KR20220133109A - Composition for Differential Diagnosis of Malignant Peripheral Nerve Sheath Tumor - Google Patents

Abstract

The present invention relates to a composition for the differential diagnosis of malignant peripheral nerve sheath tumor (MPNST), a differential diagnosis kit including the same, a method for providing information necessary for the differential diagnosis of MPNST using the same, and a method for screening a composition for preventing, alleviating, and treating MPNST. By using the composition capable of detecting a protein marker of the present invention, it is expected to predict and diagnose the occurrence of malignant tumors in patients with type 1 neurofibromatosis.

Description

Composition for Differential Diagnosis of Malignant Peripheral Nerve Sheath Tumor

The present invention provides a composition for differential diagnosis of malignant peripheral nerve sheath tumor (MPNST), a kit for differential diagnosis comprising the same, a method for providing information necessary for differential diagnosis of malignant peripheral nerve sheath tumor using the same, prevention and improvement of malignant peripheral schwannoma And it relates to a screening method of a composition for treatment, and the like.

Neurofibromatosis type I (hereinafter referred to as 'NF-1') was first described by Fredrich von Recklinghausen in 1882 and is one of the most common genetic diseases. NF-1 is inherited through autosomal dominant inheritance, affecting 1:3,000 people worldwide. NF-1 is characterized by multiple milk coffee spots of the skin and neurofibromas of the skin. In addition, varying levels of nervous and skeletal expression and neoplastic expression may affect patients. NF-1 patients include plexiform neurofibromas (PN; 27%), optic nerve glioma (15-20%), pheochromocytoma (1%), and malignant peripheral nerve sheeth tumors. , MPNST; 8-13%) at risk of developing central and peripheral nervous system tumors. This is about 1,000 times higher than the general population.

Malignant peripheral schwannoma (hereinafter referred to as 'MPNST') is one of the complications that has the greatest impact on the survival rate of NF-1 patients. It is known to be caused by a malignant transformation of plexiform neurofibroma, a benign nerve myelin tumor, and should be suspected if severe pain, rapid increase in the size of the fibroma, and neurological symptoms are accompanied.

To date, 18F-FDG-PET (18F-fluorodeoxyglucose positron emission computerized tomography) has been used to differentiate between benign tumors and MPNST, and if malignancy is suspected, MPNST is diagnosed based on histological findings.

Most MPNST histologically, spindle cells form a bundle, nuclear differentiated cells are observed, and have a pattern of infiltrating surrounding tissues. S100 protein staining is used for the diagnosis of MPNST, but 50-90% of MPNST stains are positive, and Leu-7 protein and myelin basic protein are positive in about 50% of MPNST. Vimentin (vimentin) is also a pathological marker used for the diagnosis of MPNST, but is specific to spindle cells and is limited in the specific diagnosis of MPNST. Therefore, there is a need for pathological indicators that can clearly determine the progression of MPNST in NF-1.

MPNST is a disease requiring surgical resection after diagnosis. Even after surgery, there is a risk of recurrence in about 50%, and chemotherapy and radiation therapy may be required if complete resection is not achieved through surgery. Nevertheless, the 5-year survival rate for MPNST patients remains 20-50%.

In the absence of a treatment that can target MPNST so far, finding a biomarker that can detect MPNST early in blood or urine could contribute to improving the survival rate of patients.

WO 2013142427 A1

Accordingly, the present inventors performed extensive quantitative and qualitative proteomic analysis using malignant peripheral nerve sheath tumor (MPNST) and benign neurofibroma tissues. As a result, the present invention identified proteins specifically expressed in MPNST tissues. was completed.

Accordingly, it is an object of the present invention to provide a composition for differential diagnosis of malignant peripheral schwannoma (MPNST).

Another object of the present invention is to provide a kit for differential diagnosis of malignant peripheral schwannoma comprising the composition according to the present invention.

Another object of the present invention is to provide a method for providing information necessary for differential diagnosis of malignant peripheral schwannoma.

Another object of the present invention is to provide a composition for preventing, improving or treating malignant peripheral schwannoma.

Another object of the present invention is to provide a screening method for a substance for the prevention, improvement or treatment of malignant peripheral schwannoma.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase, or It provides a composition for differential diagnosis of malignant peripheral nerve sheath tumor (MPNST), comprising an agent for measuring its mRNA expression level as an active ingredient.

In one embodiment of the present invention, the composition can be diagnosed by distinguishing malignant peripheral schwannoma from type 1 neurofibromatosis.

In another embodiment of the present invention, the agent for measuring the expression level of the protein may be an antibody or aptamer specific for the protein, but is not limited thereto.

In another embodiment of the present invention, the agent for measuring the expression level of the mRNA may be a primer set or a probe that specifically binds to the mRNA, but is not limited thereto.

In addition, the present invention provides a kit for differential diagnosis of malignant peripheral schwannoma comprising the composition according to the present invention.

In one embodiment of the present invention, the kit is one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1, and neutrophil collagenase, or its If the expression of mRNA is detected or when the expression level thereof is higher compared to the expression level of the benign neurofibroma sample, instructions may include instructions to determine that it is malignant peripheral schwannoma.

In addition, the present invention provides one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1, and neutrophil collagenase in a biological sample isolated from a subject. Or it provides a method for providing information necessary for differential diagnosis of malignant peripheral schwannoma, comprising the step of measuring its mRNA expression level.

In one embodiment of the present invention, the method comprises one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase, or its The method may further include the step of diagnosing malignant peripheral schwannoma when mRNA expression is detected or when the expression level thereof is higher than the expression level in a biological sample isolated from a type 1 neurofibromatosis patient.

In another embodiment of the present invention, the subject may be a patient suspected of malignant peripheral schwannoma or a patient diagnosed with type 1 neurofibromatosis.

In another embodiment of the present invention, the biological sample is blood, whole blood, plasma, urine, tissue, cell, organ, bone marrow, microneedle aspiration sample, microneedle wash, core needle biopsy sample and saliva. It may be one or more selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the protein expression level is determined by Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunostaining, It may be measured by one or more methods selected from the group consisting of immunoprecipitation assay, complement fixation assay, mass spectrometry, FACS, and protein chip, but is not limited thereto.

In another embodiment of the present invention, the mRNA expression level is at least one selected from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization and DNA chip. It may be measured by a method, but is not limited thereto.

In addition, the present invention provides an inhibitor of activity of one or more signaling proteins selected from the group consisting of TGF-β signaling system, ILK signaling system, actin-cytoskeleton signaling system, and RhoGDI signaling system, or a gene encoding the protein It provides a pharmaceutical composition for the prevention or treatment of malignant peripheral schwannoma, comprising an expression inhibitor as an active ingredient.

Furthermore, the present invention relates to an inhibitor of the activity of one or more signaling proteins selected from the group consisting of TGF-β signaling, ILK signaling, actin-cytoskeleton signaling, and RhoGDI signaling, or a gene encoding the protein. It provides a method for preventing or treating malignant peripheral schwannoma, comprising administering an expression inhibitor to an individual in need thereof.

In addition, the present invention provides an inhibitor of activity of one or more signaling proteins selected from the group consisting of TGF-β signaling system, ILK signaling system, actin-cytoskeleton signaling system, and RhoGDI signaling system, or encoding the protein To provide a preventive or therapeutic use of a gene expression inhibitor for malignant peripheral schwannoma.

In addition, the present invention provides an inhibitor of activity of one or more signaling proteins selected from the group consisting of TGF-β signaling system, ILK signaling system, actin-cytoskeleton signaling system, and RhoGDI signaling system, or a gene encoding the protein It provides a health functional food composition for preventing or improving malignant peripheral schwannoma, comprising an expression inhibitor as an active ingredient.

In one embodiment of the present invention, the signal transduction system protein may be a kinase (kinase), but is not limited thereto.

In another embodiment of the present invention, the kinase may be a kinase encoded by one or more genes selected from the group consisting of CDK4, STK10, CDK5, MAP2K1, ALPK3, PNKP, PDK3, PHKG1, CFL1, and PACSIN3, but is limited thereto. it's not going to be

In another embodiment of the present invention, the protein activity inhibitor may be one or more selected from the group consisting of peptides, antibodies, aptamers and compounds that specifically bind to the protein, but is not limited thereto.

In another embodiment of the present invention, the inhibitor of the activity of the signaling system protein is selumetinib, albocidib, purvalanol, palbociclib, ribociclib ), abemaciclib, fostamatinib, binimetinib, radicicol, AZD-8330, dihydrolipoic acid, alsterpaullone, hime Hymenialdisine, indirubin-3'-monoxime, olomoucine, SU9516, and 6-phenyl[5H]pyrrolo[2,3-B]pyrazine It may be one or more selected from the group consisting of, but is not limited thereto.

In another embodiment of the present invention, the gene expression inhibitor is miRNA, siRNA, shRNA, ribozyme, DNAzyme, PNA (peptide nucleic acid) and antisense oligonucleotides that complementarily bind to the mRNA of the gene. It may be one or more selected from the group consisting of, but is not limited thereto.

In addition, the present invention comprises the steps of (a) contacting a candidate material to the isolated cell or tissue; (b) the activity or encoding of one or more signaling proteins selected from the group consisting of TGF-β signaling pathway, ILK signaling pathway, actin-cytoskeleton signaling pathway and RhoGDI signaling pathway in the cell or tissue measuring the expression level of the gene; And (c) when the activity of the protein or the expression level of the gene encoding the protein in the cell or tissue contacted with the candidate substance is reduced compared to the level before contact with the candidate substance, the candidate substance is used to prevent malignant peripheral schwannoma. It provides a screening method of a substance for prevention, improvement or treatment of malignant peripheral schwannoma, comprising the step of determining the substance for improvement or treatment.

In one embodiment of the present invention, the signal transduction system protein may be a kinase (kinase), but is not limited thereto.

In another embodiment of the present invention, the kinase may be a kinase encoded by one or more genes selected from the group consisting of CDK4, STK10, CDK5, MAP2K1, ALPK3, PNKP, PDK3, PHKG1, CFL1, and PACSIN3, but is limited thereto. it's not going to be

In another embodiment of the present invention, the isolated cell or tissue may be a nerve cell or nerve tissue, but is not limited thereto.

The present invention relates to a composition for differential diagnosis of malignant peripheral nerve sheath tumor (MPNST), and the like, and whether or not a malignant tumor occurs in a type 1 neurofibromatosis patient using the composition capable of detecting a protein marker of the present invention It can be predicted, diagnosed, and used as a therapeutic target.

1A and 1B are time series flow charts of quantitative proteomic analysis using benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissues.
2 is a diagram schematically illustrating a proteomic data analysis method.
Figure 3 is a protein abundance distribution (distribution of protein abundance) graph showing the protein quantification results for benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissue.
4 is a diagram showing the results of qualitative proteomic analysis for two groups of neurofibroma (NF) and malignant peripheral schwannoma (MPNST) using Venn diagram analysis.
Figures 5a and 5b is a road showing the protein investigation results showing a 10-fold increase in expression in benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissue, Figure 5a is a protein expressed in the extracellular exosome, Figure 5b is the result of selecting a protein that degrades the extracellular matrix.
6a to 6j are graphs showing the results of examining the expression patterns of S-100 isoform protein and vimentin in benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissues, FIG. 6a is S100-A1 protein, FIG. 6b is S100-A4 protein, FIG. 6c is S100-A6 protein, FIG. 6d is S100-A7 protein, FIG. 6e is S100-A8 protein, FIG. 6f is S100-A9 protein, FIG. 6g is S100-A10 protein, FIG. 6h is S100 -A13 protein, Figure 6i is a S100-B protein, Figure 6j is a diagram showing the results for vimentin (vimentin). (F1-F10 denotes a sample of a patient's MPNST region, and F11-F20 denotes a sample of a positive NF region of a patient. Specifically, F1 and F2 denote a sample of the MPNST region of Patient 1; F3, F4 denote a sample of the MPNST region of a patient 2; F5, F6 are MPNST site samples from patient 3; F7, F8 are MPNST site samples from patient 4; F9, F10 are MPNST site samples from patient 5; F11, F12 are samples from patient 1's positive NF site; F13, F14 are patient Positive NF site samples from 2; F15 and F16 are positive NF site samples from Patient 3; F17, F18 are positive NF site samples from Patient 4; F19, F20 refer to positive NF site samples from Patient 5 (see Table 3) )
7 is a graph showing the results of examining the expression pattern of endosialin in benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissues.
8 is a graph showing the results of examining the expression pattern of SPARC-like protein 1 in benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissues.
9 is a graph showing the results of examining the expression pattern of neutrophil collagenase in benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissues.
10A to 10C are diagrams showing the results of hierarchical cluster analysis of proteomic expression changes in benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissues. One diagram, FIG. 10b is a diagram showing a cluster of proteins with increased expression in MPNST, and FIG. 10c is a diagram showing a cluster of proteins with decreased expression in MPNST.
11 is a diagram showing the results of gene ontology analysis for malignant peripheral schwannoma (MPNST) specific protein.
12 is a diagram showing the results of ingenuity pathway analysis (IPA) for malignant peripheral schwannoma (MPNST)-specific protein.
13 to 21 are graphs comparing and analyzing the abundance of kinases with increased expression or activity specifically in malignant peripheral schwannoma (MPNST) compared to benign neurofibroma (NF), FIG. 13 is CDK4, FIG. 14 is MAP2K1, Figure 15 shows the results for the abundance of PDK3, Figure 16 is STK10, Figure 17 is ALPK3, Figure 18 is PHKG1, Figure 19 is CDK5, Figure 20 is PNKP, Figure 21 shows the results for the abundance of PACSIN3.
22 is a view showing the results of IHC (Immunohistochemistry) for MMP8 protein (neutrophyll collagenase) in benign neurofibroma (NF) and malignant peripheral schwannoma (MPNST) tissues (A1 and B1 are NF tissues, A2 and B2 are MPNST organization).
23 is a diagram showing the results of analyzing the difference in MMP8 protein (neutropyl collagenase) expression according to the presence or absence of tumor removal using the blood of a patient with malignant peripheral schwannoma (MPNST) by ELISA (enzyme-linked immunosorbent assay).
24 shows ILK signaling with increased expression or activity specifically in malignant peripheral schwannoma (MPNST) compared to benign neurofibroma (NF), actin-cytoskeleton signaling pathway, and IHC for kinase (Immunohistochemistry) is a diagram showing the results (A1 to J1 are NF tissues, A2 to J2 are MPNST tissues).

The present invention relates to a plurality of biomarkers for differential diagnosis of transition from Neurofibromatosis type I to Malignant peripheral nerve sheath tumor (MPNST) and a signal transduction pathway that specifically promotes MPNST or The present invention relates to a screening method for an MPNST therapeutic agent targeting a kinase whose expression is increased, and the present inventors have conducted intensive studies to discover MPNST-specific diagnostic markers and new therapeutic targets. As a result, a plurality of proteins disclosed herein and The present invention was completed by discovering the signaling pathway.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for differential diagnosis of malignant peripheral nerve sheath tumor (MPNST) comprising, as an active ingredient, an agent for measuring the mRNA expression level of one or more proteins selected from the group consisting of the proteins shown in Table 1 below. it's about

In one embodiment of the present invention, proteins specific for MPNST were identified through proteomic analysis using malignant peripheral schwannoma (MPNST) and benign neurofibroma (NF) and tissues, and among them, 3 that can be detected in blood or urine Species proteins - endosialin, SPARC-like protein 1 and neutrophil collagenase - were selected and shown to be used as biomarkers for MPNST (see Examples 2 to 4 and Example 6).

