrocytes, and muscle cells.

The peptide of the present invention can be manufactured so that the purity of each peptide is 90% or more through a conventional peptide synthesis method or manufacturing method known to those skilled in the art. . The peptide can be used by producing both D-form or L-form, a peptide composed of only a part of the D-form or L-form sequence, or a racemate form thereof through a conventional peptide synthesis method or manufacturing method known to those skilled in the art. can In addition, in order to increase the stability of the peptide, other conventional modifications known in the art are possible. In the present invention, the peptide was preferably synthesized using a solid state peptide synthesis method, but as described above, the peptide synthesis method and conditions are not limited thereto.

The present inventors have discovered cell-penetrating peptides having excellent cell-penetrating ability, and in order to additionally discover peptides having excellent cell-penetrating ability, modify or substitute amino acids at specific positions based on the sequence and structure of the discovered peptides, and their By modifying the length, various peptide candidate groups were designed and synthesized, and various cell-penetrating peptides having excellent cell-penetrating ability according to the present invention were discovered by analyzing their cell permeability.

More specifically, in this example, based on the peptide (reference peptide) consisting of 16 amino acids of No. 2020-0049621, the 4th, 6th, 9th, 10th, 12th, and 13th amino acids in the reference peptide With the essentially conserved peptide as a basic backbone, i) the first peptide candidate group in which the amino acids at the 1st and 2nd positions from the N-terminal are AH or absent based on the amino acid position of the reference peptide, ii) the amino acid position of the reference peptide A second peptide candidate group in which the amino acids at the 1st and 2nd positions from the N-terminus are GH based on It was classified into 3 peptide candidate groups.

Subsequently, in the first peptide candidate group, ① no additional substitution of amino acids based on the amino acid position of the reference peptide, or ② at least one or more amino acid substitutions at the 3rd, 8th, 14th, and 15th positions For peptides, a peptide candidate group with modified lengths was designed. Thereafter, as a result of in vitro cell permeability analysis, in the first peptide candidate group according to an embodiment, the 3rd, 8th, 14th, and 15th positions from the unsubstituted or N-terminus based on the amino acid position of the reference peptide It was found that even when the length of the peptide was changed along with the amino acid substitution in , the unique structure of the peptide exhibiting excellent cell permeability was maintained.

In addition, in the second peptide candidate group, based on the amino acid position of the reference peptide, ① 3 to 6 (amino acid substitution at the first region, 3rd or 5th position), ② 7 to 10 (second region, an amino acid substitution at the 7th and/or 8th position), or ③ a peptide comprising a substitution in the position region 11-16 (amino acid substitution at the 3rd region, 11th, 14th, or 15th position). For the target, a peptide candidate group with modified length was designed. Thereafter, as a result of in vitro cell permeability analysis, in the second peptide candidate group according to an embodiment, the 3rd, 5th, 7th, 8th, 11th, 14th from the N-terminus based on the amino acid position of the reference peptide It was found that even when the length of the peptide was changed along with the amino acid substitution at the th and 15th positions, the unique structure of the peptide exhibiting excellent cell permeability was maintained.

In addition, in the third peptide candidate group, ① no additional amino acid substitution is made based on the amino acid position of the reference peptide, ② contains one amino acid substitution at the 8th or 14th position, or ③ the 8th, 14th, and 16th positions A peptide candidate group whose length was modified was designed for peptides containing two amino acid substitutions at the 1st position, or 4 amino acid substitutions containing 3 amino acid substitutions at the 8th, 14th, and 16th positions. Thereafter, as a result of in vitro cell permeability analysis, in the third peptide candidate group according to an embodiment, unsubstituted based on the amino acid position of the reference peptide, or at the 8th, 14th, and 16th positions from the N-terminus It was found that even when the length of the peptide was changed along with the amino acid substitution of the peptide, the unique structure of the peptide exhibiting excellent cell permeability was maintained.

From the results of the above examples, it was found that a total of 162 types of peptides according to the present invention have excellent cell permeability, and based on this, it can be used as a carrier that can effectively introduce any substance bound to the peptide into cells.

Another aspect provides a complex comprising the cell penetrating peptide and a biologically active substance.

The complex includes all of a compound formed by simply mixing a peptide and a substance, a compound formed by mixing a peptide and a substance, or a compound formed by connecting or conjugating them by a chemical bond. In addition, the complex may be linked by a physical bond, a chemical bond, a covalent bond, a non-covalent bond, self-assembly, or may be linked in an integrated or fused form using a mediator.

In addition, the complex may be a complex formed by expressing the peptide and the biologically active material in a fusion state with each other. For example, when a gene expressing the peptide and a biologically active substance is inserted into one vector, and then an organism is transformed with the vector to express the gene inserted into the vector, the peptide and the biologically active substance are fusion proteins (fusion protein) can be expressed. When expressed as a fusion protein, any linker may be included between the peptide and the biologically active material.

In addition, in the complex, the cell-penetrating peptide may include both single or plural combined forms in order to efficiently deliver a biologically active substance into a cell, and the cell-penetrating peptide binds according to the biologically active substance to be delivered. The number can be easily selected or adjusted by those skilled in the art.

The biologically active substance capable of forming a complex by binding to the cell-penetrating peptide preferably means 'a substance having biological or pharmaceutical activity', which penetrates into cells (in the cytoplasm or nucleus) and is used to regulate physiological activity. It refers to a substance that has biological activity in various parts of the body, such as in cells, tissues, interstitial cells, blood, etc. that are involved or that can express pharmacological effects, or that need to be transported and acted upon. For example, but not limited to, a compound (chemical compound), protein, glycoprotein, peptide antibody (antibody), enzyme (enzyme), nuclease (nuclease), hormone, cytokine (cytokine), transcription factor (transcription factor) ), toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and fluorescence It may be at least one selected from the group consisting of dyes (fluorochromes).

The biologically active material is, for example, a chemical compound, protein, glycoprotein, peptide, antibody, enzyme, nuclease, hormone, cytokine, transcription factor (transcription) factor), toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, microparticles, nanoparticles, liposomes, viruses, quantum dots, and It may be at least one selected from the group consisting of fluorochromes. The compound is a broad concept that includes chemicals that can function as drugs, including natural or synthetic chemicals.

The nuclease is, for example, CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CASΦ, CAS variants, CxxC-finger protein-1 (Cfp1), Zinc-finger nucleases (ZEN) and transcription activator-TALEN- like effector nuclease) may be selected from the group consisting of, but is not limited thereto.

The nucleic acid is, for example, DNA, RNA, antisense oligonucleotide (ASO), microRNA (miRNA), small interfering RNA (siRNA), circular RNA (circular RNA), long noncoding RNA (long noncoding RNA) lncRNA), small activating RNA (saRNA), messenger RNA (MRNA), aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino ) may be selected from the group consisting of, and may additionally include decoy DNA, plasmid, shRNA, antisense RNA, oligoribonucleotide, or transfer RNA, but is not limited thereto.

The drug (drug) is a compound drug (chemical drug), bio drug (bio drug), nucleic acid drug (nucleic acid drug), peptide drug (peptide drug), protein drug (protein drug), natural product drug (natural product drug), It may be selected from the group consisting of a hormone, a contrast agent, and an antibody, but is not limited thereto.

As used herein, the term "contrast agent" is a broad concept including all substances used for imaging of structures or fluids in a living body in medical imaging. Suitable contrast agents include, but are not limited to, radiopaque contrast agents, paramagnetic contrast agents, superparamagnetic contrast agents, computed tomography (CT) contrast agents, and other contrast agents.

