Pharmaceutical preparations of inactivated polypeptide TRP

Abstract

The present invention relates to a pharmaceutical preparation comprising an inactivated polypeptide TRP and a phospholipid dispersant. The pharmaceutical preparation according to the present invention can inhibit the aggregation of inactivated TRP, increase intracellular drug delivery, reduce cytotoxicity, increase drug safety and improve therapeutic efficacy.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical preparation comprising an inactivated polypeptide TRP, and more particularly to a pharmaceutical preparation comprising an inactivated polypeptide TRP and a phospholipid dispersant. Background Art

[0002] Unactivated polypeptide (TRP: tissue regeneration polypeptide) is a substance with a novel mechanism capable of promoting the formation or regeneration of tissues such as bone, cartilage, etc., or preventing fibrosis and sclerosis of organs such as kidney, liver, lung, heart, etc., and has a structure containing: PTD (protein transduction domain), which achieves cell membrane penetration without the help of cell membrane receptors; FAD (furin activation domain), which has at least one proprotein convertase cleavage site and is cleaved by proprotein convertase to activate the unactivated TRD (tissue regeneration domain) in the cell; and TRD, which is activated by being cleaved by the proprotein convertase of FAD to promote tissue growth or formation in the cell or induce tissue regeneration (Korean Patent No. 10-00775958 and U.S. Patent No. 8,268,590).

[0003] Unactivated TRP has a completely different structure and properties from conventionally known active proteins such as rhBMP or TGF-β. Specifically, all conventionally known rhBMP and TGF-β are biochemically activated before being introduced into human or mammalian cells and have a three-dimensional structure (Eur. J. Biochem., 237: 295, 1996; J. Mol. Biol., 287: 103, 1999).

[0004] Therefore, in order to produce rhBMP and TGF-β as conventional activators, the problem is that the recombinant culture of mammalian cells such as CHO must be relied on, and these mammalian cells have very low productivity. Some rhBMPs, such as rhBMP14 (MP-52) from BioPharm, are produced by cultivating cheap recombinant Escherichia coli rather than animal cells, but even in this case, there are many restrictions on the biochemical structure of the BMP to be produced. Specifically, active MP-52 can be produced using recombinant Escherichia coli, but recombinant Escherichia coli is not easy to produce other active BMPs or active TGF-β that can be applied to the human body to promote bone or cartilage formation and regenerate various tissues, such as active rhBMP2, rhBMP4, rhBMP7, etc., and its tertiary or quaternary structure is required for pharmacological action. In addition, these conventional recombinant growth factors are sensitive to temperature changes, and have the disadvantage of low stability at room temperature (Table 1 (comparison of differences between recombinant growth factors and TRP)).

[0005] In contrast, the inactivated TRP exists in the form of poorly soluble inclusion bodies and thus has very high stability even at room temperature and is very robust even under changes in acidity (Table 1).

[0006] [Table 1]

[0007]

[0008] However, in order to use unactivated TRP as a therapeutic agent, the conventional problem is that dispersibility must be improved by suppressing aggregation and precipitation caused by the poor solubility of TRP, and low cell permeability and cytotoxicity when applied at high doses must be overcome. In addition, when PTD is used to deliver proteins into cells, the size of the protein delivery system (cargo) is very limited, and it is difficult to deliver proteins of 10kd or larger (J. Cell Sci., 129: 893-897, 2016). For example, TRD and FAD in TRP correspond to protein cargoes, and BMP-2 is 44.7kd, BMP-7 is 49.3kd, and HGF is 83.1kd, so the intracellular penetration of therapeutic proteins using PTD is very limited. Therefore, when a method for preventing hydrophobic protein aggregation and increasing the intracellular permeability of large-size cargoes is developed, it is possible to increase the effect of TRP on the treatment of diseases.

[0009] Therefore, the inventors have made extensive efforts to develop new formulations using inactivated TRP for therapeutic purposes, and thus determined that when inactivated TRP is used together with a phospholipid dispersant such as egg lecithin, soybean lecithin, triglyceride (triolein), LysoPC (L-α-lysophosphatidylcholine), Cremophor, etc., (1) TRP aggregation is inhibited, (2) the cell permeability of TRP with a large protein cargo size is increased, (3) cytotoxicity is reduced, (4) protein stability is increased, and (5) the therapeutic effect is also improved, thereby completing the present invention. Summary of the invention

[0010] An object of the present invention is to provide a pharmaceutical preparation which can ensure dispersibility by inhibiting the aggregation of inactivated TRP, increase the stability of protein drugs, and improve cell permeability, thereby improving therapeutic efficiency.

[0011] Another object of the present invention is to provide a composition for treating fibrotic diseases comprising TRP2 and a phospholipid dispersant.

[0012] Still another object of the present invention is to provide a method for treating or preventing fibrotic diseases, which comprises administering a composition comprising TRP2 and a phospholipid dispersant.

[0013] Still another object of the present invention is to provide a composition comprising TRP2 and a phospholipid dispersant for use in treating or preventing fibrotic diseases.

[0014] Still another object of the present invention is to provide a use of a composition comprising TRP2 and a phospholipid dispersant for manufacturing a medicament for treating or preventing fibrotic diseases.

[0015] In order to achieve the above-mentioned purpose, the present invention provides a pharmaceutical preparation, which comprises (a) an inactivated polypeptide TRP containing the following domains: PTD (protein transduction domain), which achieves cell membrane penetration without the help of cell membrane receptors; FAD (furin activation domain), which has at least one protein convertase cleavage site and is cleaved by protein convertase to activate the inactivated TRD (tissue regeneration domain) in the cell; and TRD, which is activated by being cleaved by the protein convertase of FAD to promote tissue growth or formation in the cell or induce tissue regeneration, and (b) a phospholipid dispersant.

[0016] In addition, the present invention provides a composition for treating fibrotic diseases, comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.

[0017] In addition, the present invention provides a method for treating or preventing a fibrotic disease, comprising administering a composition comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.

[0018] In addition, the present invention provides a use of a composition comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for treating or preventing fibrotic diseases.

[0019] In addition, the present invention provides a use of a composition comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for manufacturing a medicament for treating or preventing a fibrotic disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of DLS analysis of TRP (A) and its intracellular permeability confirmed using confocal fluorescence microscopy (B and C) are shown;

[0021] Figure 2 The results of the aggregation of TRP when no dispersant is used and the dispersion effect on TRP2 when a dispersant is used are shown by DLS;

[0022] Figure 3Scanning electron microscope images of TRP2 particles without a dispersant (TRP2), TRP2 particles dispersed with egg lecithin (TRP2-EL), TRP2 particles dispersed with soybean lecithin (TRP2-SL), TRP2 particles dispersed with LysoPC (TRP-LysoPC), TRP2 particles dispersed with triglyceride (TRP2-triolein), and TRP2 particles dispersed with DSPE-PEG-amine (TRP2-DSPE-PEG-amine) are shown;

[0023] Figure 4 Shown are images of the TRP used in Example 3 and TRP2 in a dispersion preparation observed in three dimensions using a confocal microscope after treating 293 cells with the TRP used in Example 3 and TRP2 in a dispersion preparation for 4 hours (upper images), and images confirming the distribution of TRP2 by Z-stack (lower images);

[0024] Figure 5 Shown are images confirming the intracellular permeability of TRP and TRP2 in the dispersion preparation used in Example 3, which were taken using a transmission electron microscope after 293 cells were treated therewith for 4 hours;

[0025] Figure 6 Shows images of the TRP used in Example 3 and TRP2 in a dispersion preparation observed for the endosomal marker Rab7-mCherry fluorescence using a confocal microscope after 293 cells were treated with the TRP used in Example 3 and TRP2 in a dispersion preparation for 4 hours;

[0026] Figure 7 Results demonstrating the cytotoxicity of TRP formulations containing a phospholipid emulsifier as a dispersant are shown;

[0027] FIG8 shows the results confirming rapid aggregation of TRP when no dispersant is used (A) and the DLS measurement results of the aggregation inhibition effect according to time and storage temperature when various dispersants are used (B to D);

[0028] Fig. 9 DLS measurements showing the stability of TRP2 as a function of egg lecithin concentration and as a function of storage time and temperature;

[0029] Fig.10 shows the DLS measurement results of the stability of TRP2 according to storage time and temperature after dispersing various amounts of TRP2 using egg lecithin;

[0030] Fig.11Confocal microscopy images (A) and Western blot results (B) showing the intracellular permeability of TRP2 after being dispersed in various ways using egg lecithin and stored in a refrigerated state for 108 days;

[0031] Fig.12 The present invention shows the observation results of the inhibitory effect of TRP2 on peritoneal fibrosis according to the dose of TRP2 without a dispersant in an in vivo animal model;

[0032] Fig.13 The present invention shows the observation results of the inhibitory effect of the TRP2 preparation containing Cremophor on peritoneal fibrosis according to the dose in an in vivo animal model;

[0033] Fig.14 The present invention shows the observation results of the inhibitory effect of TRP2 preparations containing egg lecithin on peritoneal fibrosis according to the dosage in an in vivo animal model;

[0034] Fig.15 showing the observation results of the inhibitory effect of TRP2 preparations containing soybean lecithin on peritoneal fibrosis according to the dose in an in vivo animal model; and

[0035] Fig.16 The 50% effective dose (ED50) results based on the results of experiments using TRP2 without a dispersant and TRP2 containing a dispersant on animals are shown. DETAILED DESCRIPTION

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Generally, the nomenclature used herein is well known and typical in the art.

[0037] The inactivated polypeptide (TRP: tissue regeneration polypeptide) of the present invention has a structure containing: PTD (protein transduction domain), which achieves cell membrane penetration without the help of cell membrane receptors; FAD (furin activation domain), which has at least one proprotein convertase cleavage site and is cleaved by proprotein convertase to activate the inactivated TRD (tissue regeneration domain) in the cell; and TRD, which is activated by being cleaved by the proprotein convertase of FAD to promote tissue growth or formation in the cell or induce tissue regeneration, and the inactivated polypeptide of the present invention is a substance with a new mechanism that can promote the formation or regeneration of tissues such as bones, cartilage, etc., or prevent fibrosis and sclerosis of organs such as kidneys, liver, lungs, heart, etc. (Korean Patent No. 10-00775958 and U.S. Patent No. 8,268,590).

[0038] Inactivated TRP exists in the form of poorly soluble inclusion bodies and therefore has very high stability even at room temperature and is very robust even under changes in acidity. In order to use inactivated TRP as a therapeutic agent, aggregation and precipitation due to the poor solubility of TRP should be suppressed, and low cell permeability and cytotoxicity when administered at high doses should be overcome.

[0039] TRP is a substance with a mechanism to permeate cells and is activated by refolding and processing in the cytoplasm. In order to increase its pharmacological effect, it is absolutely necessary to ensure physicochemical homogeneity by inhibiting the aggregation of peptide preparations and increase the intracellular permeability of proteins with very large molecular weight (about 50 kd) such as TRP2.

[0040] In the present invention, with the goal of developing a preparation that inhibits the aggregation of unactivated TRP, the TRP aggregation inhibitory effects of various aggregation inhibitors and dispersants were confirmed. As a result, it was confirmed that TRP preparations containing phospholipid dispersants such as egg lecithin, soybean lecithin, triglycerides (triolein), LysoPC (L-α-lysophosphatidylcholine), Cremophor, etc. imparted a high aggregation inhibitory effect, improved the cell permeability of TRP, reduced cytotoxicity, and increased in vivo efficacy.