The endosialin is encoded by the CD248 gene, and may consist of the amino acid sequence of SEQ ID NO: 1, and includes isoform proteins in which amino acids 1 to 324 are deleted from the amino acid sequence of SEQ ID NO: 1. The SPARC-like protein 1 is encoded by the SPARCL1 gene and may consist of the amino acid sequence of SEQ ID NO: 2, and includes isoform proteins in which amino acids 1 to 125 are deleted from the amino acid sequence of SEQ ID NO: 2. In addition, the neutrophyll collagenase may be encoded by the MMP8 gene and consist of the amino acid sequence of SEQ ID NO: 3.

As used herein, the term "protein" is used interchangeably with a polypeptide or a peptide, for example, refers to a polymer of amino acid residues as commonly found in proteins in their natural state. "mRNA" refers to RNA that transfers genetic information (gene-specific nucleotide sequence) to ribosomes that specify an amino acid sequence from a specific gene during protein synthesis.

As used herein, the term "expression" refers to the production of a protein or nucleic acid in a cell.

As used herein, the term “differential diagnosis” refers to distinguishing a particular disease or condition from others exhibiting similar symptoms. The differential diagnostic method is a systematic diagnostic method used to confirm the existence of a condition for which multiple alternatives are possible. These methods are essentially elimination processes or information-gathering processes that reduce the probability of candidate states to negligible levels. The term is also used to refer to determining whether to transition from benign neurofibromatosis to malignant peripheral schwannoma.

In the present invention, the composition can be diagnosed by distinguishing malignant peripheral schwannoma (MPNST) from type 1 neurofibromatosis (NF-1).

As used herein, the term "Neurofibromatosis type I" or "benign neurofibroma" is a genetic disease showing various clinical symptoms in the skin, skeletal system, nervous system, etc. ) that was first reported by Neurofibromatosis type 1 has milky coffee-colored spots on the skin (cafe-au-lait-spot), axillary freckling, inguinal freckling, neurofibroma, and small, tinted iris. It shows characteristic symptoms of Lisch nodule, optic glioma, and osteogenesis disorders, which are haplotypes. In addition, short stature, scoliosis, and learning disabilities may appear. Most tumors seen in patients with type 1 neurofibromatosis are benign, but in rare cases they can progress to malignancy and can occur anywhere in the body with nerves. According to the study, it is reported that it is detected before the age of 1 year in 67% of cases, is accompanied by a characteristic skin lesion, milk coffee-colored spots, in 25-90%, and becomes malignant in up to 16%.

As used herein, the term "malignant peripheral nerve sheath tumor (MPNST)" is used interchangeably with "malignant peripheral nerve sheath tumor", generally occurs in the extremities and head and neck, and rarely in the abdominal cavity, pelvis, genitourinary system, etc. Occurs. It is mainly associated with type 1 neurofibromatosis, and the lifetime risk of malignant peripheral schwannoma among neurofibromatosis patients is known to be around 10%, and it is one of the intractable diseases with a very poor prognosis and high malignancy.

When the composition of the present invention is for measuring the expression level of a protein, the agent for measuring the expression level of the protein may be an antibody or aptamer specific for the protein, but is not limited thereto.

As used herein, the term "antibody" refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. In addition, as long as it has antigen-antibody binding properties, a part of the whole antibody is also included in the antibody of the present invention, and all kinds of immunoglobulin antibodies that specifically bind to the protein of the present invention are included. For example, a complete antibody having two full-length light chains and two full-length heavy chains, as well as functional fragments of the antibody molecule, i.e. Fab, F(ab'), F(ab') with antigen-binding function. )2 and Fv, and the like. Furthermore, the antibody of the present invention includes special antibodies such as humanized antibodies and chimeric antibodies and recombinant antibodies as long as they can specifically bind to the above protein.

As used herein, the term "aptamer" refers to a substance capable of specifically binding to an analyte to be detected in a sample, and a single-stranded nucleic acid (DNA, RNA, or modified) having a stable tertiary structure by itself. nucleic acid), it is possible to specifically confirm the presence of the target protein in the sample. According to the general aptamer preparation method, the aptamer is synthesized by determining the sequence of an oligonucleotide having a selective and high binding affinity to the target protein to be identified, and then the 5' end or 3' end of the oligonucleotide is applied to the aptamer. To be able to bind to the functional group of the tamer chip, -SH, -COOH, -OH or NH ₂ It may be made by transforming it, but is not limited thereto.

The composition of the present invention comprising an antibody specific for the protein described in Table 1 may additionally include an agent necessary for a method for detecting a known protein, and there is no limitation on a method for detecting a known protein using the present composition. It can be used to measure the expression level of a protein in a subject (in the present invention, a biological sample obtained from the subject).

In addition, when the composition of the present invention is for measuring the expression level of mRNA, the agent for measuring the expression level of the mRNA may be a primer set or probe that specifically binds to the mRNA, but is not limited thereto. .

As used herein, the term "primer" refers to a nucleic acid sequence having a short free three-terminal hydroxyl group, capable of base pairing with a complementary template and serving as a starting point for template strand copying. . Primers are capable of initiating DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) in an appropriate buffer and temperature.

As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to several bases to several hundred bases as short as possible to achieve specific binding to mRNA, and is labeled with a specific mRNA existence can be checked. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art.

The "primer" or "probe" may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, primers or probes can be variously modified according to methods known in the art within a range that does not interfere with hybridization with mRNA. Examples of such modifications include methylation, encapsulation, substitution of one or more homologues of natural nucleotides, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc. ) or charged linkages (eg, phosphorothioate, phosphorodithioate, etc.), and fluorescence or enzymatic binding of a labeling material.

The composition of the present invention comprising a primer set or probe specific for the mRNA of the protein described in Table 1 above may further include an agent required for a known method for detecting RNA. Using the composition of the present invention, it is possible to measure the level of mRNA of the protein markers in a subject by using a known method for detecting RNA without limitation.

In addition, in the present invention, since the nucleic acid sequence of the gene encoding the mRNA of the protein is registered in the gene bank, those skilled in the art can use antisense oligonucleotides, primer pairs, or You can design the probe.

The antisense oligonucleotide can be chemically synthesized using the phosphoramidite solid support method or other well-known methods, and includes all functional equivalents. In addition, the above-mentioned "primer" or "probe" may be equally applied.

The term "functional equivalent" refers to, for example, one or more substitutions, deletions or additions from a reference sequence, the actual effect that does not result in various functional dissimilarities between the reference sequence and the subject sequence. (net effect) refers to both the nucleotide and nucleic acid sequences of the changed mutant sequence. Typically, such substantially equivalent sequences are only about 35% (i.e., the number of substitutions, additions, and deletions of each residue in the substantially equivalent sequence compared to the corresponding reference sequence and the remaining total number in the substantially equivalent sequence) is about 0.35 or less when divided by ), which varies from those listed herein. Such sequences have 65% sequence identity to the listed sequences. Substantially equivalent, eg mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity, more preferably at least 90% sequence identity with the listed amino acid sequences. Substantially equivalent nucleotide sequences of the invention may have a lower percentage of sequence identity, for example, given the redundancy or degeneracy of the genetic code. Preferably, the nucleotide sequence should have at least about 65% identity, more preferably at least about 75% identity, and most preferably about 95% identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent synthetic characteristics are treated as substantially equivalent. To determine equivalents, truncation of the mature sequence (eg, via a mutation that produces a spurious stop codon) should be neglected.

As another aspect of the present invention, the present invention may provide a kit for differential diagnosis of malignant peripheral schwannoma comprising the composition according to the present invention.

In the present invention, the kit is one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase, or expression of mRNA thereof If these are detected or their expression level is higher compared to the expression level of the benign neurofibroma sample, instructions may include instructions to determine a malignant peripheral schwannoma.

The kit of the present invention may include an antibody that recognizes a target protein as a marker or a primer set and probe that recognizes mRNA as a marker, as well as one or more other component compositions, solutions, or devices suitable for the analysis method.

In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each primer set specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of the control gene. Other Reverse Transcription Polymerase Reaction Kits include test tubes or other suitable containers, reaction buffers (varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterile water, etc. may be included.

In another specific embodiment, it may be a diagnostic kit comprising essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotide corresponding to a gene or fragment thereof is attached, and reagents, agents, enzymes, etc. for preparing a fluorescent probe. The substrate may also contain cDNA or oligonucleotides corresponding to control genes or fragments thereof.

As another aspect of the present invention, the present invention is composed of endosialin, SPARC-like protein 1 and neutrophil collagenase in a biological sample isolated from a subject. It is possible to provide a method for providing information necessary for differential diagnosis of malignant peripheral schwannoma, comprising measuring the expression level of one or more proteins selected from the group or mRNA thereof.

In the present invention, the method is one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase, or expression of mRNA thereof The method may further include the step of diagnosing malignant peripheral schwannoma when the detected or their expression level is higher than the expression level in a biological sample isolated from a type 1 neurofibromatosis patient.

As used herein, the term "detection" refers to measuring and confirming the presence (expression) of a target substance (marker protein in the present invention), or measuring a change in the presence level (expression level) of a target substance. And it is meant to include all of the confirmation. In the same context, measuring the expression level of the protein means measuring whether it is expressed (ie, measuring the presence or absence of expression), or measuring the level of qualitative and quantitative change of the protein. The measurement may be performed without limitation, including both a qualitative method (analysis) and a quantitative method. Types of qualitative and quantitative methods for measuring protein levels are well known in the art, and the experimental methods described herein are included therein. A specific protein level comparison method for each method is well known in the art. Therefore, the detection of the target protein is meant to include detecting the presence or absence of the protein described in Table 1, or confirming the increase (up-regulation) or decrease (down-regulation) of the protein expression level.

In addition, the term "differential diagnosis" refers to, in addition to the above meaning, determining the susceptibility of a subject to a specific disease or disorder, determining whether the subject currently has a specific disease or disorder, determining the prognosis of a subject afflicted with a particular disease or condition, or therametrics (eg, monitoring a subject's condition to provide information about the efficacy of treatment).

In the present invention, the subject may be a patient suspected of malignant peripheral schwannoma or a patient diagnosed with type 1 neurofibromatosis.

In the present invention, the biological sample can be used without limitation as long as it is collected from a subject for differential diagnosis of malignant peripheral schwannoma, for example, tissue, cells, blood, plasma, serum, saliva obtained by biopsy, etc. , nasal fluid, sputum, joint capsule fluid, amniotic fluid, ascites, cervical secretions, vaginal secretions, urine, cerebrospinal fluid, and other biological samples, as well as microneedle aspiration samples and microneedle washing fluids. Preferably from the group consisting of biological liquid samples, for example, blood, whole blood, plasma, urine, tissue, cells, organs, bone marrow, microneedle aspiration sample, microneedle lavage fluid, core needle biopsy sample and saliva It can be measured in one or more selected. The present invention has great technical significance in that, in addition to the lesion tissue in which malignant peripheral schwannoma has occurred, diagnosis can be made using a body fluid sample (eg, urine or plasma), thereby increasing the utility, speed, convenience and safety of diagnosis.

The biological sample may be pretreated prior to use for detection or diagnosis. For example, it may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. The sample may be prepared to increase the detection sensitivity of the protein marker. For example, a serum sample obtained from a patient may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, It may be pre-treated using a method such as sequential extraction or gel electrophoresis.

The protein expression level measurement is not particularly limited as long as it is by a protein expression measurement method known in the art, but for example, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, Ouchterlony immune diffusion method (Ouchterlony). immunodiffusion), rocket immunoelectrophoresis, immunostaining, immunoprecipitation, complement fixation, mass spectrometry, FACS, and protein chip.

The mRNA expression level measurement is from the group consisting of PCR, RNase protection assay, northern blotting, southern blotting, in situ hybridization and DNA chip by a gene expression measurement method known in the art. It may be one or more selected, but is not limited thereto.

As used herein, the term 'analysis' may preferably mean 'measurement', and the qualitative analysis may mean measuring and confirming the presence or absence of a target substance, and the quantitative analysis is It may mean measuring and confirming a change in the presence level (expression level) or amount of a desired substance. In the present invention, analysis or measurement may be performed without limitation, including both qualitative and quantitative methods, and preferably quantitative measurement may be performed.

On the other hand, the mechanisms of MPNST in NF-1 are: complete loss of intratumoral NF-1, excessive activation of RAS-MAPT signal, loss of CDKN2A gene, loss of PRC2 (polycomb repressive complex 2) constituent genes SUZ12 and EED, and tumor Loss of the suppressor gene TP53, abnormal hyperactivity of the PI3K-AKT-mTOR signaling system, and abnormal activation of epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGF-R), vascular endothelial growth factor receptor (VEGFR), etc. has been known to be involved. Based on this, various clinical studies have been conducted in MPNST, but there has been no study showing a clear therapeutic effect. Therefore, there was a need to explore new therapeutic targets and candidates using new approaches.

According to this requirement, the present invention provides four types of signaling pathways that are specifically enhanced in malignant peripheral schwannoma (MPNST) - ILK signaling system, actin-cytoskeleton signaling system, RhoGDI signaling system, TGF- β signaling system - and 10 kinases with increased expression or activity specifically in MPNST - Kinases expressed by CDK4, MAP2K1, PDK3, STK10, ALPK3, PHKG1, CDK5, PNKP, CFL1, and PACSIN3 genes - Discovered and it was confirmed that they can be utilized as new therapeutic targets of MPNST (see Examples 5 and 7).

Accordingly, as another aspect of the present invention, the present invention relates to the activity of one or more signaling proteins selected from the group consisting of TGF-β signaling, ILK signaling, actin-cytoskeleton signaling, and RhoGDI signaling. It is possible to provide a pharmaceutical composition for preventing or treating malignant peripheral schwannoma, comprising an inhibitor or an expression inhibitor of a gene encoding the protein as an active ingredient.

In another aspect of the present invention, the present invention provides an inhibitor of activity of one or more signaling proteins selected from the group consisting of TGF-β signaling, ILK signaling, actin-cytoskeleton signaling, and RhoGDI signaling, or It is possible to provide a health functional food composition for preventing or improving malignant peripheral schwannoma, comprising an expression inhibitor of the gene encoding the protein as an active ingredient.

The "TGF-β signaling protein" includes all proteins directly or indirectly involved in TGF-β signaling. For example, SMAD1, SMAD2, SMAD3, SMAD5, SMAD9, Thrombospondin-1, Rock, Cdc42/RAC, PAK, LIMK, PI3K, Ras, Raf, MEK1/2, ERK1/2, JNK, P38, Cofilin, mTOR, There is AKT.

