Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

• the present invention relates to a peptide having cell membrane penetrating activity, a transmembrane carrier comprising the peptide having cell membrane penetrating activity as an effective component, a transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance, a transfection kit comprising the peptide having cell membrane penetrating activity and the target substance, use of the peptide having cell membrane penetrating activity for the manufacture of a transmembrane complex, use of the transmembrane complex for the manufacture of a medicament, and a method for delivering a target substance into cell interior which comprises administrating to a subject with a transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance to induce transduction of the transmenbrane complex into cell interior.
• In vitro methods include electroporation, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, incubation with calcium-phosphate-DNA precipitate, DEAE-dextran mediated transfection, infection with modified viral nucleic acids, and direct micro-injection into single cells. But such methods are of extremely limited usefulness for delivery of proteins.
• Folic acid has been used as a transport moiety (C. P. Leamon and Low, 1991). However, these methods have not proved to be highly reliable or generally useful. Recently to introduce macromolecules such as a protein into a cell interior, gene therapy becomes in the limelight but this have also problems in that targeting is incorrect. As a alternative, research on protein transduction or protein therapy is actively progressed.
• Protein transduction domain was first reported that purified human immunodeficiency virus type-1 ("HIV") TAT protein is taken up from the surrounding medium by adding it to human cells growing in culture medium(Green et al., 1988, Frankel et al, 1988). After this report, drosophila homeotic transcription factor, antennapedia(Antp)(Joliot et al., 1991) and herpes simplex virus- 1 DNA-binding protein, VP22(Elliot et al,, 1997) were also identified.
• HAV human immunodeficiency virus type-1
• PTDs protein transduction domains
• recombinant expression vector was developed by using a peptide containing 1 1 amino acids of TAT 47-57 and TAT fusion proteins were prepared by linking the TAT peptide to other peptides or proteins and so introduction of full-length protein into intracellular compartment became possible without the limitation of size or function (Nagahara et al., 1988).
• PTDs can be linked with other peptide or proteins to form fusion protein and then be transduced into cell interior, there are many attempts to transduce therapeutic drug, peptide, protein etc. into cell interior using PTDs.
• PTDs which do not contain lots of basic amino acid residues. Also, it has been reported that PTDs penetrate phosphoelipid bilayer of cell membrane by helix conformation.
• TCTP tumor protein
• HRF histamine-releasing factor
• TCTP had been known as tumor-specific protein until 1980' and the synthesis thereof was assumed to be related to proliferative stage of tumor.
• TCTP was reported as a tumor protein of 2I kDa, p21 in mouse erythroleukemia cell line (Chitpatima et al., 1988). Also, it was revealed that p23, relating to cell growth in Ehrlich ascites tumor is the same as TCTP/HRF (Bohm et al, 1989).
• TCTP is frequently found in tumor cell, particularly growing vigorously, and exists in cytoplasm. It is a known protein consisting of 172 amino acids (NCBI accession #P 13693 (Homo sapiens)) and shows high homology between species. 45 amino acids at its C-terminal form basic domain. Because such domain has about 46% homology with MAP- I B, microtubule-associated protein, it was also assumed that HRF is a microtubule-associated protein. Gachet, et al. (1997) observed that HRF is distributed consistently along with the cytoskeletal network to some extent using confocal microscope, which suggests that HRF binds to the cytoskeleton.
• TCTP expression is characterized by that mRNA is maintained in regular level, but in case that exterior stimulus such as serum exists, it is transformed to polysome to be translated. According to the characteristic, it was named as 'Translationally Controlled Tumor Protein(TCTP)' (Thomas et al., 1981 ; Thomas and Thomas., 1986). It was also reported that TCTP mRNA is suppressed during translation, but when it receives cell division signal, it is activated and translated to protein (Thomas and Thomas, 1986).
• TCTP/HRF is considered as a histamine releasing material interacting with basophil or mast cell and related to allergic inflammatory response.
• TCTP/HRF can pass through cell membrane. Since the amino acid sequence of TCTP/HRF has no the part consisting of plenty of basic amino acids, arginine or lysine, which is a characteristic of representative PTDs, and no similar amino acid sequences to those of other PTDs, the present inventors considered TCTP has a domain which is different to other known PTDs in aspect of the protein structures.