For example, radiopaque contrast agents (for X-ray imaging) include inorganic iodine compounds and organic iodine compounds (eg diatrizoart), radiopaque metals and salts thereof (eg silver, gold, platinum, etc.) ) and other radiopaque compounds (eg, calcium salts, barium salts such as barium sulfate, tantalum and tantalum oxide). Suitable paramagnetic contrast agents (for MR imaging) include gadolinium diethylene triaminepentaacetic acid (Gd-DTPA) and its derivatives, and other gadolinium, manganese, iron, dysprosium, copper, europium (europium), erbium, chromium, nickel and cobalt complexes such as 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid ( DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,-N',N"-triacetic acid (D03A), 1,4,7-triazacyclononane -N,N',N"-triacetic acid (NOTA), 1,4,8,10-tetraazacyclotetradecane-N,N',N",N"'-tetraacetic acid (TETA), hydroxybenzyl ethylene-diamine diacetic acid (HBED) and the like. Suitable superparamagnetic contrast agents (for MR imaging) include magnetite, super-paramagnetic iron oxide (SPIO), ultrasmall superparamagnetic iron oxide (USPIO) and monocrystalline iron oxide . Other suitable contrast agents are iodinated and non-iodinated, ionic and non-ionic CT contrast agents, and contrast agents such as spin-labels, or other diagnostically effective agents.

The "bio-drug" refers to various biopharmaceuticals such as (original) biologics, biogenerics, biobetters, and biosuperiors. The bio-drug refers to any drug manufactured, secreted, or semi-synthesized from a biological origin, and includes, but is not limited to, vaccines, blood products, antigens, cell products, gene therapy products, stem cells, and the like.

The nanoparticles may be selected from the group consisting of iron oxide, gold, carbon nanotubes, and magnetic beads, but is not limited thereto.

Another aspect provides a composition for mass delivery comprising the complex as an active ingredient.

The composition for mass delivery may be used to deliver a biologically active material to a living tissue or blood or to promote cell permeation. The composition may be delivered through cells constituting a living tissue or through cell-to-cell junctions, but there is no limitation on the delivery method.

The living tissue means one or more epithelial tissue, muscle tissue, nervous tissue, and connective tissue, and each organ may consist of one or more tissues, so mucosa, skin, brain, lung, liver, kidney, spleen, lung, heart, stomach , large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genitalia, muscle, blood, blood vessels, lymphatic vessels, lymph nodes, thymus, pancreas, adrenal gland, thyroid gland, parathyroid gland, larynx, tonsils, bronchi, alveoli However, it is not limited thereto.

When the complex is to be delivered to a specific cell, tissue or organ, the material having the biological activity is an extracellular partial protein of a ligand capable of selectively binding to a receptor specifically expressed in a specific cell, tissue or organ, or A complex can be formed by binding to a monoclonal antibody (mAb) capable of specifically binding to these receptors or ligands and a modified form. The binding between the peptide and the biologically active substance is by indirect linking by cloning technique using an expression vector at the nucleotide level, or by direct linking by chemical or physical covalent or non-covalent binding between the peptide and the biologically active substance. can do.

When the composition including the complex is used as a pharmaceutical composition, the composition may include the active ingredient in an amount of about 0.0001 to 50% by weight based on the total weight of the composition.

The composition may contain one or more active ingredients having the same or similar functions in addition to the active ingredients.

The composition may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and one or more of these components, and, if necessary, an antioxidant , buffers, bacteriostatic agents, and other conventional additives may be added. In addition, by additionally adding diluents, dispersants, surfactants, binders and lubricants, it can be formulated into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets, and can act specifically on target organs. A target organ-specific antibody or other ligand may be used in combination with the carrier. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's literature.

The composition comprising the complex as an active ingredient is intravenous, intraperitoneal, intramuscular, intrathecal, intracerebroventricular, subcutaneous, intradermal. , intranasal (nasal), intramucosal (mucosal), inhalation (inhalation), it can be delivered into the living body by injecting orally (oral). The dosage varies according to the subject's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions, or emulsions. Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays, but is not limited thereto.

Another aspect provides a method for delivering a substance into a cell, comprising the step of treating the cell with the composition for substance delivery.

Since the cell-penetrating peptide having a mass transfer function according to the present invention is a very small peptide, it is possible to minimize any biological interference with the active material that may occur.

Another aspect provides a polynucleotide encoding the peptide.

In one embodiment, the polynucleotide may be in the form of RNA or DNA, which includes cDNA and synthetic DNA. DNA can be single-stranded or double-stranded. If single-stranded, it may be the coding strand or the non-coding (antisense) strand, wherein the coding sequence encodes the same polypeptide, as a result of the degeneracy or redundancy of the genetic code.

The polynucleotide may also include a variant of the polynucleotide described herein, wherein the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide. can Allelic variants are alternate forms of a polynucleotide sequence that may have substitutions, deletions, or additions of one or more nucleotides that do not substantially alter the function of the polynucleotide being encoded (encoded). It is well known in the art that a single amino acid can be encoded by more than one nucleotide codon and that the polynucleotide can be readily modified to produce alternating polynucleotides encoding the same peptide.

Since the novel cell-penetrating peptide according to an aspect has excellent cell-penetrating ability, it can effectively deliver a biologically active substance into a living body, such as cells and tissues, so that it can be usefully used in the field of basic research, diagnosis and treatment of various diseases, etc. there is.

1 shows the cell permeability analysis results for the peptide candidate group (#1 to #4, and #6 to #11) in which only the length of the peptide was modified without amino acid substitution in the first peptide candidate group according to an embodiment.
2 is a peptide comprising at least one amino acid substitution at the 3rd, 8th, 14th, and 15th positions from the N-terminus based on the amino acid position of the reference peptide in the first peptide candidate group according to an embodiment; Shows the results of cell permeability analysis for the peptide candidate group (#5, and #12 to #23) in which the length of the was modified.
3 is a peptide candidate group in which the length of the peptide containing the amino acid substitution at the 3rd or 5th position from the N-terminus is modified based on the amino acid position of the reference peptide in the second peptide candidate group according to an embodiment (#24 to #38) shows the results of cell permeability analysis.
4 is a peptide candidate group in which the length of a peptide containing amino acid substitutions at the 7th and/or 8th positions from the N-terminus is modified based on the amino acid position of the reference peptide in the second peptide candidate group according to an embodiment ( It shows the results of cell permeability analysis for #39 to #49).
Figure 5a is a peptide candidate group in which the length of the peptide containing the amino acid substitution at the 8th position from the N-terminus is modified from the amino acid position of the reference peptide in the second peptide candidate group according to an embodiment (#50 to #60 ) shows the results of cell permeability analysis.
Figure 5b is a peptide candidate group (#61 to #70) in which the length of the peptide containing the amino acid substitution at the 8th position from the N-terminus is modified based on the amino acid position of the reference peptide in the second peptide candidate group according to an embodiment; ) shows the results of cell permeability analysis.
5c is a peptide candidate group (#71 to #82) in which the length of the peptide containing the amino acid substitution at the 8th position from the N-terminus is modified based on the amino acid position of the reference peptide in the second peptide candidate group according to an embodiment; ) shows the results of cell permeability analysis.
6 is a peptide candidate group (#83 to #91 in which the length of the peptide containing the amino acid substitution at the 11th position from the N-terminus is modified based on the amino acid position of the reference peptide in the second peptide candidate group according to an embodiment; ) shows the results of cell permeability analysis.
7 is a second peptide candidate group according to an embodiment, based on the amino acid position of the reference peptide, the length of the peptide comprising the amino acid substitution at the 14th position, or the 8th and 14th positions from the N-terminus is modified. It shows the results of cell permeability analysis for the peptide candidate group (#92 to #103).
8 is a second peptide candidate group according to an embodiment, the length of the peptide containing amino acid substitutions at the 15th position, or the 8th and 15th positions from the N-terminus based on the amino acid position of the reference peptide is modified. It shows the results of cell permeability analysis for the peptide candidate group (#104 to #110).
9 shows the cell permeability analysis results for the peptide candidate group (#111 to #119) in which only the length of the peptide was modified without amino acid substitution in the third peptide candidate group according to an embodiment.
10 is a peptide candidate group in which the length of the peptide containing the amino acid substitution at the 8th or 14th position from the N-terminus is modified based on the amino acid position of the reference peptide in the third peptide candidate group according to an embodiment (#129) , and #137 to #145) show the results of cell permeability analysis.
11 is a third peptide candidate group according to an embodiment, with respect to the amino acid position of the reference peptide, the length of the peptide comprising two amino acid substitutions at the 8th, 14th, and 16th positions from the N-terminus is modified. The results of cell permeability analysis for the selected peptide candidates (#120 to #128, #146 to #152, #154, and #162) are shown.
12 is a third peptide candidate group according to an embodiment, with respect to the amino acid position of the reference peptide, the length of the peptide comprising three amino acid substitutions at the 8th, 14th, and 16th positions from the N-terminus is modified. The results of cell permeability analysis for the selected peptide candidate groups (#130 to #136, #153, and #155 to #161) are shown.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example 1]