[0041] Therefore, one aspect of the present invention relates to a pharmaceutical preparation comprising (a) an inactivated polypeptide TRP containing: a PTD (protein transduction domain) which achieves cell membrane penetration without the help of cell membrane receptors; a FAD (furin activation domain) which has at least one proprotein convertase cleavage site and is cleaved by a proprotein convertase to activate the inactivated TRD (tissue regeneration domain) in cells; and a TRD which is activated by cleavage by the proprotein convertase of FAD to promote tissue growth or formation or induce tissue regeneration in cells, and (b) a phospholipid dispersant.

[0042] In the present invention, the phospholipid dispersant may be selected from soybean lecithin, triglyceride (triolein), LysoPC (L-α-lysophosphatidylcholine) and Cremophor, but is not limited thereto.

[0043] In the present invention, lecithin can be egg lecithin, soybean lecithin, sunflower oil lecithin, rapeseed lecithin (canola lecithin), cottonseed lecithin or lecithin extracted from animal fat, but is not limited thereto. Preferably, egg lecithin or soybean lecithin is used, and egg lecithin is more preferably used.

[0044] In one embodiment of the present invention, the aggregation of TRP is confirmed by DLS (dynamic light scattering, Otsuka, ELSZ-2000Zs) and confocal fluorescence microscopy. Most TRP particles exist in a size of 300 μm or larger due to aggregation, and are observed as very large aggregates even when using confocal microscopy. Therefore, these particles exhibit low intracellular penetration efficiency and tend to exist mostly outside the cell membrane ( Figure 1 ).

[0045] TRP is a substance with a mechanism of permeating cells and is activated by refolding and processing in the cytoplasm. In order to increase its pharmacological effect, it was confirmed that it was necessary to ensure physicochemical homogeneity by inhibiting the aggregation of peptide preparations and increase the intracellular permeability of proteins with very large molecular weight (about 50kd) such as TRP2.

[0046] Since normal cells have a size of 10-20 μm, it is necessary to prepare a preparation having a uniform size of 5 μm or less in order to use TRP for therapeutic purposes. From a pharmacological point of view for patient treatment, a preparation having a uniform size of 0.5 μm or less is preferred.

[0047] In another embodiment of the present invention, the TRP2 aggregation inhibitory effect was confirmed using sugars, amino acids, ammonium sulfate, and emulsifiers conventionally used for the formulation of peptides or antibodies. For conventional aggregation inhibitors, it was confirmed that the TRP aggregation inhibitory effect was not significant (Table 2), and when 10% glycerol was used, the effect of slightly reducing the size of TRP aggregates was confirmed. However, even in this case, most of the TRPs still existed in a very large size, and the TRPs existed outside the cell membrane rather than permeating the cell ( Figure 2 ).

[0048] In another embodiment of the present invention, the TRP aggregation inhibitory effect of a TRP preparation was confirmed, and the TRP preparation contained a dispersant such as Cremophor, egg lecithin, soybean lecithin, LysoPC (lysophosphatidylcholine), triolein, DOTAP, DSPE-PEG-amine and soybean oil, and phospholipid emulsifiers such as Cremophor, egg lecithin, soybean lecithin, LysoPC and triolein effectively inhibited the aggregation of TRP2 and kept TRP2 in the form of micelles. However, soybean oil, which is mainly used for weakly soluble low molecular weight compounds, was confirmed to have no aggregation inhibitory effect (Table 3 and Table 4). Figure 2 and Figure 3 ).

[0049] In another embodiment of the present invention, for Cremophor, egg lecithin, soybean lecithin, LysoPC and triolein, which have been shown to exhibit aggregation inhibition, the effect of increasing the cell membrane permeability of TRP was confirmed by confocal fluorescence microscopy and transmission electron microscopy. For cells to which only TRP was administered, TRP was mostly present on the cell membrane, but the TRP2 preparation to which the emulsifier (Cremophor, egg lecithin (EL), soybean lecithin (SL), LysoPC and triolein) was added was in a roughly uniform form due to the reduced particle size, so it passed through the cell membrane and was thus observed in the cytoplasm ( Figure 4 and Figure 5 ), indicating that the cell membrane permeability of TRP was greatly increased by reducing the particle size of TRP2 preparations due to aggregation inhibition. It was confirmed that TRP2 preparations containing this dispersion agent were effectively delivered to the cytoplasm through endosomes ( Figure 6 ).

[0050] In another embodiment of the present invention, the cytotoxicity of TRP preparations containing Cremophor or egg lecithin in 293A cells was analyzed using the JuLI Stage automated cell imaging system, and when no dispersant was used, cytotoxicity appeared as the amount of preparation used (1 μg and 5 μg) increased, and egg lecithin (EL), soybean lecithin (SL), triglyceride (triolein), and LysoPC did not exhibit cytotoxicity even when the amount of preparation used increased ( Figure 7 ). Cremophor showed low cytotoxicity compared to when no dispersant was used, but cytotoxicity occurred at a high dose (5 μg). Therefore, egg lecithin, soybean lecithin (SL), triglyceride (triolein), and LysoPC were confirmed to be suitable for TRP preparation.

[0051] In another embodiment of the present invention, in order to confirm the stability of the preparation containing TRP2 and egg lecithin, the preparation was frozen at -20°C and then allowed to stand at room temperature to evaluate whether the aggregation inhibition ability was maintained over time. TRP2 without a dispersant rapidly aggregated within a few minutes, and the size of the aggregates increased over time. Even after 56 days, the average size of the TRP2 preparation containing egg lecithin was 150nm or less, indicating that the aggregation inhibition ability remained constant (Figure 8). In addition, it was confirmed that the preparation containing a dispersant was stable for a very long time at various storage temperatures of TRP.

[0052] In another embodiment of the present invention, when various concentrations of egg lecithin were used and various concentrations of TRP2 were used, it was confirmed that the dispersibility was maintained according to the storage conditions and long-term storage ( Fig. 9 and Fig.10In addition, it was confirmed that the increased intracellular permeability of TRP2 containing the dispersant was maintained under various storage conditions ( Fig.11 ).

[0053] In another embodiment of the present invention, when a TRP formulation containing a dispersant (Cremophor, egg lecithin or soy lecithin) was administered to an animal model, the in vivo effect of a TRP2 formulation containing a dispersant in a peritoneal dialysis (PD) animal model was confirmed. Regarding the inhibition of peritoneal fibrosis in the animal model, the formulation containing a dispersant showed a very low ED50 (Tables 5 to 9 and Tables 10 to 11) compared to TRP2 without a dispersant. Figures 12 to 16 ).

[0054] The preparation of the present invention can be suitable for injection, and a dispersant such as egg lecithin, soybean lecithin, triglyceride or Cremophor is contained in an amount of 100 to 50,000 parts by weight based on 100 parts by weight of the inactivated polypeptide TRP, preferably 200 to 10,000 parts by weight based on 100 parts by weight of TRP, and more preferably 500 to 5,000 parts by weight based on 100 parts by weight of TRP.

[0055] The inactivated TRP of the present invention can be prepared in the following manner, wherein a recombinant vector is constructed in which a nucleotide sequence encoding FAD, a nucleotide sequence of PTD, a nucleotide sequence for labeling, and a nucleotide sequence encoding four or more histidines for separation and purification are inserted 5' upstream of a DNA encoding TRD, and bacteria transformed with the recombinant vector are cultured and thus express the [PTD-FAD-TRD] polypeptide, and then a urea solution is added to the culture solution, thereby removing the two-dimensional structure and the three-dimensional structure of the polypeptide or converting the polypeptide into a one-dimensional linear structure, and then purifying the [PTD-FAD-TRD] polypeptide. For example, it can be prepared by the method disclosed in Korean Patent Application Publication No. 2006-0106606.

[0056] In the present invention, TRD can be represented by an amino acid sequence selected from SEQ ID NO: 1 to 13, but TRD is not limited thereto, as long as it is a protein having the activity of promoting tissue growth or formation in cells or inducing tissue regeneration. Examples of such TRD are polypeptides selected from BMP, TGF-β superfamily, β-NGF (β-nerve growth factor), β-amyloid protein, ADAM (a disintegrin-like metalloproteinase), TNF-α, MMP (matrix metalloproteinase), insulin-like growth factor-1 (IGF-1), hepatocyte growth factor and Dickkopf.

[0057] In the present invention, FAD may be represented by an amino acid sequence selected from SEQ ID NO: 14 to 26, but FAD is not limited thereto, as long as it has a proprotein convertase cleavage site and is cleaved by a proprotein convertase in a cell, thereby activating TRD. In addition, PTD may be selected from TAT, Antp peptide derived from Drosophila, VP22 peptide, and mph-1-btm, but is not limited thereto.

[0058] The PTD used in the present invention is TAT ​​(YGRKKRRQRRR: SEQ ID NO: 27), but the present invention is not limited thereto. Examples of PTD may include Antp peptides derived from Drosophila, VP22 peptides (Gene Therapy, 8: 1, Blackbirch Press, 2001), mph-1-btm (US 2005 / 0147971), PTD-3, PTD-4 (Cancer Research, 2001, 61, 474-477), TAT48-60, Penetratin, polyarginine, CADY (FEBS Letters 2013, 587-1702), etc. In addition, the nucleotide sequence used for labeling is an X-press tag, but Flag, Myc, Ha, GST, etc. may be used instead.

[0059] In the method for producing inactivated TRP according to the present invention, the purification step includes binding the polypeptide to nickel-titanium beads, followed by washing with the same solution and then eluting with imidazole and a high salt buffer solution, but the present invention is not limited thereto.

[0060] In the present invention, the proprotein convertase may be furin, but is not limited thereto. Examples thereof may include PC7, PC5 / 6A, PC5 / 6B, PACE4, PC1 / 3, PC2, PC4, and the like.

[0061] The unactivated TRP according to the present invention does not have the three-dimensional stereoregularity common to conventional active BMPs, and does not have biochemical activity itself, but when administered in humans or mammals, the proprotein convertase cleavage site of FAD is cleaved in vivo by the proprotein convertase present in the cells, thereby activating TRD, and the activated TRD is secreted outside the cells, thereby exhibiting the expected efficacy. The TRP according to the present invention preferably takes the form of a polypeptide in which PTD, FAD and TRD are fused. The TRP according to the present invention has the function of promoting the formation or regeneration of tissues such as bones, cartilage, etc. in the human body, or preventing fibrosis and sclerosis of organs such as kidneys, liver, lungs, heart, etc., and further induces the regeneration of original tissues.

[0062] The TRP according to the present invention can be mass-produced for practical use by culturing bacteria such as recombinant Escherichia coli without being restricted by the biochemical structure of the protein, and because its biochemically inactive state is maintained before in vivo administration, its production cost is only a few tenths of that of conventionally known active proteins (rhBMP, TGF-β, etc.) used for similar uses, and its separation, purification, processing, storage and administration are extremely simple and convenient.

[0063] In addition, the inactivated TRP according to the present invention is a polypeptide containing: PTD, which achieves cell membrane penetration without the help of cell membrane receptors; FAD, which has at least one proprotein convertase cleavage site and is cleaved by a proprotein convertase to activate the inactivated TRD in the cell; and TRD, which is activated in the cell by being cleaved by the proprotein convertase of FAD to promote tissue growth or formation or tissue regeneration. Although TRP itself has no activity, it penetrates organisms or cells, and then FAD is cleaved by a proprotein convertase that exists in large quantities in almost all cells, thereby activating TRD, and the activated protein is secreted outside the cell, thereby exerting its effectiveness. The TRP according to the present invention can solve all the problems of conventional active BMP-like proteins, which are expensive to prepare, difficult to store and handle, and essentially require receptors.