The "ILK signaling system (Integrin-linked kinase signaling) protein" includes all proteins directly or indirectly involved in ILK signaling. For example, mTOR, AKT, VEGF, NF-κGSK3β, SNAIL, AP-1, MMP9, JAK2, Cdc42/RAC, SNAI1, JNK, PI3K, AP-1, C-Jun, HIF1α, COX2, ß-Catenin have.

The "actin-cytoskeleton signaling protein" includes all proteins directly or indirectly involved in actin-cytoskeleton signaling. For example, PI3K, CaM, PKA, Rho, ROCK, Ras, Rac, Paxillin, FAK, Cofilin, Cortactin, WAVE, PIX, TIAM1, Vav, PAK1, GEF, VASP, MEKK, MEK, MLCK, MLC, LIMK, ADF, CapZ, Hsp90, Gelsolin, ADF, Filamin, Profilin, RhoGEF, Myosin, ACTN, Vinculin, ACTN, PFN, MEK1/2, ERK1/2.

The "RhoGDI signaling system (Rho GDP-dissociation inhibitor signaling) protein" includes all proteins directly or indirectly involved in RhoGDI signaling. For example, MKK4/7, JNK, C-Jun, PARP, ROCK1, ROCK2, Cdc42, eNOS, Tau, MLCP, MLC, CRMP2, Adducin, LIMK, PKN, BORGs, Citron, WASP, MRCK, PI3K, PAK, There are PAR6 and PAR3.

In the present invention, the signal transduction protein may be a kinase, and the kinase may be one or more kinases selected from the group consisting of the proteins shown in Table 2 below, but is not limited thereto.

In the present invention, the kinases in Table 2 below are "CDK4", "STLK10" and "PHKG1" are ILK signaling pathways; "CDK5" refers to the actin-cytoskeleton signaling pathway and the RhoGDI signaling pathway; “MAP2K1” and “CFL1” refer to the actin-cytoskeleton signaling pathway and the TGF-β signaling pathway; "ALPK3" stands for Heart development; PNKP is DNA damage &repair; “PDK3” refers to pyruvate metabolism, citric acid (TCA) cycle; "PACSIN3" may belong to the actin-cytoskeleton signaling system, but is not limited thereto.

In the present invention, it was confirmed that the proteins listed in Table 2 (including p-MEK) are specifically up-regulated in MPNST, substances capable of inhibiting them, for example, antagonists to the protein may be used as a treatment for MPNST.

In the present invention, the protein activity inhibitor may be at least one selected from the group consisting of peptides, antibodies, aptamers and compounds that specifically bind to the protein, but is not limited thereto.

As used herein, the definitions of "antibody" and "aptamer" are the same as described above.

In the present invention, the inhibitor of the activity of the signaling system protein is selumetinib, albocidib, purvalanol, palbociclib, ribociclib, abemaci Clip (abemaciclib), fostamatinib (fostamatinib), binimetinib, radicicol (radicicol), AZD-8330, dihydrolipoic acid (dihydrolipoic acid), alsterpaullone ( hymenialdisine), indirubin-3'-monoxime, olomoucine, SU9516, and 6-phenyl[5H]pyrrolo[2,3-B]pyrazine from the group consisting of It may be one or more selected, but is not limited thereto.

In the present invention, the gene expression inhibitor is selected from the group consisting of miRNA, siRNA, shRNA, ribozyme, DNAzyme, PNA (peptide nucleic acid) and antisense oligonucleotides complementary to the mRNA of the gene It may be one or more, but is not limited thereto.

As used herein, the term "antisense oligonucleotide" refers to a nucleic acid-based molecule capable of forming a duplex with an mRNA by having a sequence complementary to the seed sequence of the target mRNA, particularly the mRNA. encompasses Thus, the term “antisense oligonucleotide” herein may be described as a “complementary nucleic acid-based inhibitor”.

The term "complementary" as used herein in reference to an antisense oligonucleotide is sufficiently complementary to selectively hybridize an antisense oligonucleotide to the mRNA target under certain hybridization or annealing conditions, preferably physiological conditions. It means that it has a meaning encompassing both substantially complementary and perfectly complementary, and preferably means completely complementary.

Antisense oligonucleotides in the present invention include various molecules. The antisense oligonucleotide is a DNA or RNA molecule, more preferably an RNA molecule. Optionally, the antisense oligonucleotide used in the present invention is ribonucleotide (RNA), deoxyribonucleotide (DNA), 2'-O-modified oligonucleotide, phosphorothioate-backbone deoxyribonucleotide, PNA (peptide nucleic acid) or LNA (locked nucleic acid). The 2'-O-modified oligonucleotide may be, for example, a 2'-O-alkyl oligonucleotide, a 2'-O-C1-3 alkyl oligonucleotide, or a 2'-O-C1-3 methyl oligonucleotide.

As used herein, the terms "miRNA, siRNA and shRNA" refer to nucleic acid molecules that bind to mRNA transcribed from a target gene mainly to mediate RNA interference or gene silencing, thereby inhibiting the translation of the mRNA. do. Since the miRNA, siRNA and shRNA can inhibit expression of a target gene at a translational level, they can be used in an efficient gene knockdown method or gene therapy method.

As used herein, the term "ribozyme" may inhibit protein expression of a target gene by recognizing a specific nucleotide sequence in a target RNA molecule and cleaving it site-specifically.

As used herein, the term "PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, wherein the deoxyribose phosphate backbone is replaced with a pseudopeptide backbone and only the original four nucleobases are retained. . The neutral backbone of PNA is known to provide specific hybridization to DNA and RNA under conditions of low ionic strength, inducing transcriptional or translational repression or inhibiting replication, resulting in antisense or antisense to sequence-specific regulation of gene expression. It can be used as an antigen preparation.

On the other hand, the content of the inhibitor of the activity of the signaling system protein or the expression inhibitor of the gene encoding the protein in the composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, the entire composition It may be 0.0001 to 99.9 wt%, or 0.001 to 50 wt%, based on the weight, but is not limited thereto. The content ratio is a value based on the dry amount from which the solvent is removed.

The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.

The pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, spirits, troches, fragrances, and limonaade. , tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.

Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

As additives for tablets, powders, granules, capsules, pills, and troches according to the present invention, corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid Calcium monohydrogen, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodite, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, A lubricant such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

As additives for the liquid formulation according to the present invention, water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

In the syrup according to the present invention, a sucrose solution, other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifier, thickening agent, etc. may be used.

Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.

Suspension agents according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Agents may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injectables according to the present invention include distilled water for injection, 0.9% sodium chloride injection solution, ring gel injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG (PEG), lactated ring gel injection solution, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin, peptone and gum; isotonic agents such as sodium chloride; sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), stabilizers such as ethylenediaminetetraacetic acid; sulphating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesic agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as SiMC sodium, sodium alginate, Tween 80, and aluminum monostearate.

The suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposhiro (OSI, OSIX, A, B, C, D, H, L), Suppository IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), supostal (N, Es), Wecobi (W, R, S, M, Fs), tester triglyceride base (TG-95, MA, 57) and The same mechanism may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention may be administered to an individual by various routes. Any mode of administration can be contemplated, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal administration. It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.

The pharmaceutical composition of the present invention is determined according to the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

In the present invention, "individual" means a subject in need of treatment of a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. means the mammals of

In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, "prevention" means any action that suppresses or delays the onset of a desired disease, and "treatment" means that the desired disease and metabolic abnormalities are improved or It means any action that is changed for the better.

As used herein, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological effects. The health functional food of the present invention may include normal food additives, and unless otherwise specified, whether it is suitable as a food additive is related to the item according to the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety. It is judged according to the standards and standards.

As another aspect of the present invention, the present invention comprises the steps of (a) contacting a candidate material to the isolated cell or tissue; (b) the activity or encoding of one or more signaling proteins selected from the group consisting of TGF-β signaling pathway, ILK signaling pathway, actin-cytoskeleton signaling pathway and RhoGDI signaling pathway in the cell or tissue measuring the expression level of the gene; And (c) when the activity of the protein or the expression level of the gene encoding the protein in the cell or tissue contacted with the candidate substance is reduced compared to the level before contact with the candidate substance, the candidate substance is used to prevent malignant peripheral schwannoma. , It is possible to provide a screening method of a substance for the prevention, improvement or treatment of malignant peripheral schwannoma, comprising the step of determining the substance for improvement or treatment.

In the present invention, the candidate material may include, but is not limited to, a low molecular weight compound, a high molecular weight compound, a microbial culture medium or extract, a natural product extract, a nucleic acid, a protein, a sugar, and a lipid, but is not limited thereto, TGF-β signaling system, Any substance capable of reducing the activity of one or more signaling proteins selected from the group consisting of the ILK signaling system, the actin-cytoskeleton signaling system, and the RhoGDI signaling system or the expression level of the gene encoding the protein may be included.

The nucleic acid may be selected from the group consisting of microRNA, siRNA, shRNA, antisense RNA, aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino. not limited

In the present invention, the signaling system protein may be a kinase, and the kinase is CDK4, STK10, CDK5, MAP2K1, ALPK3, PNKP, PDK3, PHKG1, CFL1, and one or more genes selected from the group consisting of PACSIN3. It may be a kinase encoded by, but is not limited thereto.

In the present invention, the isolated cell or tissue may be a nerve cell or nerve tissue, but is not limited thereto.

The terms used in the present invention have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Throughout the specification of the present invention, when a part "includes" a component, it means that other components may be further included, rather than excluding other components, unless otherwise stated. The terms "about," "substantially," and the like, to the extent used throughout the specification of the present invention are used in or close to the numerical value when the manufacturing and material tolerances inherent in the stated meaning are presented, and the present invention It is used to prevent an unconscionable infringer from using the disclosure in which exact or absolute figures are mentioned to help understand.

Throughout the specification of the present invention, the term "combination of these" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, It means to include one or more selected from the group consisting of components.

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

[Example]

Example 1. Sample preparation and proteomic analysis

1.1. Sample preparation

Malignant peripheral schwannoma (MPNST) tissues were obtained from 5 patients diagnosed with type 1 neurofibromatosis, and benign neurofibroma tissues obtained from 5 patients were used as a control group. Information on MPNST patients is shown in Table 3 below.

The obtained tissue sample was fixed with formalin and then embedded with paraffin. For proteomic isolation, as shown in the flowchart of FIGS. 1A and 1B , the FFPE (formalin-fixed paraffin-embedded) tissue slide was separated with a razor, put into a tube, and 200 μl of heptane was used for 1 hour. Deparaffinized at °C. Then, for crushing, extraction and homogenization of the sample, 100 μl of 5% SDS, 50 mM Triethylammonium biocarbonate buffer was added using an AFA ^® (adaptive focused acoustics ^® ) ultrasonic grinder, and then the sample was lysed. Thereafter, the sample was digested using Trypsin/LysC protease to prepare a peptide, and then the protein was analyzed using LC-MS.

1.2. proteomic analysis

Proteomic analysis was performed using LC-MS. The mass spectrometry (hereinafter referred to as 'MS') equipment is Q-Exactive Plus BioPharm version (ThermoFisher, USA), and liquid chromatography (hereinafter referred to as 'LC') is the UltiMate™ RSLCnano System (ThermoFisher, USA) was used. Samples were injected by 5.0 μl using an automatic sample injection device and analyzed with an ion trap mass spectrometer linked to NanoLC at a flow rate of 250 nl/min.

As a column used for LC, a fused silica capillary column C18 having an inner diameter of 75 μm and an outer diameter of 360 μm was used. Samples were analyzed at a flow rate of 250 nl/min for 200 min, and the concentration gradient was separated from 5% solution B to 50% solution B (80% acetonitrile, 0.1% formic acid, 5% DMSO) for 90 min.

As parameters for MS, MS1 resolution 70000, MS1 maximum fill time 20 msec, data dependent acquisition (DDA) method (top 10) were used, MS2 resolution 17500, MS2 maximum fill time 100 msec, AGC (auto gain control) 1e6 was performed.

Example 2. Analysis of proteomic data and identification of malignant peripheral schwannoma (MPNST) specific protein through it

Proteomic data analysis was performed on the malignant peripheral schwannoma (MPNST) and benign neurofibroma (NF) tissue samples obtained in Example 1.

First, as shown in Figure 2, each raw data was used using Proteome Discover 2.2 software (Thermo, USA). Using the human proteomic reference database provided by Uniprot, the qualitative information and quantitative information of proteins were extracted with a label free quantitation algorithm. For qualitative analysis, carbamidomethylation was used for cysteine as a fixed modification condition among peptide search conditions, and n-terminal acetylation and methionine oxidation were applied as a variable modification condition. The initial mass error of precursor ions was adjusted to 10 ppm and the mass error for fragment ions was adjusted to 20 ppm, and both unique peptide and razor peptide were used when mapping peptides to proteins. For label-free quantification, the peak intensity of the unique peptide was used.

As a result, as shown in FIG. 3 , it was confirmed that the distribution of quantitative values of proteomic values obtained from each sample was uniform.

In addition, principal component analysis (PCA) was performed using the proteomic quantitative information of the sample. As a result, as shown in FIG. 4 , it was confirmed that the two groups could be distinguished through quantitative and qualitative differences in proteomic bodies between the two groups of MPNST and NF. More specifically, referring to the graph on the right of FIG. 4 , 3,483 proteins were identified in the positive NF tissue and 3,601 proteins were identified in the MPNST tissue, and 141 proteins were not identified in the positive NF tissue but only identified in the MPNST tissue. 23 cases were found.

Example 3. Derivation of protein with increased expression in malignant peripheral schwannoma (MPNST)

As shown in FIG. 5a through quantitative analysis of the proteomic using volcano plot analysis, there was a 10-fold increase in expression in MPNST compared to positive NF, and the protein mainly expressed in extracellular exosomes. irradiated (indicated by a red box). This is because the protein excreted in exosomes can be detected in blood or urine, which is advantageous for collecting samples for diagnosis.

As a result, as shown on the right side of FIG. 5A , 24 proteins such as CD248, DNAJB4, POTEE, SPARCL1, and TBC1D4 were selected.

In addition, as shown in FIG. 5b , a protein (proteinaceous extracellular matrix) that decomposes the extracellular matrix was investigated (indicated by a red box). This is because proteins expressed in the extracellular matrix can also be detected in blood or urine. In addition, since matrix degradation is a phenomenon that occurs when cancer cells infiltrate surrounding tissues, it is a protein based on cell malignancy.

As a result, as shown on the right side of FIG. 5B , CD248, SPARCL1, CILP2, MMP8, and SPON1 proteins were selected.

Comparative Example 1. Investigation of expression patterns of S-100 protein in malignant peripheral schwannoma (MPNST) and benign neurofibroma (NF)

The expression pattern of S-100 protein, which has been used as an important pathological index for the distinction between positive NF and MPNST, was investigated.