Example 1. Preparation of cell-penetrating peptide candidates

1-1. Cell-penetrating peptide candidate group design

In this example, cell-penetrating peptides having variously modified sequences and lengths were designed and synthesized using the Korean Patent Application No. 2020-0049621 peptide (GHHERLKSDEWSVTSG, hereinafter referred to as a reference peptide) as a basic backbone. Considering the sequence and structure of the reference peptide, the amino acid at the 4th position (E), the amino acid at the 6th position (L), the amino acid at the 9th position (D), the amino acid at the 10th position (E), the 12th position The amino acid (S) of and the amino acid (V) at the 13th position are not modified, by substituting or deleting the amino acids at the remaining positions and adding an amino acid sequence to their N- and/or C-terminus. A group of permeable peptide candidates was designed.

Together with this, in the reference peptide, the first peptide candidate group in which the amino acid at the 1st and 2nd positions is AH or absent, the second peptide candidate group in which the amino acid at the 1st and 2nd position is GH, and the first and the second amino acid at the 2nd position is SH Cell-permeable peptide candidate groups were designed and synthesized by classifying them into 3 peptide candidate groups.

1-2. Synthesis, separation and purification of peptide candidates

The present inventors used a solid state peptide synthesis (SPPS) method to synthesize each of the peptides described in Example 1-1. The method is an organic synthesis method in which the C-terminus of an amino acid whose N-terminus is protected with F-moc is joined to the N-terminus of the resin one by one. The solvent for all reactions was N,N -dimethylformamide ( N,N -dimethylformamide; DMF), and the amino acid coupling condition was 0.5M DIC ( N,N- Diisopropylcarbodiimide, DIC) 1 in an amino acid solution with a concentration of 2M. ml, 1 M Ethyl cyanohydroxyiminoacetate (Oxyma) was mixed with 0.5 ml and reacted in a microwave synthesizer. In addition, amino acids were prepared by varying the reaction time, temperature, or microwave voltage for each amino acid sequence. In order to synthesize the next amino acid, the F-moc of the previous amino acid must be removed. For this, deprotect the F-moc protecting group twice at 80 °C for 2 minutes using 80% DMF and 20% piperidine solution ( deprotecting). Between all the coupling processes and the deprotecting process, a process of washing three times with DMF and methylene chloride (dichloromethane, DCM) was alternately performed.

For the peptide synthesized through the above method, a fluorescent substance including a carboxyl group (-COOH) at the N-terminus of the peptide may be linked by a chemical bonding method in order to later observe and quantify cell permeability. In this case, the fluorescent material that can be used is 5-FAM (5-carboxyfluorescein), FITC (Fluorescein-5-isothiocyanate), cyanine 3 carboxylic acid, cyanine 5 carboxylic acid, cyanine 7 carboxylic acid ( Cyanine 7 carboxylic acid), and the like, and at least one of the above fluorescent materials may be used.

To this end, the present inventors first synthesized the last amino acid of the peptide synthesized on a solid resin, and then mixed well with 5-FAM: DIC: Oxyma: resin in a 2: 2.5: 4: 1 ratio, and then reacted in the resin. It proceeded at room temperature for 2 hours, but a magnetic stirrer was used. Next, when the color of the resin changed from yellow to dark yellow or orange during the synthesis process, the process of washing DMF and methylene chloride three times was alternately performed. Then, to separate the peptide/5-FAM from the solid resin, the synthesis was completed in a cleavage solution in which trifluoroacetic acid (TFA): triisopropylsilane (TIS): distilled water was mixed in a ratio of 95: 2.5: 2.5. After the resin was reacted for 2 hours using a stirrer, the resin was filtered out with an asbestos filter. From the filtered solution, the solution was evaporated under nitrogen gas, and if a precipitate was formed, it was precipitated with diethyl ether stored cold. The precipitated peptide/5-FAM was dried under vacuum and then dissolved in distilled water and freeze-dried.

The lyophilized peptide was dissolved in distilled water or acetonitrile (ACN), etc., and separated and purified using reverse phase high-performance liquid chromatography. At this time, solvent A (distilled water 99.9%, TFA 0.1%) and solvent B (distilled water 9.9%, acetonitrile 90%, TFA 0.1%) were used as mobile phase solvents of the HPLC. The HPLC mobile phase started with Solvent A 90% and Solvent B 10%, and the separation proceeded while increasing the solvent B to 1%/min gradient. Thereafter, the separated peptide solution was lyophilized to remove the solvent and then dissolved in a desired solvent to conduct the experiment.

Example 2. In vitro cell permeability assay

2-1. experimental method

In order to evaluate the cell permeability of the peptide candidates synthesized in Example 1, the present inventors performed in vitro permeability analysis for blood-brain barrier cells.

Specifically, hCMEC/D3 cells, which are human blood-brain barrier cells, are seeded in a 384-well plate, and in EGM (endothelial cell growth medium), an epithelial cell growth medium, 37° C. until the cells reach 80-90% of the plate area. , and incubated overnight under CO ₂ conditions. Then, the FAM-linked candidate peptides prepared in Example 1-2 and the negative control group FAM alone were diluted to 10 μM in the growth factor-free medium to prepare an amount to be treated by 10 μL per well. Subsequently, 10 μL of a solution prepared in advance by diluting the peptide in a plate in which 40 μL of cells were cultured was treated at a rate of 10 μL per well, and then incubated at 37° C. and CO ₂ conditions for 2 hours. Next, all the peptide-treated solutions from each well were removed with an automatic plate washer, washed 3 times with 1x PBS, and then treated with 4% paraformaldehyde at 75 μL per well, and the cells were fixed at room temperature for 15 minutes. did. After 15 minutes, washing with 1x PBS was repeated, and then 40 μL of Hoechst 33342 was diluted 1:5000 in 1x PBS and added to each well, treated at room temperature for 15 minutes, and then washed 3 times. Next, after focusing based on DAPI with Cytation 5 equipment, divide cells with a size of 20 μm centered on DAPI, select only cells of this size, perform image reading of FAM fluorescence, and process the image to mean -FAM value was obtained and a graph was made in which the FAM experimental group was corrected with a negative control group, and the blood-brain barrier cell permeability was calculated. Statistics were performed by ONE-WAY ANOVA analysis, and multiple comparisons were obtained using the FAM value as a control and corrected by the Bonferroni method (95% confidence) (mean±SEM, *p<0.1, **p<0.01, ***p<0.001, ****p<0.0001).

2-2. Cell permeability analysis for the first peptide candidate group

As designed in Example 1-1, in the first peptide candidate group, a peptide candidate group in which there was no additional amino acid substitution or whose length was modified for a peptide in which at least one amino acid was substituted was designed, and their cell The permeability was checked.