• N- and C-terminus get loose and exposed and middle part forms a spherical shape.
• the present inventors made a constant effort for looking for PTD in TCTP and, as a result, isolated protein transduction domain composed of very different amino acids in comparison with well-known PTDs. On the basis of this result, the present inventors have established the present invention by confirming that this domain shows remarkably high cell penetrating activity than well-known PTDs.
• This invention provides a peptide having cell membrane penetrating activity, composed of the following amino acid sequence:
• Rl may be deleted or one amino acid selected from M, A, Q, C, F, L or W, R2 may be deleted or one amino acid selected from I or A, R3 may be one amino acid selected from I or A, R4 may be one amino acid selected from Y, A, F, S or R, R5 may be one amino acid selected from R, A or K, R6 may be one amino acid selected from D, A, I or R, R7 may be deleted or one amino acid selected from L, K, A, E or R, R8 may be deleted or one amino acid selected from I, K or A, R9 may be deleted or one amino acid selected from A, S, E, Y or T, RlO may be deleted or one amino acid selected from A, H, K or E, and if RlO is K or H, the amino acid(s) selected from K, KK, R, RR or HH may be added thereto.
• amino acid sequence may be SEQ ID No.: 1.
• the amino acid sequence may be SEQ ID Nos.: 2-7.
• the amino acid sequence may be an amino acid sequence which one amino acid of SEQ ID No.: 2 is substituted with alanine.
• the above amino acid sequence may be, for example, an amino acid sequence selected from SEQ ID Nos.: 8-16, particularly SEQ ID No.: 13.
• the amino acid sequence may be an amino acid sequence selected from SEQ ID Nos.: 20-54.
• the above sequence may be, for example, an amino acid sequence selected from SEQ ID Nos.: 22, 26, 27 or SEQ ID Nos.: 31 -54.
• 'cell membrane penetrating protein domain means protein sequence having penetrating activity into cell interior (cytoplasm, nucleus) across plasma membrane.
• a peptide having cell membrane penetrating activity of the present invention is a novel cell membrane penetrating protein domain that has no similarity in sequences with well-known TAT, VP22 and Antp PTDs(Protein Transduction Domains).
• the present invention provides a peptide having cell membrane penetrating activity consisting of the amino acid sequence of SEQ ID No.: 1.
• the present invention also provides a peptide having cell membrane penetrating activity consisting of one amino acid sequence selected from SEQ ID Nos.: 2-7.
• the peptide having cell membrane penetrating activity consisting of the amino acid sequence of SEQ ID No.: 1, 2, 3 or 4 shows excellent cell penetrating activity in comparison with conventional TAT, and intracellular penetrating efficiency shows a rapidly increasing mode when treatment concentration becomes high and incubation time becomes long.
• cell penetrating activity of TCTP(I -I O) show over 3 times activity when treated for 15 minutes in 50 ⁇ M and 6 times activity when treated for 15 minutes in 100 ⁇ M, compared to that of TAT.
• cell penetrating activity at concentration of 50 ⁇ M and 100 ⁇ M of TCTP(I-IO) were higher than those of TAT about 29 times and 30 times, respectively.
• cell penetrating activity showed an increased fashion in the incubation time of 2 hours.
• a peptide comprising amino acid residues of TCTP(I -9)(SEQ ID No.: 2), TCTP(1-8)(SEQ ID No.: 3) or TCTP(2-10)(SEQ ID No.: 4) showed more excellent penetrating activity than well-known TAT(47-58) peptide.
• cell penetrating activity was excellent in the order ofTCTP(l-10)(SEQ ID No.: 1), TCTP(1 -9)(SEQ ID No.: 2), TCTP(1-8)(SEQ ID No.: 3) and TCTP(2-10)(SEQ ID No.: 4), and when 1 st amino acid of TCTP was existing, cell penetrating activity was more excellent.
• Length of the peptides may vary within the scope of, preferably, 9-15 residues, and more preferably, 9-10 residues.