[0064] Depending on the method of administration, dosage form and therapeutic purpose, it is preferred to prepare a composition containing the inactivated TRP according to the present invention as an active ingredient by mixing and diluting the active ingredient with a pharmaceutically acceptable excipient or matrix as a carrier, or by encapsulating the active ingredient in a carrier in the form of a container. In addition, the composition can be used in combination with other drugs that are beneficial for treating other bone defects. Here, the preparation of a physiologically acceptable protein composition having the desired pH, isotonicity, stability, etc. can be carried out using any typical method known in the art to which the present invention belongs.

[0065] In the present invention, according to biocompatibility, biodegradability, mechanical properties, decorative appearance and interface properties, preferred examples of the matrix include biodegradable chemical materials such as calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid or polyanhydride; biodegradable biological materials such as bone or skin collagen, other pure proteins or cell matrix components; non-biodegradable chemical materials such as sintered hydroxyapatite, bioglass, aluminate or other ceramics; and combinations of the above materials such as polylactic acid, hydroxyapatite, collagen and tricalcium phosphate. However, the present invention is not limited to the carriers described above.

[0066] In the present invention, examples of excipients may include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, magnesium stearate, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, mineral oil and the like.

[0067] Meanwhile, it is preferred to use the composition containing inactivated TRP according to the present invention by encapsulation or injection in a viscous form so as to be applied to the bone injury site. The dosage of the composition according to the present invention is not limited because it can be adjusted in consideration of the type of excipient or carrier used such as matrix, etc., the patient's bone weight, the bone injury site, the condition of the injured bone, the patient's age, sex and diet, the degree of suspected infection, the administration time and other clinical factors. However, for the generally known effective amount of the composition, in consideration of the bone weight, an appropriate amount may be administered continuously or separately, and additional administration may be determined while observing bone growth.

[0068] In another embodiment of the present invention, when a TRP formulation containing a phospholipid dispersant (Cremophor, egg lecithin or soy lecithin) was administered to an animal model, the in vivo effect of a TRP2 formulation containing a dispersant in a peritoneal dialysis (PD) animal model was confirmed. Regarding the inhibition of peritoneal fibrosis in the animal model, the formulation containing a dispersant showed a very low ED50 (Tables 5 to 9 and Tables 10 to 11) compared to TRP2 without a dispersant. Figures 12 to 16 ).

[0069] Accordingly, another aspect of the present invention relates to a composition for treating fibrotic diseases, comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.

[0070] Still another aspect of the present invention relates to a method for treating or preventing a fibrotic disease, comprising administering a composition comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant.

[0071] Yet another aspect of the present invention relates to use of a composition comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for treating or preventing fibrotic diseases.

[0072] Yet another aspect of the present invention relates to use of a composition comprising 100 parts by weight of TRP2 and 100 to 50,000 parts by weight of a phospholipid dispersant for the manufacture of a medicament for treating or preventing a fibrotic disease.

[0073] In the present invention, fibrotic diseases may include liver fibrosis, muscle fibrosis, pulmonary fibrosis, renal fibrosis, cardiac fibrosis, peritoneal fibrosis, various eye diseases caused by fibrosis, etc. This fibrosis inhibition can be applied to various cancers such as liver cancer and chronic diseases. In addition, since BMP-7 inhibits TGF-β signaling (Nature Med.2003,9,964-968), it can be used in combination with immune checkpoint anticancer drugs such as PD1 / PDL-1 antibodies (Nature 2018,554,538-543). It can also be used for various metabolic diseases, inflammation, hyperphosphatemia, and Alport syndrome.

[0074] In the present invention, the phospholipid dispersant may be selected from lecithin, Cremophor, triglyceride and LysoPC (lysophosphatidylcholine), wherein the lecithin may be egg lecithin or soybean lecithin.

[0075] Example

[0076] A better understanding of the present invention can be obtained through the following examples. These examples are only used to illustrate the present invention and should not be interpreted as limiting the scope of the present invention, as will be clear to those skilled in the art.

[0077] In particular, in the following embodiments, as TRDs constituting TRP, only hBMP2 and hBMP7 are exemplified, but it will be clear to those skilled in the art that not only BMPs derived from humans such as hBMP3, hBMP4, hBMP6, and hBMP14 (MP-52) can be used, but also BMPs derived from mammals such as rats, cattle, pigs, etc. and other higher animals can be used. In addition to BMPs, it will be clear to those skilled in the art that polypeptides such as TGF-β superfamily, β-NGF (β-nerve growth factor), β-amyloid protein, ADAM (a disintegrin-like metalloproteinase), TNF-α family (including exoprotein-A (Eda-1)), MMP (including MT1-MMP (membrane-matrix metalloproteinase) and MMP-2), and insulin-like growth factor-1 (IGF-1) can also be used.

[0078] In addition, in the following embodiments, only the prodomain of BMP is exemplified as FAD constituting TRP, but it will be clear to those skilled in the art that FAD is not limited as long as it has a cleavage site for proprotein convertase and is cleaved by proprotein convertase in the cell to activate TRD.

[0079] In addition, in the following examples, phospholipids and ultrasound were used to disperse the TRP protein, but it will be clear to those skilled in the art that a high-pressure homogenizer or disperser commonly used in drug production may be used.

[0080] Example 1: Demonstration of TRP aggregation and cell permeability

[0081] To confirm the aggregation of TRP, DLS (dynamic light scattering, Otsuka, ELSZ-2000Zs) and confocal fluorescence microscopy were performed.

[0082] The TRP used was the following TRP2 having the BMP7 sequence as TRD.

[0083] TRP2 amino acid sequence (SEQ ID NO: 28):

[0084]

[0085] Bold: PTD, Italic Underline: Human BMP-7, Underline: FAD

[0086] The particle size of TRP2 isolated and purified from the inclusion bodies of transformed E. coli was measured using DLS. Figure 1 As shown in A, most of the TRP particles exhibited very large and varied particle sizes due to aggregation, and in some cases, TRPs were present at 300 μm or larger.

[0087] 293A cells were treated with 100 ng of this TRP2 for 4 hours, and the cells were fixed in 10% formalin, stained with X-press monoclonal antibody (Thermo Fisher R910-25) and Alexa-594-labeled secondary antibody (Thermo Fisher A32744) to visualize TRP2, and observed using a confocal microscope (Carl Zeiss, LSM700).

[0088] Based on its results, even in Figure 1 Very large aggregates were also observed on low-magnification confocal microscopy of B, and multi-layer imaging and stereoscopic analysis (Z stacks) were performed to observe the three-dimensional distribution pattern. Based on their results, Figure 1 As shown in C, most TRP2 exists in aggregated form outside the cell membrane rather than inside the cell. Therefore, TRP2 exhibits low intracellular permeation efficiency and tends to exist outside the cell membrane.

[0089] TRP is a substance with a mechanism to permeate cells and is activated by refolding and processing in the cytoplasm. In order to increase its pharmacological effect, it is necessary to ensure physicochemical homogeneity by inhibiting the aggregation of peptide preparations and increase the intracellular permeability of proteins with very large molecular weight (about 50 kd) such as TRP2.

[0090] Example 2: Analysis of TRP aggregation inhibition using conventional peptide and antibody drug preparations

[0091] TRP2 aggregation inhibition was evaluated using sugars, amino acids, ammonium sulfate, and emulsifiers conventionally used for formulating peptides or antibodies.

[0092] [Table 2]

[0093]

[0094] To this end, the recombinant protein TRP2 at a concentration of 1 mg / ml in PBS was diluted with various dispersants at the concentrations shown in Table 2 to obtain 100 μg / ml of TRP2, which was then allowed to stand at room temperature for 1 hour and subsequently subjected to DLS measurement. Here, the effect of each dispersant was evaluated in a semi-quantitative manner based on the average size of the stabilized particles in DLS (0: >4 μm; 1+: <4 μm; 2+: <3 μm; 3+: <2 μm; 4+: <1 μm; and 5+: 250 μm). In addition, when the presence of a dispersing effect was determined, the stability of the particles was evaluated by additionally measuring the zeta potential.

[0095] [Table 3]

[0096]

[0097] Based on the results, as shown in Table 3, for aggregation inhibitors conventionally used in recombinant proteins or antibodies, it was confirmed that the TRP aggregation inhibition effect was not significant, and for Tween 80, the presence of aggregation was confirmed, but the ζ potential was very low (1.63 mV), indicating that the particle stability was very poor.

[0098] Furthermore, 10% glycerol, which is most commonly used to prevent aggregation of recombinant proteins or antibodies, slightly reduced the aggregate size of TRP, but even in this case, most of TRP2 still existed in a very large size, thus finding that 10% glycerol is not suitable as a primary dispersant for TRP2.

[0099] Example 3: Analysis of the Aggregation Inhibition Effect of TRP Formulations Using Phospholipid Emulsifiers as Dispersants

[0100] The TRP aggregation inhibition effect of TRP preparations containing Cremophor EL (Merck, 238470), egg lecithin (BOC Sciences, BS18J04211), soybean lecithin (TCI, L0023), LysoPC (lysophosphatidylcholine) (Avanti, 830071P), triolein (Sigma, T7140), DOTAP (Avanti, 890890P), DSPE-PEG-amine (Avanti, 880128P) and soybean oil (CJ Cheiljeang) as emulsifiers was evaluated.

[0101] To this end, the recombinant protein TRP2 at a concentration of 1 mg / ml in PBS was sonicated in a 10% glycerol solution and dispersed using various dispersants shown in Table 3, and then the formation of its micelles was induced using weak ultrasound. Here, the appropriate concentration of the dispersing emulsifier was determined using an emulsifier at a concentration of 0.1%. The dispersed TRP2 was allowed to stand at room temperature for 1 hour to induce aggregation, and the average size and dispersion of the particles were measured by DLS. In addition, the aggregation inhibition effect of these dispersants was observed using a scanning electron microscope (Carl Zeiss, model name: Merlin).

[0102] [Table 4]

[0103]

[0104] Based on the results, as shown in Table 4 and Figure 2 As shown in, when dispersed using 10% Cremophor (TRP-Cre), 0.1% egg lecithin (TRP2-EL), soybean lecithin (TRP2-SL), LysoPC (TRP2-LysoPC) or triolein (TRP2-triolein) as a phospholipid emulsifier, the aggregation of TRP2 was effectively inhibited, and the micelle form with an average size of 250 nm or less was maintained, thereby exhibiting an excellent dispersion effect as measured by DLS. However, it was confirmed that soybean oil, which is mainly used for weakly soluble low molecular weight compounds, had no aggregation inhibitory effect.

[0105] In addition, if Figure 3 As shown in , stable dispersions of a certain size using a phospholipid emulsifier were directly confirmed by scanning electron microscopy (TRP2 / TRP2-LysoPC formulation × 10,000, scale increment 1 μm; other formulations × 20,000, scale increment 20 nm).

[0106] Therefore, it was confirmed that phospholipid emulsifiers can be used as aggregation inhibitors and dispersants, and unlike conventional recombinant proteins or antibodies, phospholipid emulsifiers can effectively disperse TRPs.

[0107] Example 4: Analysis of the Effect of Increasing Cell Membrane Permeability of TRP Formulations Containing Dispersants

[0108] TRP is developed as a precursor protein prodrug of BMP-7. Therefore, in order for TRP to exhibit effective pharmacological activity, it is very important to effectively deliver it to the cytoplasm through endosomal transport. Therefore, it is attempted to confirm that the preparation showing the excellent dispersion effect of TRP2 enhances the intracytoplasmic permeability of TRP2. For Cremophor, egg lecithin, soybean lecithin, LysoPC and triolein, which were confirmed to exhibit aggregation inhibition in Example 3, the effect of enhancing the cell membrane permeability of TRP was evaluated by confocal fluorescence microscopy. To this end, egg lecithin (EL), soybean lecithin (SL), LysoPC and triolein dispersants with a concentration of 0.1% were prepared, and 10% Cremophor (Cre) was used. TRP2 was dispersed using each dispersant and ultrasound to a concentration of 100 μg / ml, and 293A cells were treated therewith for 6 hours in the same manner as in Example 1, fixed and stained with X-press monoclonal antibody (Thermo Fisher R910-25) and secondary antibody labeled with Alexa-594 (Thermo Fisher A32744), followed by multi-layer imaging and stereoscopic analysis using a confocal microscope (Carl Zeiss, LSM700).