(1) Peptide candidate group in which only the length of the peptide is modified without amino acid substitution

In the first peptide candidate group in which the amino acids at the 1st and 2nd positions in the reference peptide were AH or absent, a peptide candidate group in which only the length of the peptide was modified without amino acid substitution was designed, and detailed sequence information is shown in Table 1 below. In Table 1, the bolded portion indicates a region corresponding to the reference peptide, and the underlined portion indicates a region in which amino acids are substituted or added.

number designation order SEQ ID NO: note One #One A HHERLKSDEWSVTSG G One 2 #2 A HHERLKSDEWSVTSG GL 2 3 #3 A HHERLKSDEWSVTSG GLIES 3 4 #4 A HHERLKSDEWSVTSG GLIESESAET 4 5 #6 ESKAVKWSAL A HHERLKSDEWSVTSG G 6 6 #7 ESKAVKWSAL A HHERLKSDEWSVTSG GL 7 7 #8 ESKAVKWSAL A HHERLKSDEWSVTSG GLIES 8 8 #9 ESKAVKWSAL A HHERLKSDEWSVTSG GLIESESAET 9 9 #10 ESKAVKWSAL A HHERLKSDEWSVTSG 10 10 #11 A HHERLKSDEWSVT 11 15, 16 fruit

As a result of confirming the cell permeability in the peptide candidate group, as shown in FIG. 1, the peptides of sequences #1 to #4, and #6 to #11 in Table 1 showed an excellent effect of influx into the blood-brain barrier cells. was confirmed to be indicated.

(2) Peptide candidate group in which the length of the peptide containing substitution of one or more amino acids is modified

In the first peptide candidate group in which the amino acid at the 1st and 2nd positions in the reference peptide is AH or absent, at least at the 3rd, 8th, 14th, and 15th positions from the N-terminus with respect to the amino acid position of the reference peptide A peptide candidate group in which the length of a peptide containing one or more amino acid substitutions was modified was designed, and detailed sequence information is shown in Table 2 below. In Table 2, the bolded portion indicates the region corresponding to the reference peptide, and the underlined portion indicates the region in which amino acids are substituted or added.

number designation order SEQ ID NO: note One #5 A HHERLK C DEWSVTSG GLIECESAET 5 2 #12 A ERLKSDEWSVTSG 12 1, 2 results 3 #13 A HHERLK A DEWSVT Q G G 13 4 #14 A HHERLK A DEWSVTQG GL 14 5 #15 A HHERLK A DEWSVT Q G GLIES 15 6 #16 A HHERLK A DEWSVT Q G GLIECESAET 16 7 #17 ESKAVKWSAL A HHERLK A DEWSVT Q G G 17 8 #18 ESKAVKWSAL A HHERLK A DEWSVT Q G GL 18 9 #19 ESKAVKWSAL A HHERLK A DEWSVT Q G GLIES 19 10 #20 ESKAVKWSAL A HHERLK A DEWSVT Q G GLIESESAET 20 11 #21 ESKAVKWSAL A HHERLK A DEWSVT Q G GLIECESAET 21 12 #22 ESKAVKWSAL A HHERLK A DEWSVT Q G 22 13 #23 A HHERLK A DEWSV R 23 15, 16 fruit

As a result of confirming the cell permeability in the peptide candidate group, as shown in FIG. 2, it was confirmed that the peptides having sequences corresponding to #5 and #12 to #23 in Table 2 exhibit an excellent influx effect into the blood-brain barrier cells. did

From the above results, in the first peptide candidate group according to an embodiment, with amino acid substitutions at the 3rd, 8th, 14th, and 15th positions from the N-terminus or unsubstituted based on the amino acid position of the reference peptide, Even when the length of the peptide was modified, it was found that the unique structure of the peptide exhibiting excellent cell permeability was maintained.

2-3. Cell permeability analysis for the second peptide candidate group

As designed in Example 1-1, in the second peptide candidate group, the peptide was divided into three regions, ie, 3 to 6 (first region) and 7 from the N-terminus based on the amino acid position of the reference peptide. Divided into ~10 (2nd region), 11 ~ 16 (3rd region) position region, and target peptides in which one or more amino acids are substituted for each region or in two or more regions, a peptide candidate group whose length is modified designed, and to check their cell permeability.

(1) Peptide candidate group in which the length of the peptide containing the first region substitution is modified

In the second peptide candidate group in which the amino acid at the 1st and 2nd positions in the reference peptide is GH, the amino acid substitution at the 3rd or 5th position from the N-terminus with respect to the first region, specifically, the amino acid position of the reference peptide A peptide candidate group in which the length of the containing peptide was modified was designed, and specific sequence information is shown in Table 3 below. In Table 3, bolded portions indicate regions corresponding to the reference peptide, and underlined portions indicate regions in which amino acids have been substituted or added.

number designation order SEQ ID NO: note One #24 ESKAVKWSAL GH A ERLKSDEWSVTSG G 24 2 #25 ESKAVKWSAL GH A ERLKSDEWSVTSG GL 25 3 #26 ESKAVKWSAL GH A ERLKSDEWSVTSG GLIES 26 4 #27 ESKAVKWSAL GH A ERLKSDEWSVTSG GLIESESAET 27 5 #28 ESKAVKWSAL GH A ERLKSDEWSVTSG 28 6 #29 GHH A LKSDEWSVTSG G 29 7 #30 GHH A LKSDEWSVTSG GL 30 8 #31 GHH A LKSDEWSVTSG GLIES 31 9 #32 GHH A LKSDEWSVTSG GLIESESAET 32 10 #33 ESKAVKWSAL GHH A LKSDEWSVTSG G 33 11 #34 ESKAVKWSAL GHH A LKSDEWSVTSG GL 34 12 #35 ESKAVKWSAL GHH A LKSDEWSVTSG GLIES 35 13 #36 ESKAVKWSAL GHH A LKSDEWSVTSG GLIESESAET 36 14 #37 ESKAVKWSAL GHH A LKSDEWSVTSG 37 15 #38 GHH A LKSDEWSVT 38 15, 16 fruit

As a result of confirming cell permeability in the peptide candidate group, as shown in FIG. 3 , it was confirmed that the peptides having sequences corresponding to #24 to #38 in Table 3 exhibited an excellent effect on influx into the blood-brain barrier cells.

(2) Peptide candidate group in which the length of the peptide containing the second region substitution is modified

In the second peptide candidate group in which the amino acid at the 1st and 2nd positions in the reference peptide is GH, the second region, specifically, the amino acid at the 7th and/or 8th position from the N-terminus with respect to the amino acid position of the reference peptide A peptide candidate group in which the length of the peptide containing the substitution was modified was designed, and specific sequence information is shown in Tables 4 and 5 below. In Tables 4 and 5, bolded portions indicate regions corresponding to the reference peptide, and underlined portions indicate regions in which amino acids have been substituted or added.

number designation order SEQ ID NO: note One #39 GHHERL R SDEWSVTSG G 39 2 #40 GHHERL R SDEWSVTSG GL 40 3 #41 GHHERL R SDEWSVTSG GLIES 41 4 #42 GHHERL RC DEWSVTSG GLIECESAET 42 5 #43 ESKAVKWSAL GHHERL R SDEWSVTSG G 43 6 #44 ESKAVKWSAL GHHERL R SDEWSVTSG GL 44 7 #45 ESKAVKWSAL GHHERL R SDEWSVTSG GLIES 45 8 #46 ESKAVKWSAL GHHERL R SDEWSVTSG GLIESESAET 46 9 #47 ESKAVKWSAL GHHERL RC DEWSVTSG GLIECESAET 47 10 #48 ESKAVKWSAL GHHERL R SDEWSVTSG 48 11 #49 GHHERL R SDEWSVT 49 15, 16 fruit