As a result of examining a total of 9 types of S-100 isoforms, as shown in FIGS. 6a to 6i , there was a difference in the expression pattern between positive NF and MPNST according to individual patients. These results clearly reveal the limitations of the S-100 protein as a specific marker for the differentiation of positive NF and MPNST.

In addition, as a result of examining vimentin as another indicator to distinguish between positive NF and MPNST, there was no difference in the expression pattern between positive NF and MPNST.

Taken together, the above results suggest that S-100 and vimentin, which are previously used markers, have clear limitations in clinical application as MPNST-specific biomarkers.

Example 4. Selection of diagnostic markers for malignant peripheral schwannoma (MPNST)

4.1. Endsialin (CD248)

Endsialin, a protein encoded by the CD248 gene, is a molecule involved in vascular regeneration of tumors, and is secreted into the extracellular matrix and is also found in exosomes.

As can be seen in FIG. 7 , endosialin was specifically found in all MPNST tissues obtained from 5 patients. In particular, since endosialin is found in exosomes, it is expected to have high utility as a diagnostic marker for MPNST in that it is expected to be detectable in blood and urine samples.

4.2. SPARC-Like Protein 1 (SPARCL1)

SPARC-like protein 1 encoded by the SPARCL1 gene is mainly secreted extracellularly and is a protein that binds to calcium, extracellular matrix, collagen, and the like.

As shown in FIG. 8 , SPARC-like protein 1 was not detected in positive NF, but was specifically expressed in MPNST. Since this protein is also found extracellularly and is also present in exosomes, it is expected that it can be detected in blood and urine samples, and thus its utility as a diagnostic marker for MPNST is expected to be high.

4.3. Neutrophil Collagenase (MMP8)

Neutrophil collagenase encoded by the MMP8 gene is a protease that degrades the extracellular matrix, and is known to be involved in cancer metastasis and various inflammatory responses.

As shown in FIG. 9 , it was confirmed that neutrophyll collagenase was specifically expressed in all MPNST tissues obtained from 5 patients. In particular, since neutrophilic collagenase is secreted into the extracellular matrix, it is expected to be highly useful as a diagnostic marker for MPNST in that it is expected to be detectable in blood and urine samples.

Example 5. New therapeutic target discovery of malignant peripheral schwannoma (MPNST)

5.1. hierarchical cluster analysis

The proteomic whose expression is specifically increased in MPNST was divided into two clusters through hierarchical clustering analysis. More specifically, as shown in FIG. 10a, from a total of 3,624 proteins identified in both groups, 3,455 proteins identified in 80% or more samples were extracted from each group, and Student's T test was performed for both groups to perform Benjamini-Hochberg FDR. 292 proteins satisfying < 0.01 were selected. As a result of calculating the Z score for the quantitative information of the corresponding proteins and performing K-means clustering analysis, it was confirmed that two clusters were formed. For proteins corresponding to each cluster, the biological process, cellular compartment, and molecular function were investigated by performing g:Profiler (https://biit.cs.ut.ee/gprofiler/).

Referring to FIG. 10B , cluster 1 is a protein with increased expression in MPNST, and a protein that functions by binding to the extracellular matrix, such as actin and musculoskeletal protein, was found.

Referring to FIG. 10c , cluster 2 is a protein with reduced expression in MPNST, and proteins mainly secreted into the extracellular matrix or present in exosomes were found.

5.2. Discovery of target signaling system through GO analysis and IPA (ingenuity pathway analysis)

Gene ontology analysis and QIAGEN Ingenuity Pathway Analysis were performed on 144 MPNST-specific proteins to identify pathways involving proteins whose expression specifically changes in MPNST. Biological processes, cellular compartments, and molecular functions related to 144 MPNST-specific proteins were investigated by inputting 144 MPNST-specific protein accession numbers to ShinyGO and IPA server.

As a result, as shown in FIGS. 11 and 12 , it was confirmed that the ILK signaling system, the actin-cytoskeleton signaling system, and the RhoGDI signaling system were specifically promoted in MPNST. In addition, when inferring the upstream signaling of the signaling system, it was predicted that the enhancement of TGF-β would be the basis.

5.3. Discovering kinases as new therapeutic targets

The kinase specifically expressed or increased in activity in MPNST in association with the signaling system discovered in Example 5.2 can be utilized as a therapeutic target. Accordingly, the present inventors have discovered a kinase that is increased in MPNST.

As a result, as shown in FIGS. 13 to 21 , it was confirmed that kinases such as CDK4, MAP2K1, PDK3, STK10, ALPK3, PHKG1, CDK5, PNKP and PACSIN3 were increased in MPNST. Accordingly, inhibitors of these kinases, for example antagonists to these kinases, may be used in the treatment of MPNST.

In particular, fostamatinib (fostamatinib) is expected to be utilized as a therapeutic agent for MPNST because it exhibits multiple antagonistic action against CDK4, MAP2k1, STK1 and PHKG1.

Example 6. Verification of diagnostic markers of malignant peripheral schwannoma (MPNST)

In order to verify the expression of MMP8 proteome among the MPNST diagnostic markers discovered in Example 4, IHC staining was performed on the tissues of MPNST patients.

As a result, as shown in Table 4 and FIG. 22, it was confirmed that the MMP8 protein (neutrophyll collagenase) was overexpressed in the MPNST tissue as compared to the NF tissue.

In addition, the difference in MMP8 protein expression in blood, which is easy to obtain a patient's sample, was confirmed by ELISA analysis. Specifically, among the normal group (WT00), NF1 patients (SEL00), MPNST patients, patients with post-OP tumor removal completely (clear), patients with tumors that may exist because they are not completely removed depending on the tumor removal pattern (intermediate), the plasma from patients with no clearance or metastasis (involvement) was analyzed by ELISA assay.

As a result, as shown in Table 5 and FIG. 23, it was confirmed that the expression of MMP8 protein was increased in the plasma of a patient whose tumor was not completely removed (intermediate), metastasized, or not removed at all (involvement). From the above results confirming the difference in MMP8 protein expression depending on whether or not tumor was removed, it was confirmed that MMP8 protein can be utilized as a diagnostic marker for MPNST.

Example 7. Validation of the therapeutic target of malignant peripheral schwannoma (MPNST)

ILK, Cofilin, MEK, p in MPNST patient tissues to present the ILK signaling system, actin-cytoskeleton signaling system, and abnormal activity of various kinases, which were discovered in Example 5 above. -MEK, and IHC staining of the PACSIN3 proteome was performed.

As a result, as shown in Table 6 and FIG. 24, it was confirmed that the expression of ILK, Cofilin, MEK, p-MEK, and PACSIN3 proteomics was increased in MPNST tissues compared to NF tissues.