[0109] Based on its results, such as Figure 4 As shown in A, in the case of TRP2 without a dispersant, the aggregated protein was mostly attached to the outside of the cell membrane, similar to Example 1. When 25% glycerol was used as a dispersant, intracytoplasmic permeability increased slightly, but the protein was mostly present outside the cell membrane, so there was no significant effect ( Figure 4 B). However, for egg lecithin (TRP2-EL), soybean lecithin (TRP2-SL), triolein (TRP2-triolein), Cremophor (TRP-Cre), and LysoPC (TRP2-LysoPC), which were phospholipid emulsifiers showing excellent protein dispersion in Example 3, it was confirmed that a very large amount of protein was delivered to the cytoplasm ( Figure 4 C to Figure 4 G). However, soybean oil, which is commonly used to disperse water-insoluble drugs, did not increase intracytoplasmic permeability ( Figure 4 H).

[0110] In addition, in order to directly confirm the cell permeability of TRP protein, the protein was stained with 2.5% uranyl acetate (Electron Microscopy Sciences, #22400) and dispersed using each dispersion preparation as in Example 1. 293A cells were treated with 50 ng of TRP2 contained in each dispersion preparation, embedded in resin, and observed using an electron microscope (JEOL, JEM1011).

[0111] Based on its results, such as Figure 5 As shown in , TRP (TRP2) without a dispersant was present in aggregated form on the outer surface of the cell membrane, and when a dispersion preparation was used, it was confirmed that the protein was well delivered to the cytoplasm through the endosome (indicated by a red circle).

[0112] These results indicate that TRP2 formulations containing Cremophor, egg lecithin, soybean lecithin, LysoPC, or triolein significantly improved the cell membrane permeability and aggregation inhibition of TRP.

[0113] Example 5: Demonstration of the effect of TRP formulations containing dispersants on increasing intracellular protein delivery using endosomes

[0114] In Examples 3 and 4, it was confirmed that phospholipid dispersants significantly increased the delivery of TRP2 to cells and the aggregation inhibition effect. In addition, it can be observed from scanning electron micrographs that the TRP delivered to the cytoplasm is surrounded by a double membrane structure. This indicates the possibility that the effective intracytoplasmic delivery of TRP2 using phospholipid dispersants will utilize the endosomal pathway. To this end, according to the manufacturer's instructions, the TRP protein was fluorescently labeled using Alexa Fluor 488 (excitation: 494nm, emission: 519nm) protein labeling kit (Invitrogen, MP10235). As in Example 4, egg lecithin (TRP2-EL), soybean lecithin (TRP2-SL), triolein (TRP2-triolein) and LysoPC (TRP2-LysoPC) were used to disperse the fluorescently labeled TRP and form micelles. In addition, in order to detect endosomes by fluorescence, the fusion expression vector of mCherry (excitation: 587 nm, emission: 610 nm) fluorescence gene and endosomal marker Rab7a (Ras-associated protein Rab-7a) gene was inserted into pcDNA3.1 (Invitrogen) to construct the mCherry-Rab7a expression vector containing mCherry fluorescence and human Rab7a gene (SEQ ID NO: 29).

[0115] The mCherry-Rab7a expression vector thus constructed was transfected into 293A cells to induce expression, and 48 hours later, the cells were treated with 100 ng of fluorescently labeled TRP2 for 4 hours and observed using a confocal microscope. Here, the cell nuclei were stained with NucBlue (Invitrogen, NucBlue Live ReadyProbes, R37605).

[0116] Based on its results, such as Figure 6 As shown in A, in the case of TRP2 without a dispersant, a very small amount of TRP2 was delivered to the cytoplasm and was found to be almost not bound to the endosomal marker Rab7 (scale increment is 5 μm). However, it was found that TRP dispersed by various phospholipid preparations was delivered to the cytoplasm in large amounts and existed (co-localized) at the same site as Rab7a similar to Example 4 ( Figure 6 B) Therefore, it can be concluded that, unlike conventional TRP2, the use of micellar dispersions of egg lecithin, soybean lecithin, triolein, and LysoPC enabled the efficient delivery of TRP into the cytoplasm via endosomes.

[0117] Example 6: Analysis of cytotoxicity of TRP formulations containing dispersants

[0118] It was found in Examples 1 and 4 that the water-insoluble TRP protein exists in the form of attachment to the cell membrane surface. In addition, it was found in Example 5 that the micelles formed using the dispersion preparation were delivered to the cytoplasm through the endosome. Cytotoxicity occurs due to the destruction of the cell membrane when TRP without dispersant is overdosed, and when the dispersion preparation is used, it is possible to prevent cell membrane damage by endosomal recycling. Therefore, the cytotoxicity of each TRP preparation in 293A cells (Thermo Fisher, R70507) was analyzed using the JuLI Stage automatic cell imaging system to confirm the cytotoxicity reduction effect caused by the dispersant. Here, 5000 293A cells were spread in a 24-well plate and the cells were allowed to adhere for 16 hours. Then, cells were treated with TRP2 dispersed in various forms at various concentrations, and cell proliferation was observed using a real-time microscope mounted on the JuLI Stage to analyze cytotoxicity.

[0119] Based on its results, such as Figure 7 As shown in A, when the area is about 2cm 2When used at high doses of 1 μg or more in 24-well cell culture plates, undispersed TRP2 caused cytotoxicity. However, 0.25% egg lecithin (TRP2-EL), soybean lecithin (TRP2-SL), triolein (TRP2-triolein) and LysoPC (TRP2-LysoPC) did not cause cytotoxicity even when used in an amount of 20 μg. However, TRP2 dispersed with 10% Cremophor exhibited slight toxicity when used in an amount of 5 μg or more. Therefore, it was confirmed that egg lecithin (TRP2-EL), soybean lecithin (TRP2-SL), triolein (TRP2-triolein) and LysoPC (TRP2-LysoPC) reduced cytotoxicity.

[0120] Example 7: Demonstration of the stability of TRP formulations containing egg lecithin

[0121] In protein drugs, protein stability is a very important issue affecting reproducibility, approval, safety, and drug efficacy. In order to confirm the stability of conventional TRP2, it was frozen at -20°C and allowed to stand at room temperature to evaluate whether the aggregation inhibition ability was maintained over time. As a result, protein aggregation occurred very quickly (within 10 minutes) due to aggregation according to the hydrophobic physicochemical properties of TRP, as described in Table 1 (Figure 8A).

[0122] To evaluate the effect of dispersion formulation on protein stability, in addition to the effects of dispersion formulation described in Examples 3 to 5, TRP2 (TRP2-EL) was dispersed using 0.1% egg lecithin, and the stability of the protein drug over time was measured by DLS.

[0123] Based on the results thereof, as shown in FIG8A , TRP2 dispersed using egg lecithin maintained a very stable state for 56 days or more.

[0124] Storage temperature has a great influence on the stability of protein drugs. To confirm this influence, TRP2 was dispersed at a concentration of 100 μg / ml using 0.1% egg lecithin, and its stability according to various storage temperatures and time periods was measured.

[0125] Based on the results thereof, as shown in FIG8C , it was confirmed that a very stable formulation having an average particle size of 200 nm or less was maintained for different storage time periods at different temperatures from −70° C. to 25° C. In particular, it was confirmed that the dispersing effect of egg lecithin on inhibiting TRP2 protein aggregation was long-lasting and stable, since the very stable form could be maintained for 108 days or more under refrigeration at 4° C.

[0126] To confirm the stability of TRP2 with dispersants other than egg lecithin, TRP2 was dispersed using 10% Cremophor, 0.1% soy lecithin (TRP2-SL), 0.5% triolein (TRP2-triolein), and 0.5% LysoPC (TRP2-LysoPC), and the stability of the particles was measured by DLS immediately after dispersion (day 0) and after storage in a refrigerated state at 4°C for 30 and 70 days.

[0127] Based on the results thereof, as shown in FIG. 8D , it was found that, similar to egg lecithin, each preparation maintained a very stable state for 70 days or more.

[0128] Egg lecithin was used in various concentrations in the drug. To confirm the stability of the formulation according to the concentration of egg lecithin, TRP2 was dispersed at a concentration of 100 μg / ml using 0.5% egg lecithin, and the stability of the protein particles according to various storage temperatures and time periods was measured by DLS.

[0129] Based on its results, such as Fig. 9 As shown in FIG. 8A , protein dispersibility was stable even when 0.5% egg lecithin was used, regardless of storage conditions, which is very similar to the results of FIG. 8C using 0.1% egg lecithin.

[0130] In addition, in order to confirm the aggregation inhibitory effect according to the protein drug dose, TRP2 at various concentrations of 100 to 500 μg / ml was dispersed using 0.1% egg lecithin and stored in a refrigerated state at 4°C for various time periods, and the egg lecithin dispersibility according to the TRP concentration was measured.

[0131] Based on its results, such as Fig.10 As shown in , when 0.1% egg lecithin was used, TRP remained in a very stable state for 20 days or more even at a concentration of 400 μg / ml. However, TRP at a concentration of 500 μg / ml or more caused some aggregation after 7 days.

[0132] To evaluate whether the cell permeability of TRP is maintained even after long-term storage using a dispersion, 293A cells were treated for 4 h with TRP2 dispersed using 0.1% egg lecithin and stored under refrigeration for 108 days and then observed using a confocal microscope.

[0133] Based on its results, such as Fig.11 As shown in A, it was found that the intracellular permeability of TRP dispersed with egg lecithin and stored for 108 days remained unchanged. In addition, to evaluate whether TRP2 protein denatured according to various egg lecithin concentrations and storage temperatures, dispersed protein (TRP2) and TRP2 treated in cells (TRP2 transduction) were confirmed.

[0134] Based on its results, such as Fig.11 As shown in B, TRP2 protein was found not to be denatured at all under various storage temperatures and egg phosphatidylcholine concentrations.

[0135] Example 8: Confirmation of the in vivo effect of TRP2 preparations

[0136] When TRP preparations containing dispersants (Cremophor, egg lecithin or soy lecithin) were applied to animal models, changes in their therapeutic effects were confirmed. In the above examples, the effects of increasing the dispersibility of TRP2 proteins containing phospholipids such as egg lecithin, Cremophor, soy lecithin, triolein and LysoPC, improving safety, enhancing drug efficacy due to increased cell penetration efficiency, and improving stability were confirmed. The enhancement of the therapeutic effects based on these effects was confirmed using a peritoneal fibrosis model.

[0137] For animal experiments, a peritoneal dialysis catheter (Access technologies, rat-O-opening, 7fr x 24" silicone catheter with a rounded tip) was inserted into the abdominal cavity of male Sprague-Dawley rats weighing 250-280 g. Every day after one week after surgery, 20 ml of saline was administered to the control group, and peritoneal dialysis fluid containing 4.25% high-concentration glucose was administered to the experimental group. In order to confirm the effect of the TRP2 preparation on the treatment of peritoneal fibrosis, various doses of TRP2 were mixed with the peritoneal dialysis fluid and injected. Therefore, the TRP2 protein drug was administered once a week. Four weeks after the administration of the peritoneal dialysis fluid, each animal was sacrificed, and the peritoneum was collected, fixed in 10% neutral formalin, embedded in paraffin, and stained with Masson's Trichrome, so the extent of histological fibrosis was determined by measuring the thickness of fibrosis at least 30 sites.