number designation order SEQ ID NO: note One #50 GHHERLK A DEWSVTSG G 50 2 #51 GHHERLK A DEWSVTSG GL 51 3 #52 GHHERLK A DEWSVTSG GLIES 52 4 #53 GHHERLK A DEWSVTSG GLIESESAET 53 5 #54 ESKAVKWSAL GHHERLK A DEWSVTSG G 54 6 #55 ESKAVKWSAL GHHERLK A DEWSVTSG GL 55 7 #56 ESKAVKWSAL GHHERLK A DEWSVTSG GLIES 56 8 #57 ESKAVKWSAL GHHERLK A DEWSVTSG GLIESESAET 57 9 #58 ESKAVKWSAL GHHERLK A DEWSVTSG GLIECESAET 58 10 #59 ESKAVKWSAL GHHERLK A DEWSVTSG 59 11 #60 GHHERLK A DEWSVT 60 15, 16 fruit 12 #61 GHHERLK L DEWSVTSG G 61 13 #62 GHHERLK L DEWSVTSG GL 62 14 #63 GHHERLK L DEWSVTSG GLIE 63 15 #64 GHHERLK L DEWSVTSG GLIESESAET 64 16 #65 ESKAVKWSAL GHHERLK L DEWSVTSG G 65 17 #66 ESKAVKWSAL GHHERLK L DEWSVTSG GL 66 18 #67 ESKAVKWSAL GHHERLK L DEWSVTSG GLIES 67 19 #68 ESKAVKWSAL GHHERLK L DEWSVTSG GLIESESAET 68 20 #69 ESKAVKWSAL GHHERLK L DEWSVTSG 69 21 #70 GHHERLK L DEWSVT 70 15, 16 fruit 22 #71 GHHERLK Q DEWSVTSG G 71 23 #72 GHHERLK Q DEWSVTSG GL 72 24 #73 GHHERLK Q DEWSVTSG GLIES 73 25 #74 GHHERLK Q DEWSVTSG GLIESESAET 74 26 #75 GHHERLK Q DEWSVTSG GLIECESAET 75 27 #76 ESKAVKWSAL GHHERLK Q DEWSVTSG G 76 28 #77 ESKAVKWSAL GHHERLK Q DEWSVTSG GL 77 29 #78 ESKAVKWSAL GHHERLK Q DEWSVTSG GLIES 78 30 #79 ESKAVKWSAL GHHERLK Q DEWSVTSG GLIESESAET 79 31 #80 ESKAVKWSAL GHHERLK Q DEWSVTSG GLIECESAET 80 32 #81 ESKAVKWSAL GHHERLK Q DEWSVTSG 81 33 #82 GHHERLK Q DEWSVT 82 15, 16 fruit

As a result of confirming cell permeability in the peptide candidate group, as shown in FIGS. 4 and 5A to 5C, the peptides of the sequences corresponding to #39 to #49 in Table 4 and #50 to #82 in Table 5 are excellent brain It was confirmed that the effect of influx into blood vessel barrier cells was exhibited.

(3) Peptide candidate group in which the length of the peptide containing the third region substitution is modified

In the second peptide candidate group in which the amino acid at the 1st and 2nd positions in the reference peptide is GH, the third region, specifically, at the 11th, 14th, or 15th position from the N-terminal based on the amino acid position of the reference peptide A peptide candidate group in which the length of the peptide containing the amino acid substitution was modified was designed, and together with this, a peptide candidate group combined with the amino acid substitution of the second region (8th position) was additionally set (#102, #109), and specific sequence information is shown in Table 6, Table 7, and Table 8 below. In Tables 6, 7, and 8, bolded portions indicate regions corresponding to the reference peptide, and underlined portions indicate regions in which amino acids have been substituted or added.

number designation order SEQ ID NO: note One #83 GHHERLKSDE I SVTSG G 83 2 #84 GHHERLKSDE I SVTSG GL 84 3 #85 GHHERLKSDE I SVTSG GLIES 85 4 #86 GHHERLKSDE I SVTSG GLIESESAET 86 5 #87 ESKAVKWSAL GHHERLKSDE I SVTSG G 87 6 #88 ESKAVKWSAL GHHERLKSDE I SVTSG GL 88 7 #89 ESKAVKWSAL GHHERLKSDE I SVTSG GLIES 89 8 #90 ESKAVKWSAL GHHERLKSDE I SVTSG GLIESESAET 90 9 #91 GHHERLKSDE I SVT 91 15, 16 fruit

number designation order SEQ ID NO: note One #92 GHHERLKSDEWSV K SG G 92 2 #93 ESKAVKWSAL GHHERLKSDEWSV K SG G 93 3 #94 ESKAVKWSAL GHHERLKSDEWSV K SG GL 94 4 #95 ESKAVKWSAL GHHERLKSDEWSV K SG GLIES 95 5 #96 ESKAVKWSAL GHHERLKSDEWSV K SG GLIESESAET 96 6 #97 ESKAVKWSAL GHHERLKSDEWSV K SG 97 7 #98 GHHERLKSDEWSV R SG G 98 8 #99 ESKAVKWSAL GHHERLKSDEWSV R SG G 99 9 #100 ESKAVKWSAL GHHERLKSDEWSV R SG GL 100 10 #101 ESKAVKWSAL GHHERLKSDEWSV R SG GLIES 101 11 #102 ESKAVKWSAL GHHERLK C DEWSV R SG GLIECESAET 102 8 substitutions 12 #103 ESKAVKWSAL GHHERLKSDEWSV R SG 103

number designation order SEQ ID NO: note One #104 GHHERLKSDEWSVT Q G G 104 2 #105 ESKAVKWSAL GHHERLKSDEWSVT Q G G 105 3 #106 ESKAVKWSAL GHHERLKSDEWSVT Q G GL 106 4 #107 ESKAVKWSAL GHHERLKSDEWSVT Q G GLIES 107 5 #108 ESKAVKWSAL GHHERLKSDEWSVT Q G GLIESESAET 108 6 #109 ESKAVKWSAL GHHERLK C DEWSVT Q G GLIECESAET 109 8 substitutions 7 #110 ESKAVKWSAL GHHERLKSDEWSVT Q G 110

As a result of confirming cell permeability in the peptide candidate group, as shown in FIGS. 6 to 8, #83 to #91 in Table 6, #92 to #103 in Table 7, and #104 to #110 in Table 8 It was confirmed that the peptide of the sequence of

From the above results, in the second peptide candidate group according to an embodiment, at the 3rd, 5th, 7th, 8th, 11th, 14th, and 15th positions from the N-terminal based on the amino acid position of the reference peptide It was found that even when the length of the peptide was changed along with the amino acid substitution of , the unique structure of the peptide exhibiting excellent cell permeability was maintained.

2-4. Cell permeability analysis for the third peptide candidate group

As designed in Example 1-1 above, in the third peptide candidate group, a peptide candidate group in which the length was modified for a peptide with no additional amino acid substitution or at least one amino acid substitution was designed, and their cell permeability was to check.

(1) Peptide candidate group in which only the length of the peptide is modified without substitution of amino acids

In the third peptide candidate group in which the amino acids at the 1st and 2nd positions in the reference peptide are SH, a peptide candidate group in which only the length of the peptide was modified without substitution of amino acids was designed, and detailed sequence information is shown in Table 9 below. In Table 9, the bolded portion indicates the region corresponding to the reference peptide, and the underlined portion indicates the region in which amino acids are substituted or added.

number designation order SEQ ID NO: note One #111 S HHERLKSDEWSVTSG G 111 2 #112 S HHERLKSDEWSVTSG GL 112 3 #113 S HHERLKSDEWSVTSG GLIES 113 4 #114 ESKAVKWSAL S HHERLKSDEWSVTSG G 114 5 #115 ESKAVKWSAL S HHERLKSDEWSVTSG GL 115 6 #116 ESKAVKWSAL S HHERLKSDEWSVTSG GLIES 116 7 #117 ESKAVKWSAL S HHERLKSDEWSVTSG GLIESESAET 117 8 #118 ESKAVKWSAL S HHERLKSDEWSVTSG 118 9 #119 S HHERLKSDEWSVT 119 15, 16 fruit

As a result of confirming cell permeability in the peptide candidate group, as shown in FIG. 9 , it was confirmed that the peptides having sequences corresponding to #111 to #119 in Table 9 exhibited an excellent effect on influx into the blood-brain barrier cells.