• a peptide having cell membrane penetrating activity of the present invention may be prepared by artificial synthesis or by isolating the sequence of TCTP(I -I O)(SEQ ID No.: 1), TCTP(1-9)(SEQ ID No.: 2), TCTP(1-8)(SEQ ID No.: 3) or TCTP(2-10)(SEQ ID No.: 4) from TCTP.
• Synthesis of the peptide of the present invention may be performed, for example, by using an instrument or by using genetic engineering.
• synthesis can be performed by using Fmoc solid-phase method on automatic peptide synthesizer (PeptrEX-R48, Peptron). After purifying the synthesized peptide from resin, the peptide can be purified and analyzed by reverse-phase HPLC (Prominence LC-20AB, Shimadzu, Japan) with Shiseido capcell pak Cl 8 analytic RP column. After synthesis is completed, the peptide can be identified by a mass spectrometer (HP 1 100 Series LC/MSD, Hewlett-Packard, Roseville, USA).
• nucleic acid sequences corresponding to a desired peptide can be introduced into recombinant vector for protein expression, then the expression of peptide coding region can be induced by IPTG in E.coli bacteria like a BL21( ⁇ DE3) or BL21 ( ⁇ DE3)pLys, that is deficient in proteases, and the peptide can be purified.
• the present invention also provides a peptide having cell membrane penetrating activity, composed of the amino acid sequence of SEQ ID Nos.: 8-16.
• alanine-substituent of 6th residue, aspartic acid (SEQ ID No.: 13) showed 2.5 times increased penetrating activity than WT(wild type) peptide at a low concentration of lO ⁇ M and alanine-substituents of 5th and 7-9th residue(R, L, I, S)(SEQ ID Nos.: 12, 14-16) showed a little decreased but still showed activity.
• a peptide having cell membrane penetrating activity of the present invention comprises the peptide consisting of one amino acid sequence selected from SEQ ID Nos.: 8-16.
• the present invention also provides a peptides having cell membrane penetrating activity, consisting of one amino acid sequence selected from SEQ ID No.: 22, 26, 27, or 31 -54.
• the peptides of SEQ ID Nos.: 20-30 were prepared by deletion, substitution or addition of one or more amino acids in SEQ ID No.: 1.
• the peptides consisting of SEQ ID No.: 22, 26 or 27 showed better penetrating activity than TAT(I OO ⁇ M).
• the peptides of SEQ ID Nos.: 31 -45 were synthesized repeatedly and these all peptides showed better penetrating activity than TAT in lO ⁇ M.
• the peptides of SEQ ID Nos.: 46-54 were prepared as various mutant forms of SEQ ID No.: 1 , then measured for cell penetrating activity.
• a peptide having cell membrane penetrating activity of the present invention comprises the peptides consisting of SEQ ID Nos.: 22, 26, 27 and 31-54.
• Length of the peptides may vary within the scope of, preferably 5-15 residues, and more preferably 8-10 residues.
• the peptide of the present invention may be prepared by artificial synthesis or by isolating the sequence of TCTP(I -1 O)(SEQ ID No.: 1), TCTP(I -9)(SEQ ID No.: 2), TCTP(I -8)(SEQ ID No.: 3) or TCTP(2-10)(SEQ ID No.: 4) and modifying these sequences.
• Synthesis of the peptides may be prepared by same synthesis methods as described above.
• the present invention also provides a transmembrane carrier comprising the peptide having cell membrane penetrating activity as an effective component.
• the peptide having cell membrane penetrating activity provides a use as a transmembrane carrier for penetrating target substance across plasma membrane.
• the present invention provides a transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance.
• target substance' of the present invention means a molecule that may be related to a regulation of physiological activity, made a pharmacological action or maintained a biological activity in intracellular compartment.
• Target substance of the present invention may comprise nucleic acid including DNA and RNA, chemical compound such as drug, carbohydrate, lipid or glycolipid etc. as non-protein range molecule, and enzyme, regulation factor, growth factor, antibody, cytoskeletal factor etc. as protein range molecule.
• a peptide having cell membrane penetrating activity of the present invention may be linked to one or more target substances by physically/chemically covalent bond or non-covalent bond, or by mediators in incorporated or fused forms.
• the target substance is a non-protein range molecule
• a peptide having cell membrane penetrating activity of the present invention may be linked to the target substance by covalent bond, then the complex may be exposed to target cell group.