[0138] In the case of conventional undispersed TRP2, as shown in Table 5 and Fig.12 As shown in , the peritoneum of normal control (con) has a thickness of about 32 μm, but when high-concentration peritoneal dialysis fluid (PDF) was administered for 4 weeks, the peritoneal thickness was measured to be 230 μm or thicker due to peritoneal fibrosis. In this procedure, when conventional TRP2 was mixed and administered, fibrosis was reduced by about 1 / 2 at a dose of 500 μg / kg.

[0139] To confirm the therapeutic effect of using a dispersant such as Cremophor, egg lecithin or soy lecithin, TRP2 was dispersed using 10% Cremophor, 0.1% egg lecithin and 0.1% soy lecithin, and various doses thereof were mixed with peritoneal dialysis fluid and administered once a week.

[0140] Based on the results, as shown in Tables 6 to 8 below, Figures 13 to 15 As shown in Table 9, the peritoneal fibrosis inhibitory effect of TRP2 was greatly increased when a dispersant was used. Fig.16 As shown in , when Cremophor, egg lecithin, and soybean lecithin were used as dispersants, the therapeutic effect was increased by about 37 times, about 18 times, and about 8 times, respectively, for 50% effective dose (effective dose 50, ED50).

[0141] [Table 5]

[0142]

[0143] [Table 6]

[0144]

[0145] [Table 7]

[0146]

[0147] [Table 8]

[0148]

[0149] [Table 9]

[0150]

[0151] Practicality

[0152] When the pharmaceutical preparation according to the present invention is used, by inhibiting the aggregation of inactivated TRP, dispersibility is improved, cell permeability is increased, cytotoxicity is reduced, stability of the protein drug is increased, and therapeutic effects are also improved.

[0153] Although the specific embodiments of the present invention have been disclosed in detail as described above, it will be apparent to those skilled in the art that the description is merely a preferred exemplary embodiment and should not be interpreted as limiting the scope of the present invention. Therefore, the essential scope of the present invention will be defined by the appended claims and their equivalents.