(2) Peptide candidate group in which the length of the peptide containing one amino acid substitution is modified

In the third candidate peptide group in which the amino acid at the 1st and 2nd positions in the reference peptide is SH, the length of the peptide containing the amino acid substitution at the 8th or 14th position from the N-terminus based on the amino acid position of the reference peptide is modified A group of peptide candidates was designed, and specific sequence information is shown in Table 10 below. In Table 10, bolded portions indicate regions corresponding to the reference peptide, and underlined portions indicate regions in which amino acids have been substituted or added.

number designation order SEQ ID NO: note One #129 S HHERLKSDEWSV N 129 15, 16 fruit 2 #137 S HHERLK C DEWSVTSG G 137 3 #138 S HHERLK C DEWSVTSG GL 138 4 #139 S HHERLK C DEWSVTSG GLIES 139 5 #140 ESKAVKWSAL S HHERLK C DEWSVTSG G 140 6 #141 ESKAVKWSAL S HHERLK C DEWSVTSG GL 141 7 #142 ESKAVKWSAL S HHERLK C DEWSVTSG GLIES 142 8 #143 ESKAVKWSAL S HHERLK C DEWSVTSG GLIESESAET 143 9 #144 ESKAVKWSAL S HHERLK C DEWSVTSG 144 10 #145 S HHERLK C DEWSVT 145 15, 16 fruit

As a result of confirming the cell permeability in the peptide candidate group, as shown in FIG. 10, it was confirmed that the peptides of the sequences corresponding to #129 and #137 to #145 in Table 10 showed excellent influx effect into the blood-brain barrier cells. did

(3) Peptide candidate group in which the length of the peptide containing two amino acid substitutions is modified

In the third peptide candidate group in which the amino acids at the 1st and 2nd positions in the reference peptide are SH, containing two amino acid substitutions at the 8th, 14th, and 16th positions from the N-terminus based on the amino acid position of the reference peptide A peptide candidate group with modified length of the peptide was designed, and specific sequence information is shown in Table 11 below. In Table 11, the bolded portion indicates a region corresponding to the reference peptide, and the underlined portion indicates a region in which amino acids are substituted or added.

number designation order SEQ ID NO: note One #120 S HHERLKSDEWSV N S V G 120 2 #121 S HHERLKSDEWSV N S V GL 121 3 #122 S HHERLKSDEWSV N S V GLIES 122 4 #123 S HHERLKSDEWSV N S V GLIESESAET 123 5 #124 ESKAVKWSAL S HHERLKSDEWSV N S V G 124 6 #125 ESKAVKWSAL S HHERLKSDEWSV N S V GL 125 7 #126 ESKAVKWSAL S HHERLKSDEWSV N S V GLIES 126 8 #127 ESKAVKWSAL S HHERLKSDEWSV N S V GLIESESAET 127 9 #128 ESKAVKWSAL S HHERLKSDEWSV N S V 128 10 #146 S HHERLKSDEWSV N S A G 146 11 #147 S HHERLKSDEWSV N S A GL 147 12 #148 S HHERLKSDEWSV N S A GLIES 148 13 #149 S HHERLKSDEWSV N S A GLIESESAET 149 14 #150 ESKAVKWSAL S HHERLKSDEWSV N S A G 150 15 #151 ESKAVKWSAL S HHERLKSDEWSV N S A GL 151 16 #152 ESKAVKWSAL S HHERLKSDEWSV N S A GLIES 152 17 #154 ESKAVKWSAL S HHERLKSDEWSV N S A 154 18 #162 S HHERLK C DEWSV N 162 15, 16 fruit

As a result of confirming cell permeability in the peptide candidate group, as shown in FIG. 11, peptides of sequences #120 to #128, #146 to #152, #154, and #162 in Table 11 have excellent brain-vascular barrier It was confirmed that the effect of influx into cells was exhibited.

(4) Peptide candidate group in which the length of the peptide containing three amino acid substitutions is modified

In the third peptide candidate group in which the amino acids at the 1st and 2nd positions in the reference peptide are SH, containing 3 amino acid substitutions at the 8th, 14th, and 16th positions from the N-terminus based on the amino acid position of the reference peptide A peptide candidate group with modified length of the peptide was designed, and specific sequence information is shown in Table 12 below. In Table 12, bolded portions indicate regions corresponding to the reference peptide, and underlined portions indicate regions in which amino acids have been substituted or added.

number designation order SEQ ID NO: note One #130 S HHERLK C DEWSV N S V G 130 2 #131 S HHERLK C DEWSV N S V GL 131 3 #132 ESKAVKWSAL S HHERLK C DEWSV N S V G 132 4 #133 ESKAVKWSAL S HHERLK C DEWSV N S V GL 133 5 #134 ESKAVKWSAL S HHERLK C DEWSV N S V GLIES 134 6 #135 ESKAVKWSAL S HHERLK C DEWSV N S V GLIESESAET 135 7 #136 ESKAVKWSAL S HHERLK C DEWSV N S V 136 8 #153 ESKAVKWSAL S HHERLK C DEWSV N S A GLIECESAET 153 9 #155 S HHERLK C DEWSV N S A G 155 10 #156 S HHERLK C DEWSV N S A GL 156 11 #157 ESKAVKWSAL S HHERLK C DEWSV N S A G 157 12 #158 ESKAVKWSAL S HHERLK C DEWSV N S A GL 158 13 #159 ESKAVKWSAL S HHERLK C DEWSV N S A GLIES 159 14 #160 ESKAVKWSAL S HHERLK C DEWSV N S A GLIESESAET 160 15 #161 ESKAVKWSAL S HHERLK C DEWSV N S A 161

As a result of confirming cell permeability in the peptide candidate group, as shown in FIG. 12, the peptides of the sequences #130 to #136, #153, and #155 to #161 of Table 12 showed excellent cerebrovascular barrier cells. It was confirmed that the inflow effect was shown.