• the target substances may be non-covalently linked to a peptide having cell membrane penetrating activity of the present invention.
• the target substance is a nucleic acid
• it may be incorporated with a peptide having cell membrane penetrating activity of the present invention, in forms of lipid based vehicle, then exposed to target cell group.
• fusion protein incorporated with a peptide having cell membrane penetrating activity of the present invention can be prepared by obtaining cDNA of the protein(the target substance) through PCR and cloning cDNA using vectors. If it is impossible, the protein may be fused chemically.
• fusion protein can be prepared by connecting the target substance to linker, then reacting with the peptide having cell membrane penetrating activity to form linkage.
• cell penetrating complex of the present invention may be prepared as follows.
• recombinant expression vector is prepared to generate a fusion gene encoding a peptide having cell membrane penetrating activity-target substances conjugate.
• Nucleic acids encoding above fusion protein include the nucleic acid sequence encoding a peptide having cell membrane penetrating activity and the nucleic acid sequence encoding a protein as target substance.
• these nucleic acid sequences may comprise sequences consisting of SEQ ID Nos.: 17-18 or 55-81.
• Nucleic acid sequences of SEQ ID Nos.: 17-18 or 55-81 are as follows.
• nucleic acid sequences encoding the peptide of the present invention include all nucleic acid sequence encoding the peptide of the present invention besides nucleic acid sequences listed in above table.
• Recombinant expression vector of the present invention may include conventional promoter for expression, termination factor, selection marker, reporter gene, tag sequence, restriction enzyme recognitions site, multi-cloning site and so on.
• Transfection methods to host using recombinant expression vector of the present invention may be a heat shock or electroporation etc. which is known in the art.
• fusion proteins which consist of a peptide having cell membrane penetrating activity and a protein as target substance, may be purified by conventional methods known in the art.
• the present invention provides a transfection kit comprising the peptide having cell membrane penetrating activity and the target substance.
• Transfection kits are optimized systems to introduce easily DNA/RNA to intracellular compartment of mammalian cell. There are up to now calcium-phosphate method, methods using lipid complex or dextran complex, but limitations are that efficiency of these methods is 1/10 6 - 1/10 2 and depend on cell type. To overcome these limitations, transfection kits using the peptide having cell membrane penetrating activity, may be utilized.
• the transfection kit of the present invention may further comprise a binding factor combining the peptide with the target substance.
• the binding factor means specific DNA/RNA sequences including transcriptional factor, virus protein, or whole body or a part of protein that are capable to bind to target substance.
• Gal4 is a DNA binding factor.
• Gal4 is a transcriptional factor widely used in eukaryote, prokaryote and virus.
• DNA/RNA binding factors may be used by vector expressing PTDs and fusion proteins in vivo and vitro. Also, incorporation between DNA/RNA binding factors and PTDs may be accomplished by chemical interaction, physical interaction or noncovalent interaction.
• fusion complexes between a peptide having cell membrane penetrating activity of the present invention and DNA/RNA are treated outside the cells, it can be overcome both efficiency and limitation depending on the cell type.
• a peptide having cell membrane penetrating activity of the present invention and DNA/RNA binding factors it is capable that DNA/RNA is introduced into cytoplasm and nucleus of various cells in vivo and in vitro.
• introduction method can be accomplished by various route including intramuscular, intraperitoneal, intravenous, oral, subcutaneous, intracutaneous, intranasal introduction and inhalation.
• target substance may include one or more biological regulation substances selected from a group consisting of protein, lipid, carbohydrate or chemical and transfection kits of the present invention can introduce above target substance into cytoplasm and nucleus of various cells in vivo and in vitro. Fusion between PTD and target substance can be accomplished by chemical, physical covalent interacation or noncovalent interaction.
• Transfection kit of the present invention provides new technology about gene therapy and DNA/RNA vaccine according to the methods of the present invention and can express transiently or permanently and be used in clinical applications such as gene therapy and DNA/RNA vaccine as well as basic research.
• the present invention provides a use of the peptide having cell membrane penetrating activity for the manufacture of a transmembrane complex and a method for preparing transmembrane complexes by combining target substance with the peptide having cell membrane penetrating activity.