[0154] Sequence Listing Free Text

[0155] Electronic files are attached. <110> Metropolitan Life Sciences Ltd. <120> Pharmaceutical preparations of inactivated polypeptide TRP <130> PP-B2415 <150> KR 10-2019-0076443 <151> 2019-06-26 <160> 29 <170> KopatentIn 2.0 <210> 1 <211> 114 <212> PRT <213> Homo sapiens <400> 1 Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg 1 5 10 15 His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile 20 25 30 Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys Pro 35 40 45 Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val Gln 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys Val 65 70 75 80 Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu 85 90 95 Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu Gly Cys Gly 100 105 110 Cys Arg <210> 2 <211> 183 <212> PRT <213> Homo sapiens <400> 2 Ser Thr Gly Val Leu Leu Pro Leu Gln Asn Asn Glu Leu Pro Gly Ala 1 5 10 15 Glu Tyr Gln Tyr Lys Lys Asp Glu Val Trp Glu Glu Arg Lys Pro Tyr 20 25 30 Lys Thr Leu Gln Ala Gln Ala Pro Glu Lys Ser Lys Asn Lys Lys Lys 35 40 45 Gln Arg Lys Gly Pro His Arg Lys Ser Gln Thr Leu Gln Phe Asp Glu 50 55 60 Gln Thr Leu Lys Lys Ala Arg Arg Lys Gln Trp Ile Glu Pro Arg Asn 65 70 75 80 Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp Ser 85 90 95 Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser Gly 100 105 110 Ala Cys Gln Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His Ala 115 120 125 Thr Ile Gln Ser Ile Val Arg Ala Val Gly Val Val Pro Gly Ile Pro 130 135 140 Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser Ile Leu Phe 145 150 155 160 Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met Thr 165 170 175 Val Glu Ser Cys Ala Cys Arg 180 <210> 3 <211> 116 <212> PRT <213> Homo sapiens <400> 3 Ser Pro Lys His His Ser Gln Arg Ala Arg Lys Lys Asn Lys Asn Cys 1 5 10 15 Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp 20 25 30 Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Asp 35 40 45 Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile 50 55 60 Val Gln Thr Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys Ala Cys 65 70 75 80 Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu 85 90 95 Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly 100 105 110 Cys Gly Cys Arg 115 <210> 4 <211> 138 <212> PRT <213> Homo sapiens <400> 4 Ala Ala Asn Lys Arg Lys Asn Gln Asn Arg Asn Lys Ser Ser Ser His 1 5 10 15 Gln Asp Ser Ser Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser Glu 20 25 30 Gln Lys Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp 35 40 45 Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe 50 55 60 Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala 65 70 75 80 Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met Phe Pro Asp 85 90 95 His Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser 100 105 110 Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg 115 120 125 Asn Met Val Val Arg Ser Cys Gly Cys His 130 135 <210> 5 <211> 139 <212> PRT <213> Homo sapiens <400> 5 Ser Ala Ser Ser Arg Arg Arg Gln Gln Ser Arg Asn Arg Ser Thr Gln 1 5 10 15 Ser Gln Asp Val Ala Arg Val Ser Ser Ala Ser Asp Tyr Asn Ser Ser 20 25 30 Glu Leu Lys Thr Ala Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gln 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala 50 55 60 Asn Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met Asn Pro 85 90 95 Glu Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 6 <211> 139 <212> PRT <213> Homo sapiens <400> 6 Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 <210> 7 <211> 133 <212> PRT <213> Homo sapiens <400> 7 Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu Pro Gln Ala Asn Arg Leu 1 5 10 15 Pro Gly Ile Phe Asp Asp Val His Gly Ser His Gly Arg Gln Val Cys 20 25 30 Arg Arg His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Leu Asp 35 40 45 Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu 50 55 60 Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala Ile 65 70 75 80 Leu Gln Ser Leu Val His Leu Met Met Pro Asp Ala Val Pro Lys Ala 85 90 95 Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp 100 105 110 Ser Ser Asn Asn Val Ile Leu Arg Lys His Arg Asn Met Val Val Lys 115 120 125 Ala Cys Gly Cys His 130 <210> 8 <211> 110 <212> PRT <213> Homo sapiens <400> 8 Ser Ala Gly Ala Gly Ser His Cys Gln Lys Thr Ser Leu Arg Val Asn 1 5 10 15 Phe Glu Asp Ile Gly Trp Asp Ser Trp Ile Ile Ala Pro Lys Glu Tyr 20 25 30 Glu Ala Tyr Glu Cys Lys Gly Gly Cys Phe Phe Pro Leu Ala Asp Asp 35 40 45 Val Thr Pro Thr Lys His Ala Ile Val Gln Thr Leu Val His Leu Lys 50 55 60 Phe Pro Thr Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser 65 70 75 80 Pro Ile Ser Val Leu Tyr Lys Asp Asp Met Gly Val Pro Thr Leu Lys 85 90 95 Tyr His Tyr Glu Gly Met Ser Val Ala Glu Cys Gly Cys Arg 100 105 110 <210> 9 <211> 108 <212> PRT <213> Homo sapiens <400> 9 Asn Ala Lys Gly Asn Tyr Cys Lys Arg Thr Pro Leu Tyr Ile Asp Phe 1 5 10 15 Lys Glu Ile Gly Trp Asp Ser Trp Ile Ile Ala Pro Pro Gly Tyr Glu 20 25 30 Ala Tyr Glu Cys Arg Gly Val Cys Asn Tyr Pro Leu Ala Glu His Leu 35 40 45 Thr Pro Thr Lys His Ala Ile Ile Gln Ala Leu Val His Leu Lys Asn 50 55 60 Ser Gln Lys Ala Ser Lys Ala Cys Cys Val Pro Thr Lys Leu Glu Pro 65 70 75 80 Ile Ser Ile Leu Tyr Leu Asp Lys Gly Val Val Thr Tyr Lys Phe Lys 85 90 95 Tyr Glu Gly Met Ala Val Ser Glu Cys Gly Cys Arg 100 105 <210> 10 <211> 108 <212> PRT <213> Homo sapiens <400> 10 Asn Ala Lys Gly Asn Tyr Cys Lys Arg Thr Pro Leu Tyr Ile Asp Phe 1 5 10 15 Lys Glu Ile Gly Trp Asp Ser Trp Ile Ile Ala Pro Pro Gly Tyr Glu 20 25 30 Ala Tyr Glu Cys Arg Gly Val Cys Asn Tyr Pro Leu Ala Glu His Leu 35 40 45 Thr Pro Thr Lys His Ala Ile Ile Gln Ala Leu Val His Leu Lys Asn 50 55 60 Ser Gln Lys Ala Ser Lys Ala Cys Cys Val Pro Thr Lys Leu Glu Pro 65 70 75 80 Ile Ser Ileu Tyr Leu Asp Lys Gly Val Val Thr Tyr Lys Phe Lys 85 90 95 Tyr Glu Gly Met Ala Val Ser Glu Cys Gly Cys Arg 100 105 <210> 11 <211> 104 <212> PRT <213> Homo sapiens <400> 11 Ser Arg Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu Gly 1 5 10 15 Trp Asp Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala Tyr His Cys 20 25 30 Glu Gly Leu Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 35 40 45 His Ala Ile Gln Thr Leu Leu Asn Ser Met Ala Pro Asp Ala Ala 50 55 60 Pro Wing Ser Cys Cys Val Pro Wing Arg Leu Ser Pro Ile Ser Ile Leu 65 70 75 80 Tyr Ile Asp Ala Ala Asn Val Val Tyr Lys Gln Tyr Glu Asp Met 85 90 95 Val Val Glu Ala Cys Gly Cys Arg 100 <210> 12 <211> 120 <212> PRT <213> Homo sapiens <400> 12 Thr Ala Phe Ala Ser Arg His Gly Lys Arg His Gly Lys Lys Ser Arg 1 5 10 15 Leu Arg Cys Ser Lys Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly 20 25 30 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys 35 40 45 Glu Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55 60 His Ala Ile Ile Gln Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr 65 70 75 80 Pro Pro Ser Cys Cys Val Pro Thr Lys Leu Thr Pro Ile Ser Ile Leu 85 90 95 Tyr Ile Asp Ala Gly Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys Gly Cys Arg 115 120 <210> 13 <211> 120 <212> PRT <213> Homo sapiens <400> 13 Ala Pro Leu Ala Thr Arg Gln Gly Lys Arg Pro Ser Lys Asn Leu Lys 1 5 10 15 Ala Arg Cys Ser Arg Lys Ala Leu His Val Asn Phe Lys Asp Met Gly 20 25 30 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys 35 40 45 Glu Gly Leu Cys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55 60 His Ala Val Ile Gln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr 65 70 75 80 Pro Pro Thr Cys Cys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu 85 90 95 Phe Ile Asp Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys Gly Cys Arg 115 120 <210> 14 <211> 281 <212> PRT <213> Homo sapiens <400> 14 Met Val Ala Gly Thr Arg Cys Leu Leu Ala Leu Leu Leu Pro Gln Val 1 5 10 15 Leu Leu Gly Gly Ala Ala Gly Leu Val Pro Glu Leu Gly Arg Arg Lys 20 25 30 Phe Ala Ala Ala Ser Ser Gly Arg Pro Ser Ser Gln Pro Ser Asp Glu 35 40 45 Val Leu Ser Glu Phe Glu Leu Arg Leu Leu Ser Met Phe Gly Leu Lys 50 55 60 Gln Arg Pro Thr Pro Ser Arg Asp Ala Val Val Pro Pro Tyr Met Leu 65 70 75 80 Asp Leu Tyr Arg Arg His Ser Gly Gln Pro Gly Ser Pro Ala Pro Asp 85 90 95 His Arg Leu Glu Arg Ala Ala Ser Arg Ala Asn Thr Val Arg Ser Phe 100 105 110 His His Glu Glu Ser Leu Glu Glu Leu Pro Glu Thr Ser Gly Lys Thr 115 120 125 Thr Arg Arg Phe Phe Phe Asn Leu Ser Ser Ile Pro Thr Glu Glu Phe 130 135 140 Ile Thr Ser Ala Glu Leu Gln Val Phe Arg Glu Gln Met Gln Asp Ala 145 150 155 160 Leu Gly Asn Asn Ser Ser Phe His His Arg Ile Asn Ile Tyr Glu Ile 165 170 175 Ile Lys Pro Ala Thr Ala Asn Ser Lys Phe Pro Val Thr Arg Leu Leu 180 185 190 Asp Thr Arg Leu Val Asn Gln Asn Ala Ser Arg Trp Glu Ser Phe Asp 195 200 205 Val Thr Pro Ala Val Met Arg Trp Thr Ala Gly His Ala Asn His Gly 210 215 220 Phe Val Val Glu Val Ala His Leu Glu Glu Lys Gln Gly Val Ser Lys 225 230 235 240 Arg His Val Arg Ile Ser Arg Ser Leu His Gln Asp Glu His Ser Trp 245 250 255 Ser Gln Ile Arg Pro Leu Leu Val Thr Phe Gly His Asp Gly Lys Gly 260 265 270 His Pro Leu His Lys Arg Glu Lys Arg 275 280 <210> 15 <211> 289 <212> PRT <213> Homo sapiens <400> 15 Met Ala Gly Ala Ser Arg Leu Leu Phe Leu Trp Leu Gly Cys Phe Cys 1 5 10 15 Val Ser Leu Ala Gln Gly Glu Arg Pro Lys Pro Pro Phe Pro Glu Leu 20 25 30 Arg Lys Ala Val Pro Gly Asp Arg Thr Ala Gly Gly Gly Pro Asp Ser 35 40 45 Glu Leu Gln Pro Gln Asp Lys Val Ser Glu His Met Leu Arg Leu Tyr 50 55 60 Asp Arg Tyr Ser Thr Val Gln Ala Ala Arg Thr Pro Gly Ser Leu Glu 65 70 75 80 Gly Gly Ser Gln Pro Trp Arg Pro Arg Leu Leu Arg Glu Gly Asn Thr 85 90 95 Val Arg Ser Phe Arg Ala Ala Ala Ala Glu Thr Leu Glu Arg Lys Gly 100 105 110 Leu Tyr Ile Phe Asn Leu Thr Ser Leu Thr Lys Ser Glu Asn Ile Leu 115 120 125 Ser Ala Thr Leu Tyr Phe Cys Ile Gly Glu Leu Gly Asn Ile Ser Leu 130 135 140 Ser Cys Pro Val Ser Gly Gly Cys Ser His His Ala Gln Arg Lys His 145 150 155 160 Ile Gln Ile Asp Leu Ser Ala Trp Thr Leu Lys Phe Ser Arg Asn Gln 165 170 175 Ser Gln Leu Leu Gly His Leu Ser Val Asp Met Ala Lys Ser His Arg 180 185 190 Asp Ile Met Ser Trp Leu Ser Lys Asp Ile Thr Gln Phe Leu Arg Lys 195 200 205 Ala Lys Glu Asn Glu Glu Phe Leu Ile Gly Phe Asn Ile Thr Ser Lys 210 215 220 Gly Arg Gln Leu Pro Lys Arg Arg Leu Pro Phe Pro Glu Pro Tyr Ile 225 230 235 240 Leu Val Tyr Ala Asn Asp Ala Ala Ile Ser Glu Pro Glu Ser Val Val 245 250 255 Ser Ser Leu Gln Gly His Arg Asn Phe Pro Thr Gly Thr Val Pro Lys 260 265 270 Trp Asp Ser His Ile Arg Ala Ala Leu Ser Ile Glu Arg Arg Lys Lys 275 280 285 Arg <210> 16 <211> 292 <212> PRT <213> Homo sapiens <400> 16 Met Ile Pro Gly Asn Arg Met Leu Met Val Val Leu Leu Cys Gln Val 1 5 10 15 Leu Leu Gly Gly Ala Ser His Ala Ser Leu Ile Pro Glu Thr Gly Lys 20 25 30 Lys Lys Val Ala Glu Ile Gln Gly His Ala Gly Gly Arg Arg Ser Gly 35 40 45 Gln Ser His Glu Leu Leu Arg Asp Phe Glu Ala Thr Leu Leu Gln Met 50 55 60 Phe Gly Leu Arg Arg Arg Pro Gln Pro Ser Lys Ser Ala Val Ile Pro 65 70 75 80 Asp Tyr Met Arg Asp Leu Tyr Arg Leu Gln Ser Gly Glu Glu Glu Glu 85 90 95 Glu Gln Ile His Ser Thr Gly Leu Glu Tyr Pro Glu Arg Pro Ala Ser 100 105 110 Arg Ala Asn Thr Val Arg Ser Phe His His Glu Glu His Leu Glu Asn 115 120 125 Ile Pro Gly Thr Ser Glu Asn Ser Ala Phe Arg Phe Leu Phe Asn Leu 130 135 140 Ser Ser Ile Pro Glu Asn Glu Ala Ile Ser Ser Ala Glu Leu Arg Leu 145 150 155 160 Phe Arg Glu Gln Val Asp Gln Gly Pro Asp Trp Glu Arg Gly Phe His 165 170 175 Arg Ile Asn Ile Tyr Glu Val Met Lys Pro Pro Ala Glu Val Val Pro 180 185 190 Gly His Leu Ile Thr Arg Leu Leu Asp Thr Arg Leu Val His His Asn 195 200 205 Val Thr Arg Trp Glu Thr Phe Asp Val Ser Pro Ala Val Leu Arg Trp 210 215 220 Thr Arg Glu Lys Gln Pro Asn Tyr Gly Leu Ala Ile Glu Val Thr His 225 230 235 240 Leu His Gln Thr Arg Thr His Gln Gly Gln His Val Arg Ile Ser Arg 245 250 255 Ser Leu Pro Gln Gly Ser Gly Asn Trp Ala Gln Leu Arg Pro Leu Leu 260 265 270 Val Thr Phe Gly His Asp Gly Arg Gly His Ala Leu Thr Arg Arg Arg 275 280 285 Arg Ala Lys Arg 290 <210> 17 <211> 316 <212> PRT <213> Homo sapiens <400> 17 Met His Leu Thr Val Phe Leu Leu Lys Gly Ile Val Gly Phe Leu Trp 1 5 10 15 Ser Cys Trp Val Leu Val Gly Tyr Ala Lys Gly Gly Leu Gly Asp Asn 20 25 30 His Val His Ser Ser Phe Ile Tyr Arg Arg Leu Arg Asn His Glu Arg 35 40 45 Arg Glu Ile Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg 50 55 60 Pro Arg Pro Phe Ser Pro Gly Lys Gln Ala Ser Ser Ala Pro Leu Phe 65 70 75 80 Met Leu Asp Leu Tyr Asn Ala Met Thr Asn Glu Glu Asn Pro Glu Glu 85 90 95 Ser Glu Tyr Ser Val Arg Ala Ser Leu Ala Glu Glu Thr Arg Gly Ala 100 105 110 Arg Lys Gly Tyr Pro Ala Ser Pro Asn Gly Tyr Pro Arg Arg Ile Gln 115 120 125 Leu Ser Arg Thr Thr Pro Leu Thr Thr Gln Ser Pro Pro Leu Ala Ser 130 135 140 Leu His Asp Thr Asn Phe Leu Asn Asp Ala Asp Met Val Met Ser Phe 145 150 155 160 Val Asn Leu Val Glu Arg Asp Lys Asp Phe Ser His Gln Arg Arg His 165 170 175 Tyr Lys Glu Phe Arg Phe Asp Leu Thr Gln Ile Pro His Gly Glu Ala 180 185 190 Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Arg Ser Asn Asn Arg 195 200 205 Phe Glu Asn Glu Thr Ile Lys Ile Ser Ile Tyr Gln Ile Ile Lys Glu 210 215 220 Tyr Thr Asn Arg Asp Ala Asp Leu Phe Leu Leu Asp Thr Arg Lys Ala 225 230 235 240 Gln Ala Leu Asp Val Gly Trp Leu Val Phe Asp Ile Thr Val Thr Ser 245 250 255 Asn His Trp Val Ile Asn Pro Gln Asn Asn Leu Gly Leu Gln Leu Cys 260 265 270 Ala Glu Thr Gly Asp Gly Arg Ser Ile Asn Val Lys Ser Ala Gly Leu 275 280 285 Val Gly Arg Gln Gly Pro Gln Ser Lys Gln Pro Phe Met Val Ala Phe 290 295 300 Phe Lys Ala Ser Glu Val Leu Leu Arg Ser Val Arg 305 310 315 <210> 18 <211> 374 <212> PRT <213> Homo sapiens <400> 18 Met Pro Gly Leu Gly Arg Arg Ala Gln Trp Leu Cys Trp Trp Trp Gly 1 5 10 15 Leu Leu Cys Ser Cys Cys Gly Pro Pro Pro Leu Arg Pro Pro Leu Pro 20 25 30 Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Leu Leu Gly Asp Gly Gly 35 40 45 Ser Pro Gly Arg Thr Glu Gln Pro Pro Pro Ser Pro Gln Ser Ser Ser 50 55 60 Gly Phe Leu Tyr Arg Arg Leu Lys Thr Gln Glu Lys Arg Glu Met Gln 65 70 75 80 Lys Glu Ile Leu Ser Val Leu Gly Leu Pro His Arg Pro Arg Pro Leu 85 90 95 His Gly Leu Gln Gln Pro Gln Pro Pro Ala Leu Arg Gln Gln Glu Glu 100 105 110 Gln Gln Gln Gln Gln Gln Leu Pro Arg Gly Glu Pro Pro Pro Gly Arg 115 120 125 Leu Lys Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr Asn Ala Leu Ser 130 135 140 Ala Asp Asn Asp Glu Asp Gly Ala Ser Glu Gly Glu Arg Gln Gln Ser 145 150 155 160 Trp Pro His Glu Ala Ala Ser Ser Ser Gln Arg Arg Gln Pro Pro Pro 165 170 175 Gly Ala Ala His Pro Leu Asn Arg Lys Ser Leu Leu Ala Pro Gly Ser 180 185 190 Gly Ser Gly Gly Ala Ser Pro Leu Thr Ser Ala Gln Asp Ser Ala Phe 195 200 205 Leu Asn Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu Tyr 210 215 220 Asp Lys Glu Phe Ser Pro Arg Gln Arg His His Lys Glu Phe Lys Phe 225 230 235 240 Asn Leu Ser Gln Ile Pro Glu Gly Glu Val Val Thr Ala Ala Glu Phe 245 250 255 Arg Ile Tyr Lys Asp Cys Val Met Gly Ser Phe Lys Asn Gln Thr Phe 260 265 270 Leu Ile Ser Ile Tyr Gln Val Leu Gln Glu His Gln His Arg Asp Ser 275 280 285 Asp Leu Phe Leu Leu Asp Thr Arg Val Val Trp Ala Ser Glu Glu Gly 290 295 300 Trp Leu Glu Phe Asp Ile Thr Ala Thr Ser Asn Leu Trp Val Val Thr 305 310 315 320 Pro Gln His Asn Met Gly Leu Gln Leu Ser Val Val Thr Arg Asp Gly 325 330 335 Val His Val His Pro Arg Ala Ala Gly Leu Val Gly Arg Asp Gly Pro 340 345 350 Tyr Asp Lys Gln Pro Phe Met Val Ala Phe Phe Lys Val Ser Glu Val 355 360 365 His Val Arg Thr Thr Arg 370 <210> 19 <211> 292 <212> PRT <213> Homo sapiens <400> 19 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30 Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser 35 40 