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

<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation Humanscape Inc. <120> Composition for Differential Diagnosis of Malignant Peripheral Nerve Sheath Tumor <130> MP20-260P1 <150> KR 10-2021-0037912 <151> 2021-03-24 <160> 13 <170> KoPatentIn 3.0 <210> 1 < 211> 757 <212> PRT <213> Homo sapiens <400> 1 Met Leu Leu Arg Leu Leu Leu Ala Trp Ala Ala Ala Gly Pro Thr Leu 1 5 10 15 Gly Gln Asp Pro Trp Ala Ala Glu Pro Arg Ala Ala Cys Gly Pro Ser 20 25 30 Ser Cys Tyr Ala Leu Phe Pro Arg Arg Arg Thr Phe Leu Glu Ala Trp 35 40 45 Arg Ala Cys Arg Glu Leu Gly Gly Asp Leu Ala Thr Pro Arg Thr Pro 50 55 60 Glu Glu Ala Gln Arg Val Asp Ser Leu Val Gly Ala Gly Pro Ala Ser 65 70 75 80 Arg Leu Leu Trp Ile Gly Leu Gln Arg Gln Ala Arg Gln Cys Gln Leu 85 90 95 Gln Arg Pro Leu Arg Gly Phe Thr Trp Thr Thr Gly Asp Gln Asp Thr 100 105 110 Ala Phe Thr Asn Trp Ala Gln Pro Ala Ser Gly Gly Pro Cys Pro Ala 115 120 125 Gln Arg Cys Val Ala Leu Glu Ala Ser Gly Glu His Arg Trp Leu Glu 130 135 140 Gly Ser Cys Thr Leu Ala Val Asp Gly Tyr Leu Cys Gln Phe Gly Phe 145 150 155 160 Glu Gly Ala Cys Pro Ala Leu Gln Asp Glu Ala Gly Gln Ala Gly Pro 165 170 175 Ala Val Tyr Thr Thr Pro Phe His Leu Val Ser Thr Glu Phe Glu Trp 180 185 190 Leu Pro Phe Gly Ser Val Ala Ala Val Gln Cys Gln Ala Gly Arg Gly 195 200 205 Ala Ser Leu Leu Cys Val Lys Gln Pro Glu Gly Gly Val Gly Trp Ser 210 215 220 Arg Ala Gly Pro Leu Cys Leu Gly Thr Gly Cys Ser Pro Asp Asn Gly 225 230 235 240 Gly Cys Glu His Glu Cys Val Glu Glu Val Asp Gly His Val Ser Cys 245 250 255 Arg Cys Thr Glu Gly Phe Arg Leu Ala Ala Asp Gly Arg Ser Cys Glu 260 265 270 Asp Pro Cys Ala Gln Ala Pro Cys Glu Gln Gln Cys Glu Pro Gly Gly 275 280 285 Pro Gln Gly Tyr Ser Cys His Cys Arg Leu Gly Phe Arg Pro Ala Glu 290 295 300 Asp Asp Pro His Arg Cys Val Asp Thr Asp Glu Cys Gln Ile Ala Gly 305 310 315 320 Val Cys Gln Gln Met Cys Val Asn Tyr Val Gly Gly Phe Glu Cys Tyr 325 330 335 Cys Ser Glu Gly His Glu Leu Glu Ala Asp Gly Ile Ser Cys Ser Pro 340 345 350 Ala Gly Ala Met Gly Ala Gln Ala Ser Gln Asp Leu Gly Asp Glu Leu 355 360 365 Leu Asp Asp Gly Glu Asp Glu Glu Asp Glu Asp Glu Ala Trp Lys Ala 370 375 380 Phe Asn Gly Gly Trp Thr Glu Met Pro Gly Ile Leu Trp Met Glu Pro 385 390 395 400 Thr Gln Pro Pro Asp Phe Ala Leu Ala Tyr Arg Pro Ser Phe Pro Glu 405 410 415 Asp Arg Glu Pro Gln Ile Pro Tyr Pro Glu Pro Thr Trp Pro Pro Pro 420 425 430 Leu Ser Ala Pro Arg Val Pro Tyr His Ser Ser Val Leu Ser Val Thr 435 440 445 Arg Pro Val Val Val Ser Ala Thr His Pro Thr Leu Pro Ser Ala His 450 455 460 Gln Pro Pro Val Ile Pro Ala Thr His Pro Ala Leu Ser Arg Asp His 465 470 475 480 Gln Ile Pro Val Ile Ala Ala Asn Tyr Pro Asp Leu Pro Ser Ala Tyr 485 490 495 Gln Pro Gly Ile Leu Ser Val Ser His Ser Ala Gln Pro Pro Ala His 500 505 510 Gln Pro Pro Met Ile Ser Thr Lys Tyr Pro Glu Leu Phe Pro Ala His 515 520 525 Gln Ser Pro Met Phe Pro Asp Thr Arg Val Ala Gly Thr Gln Thr Thr 530 535 540 Thr His Leu Pro Gly Ile Pro Pro Asn His Ala Pro Leu Val Thr Thr 545 550 555 560 Leu Gly Ala Gln Leu Pro Pro Gln Ala Pro Asp Ala Leu Val Leu Arg 565 570 575 Thr Gln Ala Thr Gln Leu Pro Ile Ile Pro Thr Ala Gln Pro Ser Leu 580 585 590 Thr Thr Thr Ser Arg Ser Pro Val Ser Pro Ala His Gln Ile Ser Val 595 600 605 Pro Ala Ala Thr Gln Pro Ala Ala Leu Pro Thr Leu Leu Pro Ser Gln 610 615 620 Ser Pro Thr Asn Gln Thr Ser Pro Ile Ser Pro Thr His Pro His Ser 625 630 635 640 Lys Ala Pro Gln Ile Pro Arg Glu Asp Gly Pro Ser Pro Lys Leu Ala 645 650 655 Leu Trp Leu Pro Ser Pro Ala Pro Thr Ala Ala Pro Thr Ala Leu Gly 660 665 670 Glu Ala Gly Leu Ala Glu His Ser Gln Arg Asp Asp Arg Trp Leu Leu 675 680 685 Val Ala Leu Leu Val Pro Thr Cys Val Phe Leu Val Val Leu Leu Ala 690 695 700 Leu Gly Ile Val Tyr Cys Thr Arg Cys Gly Pro His Ala Pro Asn Lys 705 710 715 720 Arg Ile Thr Asp Cys Tyr Arg Trp Val Ile His Ala Gly Ser Lys Ser 725 730 735 Pro Thr Glu Pro Met Pro Pro Arg Gly Ser Leu Thr Gly Val Gln Thr 740 745 750 Cys Arg Thr Ser Val 755 <210> 2 <211> 664 <212> PRT <213> Homo sapiens <400> 2 Met Lys Thr Gly Leu Phe Phe Leu Cys Leu Leu Gly Thr Ala Ala Ala 1 5 10 15 Ile Pro Thr Asn Ala Arg Leu Leu Ser Asp His Ser Lys Pro Thr Ala 20 25 30 Glu Thr Val Ala Pro Asp Asn Thr Ala Ile Pro Ser Leu Arg Ala Glu 35 40 45 Ala Glu Glu Asn Glu Lys Glu Thr Ala Val Ser Thr Glu Asp Ser 50 55 60 His His Lys Ala Glu Lys Ser Ser Val Leu Lys Ser Lys Glu Glu Ser 65 70 75 80 His Glu Gln Ser Ala Glu Gln Gly Lys Ser Ser Ser Gln Glu Leu Gly 85 90 95 Leu Lys Asp Gln Glu Asp Ser Asp Gly His Leu Ser Val Asn Leu Glu 100 105 110 Tyr Ala Pro Thr Glu Gly Thr Leu Asp Ile Lys Glu Asp Met Ser Glu 115 120 125 Pro Gln Glu Lys Lys Leu Ser Glu Asn Thr Asp Phe Leu Ala Pro Gly 130 135 140 Val Ser Ser Phe Thr Asp Ser Asn Gln Gln Glu Ser Ile Thr Lys Arg 145 150 155 160 Glu Glu Asn Gln Glu Gln Pro Arg Asn Tyr Ser His His Gln Leu Asn 165 170 175 Arg Ser Ser Lys His Ser Gln Gly Leu Arg Asp Gln Gly Asn Gln Glu 180 185 190 Gln Asp Pro Asn Ile Ser Asn Gly Glu Glu Glu Glu Glu Glu Lys Glu Pro 195 200 205 Gly Glu Val Gly Thr His Asn Asp Asn Gln Glu Arg Lys Thr Glu Leu 210 215 220 Pro Arg Glu His Ala Asn Ser Lys Gln Glu Glu Asp Asn Thr Gln Ser 225 230 235 240 Asp Asp Ile Leu Glu Glu Ser Asp Gln Pro Thr Gln Val Ser Lys Met 245 250 255 Gln Glu Asp Glu Phe Asp Gln Gly Asn Gln Glu Gln Glu Asp Asn Ser 260 265 270 Asn Ala Glu Met Glu Glu Glu Glu Asn Ala Ser Asn Val Asn Lys His Ile 275 280 285 Gln Glu Thr Glu Trp Gln Ser Gln Glu Gly Lys Thr Gly Leu Glu Ala 290 295 300 Ile Ser Asn His Lys Glu Thr Glu Glu Lys Thr Val Ser Glu Ala Leu 305 310 315 320 Leu Met Glu Pro Thr Asp Asp Gly Asn Thr Thr Pro Arg Asn His Gly 325 330 335 Val Asp Asp Asp Gly Asp Asp Asp Gly Asp Asp Gly Gly Thr Asp Gly 340 345 350 Pro Arg His Ser Ala Ser Asp Asp Tyr Phe Ile Pro Ser Gln Ala Phe 355 360 365 Leu Glu Ala Glu Arg Ala Gln Ser Ile Ala Tyr His Leu Lys Ile Glu 370 375 380 Glu Gln Arg Glu Lys Val His Glu Asn Glu Asn Ile Gly Thr Thr Glu 385 390 395 400 Pro Gly Glu His Gin Glu Ala Lys Lys Ala Glu Asn Ser Ser A sn Glu 405 410 415 Glu Glu Thr Ser Ser Glu Gly Asn Met Arg Val His Ala Val Asp Ser 420 425 430 Cys Met Ser Phe Gln Cys Lys Arg Gly His Ile Cys Lys Ala Asp Gln 435 440 445 Gln Gly Lys Pro His Cys Val Cys Gln Asp Pro Val Thr Cys Pro Pro 450 455 460 Thr Lys Pro Leu Asp Gln Val Cys Gly Thr Asp Asn Gln Thr Tyr Ala 465 470 475 480 Ser Ser Cys His Leu Phe Ala Thr Lys Cys Arg Leu Glu Gly Thr Lys 485 490 495 Lys Gly His Gln Leu Gln Leu Asp Tyr Phe Gly Ala Cys Lys Ser Ile 500 505 510 Pro Thr Cys Thr Asp Phe Glu Val Ile Gln Phe Pro Leu Arg Met Arg 515 520 525 Asp Trp Leu Lys Asn Ile Leu Met Gln Leu Tyr Glu Ala Asn Ser Glu 530 535 540 His Ala Gly Tyr Leu Asn Glu Lys Gln Arg Asn Lys Val Lys Lys Ile 5 45 550 555 560 Tyr Leu Asp Glu Lys Arg Leu Leu Ala Gly Asp His Pro Ile Asp Leu 565 570 575 Leu Leu Arg Asp Phe Lys Lys Asn Tyr His Met Tyr Val Tyr Pro Val 580 585 590 His Trp Gln Phe Ser Glu Leu Asp Gln His Pro Met Asp Arg Val Leu 595 600 605 Thr His Ser Glu Leu Ala Pro Leu Arg Ala Ser Leu Val Pro Met Glu 610 615 620 His Cys Ile Thr Arg Phe Phe Glu Glu Cys Asp Pro Asn Lys Asp Lys 625 630 635 640 His Ile Thr Leu Lys Glu Trp Gly His Cys Phe Gly Ile Lys Glu Glu 645 650 655 Asp Ile Asp Glu Asn Leu Leu Phe 660 <210> 3 <211> 467 <212> PRT <213> Homo sapiens <400> 3 Met Phe Ser Leu Lys Thr Leu Pro Phe Leu Leu Leu Leu His Val Gln 1 5 10 15 Ile Ser Lys Ala Phe Pro Val Ser Ser Lys Glu Lys Asn Thr Lys Thr 20 25 30 Val Gln Asp Tyr L eu Glu Lys Phe Tyr Gln Leu Pro Ser Asn Gln Tyr 35 40 45 Gln Ser Thr Arg Lys Asn Gly Thr Asn Val Ile Val Glu Lys Leu Lys 50 55 60 Glu Met Gln Arg Phe Phe Gly Leu Asn Val Thr Gly Lys Pro Asn Glu 65 70 75 80 Glu Thr Leu Asp Met Met Lys Lys Pro Arg Cys Gly Val Pro Asp Ser 85 90 95 Gly Gly Phe Met Leu Thr Pro Gly Asn Pro Lys Trp Glu Arg Thr Asn 100 105 110 Leu Thr Tyr Arg Ile Arg Asn Tyr Thr Pro Gln Leu Ser Glu Ala Glu 115 120 125 Val Glu Arg Ala Ile Lys Asp Ala Phe Glu Leu Trp Ser Val Ala Ser 130 135 140 Pro Leu Ile Phe Thr Arg Ile Ser Gln Gly Glu Ala Asp Ile Asn Ile 145 150 155 160 Ala Phe Tyr Gln Arg Asp His Gly Asp Asn Ser Pro Phe Asp Gly Pro 165 170 175 Asn Gly Ile Leu Ala His Ala Phe Gln Pro Gly Gln Gly Ile Gly Gly 180 185 190 Asp Ala His Phe Asp Ala Glu Glu Thr Trp Thr Asn Thr Se r Ala Asn 195 200 205 Tyr Asn Leu Phe Leu Val Ala Ala His Glu Phe Gly His Ser Leu Gly 210 215 220 Leu Ala His Ser Ser Asp Pro Gly Ala Leu Met Tyr Pro Asn Tyr Ala 225 230 235 240 Phe Arg Glu Thr Ser Asn Tyr Ser Leu Pro Gln Asp Asp Ile Asp Gly 245 250 255 Ile Gln Ala Ile Tyr Gly Leu Ser Ser Asn Pro Ile Gln Pro Thr Gly 260 265 270 Pro Ser Thr Pro Lys Pro Cys Asp Pro Ser Leu Thr Phe Asp Ala Ile 275 280 285 Thr Thr Leu Arg Gly Glu Ile Leu Phe Phe Lys Asp Arg Tyr Phe Trp 290 295 300 Arg Arg His Pro Gln Leu Gln Arg Val Glu Met Asn Phe Ile Ser Leu 305 310 315 320 Phe Trp Pro Ser Leu Pro Thr Gly Ile Gln Ala Ala Tyr Glu Asp Phe 325 330 335 Asp Arg Asp Leu Ile Phe Leu Phe Lys Gly As n Gln Tyr Trp Ala Leu 340 345 350 Ser Gly Tyr Asp Ile Leu Gln Gly Tyr Pro Lys Asp Ile Ser Asn Tyr 355 360 365 Gly Phe Pro Ser Ser Val Gln Ala Ile Asp Ala Ala Val Phe Tyr Arg 370 375 380 Ser Lys Thr Tyr Phe Phe Val Asn Asp Gln Phe Trp Arg Tyr Asp Asn 385 390 395 400 Gln Arg Gln Phe Met Glu Pro Gly Tyr Pro Lys Ser Ile Ser Gly Ala 405 410 415 Phe Pro Gly Ile Glu Ser Lys Val Asp Ala Val Phe Gln Gln Glu His 420 425 430 Phe Phe His Val Phe Ser Gly Pro Arg Tyr Tyr Ala Phe Asp Leu Ile 435 440 445 Ala Gln Arg Val Thr Arg Val Ala Arg Gly Asn Lys Trp Leu Asn Cys 450 455 460 Arg Tyr Gly 465 <210> 4 <211> 303 <212> PRT <213> Homo sapiens <400> 4 Met Ala Thr Ser Arg Tyr Glu Pro Val Ala Glu Ile Gly Val Gly Ala 1 5 10 15 Tyr Gly Thr Val Tyr Lys Ala Arg Asp Pro His Ser Gly His Phe Val 20 25 30 Ala Leu Lys Ser Val Arg Val Pro Asn Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45 Leu Pro Ile Ser Thr Val Arg Glu Val Ala Leu Leu Arg Arg Leu Glu 50 55 60 Ala Phe Glu His Pro Asn Val Val Arg Leu Met Asp Val Cys Ala Thr 65 70 75 80 Ser Arg Thr Asp Arg Glu Ile Lys Val Thr Leu Val Phe Glu His Val 85 90 95 Asp Gln Asp Leu Arg Thr Tyr Leu Asp Lys Ala Pro Pro Pro Gly Leu 100 105 110 Pro Ala Glu Thr Ile Lys Asp Leu Met Arg Gln Phe Leu Arg Gly Leu 115 120 125 Asp Phe Leu His Ala Asn Cys Ile Val His Arg Asp Leu Lys Pro Glu 130 135 140 Asn Ile Leu Val Thr Ser Gly Gly Thr Val Lys Leu Ala Asp Phe Gly 145 150 155 160 Leu Ala Arg Ile Tyr Ser Tyr Gln Met Ala Leu Thr Pro Val Val Val 165 170 175 Thr Leu Trp Tyr Arg Ala Pro Glu Val Leu Leu Gln Ser Thr Tyr Ala 180 185 190 Thr Pro Val Asp Met Trp Ser Val Gly Cys Ile Phe Ala Glu Met Phe 195 200 205 Arg Arg Lys Pro Leu Phe Cys Gly Asn Ser Glu Ala Asp Gln Leu Gly 210 215 220 Lys Ile Phe Asp Leu Ile Gly Leu Pro Pro Glu Asp Asp Trp Pro Arg 225 230 235 240 Asp Val Ser Leu Pro Arg Gly Ala Phe Pro Pro Arg Gly Pro Arg Pro 245 250 255 Val Gln Ser Val Val Pro Glu Met Glu Glu Ser Gly Ala Gln Leu Leu 260 265 270 Leu Glu Met Leu Thr Phe Asn Pro His Lys Arg Ile Ser Ala Phe Arg 275 280 285 Ala Leu Gln His Ser Tyr Leu His Lys Asp Glu Gly Asn Pro Glu 290 295 300 <210> 5 <211> 968 <212> PRT <213> Homo sapiens <400> 5 Met Ala Phe Ala Asn Phe Arg Arg Ile Leu Arg Leu Ser Thr Phe Glu 1 5 10 15 Lys Arg Lys Ser Arg Glu Tyr Glu His Val Arg Arg Asp Leu Asp Pro 20 25 30 Asn Glu Val Trp Glu Ile Val Gly Glu Leu Gly Asp Gly Ala Phe Gly 35 40 45 Lys Val Tyr Lys Ala Lys Asn Lys Glu Thr Gly Ala Leu Ala Ala Ala 50 55 60 Lys Val Ile Glu Thr Lys Ser Glu Glu Glu Leu Glu Asp Tyr Ile Val 65 70 75 80 Glu Ile Glu Ile Leu Ala Thr Cys Asp His Pro Tyr Ile Val Lys Leu 85 90 95 Leu Gly Ala Tyr Tyr His Asp Gly Lys Leu Trp Ile Met Ile Glu Phe 100 105 110 Cys Pro Gly Gly Ala Val Asp Ala Ile Met Leu Glu Leu Asp Arg Gly 115 120 125 Leu Thr Glu Pro Gln Ile Gln Val Val Cys Arg Gln Met Leu Glu Ala 130 135 140 Leu Asn Phe Leu His Ser Lys Arg Ile Ile His Arg Asp Leu Lys Ala 145 150 155 160 Gly Asn Val Leu Met Thr Leu Glu Gly Asp Ile Arg Leu Ala Asp Phe 165 170 175 Gly Val Ser Ala Lys Asn Leu Lys Thr Leu Gln Lys Arg Asp Ser Phe 180 185 190 Ile Gly Thr Pro Tyr Trp Met Ala Pro Glu Val Val Met Cys Glu Thr 195 200 205 Met Lys Asp Thr Pro Tyr Asp Tyr Lys Ala Asp Ile Trp Ser Leu Gly 210 215 220 Ile Thr Leu Ile Glu Met Ala Gln Ile Glu Pro Pro His His Glu Leu 225 230 235 240 Asn Pro Met Arg Val Leu Leu Lys Ile Ala Lys Ser Asp Pro Pro Thr 245 250 255 Leu Leu Thr Pro Ser Lys Trp Ser Val Glu Phe Arg Asp Phe Leu Lys 260 265 270 Ile Ala Leu Asp Lys Asn Pro Glu Thr Arg Pro Ser Ala Ala Gln Leu 275 280 285 Leu Glu His Pro Phe Val Ser Ser Ile Thr Ser Asn Lys Ala Leu Arg 290 295 300 Glu Leu Val Ala Glu Ala Lys Ala Glu Val Met Glu Glu Ile Glu Asp 305 310 315 320 Gly Arg Asp Glu Gly Glu Glu Glu