• the present invention provides a use of the transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance for the manufacture of a medicament and a method for manufacturing a medicament which comprises mixing the transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance, with a pharmaceutically acceptable carrier.
• the pharmaceutically acceptable carrier is well known to a skilled artisan, and the skilled artisan can select and use the pharmaceutically acceptable carrier which is proper for introduction to a living body.
• the present invention provides a method for delivering a target substance into cell interior which comprises administrating to a subject with a transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance to induce transduction of the transmenbrane complex into cell interior.
• the target substance is non-protein range molecule, it may be covalently attached to the peptide having cell membrane penetrating activity of the present invention, and the complex may be exposed to target cell group.
• the target substance may be non-covalently attached to the peptide having cell membrane penetrating activity of the present invention, for example, if the target substance is a nucleic acid, the complex may be exposed to target cell group in forms of lipid based vehicle incorporated with the peptide having cell membrane penetrating activity of the present invention.
• the 'subject' may be mammal including human.
• the transmembrane complex can be administrated by various route including intramuscular, intraperitoneal, intravenous, oral, subcutaneous, intracutaneous, mucosal administration and inhalation.
• Dose of the transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance is variable according to a therapeutically effective amount of the target substance and penetrating activity of the peptide, and so it is not limited to a specific dose. Only, for example, if the target substance is a nucleic acid, the dose of target substance may be 10 ⁇ 1000 ⁇ g/kg and the dose of the peptide of the present invention may be 0.1mg-10mg/kg.
• the present invention provides a method for treating related diseases by administrating to a subject with the transmembrane complex consisting of the peptide having cell membrane penetrating activity combined with a target substance thereby introducing the target substance into a cell.
• the kind of the disease desired to treatment may be varied depending on the target substance intended to administrate into cell interior.
• the 'subject' may be mammal including human.
• the transmembrane complex can be administrated by various route including intramuscular, intraperitoneal, intravenous, oral, subcutaneous, intracutaneous, mucosal administration and inhalation.
• the present invention provides a nucleic acid sequence encoding the peptide having cell membrane penetrating activity.
• the present invention provides a nucleic acid encoding the peptide having cell membrane penetrating activity, consisting of an amino acid sequences selected from SEQ ID No.: 1 , 2, 22, 26, 27 or 31-54.
• the nucleic acid may be DNA or RNA of single chain or double chain and be prepared by synthesizing artificially or isolating from organism-derived TCTP genes.
• the nucleic acids encoding the peptides consisting of SEQ ID Nos.: 1 , 2, 22, 26, 27 or 31 -54 represent the nucleic acid sequences of SEQ ID Nos.: 17-18, or 55-81 , respectively.
• nucleic acid sequences encoding the peptide of the present invention include all nucleic acid sequences encoding the peptide of the present invention, and are not limited to the nucleic acid sequences listed in above table.
• sequence encoding alanine in amino acid sequence may be gca, gcc, gcg or get.
• the peptide of the present invention having cell membrane penetrating activity has a prominent effect in delivery as compared with TAT-derived peptide.
• the peptide having cell membrane penetrating activity of the present invention, the transmembrane complex consisting of the peptide combined with a target substance, and the method for delivering a target substance into a cell using the transmembrane complex has applications on intracellular delivery in various research fields as well as on therapeutics of specific diseases where targeting of drugs is required at high efficiency.
• the peptide having cell membrane penetrating activity of the present invention the transmembrane complex consisting of the peptide combined with a target substance, and the method for delivering a target substance into a cell using the transmembrane complex is very useful as drug delivery systems.
• the peptide having cell membrane penetrating activity of the present invention has a prominent penetrating efficiency as compared with the activities of prior TAT-derived peptides and so the peptide has applications on intracellular delivery in various research fields as well as on therapeutics of specific diseases where targeting of drugs is required high efficiently. Accordingly, the peptide having cell membrane penetrating activity of the present invention, the transmembrane complex consisting of the peptide combined with a target substance, and the method for delivering a target substance into a cell using the transmembrane complex is very useful as drug delivery systems.