From the above results, in the third peptide candidate group according to an embodiment, the peptide is unsubstituted based on the amino acid position of the reference peptide, or with amino acid substitutions at the 8th, 14th, and 16th positions from the N-terminus. It was found that even when the length of the peptide was changed, the unique structure of the peptide showing excellent cell permeability was maintained.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> IMNEWRUN BIOSCIENCES, INC. <120> Novel cell penetrating peptides and use thereof <130> PN139683KR <160> 162 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #1 <400 > 1 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 2 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #2 <400 > 2 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #3 < 400> 3 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 4 <211> 26 <212> PRT <213> Artificial Sequence <220> < 223> #4 <400> 4 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 5 <211> 26 <212 > PRT <213> Artificial Sequence <220> <223> #5 <400> 5 Ala His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Cys Glu Ser Ala Glu Thr 20 25 <210> 6 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #6 <400> 6 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 7 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #7 <400> 7 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 8 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #8 <400> 8 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 9 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #9 <400> 9 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 10 <211> 26 <212> PRT <2 13> Artificial Sequence <220> <223> #10 <400> 10 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210 > 11 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #11 <400> 11 Ala His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr 1 5 10 <210> 12 <211 > 14 <212> PRT <213> Artificial Sequence <220> <223> #12 <400> 12 Ala Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 <210> 13 <211> 17 <212 > PRT <213> Artificial Sequence <220> <223> #13 <400> 13 Ala His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Gln Gly 1 5 10 15 Gly <210> 14 <211> 18 <212 > PRT <213> Artificial Sequence <220> <223> #14 <400> 14 Ala His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Gln Gly 1 5 10 15 Gly Leu <210> 15 <211> 21 < 212> PRT <213> Artificial Sequence <220> <223> #15 <400> 15 Ala His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Gln Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 16 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #16 <400> 16 Ala His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Gln Gly 1 5 10 15 Gly Leu Ile Glu Cys Glu Ser Ala Glu Thr 20 25 <210> 17 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #17 <400> 17 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Gln Gly Gly 20 25 <210> 18 <211> 28 <212> PRT <213> Artificial Sequence < 220> <223> #18 <400> 18 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Gln Gly Gly Leu 20 25 <210> 19 < 211> 31 <212> PRT <213> Artificial Sequence <220> <223> #19 <400> 19 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Gln Gly Gly Leu Ile Glu Ser 20 25 30 <210> 20 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #20 <400> 20 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Gln Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 21 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #21 <400> 21 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Gln Gly Gly Leu Ile Glu Cys Glu 20 25 30 Ser Ala Glu Thr 35 <210> 22 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #22 <400> 22 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ala His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Gln Gly 20 25 <210> 23 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #23 <400> 23 Ala His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Arg 1 5 10 <210> 24 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #24 <400 > 24 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Ala Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 25 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #25 <400> 25 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Ala Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 26 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #26 <400> 26 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Ala Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 27 <211> 36 <212> PRT <213 > Artificial Sequence <220> <223> #27 <400> 27 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Ala Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 28 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #28 <400> 28 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Ala Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 29 <211> 17 <212> PRT <213> Artificial Sequ ence <220> <223> #29 <400> 29 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 30 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #30 <400> 30 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 31 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #31 <400> 31 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 32 <211> 26 <212 > PRT <213> Artificial Sequence <220> <223> #32 <400> 32 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 33 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #33 <400> 33 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Ala Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 34 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #34 <400> 34 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Ala Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 35 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #35 <400> 35 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Ala Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 36 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #36 <400> 36 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Ala Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 37 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #37 <400 > 37 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Ala Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 38 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #38 <400> 38 Gly His His Glu Ala Leu Lys Ser Asp Glu Trp Ser Val Thr 1 5 1 0 <210> 39 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #39 <400> 39 Gly His His Glu Arg Leu Arg Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 40 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #40 <400> 40 Gly His His Glu Arg Leu Arg Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 41 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #41 <400> 41 Gly His His Glu Arg Leu Arg Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 42 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #42 <400> 42 Gly His His Glu Arg Leu Arg Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Cys Glu Ser Ala Glu Thr 20 25 <210> 43 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #43 <400> 43 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Arg Ser Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 44 <211> 28 <212> PRT <213 > Artificial Sequence <220> <223> #44 <400> 44 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Arg Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 < 210> 45 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #45 <400> 45 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Arg Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 46 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #46 <400> 46 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Glu Arg Leu 1 5 10 15 Arg Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 47 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #47 <400> 47 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Arg Cys Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Cys Glu 20 25 30 Ser Ala Glu Thr 35 <210> 48 <211> 26 <212> PRT <213> Artificial Sequence <22 0> <223> #48 <400> 48 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Arg Ser Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 49 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #49 <400> 49 Gly His His Glu Arg Leu Arg Ser Asp Glu Trp Ser Val Thr 1 5 10 <210> 50 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #50 <400> 50 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 51 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #51 <400> 51 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 52 <211> 21 <212 > PRT <213> Artificial Sequence <220> <223> #52 <400> 52 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 53 < 211> 26 <212> PRT <213> Artificial Sequence <220> <223> #53 <400> 53 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 54 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #54 <400> 54 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 55 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #55 <400> 55 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 56 <211> 31 <212 > PRT <213> Artificial Sequence <220> <223> #56 <400> 56 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 57 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #57 <400> 57 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 58 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #58 <400> 58 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Cys Glu 20 25 30 Ser Ala Glu Thr 35 <210> 59 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #59 <400> 59 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ala Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 60 <211> 14 <212> PRT <213> Artificial Sequence <220 > <223> #60 <400> 60 Gly His His Glu Arg Leu Lys Ala Asp Glu Trp Ser Val Thr 1 5 10 <210> 61 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #61 <400> 61 Gly His His Glu Arg Leu Lys Leu Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 62 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #62 <400> 62 Gly His His Glu Arg Leu Lys Leu Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 63 <211> 20 <212> PRT <213 > Artificial Sequence <220> <223> #63 <400> 63 Gly His His Glu Arg Leu Lys Leu Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu 20 <210> 64 <211> 26 <212 > PRT <213> Artificial Sequence <220> <223> #64 <400> 64 Gly His His Glu Arg Leu Lys Leu Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 65 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #65 <400> 65 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Leu Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 66 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #66 <400> 66 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Leu Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 67 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> # 67 <400> 67 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Leu Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 68 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #68 <400> 68 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Leu Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 69 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #69 <400> 69 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Leu Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 70 < 211> 14 <212> PRT <213> Artificial Sequence <220> <223> #70 <400> 70 Gly His His Glu Arg Leu Lys Leu Asp Glu Trp Ser Val Thr 1 5 10 <210> 71 <211> 17 < 212> PRT <213> Artificial Sequence <220> <223> #71 <400> 71 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 72 <211> 18 < 212> PRT <213> Artificial Sequence <220> <223> #72 <400> 72 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 73 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #73 <400> 73 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 74 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #74 <400> 74 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 75 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #75 <400> 75 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Cys Glu Ser Ala Glu Thr 20 25 <210> 76 <211> 27 <212> PRT <213> Artificial Sequence <220 > <223> #76 <400> 76 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Gln Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 77 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #77 <400> 77 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Gln Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 78 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #78 <400> 78 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Gln Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 79 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #79 <400> 79 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Gln Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 80 <211> 36 < 212> PRT <213> Artificial Sequence <220> <223> #80 <400> 80 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Gln Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Cys Glu 20 25 30 Ser Ala Glu Thr 35 <210> 81 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #81 <400> 81 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Gln Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 82 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #82 <400> 82 Gly His His Glu Arg Leu Lys Gln Asp Glu Trp Ser Val Thr 1 5 10 <210> 83 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #83 <400> 83 Gly His His Glu Arg Leu Lys Ser Asp Glu Ile Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 84 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #84 <400> 84 Gly His His Glu Arg Leu Lys Ser Asp Glu Ile Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 85 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #85 <400> 85 Gly His His Glu Arg Leu Lys Ser Asp Glu Ile Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 86 <211> 26 <212 > PRT <213> Artificial Sequence <220> <223> #86 <400> 86 Gly His His Glu Arg Leu Lys Ser Asp Glu Ile Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 87 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #87 <400> 87 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Ile Ser Val Thr Ser Gly Gly 20 25 <210> 88 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #88 <400> 88 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Ile Ser Val Thr Ser Gly Gly Leu 20 25 <210> 89 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #89 <400> 89 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Ile Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 90 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #90 <400> 90 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Ile Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 91 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #91 < 400> 91 Gly His His Glu Arg Leu Lys Ser Asp Glu Ile Ser Val Thr 1 5 10 <210> 92 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #92 <400> 92 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Lys Ser Gly 1 5 10 15 Gly <210> 93 <211> 27 <212> PRT <213> A rtificial Sequence <220> <223> #93 <400> 93 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Lys Ser Gly Gly 20 25 <210> 94 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #94 <400> 94 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Lys Ser Gly Gly Leu 20 25 <210> 95 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #95 <400> 95 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Lys Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 96 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #96 <400> 96 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Lys Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 97 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #97 <400> 97 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Lys Ser Gly 20 25 <210> 98 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> # 98 <400> 98 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Arg Ser Gly 1 5 10 15 Gly <210> 99 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> # 99 <400> 99 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Arg Ser Gly Gly 20 25 <210> 100 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #100 <400> 100 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Arg Ser Gly Gly Leu 20 25 <210> 101 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #101 <400> 101 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Arg Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 102 <211> 36 <212> PRT <213> Art ificial Sequence <220> <223> #102 <400> 102 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Arg Ser Gly Gly Leu Ile Glu Cys Glu 20 25 30 Ser Ala Glu Thr 35 <210> 103 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #103 <400> 103 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Arg Ser Gly 20 25 <210> 104 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #104 <400> 104 Gly His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Gln Gly 1 5 10 15 Gly <210> 105 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #105 <400> 105 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Gln Gly Gly 20 25 <210> 106 <211> 28 <212> PRT <213> Artificial Sequence <220 > <223> #106 <400> 106 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Gln Gly Gly Leu 20 25 <210> 107 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #107 <400> 107 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Gln Gly Gly Leu Ile Glu Ser 20 25 30 <210> 108 <211> 36 <212> PRT <213> Artificial Sequence < 220> <223> #108 <400> 108 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Gln Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 109 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #109 <400> 109 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Gln Gly Gly Leu Ile Glu Cys Glu 20 25 30 Ser Ala Glu Thr 35 <210> 110 <211> 26 <212> PRT <213> Artificial Sequence <220> <223 > #110 <400> 110 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Gly His His Glu Arg Leu 1 5 10 15 Lys Ser A sp Glu Trp Ser Val Thr Gln Gly 20 25 <210> 111 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #111 <400> 111 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 112 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #112 <400> 112 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 113 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #113 <400> 113 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 114 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #114 <400> 114 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 115 <211> 28 <212> PRT <213> Artificial Sequence <220> <223 > #115 <400> 115 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 116 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #116 <400> 116 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 117 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #117 < 400> 117 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 118 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #118 <400> 118 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 119 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #119 <400> 119 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Thr 1 5 10 <210> 120 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #120 <400> 120 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 Gly <210> 121 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #121 <400> 121 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 Gly Leu <210> 122 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #122 <400> 122 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 123 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #123 <400 > 123 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 124 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #124 <400> 124 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Val Gly 20 25 <210> 125 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #125 <400> 125 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Val Gly Leu 20 25 <210> 126 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #126 <400> 126 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Val Gly Leu Ile Glu Ser 20 25 30 <210> 127 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #127 <400> 127 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Val Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 128 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #128 <400> 128 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Val 20 25 <210> 129 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> # 129 <400> 129 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn 1 5 10 <210> 130 <211> 1 7 <212> PRT <213> Artificial Sequence <220> <223> #130 <400> 130 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 Gly <210> 131 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #131 <400> 131 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val 1 5 10 15 Gly Leu <210> 132 <211 > 27 <212> PRT <213> Artificial Sequence <220> <223> #132 <400> 132 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly 20 25 <210> 133 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #133 <400> 133 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Leu 20 25 <210> 134 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #134 <400> 134 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Leu Ile Glu Ser 20 25 30 <210> 135 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #135 <400> 135 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 136 <211> 26 <212> PRT <213> Artificial Sequence <220> <223 > #136 <400> 136 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Val 20 25 <210> 137 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #137 <400> 137 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly <210> 138 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #138 <400> 138 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu <210> 139 <211> 21 <212 > PRT <213> Artificial Sequence <220> <223> #139 <400> 139 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 140 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #140 <400> 140 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Ser Gly Gly 20 25 <210> 141 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #141 <400> 141 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Ser Gly Gly Leu 20 25 <210> 142 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #142 <400> 142 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser 20 25 30 <210> 143 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #143 <400> 143 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Ser Gly Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 144 <211 > 26 <212> PRT <213> Artificial Sequence <220> <223> #144 <400> 144 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Thr Ser Gly 20 25 <210> 145 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> #145 <400> 145 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Thr 1 5 10 <210> 146 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #146 <400> 146 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 Gly <210> 147 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #147 <400> 147 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 Gly Leu <210> 148 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> #148 <400> 148 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 Gly Leu Ile Glu Ser 20 <210> 149 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #149 <400> 149 Ser His His Glu Arg Leu Lys Ser Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 Gly Leu Ile Glu Ser Glu Ser Ala Glu Thr 20 25 <210> 150 <211> 27 <212> PRT <213> Artificial Sequence <220> < 223> #150 <400> 150 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Ala Gly 20 25 <210> 151 <211> 28 < 212> PRT <213> Artificial Sequence <220> <223> #151 <400> 151 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Ala Gly Leu 20 25 <210> 152 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> #152 <400> 152 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Ala Gly Leu Ile Glu Ser 20 25 30 <210> 153 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #153 <400> 153 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser V al Asn Ser Ala Gly Leu Ile Glu Cys Glu 20 25 30 Ser Ala Glu Thr 35 <210> 154 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #154 <400> 154 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Ser Asp Glu Trp Ser Val Asn Ser Ala 20 25 <210> 155 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> #155 <400> 155 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 Gly <210> 156 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> #156 <400> 156 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Ala 1 5 10 15 Gly Leu <210> 157 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> #157 <400> 157 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Ala Gly 20 25 <210> 158 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> #158 <400> 158 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Ala Gly Leu 20 25 <210> 159 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> # 159 <400> 159 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Se r Ala Gly Leu Ile Glu Ser 20 25 30 <210> 160 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> #160 <400> 160 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Ala Gly Leu Ile Glu Ser Glu 20 25 30 Ser Ala Glu Thr 35 <210> 161 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> #161 <400> 161 Glu Ser Lys Ala Val Lys Trp Ser Ala Leu Ser His His Glu Arg Leu 1 5 10 15 Lys Cys Asp Glu Trp Ser Val Asn Ser Ala 20 25 <210> 162 <211 > 14 <212> PRT <213> Artificial Sequence <220> <223> #162<400> 162 Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val Asn 1 5 10