Asp Ala Val Asp Ala Ala Ser Thr 325 330 335 Leu Glu Asn His Thr Gln Asn Ser Ser Glu Val Ser Pro Pro Ser Leu 340 345 350 Asn Ala Asp Lys Pro Leu Glu Glu Ser Pro Ser Thr Pro Leu Ala Pro 355 360 365 Ser Gln Ser Gln Asp Ser Val Asn Glu Pro Cys Ser Gln Pro Ser Gly 370 375 380 Asp Arg Ser Leu Gln Thr Thr Ser Pro Pro Val Val Ala Pro Gly Asn 385 390 395 400 Glu Asn Gly Leu Ala Val Pro Val Pro Leu Arg Lys Ser Arg Pro Val 405 410 415 Ser Met Asp Ala Arg Ile Gln Val Ala Gln Glu Lys Gln Val Ala Glu 420 425 430 Gln Gly Gly Asp Leu Ser Pro Ala Ala Asn Arg Ser Gln Lys Ala Ser 435 440 445 Gln Ser Arg Pro Asn Ser Ser Ala Leu Glu Thr Leu Gly Gly Glu Lys 450 455 460 Leu Ala Asn Gly Ser Leu Glu Pro Pro Ala Gln Ala Ala Pro Gly Pro 465 470 475 480 Ser Lys Arg Asp Ser Asp Cys Ser Ser Leu Cys Thr Ser Glu Ser Met 485 490 495 Asp Tyr Gly Thr Asn Leu Ser Thr Asp Leu Ser Leu Asn Lys Glu Met 500 505 510 Gly Ser Leu Ser Ile Lys Asp Pro Lys Leu Tyr Lys Lys Thr Leu Lys 515 520 525 Arg Thr Arg Lys Phe Val Val Asp Gly Val Glu Val Ser Ile Thr Thr 530 535 540 Ser Lys Ile Ile Ser Glu Asp Glu Lys Lys Asp Glu Glu Met Arg Phe 545 550 555 560 Leu Arg Arg Gln Glu Leu Arg Glu Leu Arg Leu Leu Gln Lys Glu Glu 565 570 575 His Arg Asn Gln Thr Gln Leu Ser Asn Lys His Glu Leu Gln Leu Glu 580 585 590 Gln Met His Lys Arg Phe Glu Gln Glu Ile Asn Ala Lys Lys Lys Phe 595 600 605 Phe Asp Thr Glu Leu Glu Asn Leu Glu Arg Gln Gln Lys Gln Gln Val 610 615 620 Glu Lys Met Glu Gln Asp His Ala Val Arg Arg Arg Glu Glu Ala Arg 625 630 635 640 Arg Ile Arg Leu Glu Gln Asp Arg Asp Tyr Thr Arg Phe Gln Glu Gln 645 650 655 Leu Lys Leu Met Lys Lys Glu Val Lys Asn Glu Val Glu Lys Leu Pro 660 665 670 Arg Gln Gln Arg Lys Glu Ser Met Lys Gln Lys Met Glu Glu His Thr 675 680 685 Gln Lys Lys Gln Leu Leu Asp Arg Asp Phe Val Ala Lys Gln Lys Glu 690 695 700 Asp Leu Glu Leu Ala Met Lys Arg Leu Thr Thr Asp Asn Arg Arg Glu 705 710 715 720 Ile Cys Asp Lys Glu Arg Glu Cys Leu Met Lys Lys Gln Glu Leu Leu 725 730 735 Arg Asp Arg Glu Ala Ala Leu Trp Glu Met Glu Glu His Gln Leu Gln 740 745 750 Glu Arg His Gln Leu Val Lys Gln Gln Leu Lys Asp Gln Tyr Phe Leu 755 760 765 Gln Arg His Glu Leu Leu Arg Lys His Glu Lys Glu Arg Glu Gln Met 770 775 780 Gln Arg Tyr Asn Gln Arg Met Ile Glu Gln Leu Lys Val Arg Gln Gln 785 790 795 800 Gln Glu Lys Ala Arg Leu Pro Lys Ile Gln Arg Ser Glu Gly Lys Thr 805 810 815 Arg Met Ala Met Tyr Lys Lys Ser Leu His Ile Asn Gly Gly Gly Ser 820 825 830 Ala Ala Glu Gln Arg Glu Lys Ile Lys Gln Phe Ser Gln Gln Glu Glu 835 840 845 Lys Arg Gln Lys Ser Glu Arg Leu Gln Gln Gln Gln Lys His Glu Asn 850 855 860 Gln Met Arg Asp Met Leu Ala Gln Cys Glu Ser Asn Met Ser Glu Leu 865 870 875 880 Gln Gln Leu Gln Asn Glu Lys Cys His Leu Leu Val Glu His Glu Thr 885 890 895 Gln Lys Leu Lys Ala Leu Asp Glu Ser His Asn Gln Asn Leu Lys Glu 900 905 910 Trp Arg Asp Lys Leu Arg Pro Arg Lys Lys Ala Leu Glu Glu Asp Leu 915 920 925 Asn Gln Lys Lys Arg Glu Gln Glu Met Phe Phe Lys Leu Ser Glu Glu 930 935 940 Ala Glu Cys Pro Asn Pro Ser Thr Pro Ser Lys Ala Ala Lys Phe Phe 945 950 955 960 Pro Tyr Ser Ser Ala Asp Ala Ser 965 <210> 6 <211> 292 <212> PRT <213> Homo sapiens <400> 6 Met Gln Lys Tyr Glu Lys Leu Glu Lys Ile Gly Glu Gly Thr Tyr Gly 1 5 10 15 Thr Val Phe Lys Ala Lys Asn Arg Glu Thr His Glu Ile Val Ala Leu 20 25 30 Lys Arg Val Arg Leu Asp Asp Asp Asp Glu Gly Val Pro Ser Ser Ala 35 40 45 Leu Arg Glu Ile Cys Leu Leu Lys Glu Leu Lys His Lys Asn Ile Val 50 55 60 Arg Leu His Asp Val Leu His Ser Asp Lys Lys Leu Thr Leu Val Phe 65 70 75 80 Glu Phe Cys Asp Gln Asp Leu Lys Lys Tyr Phe Asp Ser Cys Asn Gly 85 90 95 Asp Leu Asp Pro Glu Ile Val Lys Ser Phe Leu Phe Gln Leu Leu Lys 100 105 110 Gly Leu Gly Phe Cys His Ser Arg Asn Val Leu His Arg Asp Leu Lys 115 120 125 Pro Gln Asn Leu Leu Ile Asn Arg Asn Gly Glu Leu Lys Leu Ala Asp 130 135 140 Phe Gly Leu Ala Arg Ala Phe Gly Ile Pro Val Arg Cys Tyr Ser Ala 145 150 155 160 Glu Val Val Thr Leu Trp Tyr Arg Pro Pro Asp Val Leu Phe Gly Ala 165 170 175 Lys Leu Tyr Ser Thr Ser Ile Asp Met Trp Ser Ala Gly Cys Ile Phe 180 185 190 Ala Glu Leu Ala Asn Ala Gly Arg Pro Leu Phe Pro Gly Asn Asp Val 195 200 205 Asp Asp Gln Leu Lys Arg Ile Phe Arg Leu Leu Gly Thr Pro Thr Glu 210 215 220 Glu Gln Trp Pro Ser Met Thr Lys Leu Pro Asp Tyr Lys Pro Tyr Pro 225 230 235 240 Met Tyr Pro Ala Thr Thr Ser Leu Val Asn Val Val Pro Lys Leu Asn 245 250 255 Ala Thr Gly Arg Asp Leu Leu Gln Asn Leu Leu Lys Cys Asn P ro Val 260 265 270 Gln Arg Ile Ser Ala Glu Glu Ala Leu Gln His Pro Tyr Phe Ser Asp 275 280 285 Phe Cys Pro Pro 290 <210> 7 <211> 393 <212> PRT <213> Homo sapiens <400> 7 Met Pro Lys Lys Lys Pro Thr Pro Ile Gln Leu Asn Pro Ala Pro Asp 1 5 10 15 Gly Ser Ala Val Asn Gly Thr Ser Ser Ala Glu Thr Asn Leu Glu Ala 20 25 30 Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu Gln Gln Arg Lys 35 40 45 Arg Leu Glu Ala Phe Leu Thr Gln Lys Gln Lys Val Gly Glu Leu Lys 50 55 60 Asp Asp Asp Phe Glu Lys Ile Ser Glu Leu Gly Ala Gly Asn Gly Gly 65 70 75 80 Val Val Phe Lys Val Ser His Lys Pro Ser Gly Leu Val Met Ala Arg 85 90 95 Lys Leu Ile His Leu Glu Ile Lys Pro Ala Ile Arg Asn Gln Ile Ile 100 105 110 Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro Tyr Ile Val Gly 115 120 125 Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser Ile Cys Met Glu 130 135 140 His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys Lys Ala Gly Arg 145 150 155 160 Ile Pro Glu Gln Ile Leu Gly Lys Val Ser Ile Ala Val Ile Lys Gly 165 170 175 Leu Thr Tyr Leu Arg Glu Lys His Lys Ile Met His Arg Asp Val Lys 180 185 190 Pro Ser Asn Ile Leu Val Asn Ser Arg Gly Glu Ile Lys Leu Cys Asp 195 200 205 Phe Gly Val Ser Gly Gln Leu Ile Asp Ser Met Ala Asn Ser Phe Val 210 215 220 Gly Thr Arg Ser Tyr Met Ser Pro Glu Arg Leu Gln Gly Thr His Tyr 225 230 235 240 Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser Leu Val Glu Met 245 250 255 Ala Val Gly Arg Tyr Pro Ile Pro Pro Asp Ala Lys Glu Leu Glu 260 265 270 Leu Met Phe Gly Cys Gln Val Glu Gly Asp Ala Ala Glu Thr Pro Pro 275 280 285 Arg Pro Arg Thr Pro Gly Arg Pro Leu Ser Ser Tyr Gly Met Asp Ser 290 295 300 Arg Pro Pro Met Ala Ile Phe Glu Leu Leu Asp Tyr Ile Val Asn Glu 305 310 315 320 Pro Pro Pro Lys Leu Pro Ser Gly Val Phe Ser Leu Glu Phe Gln Asp 325 330 335 Phe Val Asn Lys Cys Leu Ile Lys Asn Pro Ala Glu Arg Ala Asp Leu 340 345 350 Lys Gln Leu Met Val His Ala Phe Ile Lys Arg Ser Asp Ala Glu Glu 355 360 365 Val Asp Phe Ala Gly Trp Leu Cys Ser Thr Ile Gly Leu Asn Gln Pro 370 375 380 Ser Thr Pro Thr His Ala Ala Gly Val 385 390 <210> 8 <211> 1907 <212> PRT <213> Homo sapiens <400> 8 Met Glu Val Ala Trp Leu Val Tyr Val Leu Gly Gln Gln Pro Leu Ala 1 5 10 15 Arg Gln Gly Glu Gly Gln Ser Arg Leu Val Pro Gly Arg Gly Leu Val 20 25 30 Leu Trp Leu Pro Gly Leu Pro Arg Ser Ser Pro Ser Trp Pro Ala Val 35 40 45 Asp Leu Ala Pro Leu Ala Pro Ala Arg Pro Arg Gly Pro Leu Ile Cys 50 55 60 His Thr Gly His Glu Gln Ala Gly Arg Glu Pro Gly Pro Gly Ser Ser 65 70 75 80 Thr Lys Gly Pro Val Leu His Asp Gln Asp Thr Arg Cys Ala Phe Leu 85 90 95 Pro Arg Pro Pro Gly Pro Leu Gln Thr Arg Arg Tyr Cys Arg His Gln 100 105 110 Gly Arg Gln Gly Ser Gly Leu Gly Ala Gly Pro Gly Ala Gly Thr Trp 115 120 125 Ala Pro Ala Pro Pro Gly Val Ser Lys Pro Arg Cys Pro Gly Arg Ala 130 135 140 Arg Pro Gly Glu Gly Gln Gln Gln Val Thr Thr Ala Arg Pro Pro Ala 145 150 155 160 Ile Asn Arg Gly Ala Arg Gln Pro Arg Ala Gly Ala Ala Ala Ala Gly 165 170 175 Arg Gly Pro Gly Ala Gly Ala Trp Arg Thr Gly Glu Ala Ala Ala Ser 180 185 190 Ala Gly Pro Ala Val Gly Glu Gly Gly Ala Met Gly Ser Arg Arg Ala 195 200 205 Pro Ser Arg Gly Trp Gly Ala Gly Gly Arg Ser Gly Ala Gly Gly Asp 210 215 220 Gly Glu Asp Asp Gly Pro Val Trp Ile Pro Ser Pro Ala Ser Arg Ser 225 230 235 240 Tyr Leu Leu Ser Val Arg Pro Glu Thr Ser Leu Ser Ser Asn Arg Leu 245 250 255 Ser His Pro Ser Ser Gly Arg Ser Thr Phe Cys Ser Ile Ile Ala Gln 260 265 270 Leu Thr Glu Glu Thr Gln Pro Leu Phe Glu Thr Thr Leu Lys Ser Arg 275 280 285 Ser Val Ser Glu Asp Ser Asp Val Arg Phe Thr Cys Ile Val Thr Gly 290 295 300 Tyr Pro Glu Pro Glu Val Thr Trp Tyr Lys Asp Asp Thr Glu Leu Asp 305 310 315 320 Arg Tyr Cys Gly Leu Pro Lys Tyr Glu Ile Thr His Gln Gly Asn Arg 325 330 335 His Thr Leu Gln Leu Tyr Arg Cys Arg Glu Glu Asp Ala Ala Ile Tyr 340 345 350 Gln Ala Ser Ala Gln Asn Ser Lys Gly Ile Val Ser Cys Ser Gly Val 355 360 365 Leu Glu Val Gly Thr Met Thr Glu Tyr Lys Ile His Gln Arg Trp Phe 370 375 380 Ala Lys Leu Lys Arg Lys Ala Ala Ala Lys Leu Arg Glu Ile Glu Gln 385 390 395 400 Ser Trp Lys His Glu Lys Ala Val Pro Gly Glu Val Asp Thr Leu Arg 405 410 415 Lys Leu Ser Pro Asp Arg Phe Gln Arg Lys Arg Arg Leu Ser Gly Ala 420 425 430 Gln Ala Pro Gly Pro Ser Val Pro Thr Arg Glu Pro Glu Gly Gly Thr 435 440 445 Leu Ala Ala Trp Gln Glu Gly Glu Thr Glu Thr Ala Gln His Ser Gly 450 455 460 Leu Gly Leu Ile Asn Ser Phe Ala Ser Gly Glu Val Thr Thr Asn Gly 465 470 475 480 Glu Ala Ala Pro Glu Asn Gly Glu Asp Gly Glu His Gly Leu Leu Thr 485 490 495 Tyr Ile Cys Asp Ala Met Glu Leu Gly Pro Gln Arg Ala Leu Lys Glu 500 505 510 Glu Ser Gly Ala Lys Lys Lys Lys Lys Asp Glu Glu Ser Lys Gln Gly 515 520 525 Leu Arg Lys Pro Glu Leu Glu Lys Ala Ala Gln Ser Arg Arg Ser Ser 530 535 540 Glu Asn Cys Ile Pro Ser Ser Asp Glu Pro Asp Ser Cys Gly Thr Gln 545 550 555 560 Gly Pro Val Gly Val Glu Gln Val Gln Thr Gln Pro Arg Gly Arg Ala 565 570 575 Ala Arg Gly Pro Gly Ser Ser Gly Thr Asp Ser Thr Arg Lys Pro Ala 580 585 590 Ser Ala Val Gly Thr Pro Asp Lys Ala Gln Lys Ala Pro Gly Pro Gly 595 600 605 Pro Gly Gln Glu Val Tyr Phe Ser Leu Lys Asp Met Tyr Leu Glu Asn 610 615 620 Thr Gln Ala Val Arg Pro Leu Gly Glu Glu Gly Pro Gln Thr Leu Ser 625 630 635 640 Val Arg Ala Pro Gly Glu Ser Pro Lys Gly Lys Ala Pro Leu Arg Ala 645 650 655 Arg Ser Glu Gly Val Pro Gly Ala Pro Gly Gln Pro Thr His Ser Leu 660 665 670 Thr Pro Gln Pro Thr Arg Pro Phe Asn Arg Lys Arg Phe Ala Pro Pro 675 680 685 Lys Pro Lys Gly Glu Ala Thr Thr Asp Ser Lys Pro Ile Ser Ser Leu 690 695 700 Ser Gln Ala Pro Glu Cys Gly Ala Gln Ser Leu Gly Lys Ala Pro Pro 705 710 715 720 Gln Ala Ser Val Gln Val Pro Thr Pro Pro Ala Arg Arg Arg His Gly 725 730 735 Thr Arg Asp Ser Thr Leu Gln Gly Gln Ala Gly His Arg Thr Pro Gly 740 745 750 Glu Val Leu Glu Cys Gln Thr Thr Thr Ala Pro Thr Met Ser Ala Ser 755 760 765 Ser Ser Ser Asp Val Ala Ser Ile Gly Val Ser Thr Ser Gly Ser Gln 770 775 780 Gly Ile Ile Glu Pro Met Asp Met Glu Thr Gln Glu Asp Gly Arg Thr 785 790 795 800 Ser Ala Asn Gln Arg Thr Gly Ser Lys Lys Asn Val Gln Ala Asp Gly 805 810 815 Lys Ile Gln Val Asp Gly Arg Thr Arg Gly Asp Gly Thr Gln Thr Ala 820 825 830 Gln Arg Thr Arg Ala Asp Arg Lys Thr Gln Val Asp Ala Gly Thr Gln 835 840 845 Glu Ser Lys Arg Pro Gln Ser Asp Arg Ser Ala Gln Lys Gly Met Met 850 855 860 Thr Gln Gly Arg Ala Glu Thr Gln Leu Glu Thr Thr Gln Ala Gly Glu 865 870 875 880 Lys Ile Gln Glu Asp Arg Lys Ala Gln Ala Asp Lys Gly Thr Gln Glu 885 890 895 Asp Arg Met Gln Gly Glu Lys Gly Met Gln Gly Glu Lys Gly Thr 900 905 910 Gln Ser Glu Gly Ser Ala Pro Thr Ala Met Glu Gly Gln Ser Glu Gln 915 920 925 Glu Val Ala Thr Ser Leu Gly Pro Pro Ser Arg Thr Pro Lys Leu Pro 930 935 940 Pro Thr Ala Gly Pro Arg Ala Pro Leu Asn Ile Glu Cys Phe Val Gln 945 950 955 960 Thr Pro Glu Gly Ser Cys Phe Pro Lys Lys Pro Gly Cys Leu Pro Arg 965 970 975 Ser Glu Glu Ala Val Val Thr Ala Ser Arg Asn His Glu Gln Thr Val 980 985 990 Leu Gly Pro Leu Ser Gly Asn Leu Met Leu Pro Ala Gln Pro Pro His 995 1000 1005 Glu Gly Ser Val Glu Gln Val Gly Gly Glu Arg Cys Arg Gly Pro Gln 1010 1015 1020 Ser Ser Gly Pro Val Glu Ala Lys Gln Glu Asp Ser Pro Phe Gln Cys 1025 1030 1035 1040 Pro Lys Glu Glu Arg Pro Gly Gly Val Pro Cys Met Asp Gln Gly Gly 1045 1050 1055 Cys Pro Leu Ala Gly Leu Ser Gln Glu Val Pro Thr Met Pro Ser Leu 1060 1065 1070 Pro Gly Thr Gly Leu Thr Ala Ser Pro Lys Ala Gly Pro Cys Ser Thr 1075 1080 1085 Pro Thr Ser Gln His Gly Ser Thr Ala Thr Phe Leu Pro Ser Glu Asp 1090 1095 1100 Gln Val Leu Met Ser Ser Ala Pro Thr Leu His Leu Gly Leu Gly Thr 1105 1110 1115 1120 Pro Thr Gln Ser His Pro Pro Glu Thr Met Ala Thr Ser Ser Glu Gly 1125 1130 1135 Ala Cys Ala Gln Val Pro Asp Val Glu Gly Arg Thr Pro Gly Pro Arg 1140 1145 1150 Ser Cys Asp Pro Gly Leu Ile Asp Ser Leu Lys Asn Tyr Leu Leu Leu 1155 1160 1165 Leu Leu Lys Leu Ser Ser Thr Glu Thr Ser Gly Ala Gly Gly Glu Ser 1170 1175 1180 Gln Val Gly Ala Ala Thr Gly Gly Leu Val Pro Ser Ala Thr Leu Thr 1185 1190 1195 1200 Pro Thr Val Glu Val Ala Gly Leu Ser Pro Arg Thr Ser Arg Arg Ile 1205 1210 1215 Leu Glu Arg Val Glu Asn Asn His Leu Val Gln Ser Ala Gln Thr Leu 1220 1225 1230 Leu Leu Ser Pro Cys Thr Ser Arg Arg Leu Thr Gly Leu Leu Asp Arg 1235 1240 1245 Glu Val Gln Ala Gly Arg Gln Ala Leu Ala Ala Ala