• Fig. I a and Fig. Ic are schematic diagrams showing various deletion forms of TCTP of the present invention
• Fig. Ib and Fig. Id are the western blot analysis results for cellular uptake of the various deletion forms of TCTP of Fig. I a and Fig. Ic in BEAS-2B cell line.
• Fig.2 shows a dose dependent cellular uptake after 15 minutes of treatment of TCTP-derived peptides and Fig.3 shows cellular uptake after 2 hours of treatment of TCTP-derived peptides at various concentrations in HeLa cell line.
• Fig.4 shows fluorescence microscope images representing cellular uptake after 2 hours of treatment of the TCTP-derived peptides at various concentrations in HeLa cell line.
• Fig.5 shows cellular uptakes after 2 hours of treatment of substituents of TCTP-derived peptide at various concentrations at the sensitivity of 75 and Fig.6 shows same result of Fig.5 at the sensitivity of 100.
• Figs.7, 8 and 9 shows mean fluorescence intensity showing a cellular uptake of mutant peptides of TCTP-derived peptides treated for 2 hours at various concentrations using FACS.
• Figs.10, 1 1 and 12 shows cytotoxicity of mutant peptides of TCTP-derived peptides treated for 24 or 48 hours at a various concentrations.
• pRSET vector that is capable of tagging 6 histidine was employed. Subcloning with DNA sequences corresponding to each deletion forms of TCTP was performed in the multicloning site of the vector. Then, the recombinant expression vector was introduced into E.coli BL21 (DE3)(Novagen) or BL21(DE3)pLysS (Novagen). The expression of the deletion forms of TCTP was induced by IPTG (isopropyl ⁇ -D-thiogalactoside) for 3 hours, then the protein was isolated and purified by using Ni column which binds to polyhistidine.
• IPTG isopropyl ⁇ -D-thiogalactoside
• BEAS-2B cell was treated with the deletion form of TCTP at the concentration of 15ug/ml for 1 hour or 24 hours. Then, supernatants and cell lysates were obtained and western blotted with anti-TCTP antibodies (Fig. Ib).
• N-terminus containing TCTP proteins of present invention can be transferred into cell interior for a short time, only several minutes to several tens minutes.
• Example 1 in order to confirm that the N-terminus of TCTP can function as a PTD, the peptides consisting of N-terminus of TCTP were constructed and examined for cell penetrating efficiency. 1) Synthesis of Various Peptides corresponding N-terminus amino acid of TCTP
• TCTP-derived peptides and control peptide, TAT 48-57 were synthesized as follow.
• N-terminus of each peptides was labeled with fluorescence dye, rhodamine and C-terminus was protected. Peptide purity (>95%) was determined by HPLC. Syntersis of the peptides was requested to PEPTRON, Inc. Negative control was a fluorescence dye, rhodamine (Molecular Probe) used to label in all peptides.
• HeLa cell line was propagated in DMEM (GIBCO) supplemented with 10% FBS (GIBCO) and 100 units/mL penicillin-streptomycin. Cells were grown in a 5% CO 2 incubator at 37 ° C .
• HeLa cells were cultured in 48-well plate until they were 70 ⁇ 80% grown up before a day of the experiment.
• the cells were washed with DMEM of 37 " C twice, and TCTP-derived peptides synthesized in Example 2-1) were treated to the culture medium in a dose dependent manner (0, 1 , 5, 10, 50, 100 ⁇ M), then the cells was incubated for 15 minutes or 2 hours in an CO 2 incubator at 37 ° C .
• the cells were washed in cool PBS three times and immediately measured by a microplate fluorescence reader (BIO-TEK instruments, Inc., Vermont, USA) at emission 530 nm and excitation 590 nm for a measurement of rhodamine of intracellular uptake marker.
• the sensitivity of reader was set at 100 as a basic mode, but was lowered to 75 if the fluorescent signals were too strong. All experiments were conducted in triplet repeats for reproducibility (Fig. 2 and Fig. 3).
• control peptide was transduced into cell in a dose and time-dependent manner as previously known.
• TCTP (1-10), (1-9), (1-8) peptides of the present invention were translocated not in 1-10 ⁇ M but in 50-100 ⁇ M at 15 minutes (Fig. 2) or 2 hours (Fig. 3). In 50-100 ⁇ M, intracellular translocation was observed to be very high and could not detect due to a strong fluorescence particularly after 2 hours treatment and thus the sensitivity of reader was lowered to 75.