45 Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu 50 55 60 Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro 65 70 75 80 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly 85 90 95 Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser 100 105 110 Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr 115 120 125 Asp Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135 140 Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 145 150 155 160 Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile 165 170 175 Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile 180 185 190 Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu 195 200 205 Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 210 215 220 Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg 225 230 235 240 His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser 245 250 255 Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn 260 265 270 Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 275 280 285 Arg Ser Ile Arg 290 <210> 20 <211> 269 <212> PRT <213> Homo sapiens <400> 20 Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 1 5 10 15 Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro 20 25 30 Gln Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gln Arg Glu Ile 35 40 45 Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Pro Pro 50 55 60 Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu 65 70 75 80 Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Pro Ala Glu 85 90 95 Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val 100 105 110 Glu Arg Asp Arg Ala Leu Gly His Gln Glu Pro His Trp Lys Glu Phe 115 120 125 Arg Phe Asp Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Thr Ala Ala 130 135 140 Glu Phe Arg Ile Tyr Lys Val Pro Ser Ile His Leu Leu Asn Arg Thr 145 150 155 160 Leu His Val Ser Met Phe Gln Val Val Gln Glu Gln Ser Asn Arg Glu 165 170 175 Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr Leu Arg Ala Gly Asp Glu 180 185 190 Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu 195 200 205 Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp 210 215 220 Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly Gln Arg Ala 225 230 235 240 Pro Arg Ser Gln Gln Pro Phe Val Val Thr Phe Phe Arg Ala Ser Pro 245 250 255 Ser Pro Ile Arg Thr Pro Arg Ala Val Arg Pro Leu Arg 260 265 <210> 21 <211> 318 <212> PRT <213> Homo sapiens <400> 21 Met Cys Pro Gly Ala Leu Trp Val Ala Leu Pro Leu Leu Ser Leu Leu 1 5 10 15 Ala Gly Ser Leu Gln Gly Lys Pro Leu Gln Ser Trp Gly Arg Gly Ser 20 25 30 Ala Gly Gly Asn Ala His Ser Pro Leu Gly Val Pro Gly Gly Gly Leu 35 40 45 Pro Glu His Thr Phe Asn Leu Lys Met Phe Leu Glu Asn Val Lys Val 50 55 60 Asp Phe Leu Arg Ser Leu Asn Leu Ser Gly Val Pro Ser Gln Asp Lys 65 70 75 80 Thr Arg Val Glu Pro Pro Gln Tyr Met Ile Asp Leu Tyr Asn Arg Tyr 85 90 95 Thr Ser Asp Lys Ser Thr Pro Ala Ser Asn Ile Val Arg Ser Phe Ser 100 105 110 Met Glu Asp Ala Ile Ser Ile Thr Ala Thr Glu Asp Phe Pro Phe Gln 115 120 125 Lys His Ile Leu Leu Phe Asn Ile Ser Ile Pro Arg His Glu Gln Ile 130 135 140 Thr Arg Ala Glu Leu Arg Leu Tyr Val Ser Cys Gln Asn His Val Asp 145 150 155 160 Pro Ser His Asp Leu Lys Gly Ser Val Val Ile Tyr Asp Val Leu Asp 165 170 175 Gly Thr Asp Ala Trp Asp Ser Ala Thr Glu Thr Lys Thr Phe Leu Val 180 185 190 Ser Gln Asp Ile Gln Asp Glu Gly Trp Glu Thr Leu Glu Val Ser Ser 195 200 205 Ala Val Lys Arg Trp Val Arg Ser Asp Ser Thr Lys Ser Lys Asn Lys 210 215 220 Leu Glu Val Thr Val Glu Ser His Arg Lys Gly Cys Asp Thr Leu Asp 225 230 235 240 Ile Ser Val Pro Pro Gly Ser Arg Asn Leu Pro Phe Phe Val Val Phe 245 250 255 Ser Asn Asp His Ser Ser Gly Thr Lys Glu Thr Arg Leu Glu Leu Arg 260 265 270 Glu Met Ile Ser His Glu Gln Glu Ser Val Leu Lys Lys Leu Ser Lys 275 280 285 Asp Gly Ser Thr Glu Ala Gly Glu Ser Ser His Glu Glu Asp Thr Asp 290 295 300 Gly His Val Ala Ala Gly Ser Thr Leu Ala Arg Arg Lys Arg 305 310 315 <210> 22 <211> 316 <212> PRT <213> Homo sapiens <400> 22 Met Gly Ser Leu Val Leu Thr Leu Cys Ala Leu Phe Cys Leu Ala Ala 1 5 10 15 Tyr Leu Val Ser Gly Ser Pro Ile Met Asn Leu Glu Gln Ser Pro Leu 20 25 30 Glu Glu Asp Met Ser Leu Phe Gly Asp Val Phe Ser Glu Gln Asp Gly 35 40 45 Val Asp Phe Asn Thr Leu Leu Gln Ser Met Lys Asp Glu Phe Leu Lys 50 55 60 Thr Leu Asn Leu Ser Asp Ile Pro Thr Gln Asp Ser Ala Lys Val Asp 65 70 75 80 Pro Pro Glu Tyr Met Leu Glu Leu Tyr Asn Lys Phe Ala Thr Asp Arg 85 90 95 Thr Ser Met Pro Ser Ala Asn Ile Ile Arg Ser Phe Lys Asn Glu Asp 100 105 110 Leu Phe Ser Gln Pro Val Ser Phe Asn Gly Leu Arg Lys Tyr Pro Leu 115 120 125 Leu Phe Asn Val Ser Ile Pro His His Glu Glu Val Ile Met Ala Glu 130 135 140 Leu Arg Leu Tyr Thr Leu Val Gln Arg Asp Arg Met Ile Tyr Asp Gly 145 150 155 160 Val Asp Arg Lys Ile Thr Ile Phe Glu Val Leu Glu Ser Lys Gly Asp 165 170 175 Asn Glu Gly Glu Arg Asn Met Leu Val Leu Val Ser Gly Glu Ile Tyr 180 185 190 Gly Thr Asn Ser Glu Trp Glu Thr Phe Asp Val Thr Asp Ala Ile Arg 195 200 205 Arg Trp Gln Lys Ser Gly Ser Ser Thr His Gln Leu Glu Val His Ile 210 215 220 Glu Ser Lys His Asp Glu Ala Glu Asp Ala Ser Ser Gly Arg Leu Glu 225 230 235 240 Ile Asp Thr Ser Ala Gln Asn Lys His Asn Pro Leu Leu Ile Val Phe 245 250 255 Ser Asp Asp Gln Ser Ser Asp Lys Glu Arg Lys Glu Glu Leu Asn Glu 260 265 270 Met Ile Ser His Glu Gln Leu Pro Glu Leu Asp Asn Leu Gly Leu Asp 275 280 285 Ser Phe Ser Ser Gly Pro Gly Glu Glu Ala Leu Leu Gln Met Arg Ser 290 295 300 Asn Ile Ile Tyr Asp Ser Thr Ala Arg Ile Arg Arg 305 310 315 <210> 23 <211> 316 <212> PRT <213> Homo sapiens <400> 23 Met Gly Ser Leu Val Leu Thr Leu Cys Ala Leu Phe Cys Leu Ala Ala 1 5 10 15 Tyr Leu Val Ser Gly Ser Pro Ile Met Asn Leu Glu Gln Ser Pro Leu 20 25 30 Glu Glu Asp Met Ser Leu Phe Gly Asp Val Phe Ser Glu Gln Asp Gly 35 40 45 Val Asp Phe Asn Thr Leu Leu Gln Ser Met Lys Asp Glu Phe Leu Lys 50 55 60 Thr Leu Asn Leu Ser Asp Ile Pro Thr Gln Asp Ser Ala Lys Val Asp 65 70 75 80 Pro Pro Glu Tyr Met Leu Glu Leu Tyr Asn Lys Phe Ala Thr Asp Arg 85 90 95 Thr Ser Met Pro Ser Ala Asn Ile Ile Arg Ser Phe Lys Asn Glu Asp 100 105 110 Leu Phe Ser Gln Pro Val Ser Phe Asn Gly Leu Arg Lys Tyr Pro Leu 115 120 125 Leu Phe Asn Val Ser Ile Pro His His Glu Glu Val Ile Met Ala Glu 130 135 140 Leu Arg Leu Tyr Thr Leu Val Gln Arg Asp Arg Met Ile Tyr Asp Gly 145 150 155 160 Val Asp Arg Lys Ile Thr Ile Phe Glu Val Leu Glu Ser Lys Gly Asp 165 170 175 Asn Glu Gly Glu Arg Asn Met Leu Val Leu Val Ser Gly Glu Ile Tyr 180 185 190 Gly Thr Asn Ser Glu Trp Glu Thr Phe Asp Val Thr Asp Ala Ile Arg 195 200 205 Arg Trp Gln Lys Ser Gly Ser Ser Thr His Gln Leu Glu Val His Ile 210 215 220 Glu Ser Lys His Asp Glu Ala Glu Asp Ala Ser Ser Gly Arg Leu Glu 225 230 235 240 Ile Asp Thr Ser Ala Gln Asn Lys His Asn Pro Leu Leu Ile Val Phe 245 250 255 Ser Asp Asp Gln Ser Ser Asp Lys Glu Arg Lys Glu Glu Leu Asn Glu 260 265 270 Met Ile Ser His Glu Gln Leu Pro Glu Leu Asp Asn Leu Gly Leu Asp 275 280 285 Ser Phe Ser Ser Gly Pro Gly Glu Glu Ala Leu Leu Gln Met Arg Ser 290 295 300 Asn Ile Ile Tyr Asp Ser Thr Ala Arg Ile Arg Arg 305 310 315 <210> 24 <211> 346 <212> PRT <213> Homo sapiens <400> 24 Met Asp Leu Ser Ala Ala Ala Ala Leu Cys Leu Trp Leu Leu Ser Ala 1 5 10 15 Cys Arg Pro Arg Asp Gly Leu Glu Ala Ala Ala Val Leu Arg Ala Ala 20 25 30 Gly Ala Gly Pro Val Arg Ser Pro Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45 Gly Gly Arg Thr Leu Ala Gln Ala Ala Gly Ala Ala Ala Val Pro Ala 50 55 60 Ala Ala Val Pro Arg Ala Arg Ala Ala Arg Arg Ala Ala Gly Ser Gly 65 70 75 80 Phe Arg Asn Gly Ser Val Val Pro His His Phe Met Met Ser Leu Tyr 85 90 95 Arg Ser Leu Ala Gly Arg Ala Pro Ala Gly Ala Ala Ala Val Ser Ala 100 105 110 Ser Gly His Gly Arg Ala Asp Thr Ile Thr Gly Phe Thr Asp Gln Ala 115 120 125 Thr Gln Asp Glu Ser Ala Ala Glu Thr Gly Gln Ser Phe Leu Phe Asp 130 135 140 Val Ser Ser Leu Asn Asp Ala Asp Glu Val Val Gly Ala Glu Leu Arg 145 150 155 160 Val Leu Arg Arg Gly Ser Pro Glu Ser Gly Pro Gly Ser Trp Thr Ser 165 170 175 Pro Pro Leu Leu Leu Leu Ser Thr Cys Pro Gly Ala Ala Arg Ala Pro 180 185 190 Arg Leu Leu Tyr Ser Arg Ala Ala Glu Pro Leu Val Gly Gln Arg Trp 195 200 205 Glu Ala Phe Asp Val Ala Asp Ala Met Arg Arg His Arg Arg Glu Pro 210 215 220 Arg Pro Pro Arg Ala Phe Cys Leu Leu Leu Arg Ala Val Ala Gly Pro 225 230 235 240 Val Pro Ser Pro Leu Ala Leu Arg Arg Leu Gly Phe Gly Trp Pro Gly 245 250 255 Gly Gly Gly Ser Ala Ala Glu Glu Arg Ala Val Leu Val Val Ser Ser 260 265 270 Arg Thr Gln Arg Lys Glu Ser Leu Phe Arg Glu Ile Arg Ala Gln Ala 275 280 285 Arg Ala Leu Gly Ala Ala Leu Ala Ser Glu Pro Leu Pro Asp Pro Gly 290 295 300 Thr Gly Thr Ala Ser Pro Arg Ala Val Ile Gly Gly Arg Arg Arg Arg 305 310 315 320 Arg Thr Ala Leu Ala Gly Thr Arg Thr Ala Gln Gly Ser Gly Gly Gly 325 330 335 Ala Gly Arg Gly His Gly Arg Arg Gly Arg 340 345 <210> 25 <211> 335 <212> PRT <213> Homo sapiens <400> 25 Met Asp Thr Pro Arg Val Leu Leu Ser Ala Val Phe Leu Ile Ser Phe 1 5 10 15 Leu Trp Asp Leu Pro Gly Phe Gln Gln Ala Ser Ile Ser Ser Ser Ser 20 25 30 Ser Ser Ala Glu Leu Gly Ser Thr Lys Gly Met Arg Ser Arg Lys Glu 35 40 45 Gly Lys Met Gln Arg Ala Pro Arg Asp Ser Asp Ala Gly Arg Glu Gly 50 55 60 Gln Glu Pro Gln Pro Arg Pro Gln Asp Glu Pro Arg Ala Gln Gln Pro 65 70 75 80 Arg Ala Gln Glu Pro Pro Gly Arg Gly Pro Arg Val Val Pro His Glu 85 90 95 Tyr Met Leu Ser Ile Tyr Arg Thr Tyr Ser Ile Ala Glu Lys Leu Gly 100 105 110 Ile Asn Ala Ser Phe Phe Gln Ser Ser Lys Ser Ala Asn Thr Ile Thr 115 120 125 Ser Phe Val Asp Arg Gly Leu Asp Asp Leu Ser His Thr Pro Leu Arg 130 135 140 Arg Gln Lys Tyr Leu Phe Asp Val Ser Met Leu Ser Asp Lys Glu Glu 145 150 155 160 Leu Val Gly Ala Glu Leu Arg Leu Phe Arg Gln Ala Pro Ser Ala Pro 165 170 175 Trp Gly Pro Pro Ala Gly Pro Leu His Val Gln Leu Phe Pro Cys Leu 180 185 190 Ser Pro Leu Leu Leu Asp Ala Arg Thr Leu Asp Pro Gln Gly Ala Pro 195 200 205 Pro Ala Gly Trp Glu Val Phe Asp Val Trp Gln Gly Leu Arg His Gln 210 215 220 Pro Trp Lys Gln Leu Cys Leu Glu Leu Arg Ala Ala Trp Gly Glu Leu 225 230 235 240 Asp Ala Gly Glu Ala Glu Ala Arg Ala Arg Gly Pro Gln Gln Pro Pro 245 250 255 Pro Pro Asp Leu Arg Ser Leu Gly Phe Gly Arg Arg Val Arg Pro Pro 260 265 270 Gln Glu Arg Ala Leu Leu Val Val Phe Thr Arg Ser Gln Arg Lys Asn 275 280 285 Leu Phe Ala Glu Met Arg Glu Gln Leu Gly Ser Ala Glu Ala Ala Gly 290 295 300 Pro Gly Ala Gly Ala Glu Gly Ser Trp Pro Pro Pro Ser Gly Ala Pro 305 310 315 320 Asp Ala Arg Pro Trp Leu Pro Ser Pro Gly Arg Arg Arg Arg Arg 325 330 335 <210> 26 <211> 381 <212> PRT <213> Homo sapiens <400> 26 Met Arg Leu Pro Lys Leu Leu Thr Phe Leu Leu Trp Tyr Leu Ala Trp 1 5 10 15 Leu Asp Leu Glu Phe Ile Cys Thr Val Leu Gly Ala Pro Asp Leu Gly 20 25 30 Gln Arg Pro Gln Gly Thr Arg Pro Gly Leu Ala Lys Ala Glu Ala Lys 35 40 45 Glu Arg Pro Pro Leu Ala Arg Asn Val Phe Arg Pro Gly Gly His Ser 50 55 60 Tyr Gly Gly Gly Ala Thr Asn Ala Asn Ala Arg Ala Lys Gly Gly Thr 65 70 75 80 Gly Gln Thr Gly Gly Leu Thr Gln Pro Lys Lys Asp Glu Pro Lys Lys 85 90 95 Leu Pro Pro Arg Pro Gly Gly Pro Glu Pro Lys Pro Gly His Pro Pro 100 105 110 Gln Thr Arg Gln Ala Thr Ala Arg Thr Val Thr Pro Lys Gly Gln Leu 115 120 125 Pro Gly Gly Lys Ala Pro Pro Lys Ala Gly Ser Val Pro Ser Ser Phe 130 135 140 Leu Leu Lys Lys Ala Arg Glu Pro Gly Pro Pro Arg Glu Pro Lys Glu 145 150 155 160 Pro Phe Arg Pro Pro Pro Ile Thr Pro His Glu Tyr Met Leu Ser Leu 165 170 175 Tyr Arg Thr Leu Ser Asp Ala Asp Arg Lys Gly Gly Asn Ser Ser Val 180 185 190 Lys Leu Glu Ala Gly Leu Ala Asn Thr Ile Thr Ser Phe Ile Asp Lys 195 200 205 Gly Gln Asp Asp Arg Gly Pro Val Val Arg Lys Gln Arg Tyr Val Phe 210 215 220 Asp Ile Ser Ala Leu Glu Lys Asp Gly Leu Leu Gly Ala Glu Leu Arg 225 230 235 240 Ile Leu Arg Lys Lys Pro Ser Asp Thr Ala Lys Pro Ala Ala Pro Gly 245 250 255 Gly Gly Arg Ala Ala Gln Leu Lys Leu Ser Ser Cys Pro Ser Gly Arg 260 265 270 Gln Pro Ala Ser Leu Leu Asp Val Arg Ser Val Pro Gly Leu Asp Gly 275 280 285 Ser Gly Trp Glu Val Phe Asp Ile Trp Lys Leu Phe Arg Asn Phe Lys 290 295 300 Asn Ser Ala Gln Leu Cys Leu Glu Leu Glu Ala Trp Glu Arg Gly Arg 305 310 315 320 Ala Val Asp Leu Arg Gly Leu Gly Phe Asp Arg Ala Ala Arg Gln Val 325 330 335 His Glu Lys Ala Leu Phe Leu Val Phe Gly Arg Thr Lys Lys Arg Asp 340 345 350 Leu Phe Phe Asn Glu Ile Lys Ala Arg Ser Gly Gln Asp Asp Lys Thr 355 360 365 Val Tyr Glu Tyr Leu Phe Ser Gln Arg Arg Lys Arg Arg 370 375 380 <210> 27 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> PTD <400> 27 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 28 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> TRP2 <400> 28 Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Arg Trp Gly Ser Lys Leu Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg 35 40 45 Arg Arg Gly Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly 50 55 60 Trp Glu Gly Met Ile Asp Gly Gly Ser Thr Met Ala Gly Thr Met His 65 70 75 80 Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp 85 90 95 Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp 100 105 110 Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu 115 120 125 Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His 130 135 140 Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe 145 150 155 160 Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro 165 170 175 Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln 180 185 190 Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala 195 200 205 Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe 210 215 220 Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys 225 230 235 240 Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys 245 250 255 Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val 260 265 270 Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu 275 280 285 Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe 290 295 300 Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn 305 310 315 320 Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn 325 330 335 Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln 340 345 350 Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser 355 360 365 Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr 370 375 380 Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser 385 390 395 400 Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser 405 410 415 Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr 420 425 430 Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr 435 440 445 Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile 450 455 460 Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn 465 470 475 480 Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys 485 490 495 Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His His Tyr Pro 500 505 510 Tyr Asp Val Pro Asp Tyr Ala 515 <210> 29 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> mCherry-Rab7a vector <400> 29 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaaggg aggtaagggt 720 aaaggtggta tgacctctag gaaaagtg ttgctgaagg ttatcatcct gggagattct 780 840 aaagccacaa taggagctga ctttctgacc aaggaggtga tggtggatga caggctagtc 900 acaatgcaga tatgggacac agcaggacag gaacggttcc agtctctcgg tgtggccttc 960 tacagaggtg cagactgctg cgttctggta tttgatgtga ctgcccccaa cacattcaaa 1020 accctagaga gctggagaga tgagtttctc atccaggcca gtccccgaga tcctgaaaac 1080 ttcccatttg ttgtgttggg aaacaagatt gacctcgaaa acagacaagt ggccacaaag 1140 cgggcacagg cctggtgcta cagcaaaaac aacattccct actttgagac cagtgccaag 1200 gaggccatca acgtggagca ggcgttccag acgattgcac ggaatgcact taagcaggaa 1260 acggaggtgg agctgtacaa cgaatttcct gaacctatca aactggacaa gaatgaccgg 1320 gccaaggcct cggcagaaag ctgcagttgc tga 1353