Arg Gly Ser Trp 1250 1255 1260 Gly Pro Gly Pro Ser Ser Leu Thr Val Pro Ala Ile Val Val Asp Glu 1265 1270 1275 1280 Glu Asp Pro Gly Leu Ala Ser Glu Gly Ala Ser Glu Gly Glu Gly Glu 1285 1290 1295 Val Ser Pro Glu Gly Pro Gly Leu Leu Gly Ala Ser Gln Glu Ser Ser 1300 1305 1310 Met Ala Gly Arg Leu Gly Glu Ala Gly Gly Gln Ala Ala Pro G ly Gln 1315 1320 1325 Gly Pro Ser Ala Glu Ser Ile Ala Gln Glu Pro Ser Gln Glu Glu Lys 1330 1335 1340 Phe Pro Gly Glu Ala Leu Thr Gly Leu Pro Ala Ala Thr Pro Glu Glu 1345 1350 1355 1360 Leu Ala Leu Gly Ala Arg Arg Lys Arg Phe Leu Pro Lys Val Arg Ala 1365 1370 1375 Ala Gly Asp Gly Glu Ala Thr Thr Pro Glu Glu Arg Glu Ser Pro Thr 1380 1385 1390 Val Ser Pro Arg Gly Pro Arg Lys Ser Leu Val Pro Gly Ser Pro Gly 1395 1400 1405 Thr Pro Gly Arg Glu Arg Arg Ser Pro Thr Gln Gly Arg Lys Ala Ser 1410 1415 1420 Met Leu Glu Val Pro Arg Ala Glu Glu Glu Leu Ala Ala Gly Asp Leu 1425 1430 1435 1440 Gly Pro Ser Pro Lys Ala Gly Gly Leu Asp Thr Glu Val Ala Leu Asp 1445 1450 1455 Glu Gly Lys Gln Glu Thr Leu Ala Lys Pro Arg Lys Ala Lys Asp Leu 1460 1465 1470 Leu Lys Ala Pro Gln Val Ile Arg Lys Ile Arg Val Glu Gln Phe Pro 1475 1480 1485 Asp Ala Ser Gly Ser Leu Lys Leu Trp Cys Gln Phe Phe Asn Ile Leu 1490 1495 1500 Ser Asp Ser Val Leu Thr Trp Ala Lys Asp Gln Arg Pro Val Gly Glu 1505 1510 1515 1520 Val Gly Arg Ser Ala Gly Asp Glu Gly Pro Ala Ala Leu Ala Ile Val 1525 1530 1535 Gln Ala Ser Pro Val Asp Cys Gly Val Tyr Arg Cys Thr Ile His Asn 1540 1545 1550 Glu His Gly Ser Ala Ser Thr Asp Phe Cys Leu Ser Pro Glu Val Leu 1555 1560 1565 Ser Gly Phe Ile Ser Arg Glu Glu Gly Glu Val Gly Glu Glu Ile Glu 1570 1575 1580 Met Thr Pro Met Val Phe Ala Lys Gly Leu Ala Asp Ser Gly Cys Trp 1585 1590 1595 1600 Gly Asp Lys Leu Phe Gly Arg Leu Val Ser Glu Glu Leu Arg Gly Gly 1605 1610 1615 Gly Tyr Gly Cys Gly Leu Arg Lys Ala Ser Gln Ala Lys Val Ile Tyr 1620 1625 1630 Gly Leu Glu Pro Ile Phe Glu Ser Gly Arg Thr Cys Ile Ile Lys Val 1635 1640 1645 Ser Ser Leu Leu Val Phe Gly Pro Ser Ser Glu Thr Ser Leu Val Gly 1650 1655 1660 Arg Asn Tyr Asp Val Thr Ile Gln Gly Cys Lys Ile Gln Asn Met Ser 1665 1670 1675 1680 Arg Glu Tyr Cys Lys Ile Phe Ala Ala Glu Ala Arg Ala Ala Pro Gly 1685 1690 1695 Phe Gly Glu Val Pro Glu Ile Ile Ile Pro Leu Tyr Leu Ile Tyr Arg Pro 1700 1705 1710 Ala Asn Asn Ile Pro Tyr Ala Thr Leu Glu Glu Asp Leu Gly L ys Pro 1715 1720 1725 Leu Glu Ser Tyr Cys Ser Arg Glu Trp Gly Cys Ala Glu Ala Pro Thr 1730 1735 1740 Ala Ser Gly Ser Ser Glu Ala Met Gln Lys Cys Gln Thr Phe Gln His 1745 1750 1755 1760 Trp Leu Tyr Gln Trp Thr Asn Gly Ser Phe Leu Val Thr Asp Leu Ala 1765 1770 1775 Gly Val Asp Trp Lys Met Thr Asp Val Gln Ile Ala Thr Lys Leu Arg 1780 1785 1790 Gly Tyr Gln Gly Leu Lys Glu Ser Cys Phe Pro Ala Leu Leu Asp Arg 1795 1800 1805 Phe Ala Ser Ser His Gln Cys Asn Ala Tyr Cys Glu Leu Leu Gly Leu 1810 1815 1820 Thr Pro Leu Lys Gly Pro Glu Ala Ala His Pro Gln Ala Lys Ala Lys 1825 1830 1835 1840 Gly Ser Lys Ser Pro Ser Ala Gly Arg Lys Gly Ser Gln Leu Ser Pro 1845 1850 1855 Gln Pro Gln Lys Lys Gly Leu Pro Ser Pro Gln Gly Thr Arg Lys Ser 1860 1865 1870 Ala Pro Ser Ser Lys Ala Thr Pro Gln Ala Ser Glu Pro Val Thr Thr 1875 1880 1885 Gln Leu Leu Gly Gln Pro Pro Thr Gln Glu Glu Gly Ser Lys Ala Gln 1890 1895 1900 Gly Met Arg 1905 <210> 9 <211> 521 <212> PRT <213> Homo sapiens <400> 9 Met Gly Glu Val Glu Ala Pro Gly Arg Leu Trp Leu Glu Ser Pro Pro 1 5 10 15 Gly Gly Ala Pro Pro Ile Phe Leu Pro Ser Asp Gly Gln Ala Leu Val 20 25 30 Leu Gly Arg Gly Pro Leu Thr Gln Val Thr Asp Arg Lys Cys Ser Arg 35 40 45 Thr Gln Val Glu Leu Val Ala Asp Pro Glu Thr Arg Thr Val Ala Val 50 55 60 Lys Gln Leu Gly Val Asn Pro Ser Thr Thr Gly Thr Gln Glu Leu Lys 65 70 75 80 Pro Gly Leu Glu Gly Ser Leu Gly Val Gly Asp Thr Leu Tyr Leu Val 85 90 95 Asn Gly Leu His Pro Leu Thr Leu Arg Trp Glu Glu Thr Arg Thr Pro 100 105 110 Glu Ser Gln Pro Asp Thr Pro Pro Gly Thr Pro Leu Val Ser Gln Asp 115 120 125 Glu Lys Arg Asp Ala Glu Leu Pro Lys Lys Arg Met Arg Lys Ser Asn 130 135 140 Pro Gly Trp Glu Asn Leu Glu Lys Leu Leu Val Phe Thr Ala Ala Gly 145 150 155 160 Val Lys Pro Gln Gly Lys Val Ala Gly Phe Asp Leu Asp Gly Thr Leu 165 170 175 Ile Thr Thr Arg Ser Gly Lys Val Phe Pro Thr Gly Pro Ser Asp Trp 180 185 190 Arg Ile Leu Tyr Pro Glu Ile Pro Arg Lys Leu Arg Glu Leu Glu Ala 195 200 205 Glu Gly Tyr Lys Leu Val Ile Phe Thr Asn Gln Met Ser Ile Gly Arg 210 215 220 Gly Lys Leu Pro Ala Glu Glu Phe Lys Ala Lys Val Glu Ala Val Val 225 230 235 240 Glu Lys Leu Gly Val Pro Phe Gln Val Leu Val Ala Thr His Ala Gly 245 250 255 Leu Tyr Arg Lys Pro Val Thr Gly Met Trp Asp His Leu Gln Glu Gln 260 265 270 Ala Asn Asp Gly Thr Pro Ile Ser Ile Gly Asp Ser Ile Phe Val Gly 275 280 285 Asp Ala Ala Gly Arg Pro Ala Asn Trp Ala Pro Gly Arg Lys Lys Lys 290 295 300 Asp Phe Ser Cys Ala Asp Arg Leu Phe Ala Leu Asn Leu Gly Leu Pro 305 310 315 320 Phe Ala Thr Pro Glu Glu Phe Phe Leu Lys Trp Pro Ala Ala Gly Phe 325 330 335 Glu Leu Pro Ala Phe Asp Pro Arg Thr Val Ser Arg Ser Gly Pro Leu 340 345 350 Cys Leu Pro Glu Ser Arg Ala Leu Leu Ser Ala Ser Pro Glu Val Val 355 360 365 Val Ala Val Gly Phe Pro Gly Ala Gly Lys Ser Thr Phe Leu Lys Lys 370 375 380 His Leu Val Ser Ala Gly Tyr Val His Val Asn Arg Asp Thr Leu Gly 385 390 395 400 Ser Trp Gln Arg Cys Val Thr Thr Cys Glu Thr Ala Leu Lys Gln Gly 405 410 415 Lys Arg Val Ala Ile Asp Asn Thr Asn Pro Asp Ala Ala Ser Arg Ala 420 425 430 Arg Tyr Val Gln Cys Ala Arg Ala Ala Gly Val Pro Cys Arg Cys Phe 435 440 445 Leu Phe Thr Ala Thr Leu Glu Gln Ala Arg His Asn Asn Arg Phe Arg 450 455 460 Glu Met Thr Asp Ser Ser His Ile Pro Val Ser Asp Met Val Met Tyr 465 470 475 480 Gly Tyr Arg Lys Gln Phe Glu Ala Pro Thr Leu Ala Glu Gly Phe Ser 485 490 495 Ala Ile Leu Glu Ile Pro Phe Arg Leu Trp Val Glu Pro Arg Leu Gly 500 505 510 Arg Leu Tyr Cys Gln Phe Ser Glu Gly 515 520 <210> 10 <211> 406 <212> PRT <213> Homo sapiens <400> 10 Met Arg Leu Phe Arg Trp Leu Leu Lys Gln Pro Val Pro Lys Gln Ile 1 5 10 15 Glu Arg Tyr Ser Arg Phe Ser Pro Ser Pro Leu Ser Ile Lys Gln Phe 20 25 30 Leu Asp Phe Gly Arg Asp Asn Ala Cys Glu Lys Thr Ser Tyr Met Phe 35 40 45 Leu Arg Lys Glu Leu Pro Val Arg Leu Ala Asn Thr Met Arg Glu Val 50 55 60 Asn Leu Leu Pro Asp Asn Leu Leu Asn Arg Pro Ser Val Gly Leu Val 65 70 75 80 Gln Ser Trp Tyr Met Gln Ser Phe Leu Glu Leu Leu Glu Tyr Glu Asn 85 90 95 Lys Ser Pro Glu Asp Pro Gln Val Leu Asp Asn Phe Leu Gln Val Leu 100 105 110 Ile Lys Val Arg Asn Arg His Asn Asp Val Val Pro Thr Met Ala Gln 115 120 125 Gly Val Ile Glu Tyr Lys Glu Lys Phe Gly Phe Asp Pro Phe Ile Ser 130 135 140 Thr Asn Ile Gln Tyr Phe Leu Asp Arg Phe Tyr Thr Asn Arg Ile Ser 145 150 155 160 Phe Arg Met Leu Ile Asn Gln His Thr Leu Leu Phe Gly Gly Asp Thr 165 170 175 Asn Pro Val His Pro Lys His Ile Gly Ser Ile Asp Pro Thr Cys Asn 180 185 190 Val Ala Asp Val Val Lys Asp Ala Tyr Glu Thr Ala Lys Met Leu Cys 195 200 205 Glu Gln Tyr Tyr Leu Val Ala Pro Glu Leu Glu Val Glu Glu Phe Asn 210 215 220 Ala Lys Ala Pro Asp Lys Pro Ile Gln Val Val Tyr Val Pro Ser His 225 230 235 240 Leu Phe His Met Leu Phe Glu Leu Phe Lys Asn Ser Met Arg Ala Thr 245 250 255 Val Glu Leu Tyr Glu Asp Arg Lys Glu Gly Tyr Pro Ala Val Lys Thr 260 265 270 Leu Val Thr Leu Gly Lys Glu Asp Leu Ser Ile Lys Ile Ser Asp Leu 275 280 285 Gly Gly Gly Val Pro Leu Arg Lys Ile Asp Arg Leu Phe Asn Tyr Met 290 295 300 Tyr Ser Thr Ala Pro Arg Pro Ser Leu Glu Pro Thr Arg Ala Ala Pro 305 310 315 320 Leu Ala Gly Phe Gly Tyr Gly Leu Pro Ile Ser Arg Leu Tyr Ala Arg 325 330 335 Tyr Phe Gln Gly Asp Leu Lys Leu Tyr Ser Met Glu Gly Val Gly Thr 340 345 350 Asp Ala Val Ile Tyr Leu Lys Ala Leu Ser Ser Glu Ser Phe Glu Arg 355 360 365 Leu Pro Val Phe Asn Lys Ser Ala Trp Arg His Tyr Lys Thr Thr Pro 370 375 380 Glu Ala Asp Asp Trp Ser Asn Pro Ser Ser Glu Pro Arg Asp Ala Ser 385 39 0 395 400 Lys Tyr Lys Ala Lys Gln 405 <210> 11 <211> 387 <212> PRT <213> Homo sapiens <400> 11 Met Thr Arg Asp Glu Ala Leu Pro Asp Ser His Ser Ala Gln Asp Phe 1 5 10 15 Tyr Glu Asn Tyr Glu Pro Lys Glu Ile Leu Gly Arg Gly Val Ser Ser 20 25 30 Val Val Arg Arg Cys Ile His Lys Pro Thr Ser Gln Glu Tyr Ala Val 35 40 45 Lys Val Ile Asp Val Thr Gly Gly Gly Ser Phe Ser Pro Glu Glu Val 50 55 60 Arg Glu Leu Arg Glu Ala Thr Leu Lys Glu Val Asp Ile Leu Arg Lys 65 70 75 80 Val Ser Gly His Pro Asn Ile Ile Gln Leu Lys Asp Thr Tyr Glu Thr 85 90 95 Asn Thr Phe Phe Phe Leu Val Phe Asp Leu Met Lys Arg Gly Glu Leu 100 105 110 Phe Asp Tyr Leu Thr Glu Lys Val Thr Leu Ser Glu Lys Glu Thr Arg 115 120 125 Lys Ile Met Arg Ala Leu Leu Glu Val Ile Cys Thr Leu His Lys Leu 130 135 140 Asn Ile Val His Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp Asp 145 150 155 160 Asn Met Asn Ile Lys Leu Thr Asp Phe Gly Phe Ser Cys Gln Leu Glu 165 170 175 Pro Gly Glu Arg Leu Arg Glu Val Cys Gly Thr Pro Ser Tyr Leu Ala 180 185 190 Pro Glu Ile Ile Glu Cys Ser Met Asn Glu Asp His Pro Gly Tyr Gly 195 200 205 Lys Glu Val Asp Met Trp Ser Thr Gly Val Ile Met Tyr Thr Leu Leu 210 215 220 Ala Gly Ser Pro Pro Phe Trp His Arg Lys Gln Met Leu Met Leu Arg 225 230 235 240 Met Ile Met Ser Gly Asn Tyr Gln Phe Gly Ser Pro Glu Trp Asp Asp 245 250 255 Tyr Ser Asp Thr Val Lys Asp Leu Val Ser Arg Phe Leu Val Val Gln 260 265 270 Pro Gln Asn Arg Tyr Thr Ala Glu Glu Ala Leu Ala His Pro Phe Phe 275 280 285 Gln Gln Tyr Leu Val Glu Glu Val Arg His Phe Ser Pro Arg Gly Lys 290 295 300 Phe Lys Val Ile Ala Leu Thr Val Leu Ala Ser Val Arg Ile Tyr Tyr 305 310 315 320 Gln Tyr Arg Arg Val Lys Pro Val Thr Arg Glu Ile Val Ile Arg Asp 325 330 335 Pro Tyr Ala Leu Arg Pro Leu Arg Arg Leu Ile Asp Ala Tyr Ala Phe 340 345 350 Arg Ile Tyr Gly His Trp Val Lys Lys Gly Gln Gln Gln Asn Arg Ala 355 360 365 Ala Leu Phe Glu Asn Thr Pro Lys Ala Val Leu Leu Ser Leu Ala Glu 370 375 380 Glu Asp Tyr 385 <210> 12 <211> 424 <212> PRT <213> Homo sapiens <400> 12 Met Ala Pro Glu Glu Asp Ala Gly Gly Glu Ala Leu Gly Gly Ser Phe 1 5 10 15 Trp Glu Ala Gly Asn Tyr Arg Arg Thr Val Gln Arg Val Glu Asp Gly 20 25 30 His Arg Leu Cys Gly Asp Leu Val Ser Cys Phe Gln Glu Arg Ala Arg 35 40 45 Ile Glu Lys Ala Tyr Ala Gln Gln Leu Ala Asp Trp Ala Arg Lys Trp 50 55 60 Arg Gly Thr Val Glu Lys Gly Pro G ln Tyr Gly Thr Leu Glu Lys Ala 65 70 75 80 Trp His Ala Phe Phe Thr Ala Ala Glu Arg Leu Ser Ala Leu His Leu 85 90 95 Glu Val Arg Glu Lys Leu Gln Gly Gln Asp Ser Glu Arg Val Arg Ala 100 105 110 Trp Gln Arg Gly Ala Phe His Arg Pro Val Leu Gly Gly Phe Arg Glu 115 120 125 Ser Arg Ala Ala Glu Asp Gly Phe Arg Lys Ala Gln Lys Pro Trp Leu 130 135 140 Lys Arg Leu Lys Glu Val Glu Ala Ser Lys Lys Ser Tyr His Ala Ala 145 150 155 160 Arg Lys Asp Glu Lys Thr Ala Gln Thr Arg Glu Ser His Ala Lys Ala 165 170 175 Asp Ser Ala Val Ser Gln Glu Gln Leu Arg Lys Leu Gln Glu Arg Val 180 185 190 Glu Arg Cys Ala Lys Glu Ala Glu Lys Thr Lys Ala Gln Tyr Glu Gln 195 200 205 Thr Leu Ala Glu Leu His Arg Tyr Thr Pro Arg Tyr Met Glu Asp Met 210 215 220 Glu Gln Ala Phe Glu Thr Cys Gln Ala Ala Glu Arg Gln Arg Leu Leu 225 230 235 240 Phe Phe Lys Asp Met Leu Leu Thr Leu His Gln His Leu Asp Leu Ser 245 250 255 Ser Ser Ser Glu Lys Phe His Glu Leu His Arg Asp Leu His Gln Gly Ile 260 265 270 Glu Ala Ala Ser Asp Glu Glu Asp Leu Arg Trp Trp Arg Ser Thr His 275 280 285 Gly Pro Gly Met Ala Met Asn Trp Pro Gln Phe Glu Glu Trp Ser Leu 290 295 300 Asp Thr Gln Arg Thr Ile Ser Arg Lys Glu Lys Gly Gly Arg Ser Pro 305 310 315 320 Asp Glu Val Thr Leu Thr Ser Ile Val Pro Thr Arg Asp Gly Thr Ala 325 330 335 Pro Pro Pro Gln Ser Pro Gly Ser Pro Gly Thr Gly Gln Asp Glu Glu 340 345 350 Trp Ser Asp Glu Glu Ser Pro Arg Lys Ala Ala Thr Gly Val Arg Val 355 360 365 Arg Ala Leu Tyr Asp Tyr Ala Gly Gln Glu Ala Asp Glu Leu Ser Phe 370 375 380 Arg Ala Gly Glu Glu Leu Leu Lys Met Ser Glu Glu Asp Glu Gln Gly 385 390 395 400 Trp Cys Gln Gly Gln Leu Gln Ser Gly Arg Ile Gly Leu Tyr Pro Ala 405 410 415 Asn Tyr Val Glu Cys Val Gly Ala 420 <210> 13 <211> 166 <212> PRT <213> Homo sapiens <400> 13 Met Ala Ser Gly Val Ala Val Ser Asp Gly Val Ile Lys Val Phe Asn 1 5 10 15 Asp Met Lys Val Arg Lys Ser Ser Thr Pro Glu Glu Val Lys Lys Arg 20 25 30 Lys Lys Ala Val Leu Phe Cys Leu Ser Glu Asp Lys Lys Asn Ile Ile 35 40 45 Leu Glu Glu Gly Lys Glu Ile Leu Val Gly Asp Val Gly Gln Thr Val 50 55 60 Asp Asp Pro Tyr Ala Thr Phe Val Lys Met Leu Pro Asp Lys Asp Cys 65 70 75 80 Arg Tyr Ala Leu Tyr Asp Ala Thr Tyr Glu Thr Lys Glu Ser Lys Lys 85 90 95 Glu Asp Leu Val Phe Ile Phe Trp Ala Pro Glu Ser Ala Pro Leu Lys 100 105 110 Ser Lys Met Ile Tyr Ala Ser Ser Lys Asp Ala Ile Lys Lys Lys Leu 115 120 125 Thr Gly Ile Lys His Glu Leu Gln Ala Asn Cys Tyr Glu Glu Val Lys 130 135 140 Asp Arg Cys Thr Leu Ala Glu Lys Leu Gly Gly Ser Ala Val Ile Ser 145 150 155 160Leu Glu Gly Lys Pro Leu 165