• TCTP (2-10) peptide was not translocated at a concentrarion of 1 ⁇ M to 10 ⁇ M, but was more efficiently translocated at 100 ⁇ M after 15 minutes treatment of this peptide. After 2 hours, this peptide has similar cell membrane penetrating activity to control peptide, TAT(48-57), and was more efficiently translocated at 100 ⁇ M than control peptide. So, it could be confirmed that TCTP (1-10), (1-9), (1-8) and (2-10) peptides having cell membrane penetrating activity of the prevent invention had superior ability than well-known PTD, TAT in their translocation efficiency.
• TCTP (1-10), (1-9), (1 -8) and (2-10) peptides having cell membrane penetrating activity of the present invention had superior ability than well-known PTD, TAT in their translocation efficiency. From among these peptides, translocation efficiency was superior in the order of TCTP (1-10), (1-9), (1-8) and (2-10) peptides, and existence of methionine (1 st amino acid residue) of TCTP N-terminus was important.
• the intracellular translocation of the peptide was identified by fluorescence microscope.
• HeLa cells were treated with TCTP (1-9)(SEQ ID No.: 2) at a concentration of lO ⁇ M and lOO ⁇ M by the same method of Example 2-2). A point of difference was that HeLa cells were seeded in 12 well-plate covered a glass since the plastic plate had a property of fluorescence interference. After washing, cells on cover glass attached slide glass were observed (Fig.4).
• the peptide of the present invention was weakly translocated at a low concentration of l O ⁇ M and strongly at a high concentration of l OO ⁇ M. It was found that the peptides were distributed widely in cytoplasm and nucleus of the cell.
• HeLa cell line was propagated in DMEM supplemented with 10% FBS and 100 units/mL penicillin-streptomycin. Cells were grown in a 5% CO 2 incubator at 37 ° C .
• HeLa cells were cultured in 48-well plate until they were 70-80% grown up before a day of the experiment.
• the cells were washed with DMEM of 37 ° C twice, and TCTP-derived peptides synthesized in Example 4-1) were treated to the culture medium in a dose dependent manner (0, 1 , 10, 100 ⁇ M), then the cells was incubated for 15 minutes or 2 hours in an CO 2 incubator at 37 "C .
• the cells were washed in cool PBS three times and immediately measured by a microplate fluorescence reader at emission 530 nm and excitation 590 nm for a measurement of rhodamine of intracellular uptake marker.
• the sensitivity of reader was set at 100 as a basic, but was lowered to 75 if fluorescent signals were strong. All experiments were conducted in triplet repeats for reproducibility (Fig. 5 and Fig. 6).
• HeLa cell line was propagated in DMEM supplemented with 10% FBS and 100 units/mL penicillin-streptomycin. Cells were grown in a 5% CO 2 incubator at 37 ° C .
• HeLa cells were cultured in 6-well plate until they were 70-80% grown up before a day of the experiment.
• the cells were washed with DMEM of 37 ° C twice, and TCTP-derived peptides synthesized in Example 5-1) were treated to the culture medium in a dose dependent manner (0, 1 , 10, 100 ⁇ M), then the cells was incubated for 2 hours in an CO 2 incubator at 37 ° C .
• TAT mean fluorescence intensity
• SEQ ID No.: 26 and SEQ ID NOS.: 27 were 6.1 times, 6.04 times and 1.73 times higher than WT at the concentration of 10 ⁇ M, respectively
• TAT, SEQ ID No.: 26 and SEQ ID No.: 27 were 94.75 times, 144.6 times and 342.9 times higher than WT at the concentration of lOO ⁇ M in cell penetrating activity, respectively.
• variant peptides of all 12 amino acids adding two lysines at C-terminus of WT was maintained in next designed variant peptides(from SEQ ID No.: 31) and substitution with other basic amino acids than lysine and change of number of basic amino acids were tested(SEQ ID Nos.: 48-52).
• SEQ ID Nos.: 48-52 substitution with other basic amino acids than lysine and change of number of basic amino acids
• methionine(M) with glutamine(Q) or cysteine(C)(comparison with SEQ ID No.: 23 and SEQ ID Nos.: 24-25).