Claims

1. A pharmaceutical preparation comprising: (a) an inactivated polypeptide TRP comprising: a PTD (protein transduction domain) which achieves cell membrane penetration without the aid of cell membrane receptors; a FAD (furin activation domain) which has at least one proprotein convertase cleavage site and is cleaved by a proprotein convertase to activate the inactivated TRD (tissue regeneration domain) in cells; and a TRD which is activated by cleavage by the proprotein convertase of the FAD to promote tissue growth or formation or induce tissue regeneration in cells; and (b) a dispersing agent selected from the group consisting of lecithin, Cremophor and triglycerides, Wherein the inactivated polypeptide TRP is a TRP represented by the amino acid sequence of SEQ ID NO:

28.

2. The pharmaceutical preparation according to claim 1, wherein the lecithin is selected from egg lecithin, soybean lecithin, sunflower oil lecithin, rapeseed lecithin, cottonseed lecithin and lecithin derived from animal fat.

3. The pharmaceutical preparation according to claim 1, wherein the proprotein convertase is furin.

4. The pharmaceutical preparation according to claim 1, which is for oral administration or injection. 5 . The pharmaceutical preparation according to claim 1 , wherein the content of the dispersant is in an amount of 100 to 50,000 parts by weight based on 100 parts by weight of the inactivated polypeptide TRP.

6. A composition comprising TRP2 and 100 to 50,000 parts by weight of a dispersant based on 100 parts by weight of TRP2, wherein the dispersant is selected from lecithin, Cremophor and triglyceride, wherein the TRP2 is represented by the amino acid sequence of SEQ ID NO:

28.

7. The composition according to claim 6, wherein the lecithin is egg lecithin or soybean lecithin.

8. Use of the composition according to claim 6 or 7 or the pharmaceutical preparation according to any one of claims 1 to 5 in the preparation of a medicament for treating peritoneal fibrosis.

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