Claims (16)

One or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase, or an agent for measuring the mRNA expression level thereof as an active ingredient Including, a composition for differential diagnosis of malignant peripheral nerve sheath tumor (MPNST).
According to claim 1,
The composition for differential diagnosis of malignant peripheral schwannoma, characterized in that it can distinguish and diagnose malignant peripheral schwannoma from type 1 neurofibromatosis.
According to claim 1,
The agent for measuring the expression level of the protein is an antibody or aptamer specific for the protein, a composition for differential diagnosis of malignant peripheral schwannoma.
According to claim 1,
The composition for differential diagnosis of malignant peripheral schwannoma, characterized in that the agent for measuring the expression level of the mRNA is a primer set or probe that specifically binds to the mRNA.
A kit for differential diagnosis of malignant peripheral schwannoma, comprising the composition of any one of claims 1 to 4.
6. The method of claim 5,
The kit is one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase, or the expression of mRNA thereof is detected or their A kit for differential diagnosis of malignant peripheral schwannoma, characterized in that it includes instructions for determining that the expression level is higher than the expression level of the benign neurofibroma sample.
One or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase or mRNA expression levels thereof in a biological sample isolated from a subject A method of providing information necessary for differential diagnosis of malignant peripheral schwannoma, comprising the step of measuring.
8. The method of claim 7,
In the method, the expression of one or more proteins selected from the group consisting of endosialin, SPARC-like protein 1 and neutrophil collagenase or its mRNA is detected or their When the expression level is higher than the expression level in a biological sample isolated from a type 1 neurofibromatosis patient, the information necessary for the differential diagnosis of malignant peripheral schwannoma, characterized in that it further comprises the step of diagnosing malignant peripheral schwannoma. How to provide.
8. The method of claim 7,
The method for providing information necessary for differential diagnosis of malignant peripheral schwannoma, characterized in that the subject is a patient suspected of malignant peripheral schwannoma or a patient diagnosed with type 1 neurofibromatosis.
8. The method of claim 7,
The biological sample is at least one selected from the group consisting of blood, whole blood, plasma, urine, tissue, cells, organs, bone marrow, microneedle aspiration sample, microneedle washing solution, core needle biopsy sample, and saliva. A method of providing information necessary for the differential diagnosis of malignant peripheral schwannoma.
8. The method of claim 7,
The protein expression level was determined by Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunostaining, immunoprecipitation assay, complement fixation assay, mass A method for providing information necessary for differential diagnosis of malignant peripheral schwannoma, characterized in that it is measured by at least one method selected from the group consisting of mass spectrometry, FACS, and protein chip.
8. The method of claim 7,
The mRNA expression level is PCR, RNase protection assay, Northern blotting (northern blotting), Southern blotting (southern blotting), in situ hybridization method, characterized in that measured by one or more methods selected from the group consisting of a DNA chip, A method of providing information necessary for the differential diagnosis of malignant peripheral schwannoma.
A method for screening a substance for preventing, improving or treating malignant peripheral schwannoma, comprising the steps of:
(a) contacting the candidate material with the isolated cell or tissue;
(b) the activity or encoding of one or more signaling proteins selected from the group consisting of TGF-β signaling pathway, ILK signaling pathway, actin-cytoskeleton signaling pathway and RhoGDI signaling pathway in the cell or tissue measuring the expression level of the gene; and
(c) when the activity of the protein or the expression level of the gene encoding the protein in the cell or tissue contacted with the candidate substance is reduced compared to the level before contact with the candidate substance, the candidate substance is used to prevent malignant peripheral schwannoma, Determining the substance for improvement or treatment.
14. The method of claim 13,
The signaling system protein is a kinase (kinase), characterized in that, screening method for the prevention, improvement or treatment of malignant peripheral schwannoma.
15. The method of claim 14,
The kinase is a kinase encoded by one or more genes selected from the group consisting of CDK4, STK10, CDK5, MAP2K1, ALPK3, PNKP, PDK3, PHKG1, CFL1 and PACSIN3. Prevention, improvement or treatment of malignant peripheral schwannoma, characterized in that it is a kinase Methods for screening substances for use.
14. The method of claim 13,
The isolated cell or tissue is a nerve cell or nerve tissue, characterized in that the screening method of a substance for the prevention, improvement or treatment of malignant peripheral schwannoma.

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