• substitution for tryptophan instead of methionine did not induce the important changes in the aspect of efficiency and cytotoxicity.
• Substitution for phenylalanine(SEQ ID No.: 34) or leucine(SEQ ID No.: 35) brought about the increased result of translocation efficiency at the concentration of lO ⁇ M and a decreased result at lOO ⁇ M, compared to SEQ ID No.: 31.
• Leucine substituents in SEQ ID Nos.: 31, 34, 35 and 36 caused the most increased result at 10 ⁇ M and the little decreased result at 100 ⁇ M.
• Cytotoxicity of SEQ ID No.: 35 was weaker than SEQ ID No.: 31 at 100 ⁇ M.
• SEQ ID No.: 46(3.75 times higher than MFI of WT 10 ⁇ M) and SEQ ID No.: 47(7.04 times higher than MFI of WT 10 ⁇ M) substitution for leucine caused the decreased penetrating activity but toxicity of SEQ ID No.: 46 was weaker than that of SEQ ID No.: 47.
• Aspartic acid at the position of WT-VI because SEQ ID No.: 13 had a good efficiency at the low concentration (EXAMPLE 4), was substituted by alanine or isoleucine to increase hydrophobicity.
• SEQ ID No.: 31 (WT-VhI) and 33(WT-VI:A) translocation efficiencies of both was similarly increased at lOO ⁇ M but since increased penetrating activity of SEQ ID No.: 31(29 times increase in comparison with WT) was far better than SEQ ID No.: 33(3.2 times increase in comparison with WT) at lO ⁇ M, isoleucine substitution was more effective than alanine substitution. From these results, after this experiment, isoleucine was introduced at the position of WT-VI of peptide variant (from SEQ ID No.: 31, 34-36, 39).
• Serine at the position of WT-IX when SEQ ID No.: 39(WT-IX:Y) and 41(WT-IX:T) substituted by each tyrosine and threonine only at this position were compare with SEQ ID No.: 31(WT-IX:S) in cell penetrating activity, should be maintained for the best effect. Meanwhile in all case of substitution for tryptophan instead of methionine at the position of WT-I, efficiency of SEQ ID No.: 36(WT-IX:S) was stronger than SEQ ID No.: 40(WT-IX:Y) and SEQ ID No.: 42(WT-IX:T) only at 10 ⁇ M.
• SEQ ID No.: 1 was more effective than SEQ ID No.: 2 at the concentration of 50 ⁇ M(See Figs. 2 & 3), and when histidine was substituted by glutamic acid(in comparison with SEQ ID No.: I and 28 & 30, See Figs.7, 8 & 9), SEQ ID No.: 28 and SEQ ID No.: 30 were similar with WT at lO ⁇ M and decreased 4-5 times at high concentration.
• cytotoxicity As follows. HeLa cells were cultured in 96-well plate until they were 70% grown up before a day of the experiment. Control TAT 48-57 and the mutant peptides at concentrations of 0, 1, 10, l OO ⁇ M were treated to DMEM supplemented with 10% FBS for 24 and 48 hours. After 2 hours in addition of lO ⁇ l of CCK-8 to each well, absorbance at 450nm was measured by KC4 plate reader(Figs. 10, 1 1 and 12).
• cytotoxicity of SEQ ID No.: 1 TCTP(I -I O) was about 14% compared with control, and cytotoxicities of the other peptides, TCTP-CPP#3, 7 and 8 were insignificant considering standard deviation.
• all peptides had no cytotoxicity at 1 and lO ⁇ M while cytotoxicities of TAT, TCTP(I-I O), TCTP-CPP#3, 7 and 8 were about 53.8, 28.3, 46.2, 8.2 and 25.6%, respectively.
• TCTP-CPP# 12-26 had no cytotoxicity at l ⁇ M and lO ⁇ M, but had cytotoxicity beside only TCTP-CPP#26 at lOO ⁇ M. Also, all of TCTP-CPP#27-35 had no cytotoxicity at l ⁇ M and lO ⁇ M but had cytotoxicity at lOO ⁇ M. SEQUENCE LIST FREE TEXT

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