Claims (14)

A cell-penetrating peptide consisting of an amino acid represented by the following general formula I:
[General formula Ⅰ]
X ₁ -X _a -X _b -EX _c -LX _d -X _e -DE-X _f -SV-X _g -X _h -X ₂
In Formula I, X _a is GH, AH, SH, or absent;
X _b is His(H) or Ala(A);
X _c is Arg(R) or Ala(A);
X _d is Lys(K) or Arg(R);
X _e is Ser(S), Cys(C), Ala(A), Leu(L), or Gin(Q);
X _f is Trp(W) or Ile(I);
X _g is Thr(T), Asn(N), Lys(K), or Arg(R);
X _h is SG, SV, SA, QG, or absent; and
X ₁ and X ₂ are absent or at least one or more is present, wherein X ₁ is ESKAVKWSAL and X ₂ is G, GL, GLIES, GLIESESAET, or GLIECESAET;
The cell-permeable peptide is characterized in that it consists of any one amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 162, the cell-permeable peptide.
delete delete delete delete The method according to claim 1, wherein the cells are brain blood barrier endothelial cells (brain endothelial cells), cancer cells, blood cells (blood cells), lymphocytes (lymphocyte), immune cells, stem cells, induced pluripotent stem cells (induced pluripotent stem cell; iPSC) ), neural stem cells (NSC), T cells, B cells, natural killer cells (NK cells), macrophages, neurons, glial cells, microscopic Cell-penetrating peptide, characterized in that it is selected from the group consisting of glia cells (microglia), astrocytes (astrocyte) and muscle cells (muscle cell).
A complex comprising the cell-permeable peptide of claim 1 or 6 and a biologically active substance bound to a terminus of the cell-permeable peptide.
The method according to claim 7, wherein the biologically active material is a chemical compound, protein, glycoprotein, peptide, antibody, enzyme, nuclease, hormone, cytokine, transcription factor (transcription factor), toxins, nucleic acids, carbohydrates, lipids, glycolipids, natural products, semi-synthetic drugs, drugs, liposomes, viruses, quantum dots, and fluorochromes ), characterized in that at least one selected from the group consisting of, the complex.
The method according to claim 8, wherein the nuclease is CAS9 (CRISPR associated protein 9), CAS12, CAS13, CAS14, CASΦ, CxxC-finger protein-1 (CxxC-finger protein-1), ZEN (Zinc-finger nucleases) and TALEN (Transcription activator-like) effector nuclease), characterized in that selected from the group consisting of, the complex.
The method according to claim 8, wherein the nucleic acid is DNA, RNA, antisense oligonucleotide (ASO), microRNA (miRNA), small interfering RNA (siRNA), circular RNA (circular RNA), long non-coding RNA (long noncoding RNA; lncRNA), small activating RNA (saRNA), messenger RNA (MRNA), aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino (morpholino), characterized in that selected from the group consisting of, the complex.
A composition for mass transfer comprising the complex of claim 7.
A material delivery method comprising the step of treating cells of a mammal other than a human with the composition of claim 11 .
The method according to claim 12, wherein the cells are brain endothelial cells, cancer cells, blood cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells (induced pluripotent stem cell; iPSC) ), neural stem cells (NSC), T cells, B cells, natural killer cells (NK cells), macrophages, neurons, glial cells, microscopic A material delivery method, characterized in that it is selected from the group consisting of glia cells (microglia), astrocytes (astrocyte) and muscle cells (muscle cell).
A polynucleotide encoding the peptide according to claim 1 .

Images