KR101123130B1 - Inhibitors of cell migration, invasion, or angiogenesis by blocking the function of PTK7 protein - Google Patents

Abstract

The present invention relates to inhibitors of cell migration, invasion or angiogenesis through inhibition of function of PTK7 protein, and according to the present invention, capillary-like tube formation (capillary) by VEGF. -like tube formation), cell migration, cell infiltration and cell adhesion, and inhibits the activation of PI3-kinase and Akt. In addition, by inhibiting the activation of FAK and paxillin, it is possible to effectively inhibit angiogenesis, cell migration and cell infiltration in vivo.

Description

 Inhibitors of cell migration, invasion, or angiogenesis by blocking the function of PTK7 protein

The present invention relates to inhibitors of cell migration, invasion or angiogenesis, and more particularly, cell migration and invasion, which can effectively inhibit angiogenesis, cell migration and cell invasion in vivo through the inhibition of function of PTK7 protein. Or providing angiogenesis inhibitors.

Human PTK7 (amino acid SEQ ID NO: GenBank AAN04862.1; mRNA SEQ ID NO: GenBank U40271) is an inactive type comprising an extracellular domain, a transmembrane domain, a tyrosine kinase domain, and the like. Defective receptor protein tyrosine kinase (defective RPTK), a schematic of its polypeptide structure is shown at the top of FIG. 1. Specifically, the PTK7 polypeptide is an ER signal peptide, an extracellular domain having seven immunoglobulin (loop, Ig loops), a transmembrane domain, a juxtamembrane domain, and lack of enzyme activity. Tyrosine kinase domain and C-terminal tail region.

We have identified human PTK7 (Park, SK et al., J Biochem (Tokyo) 119: 235-239) followed by mouse PTK7 (Jung, JW et al., Gene 328: 75-84), In addition, chicken KLG (Chou, YH and Hayman, MJ, Proc Natl Acad Sci USA 88: 4897-4901), Dtrk / Off-track (OTK) of Drosophila (Pulido, D. et al., EMBO J 11: 391- 404; Winberg, ML et al., Neuron 32: 53-62) and Hydra's Lemon (Miller, MA and Steele, RE, Dev Biol 224: 286-298) may be considered isotopes so PTK7 is systematically preserved It can be said.

Hydra Lemon mRNA, on the other hand, has been reported to increase expression in spermatogenesis, oogenesis, and embryoogenesis (Miller, MA and Steele, RE, Dev Biol 224: 286-298). Drosophila Dtrk / OTK has been reported to be involved in neuronal cell adhesion, regulating neuronal recognition and axon guidance (Pulido, D. et al., EMBO J 11). 391-404), together with Plexin A, acts as a receptor for Sema1A, one of the cell surface ligands of the semaphorin family, repulsive axon guidance. (Winberg, ML et al., Neuron 32: 53-62). In addition, chicken KLG binds to Sema 6D along with flexin A1 (Plexin-A1), and flexin A1-KLG-Sema 6D complex has been reported to play an important role in ventricular formation during chicken cardiac development ( Toyofuku, T. et al., Genes Dev 18: 435-447). Recently, incomplete PTK7-expressing mice consisting of only 114 N-terminal amino acids showed abnormalities in the stereoeous bundle orientation without closing neural tubes from the midbrain-back brain border to the end of the spinal cord. It dies before and after childbirth, and it has been reported that PTK7 regulates planar cell polarity (PCP) due to this feature (Lu, X. et al., Nature 430: 93-98).

Mouse PTK7 mRNA is highly expressed in the tail, ribs, segments, digestive tract, skull and facial areas early in development. Similarly, human PTK7 mRNA is elevated in the fetal colon (Mossie, K. et al., Oncogene 11: 2179-2184) and increased in metastatic colon carcinoma compared to normal colon (Saha, S. et al., Science 294: 1343-1346), reported to be reduced in metastatic melanoma (Easty, DJ et al., Int J Cancer 71: 1061-1065). Therefore, in view of the above, it can be estimated that there is a correlation between the high expression level of PTK7 in disease states such as tumors, but specifically PTK7 is associated with tumor development, cell migration, invasion, and blood vessels. It is difficult to determine whether it affects the newborn or if there is a direct causal relationship between the two.

Accordingly, the first problem to be solved by the present invention is to inhibit capillary-like tube formation, cell migration, cell invasion and cell adhesion by VEGF, PI3-kinase and Akt It is to provide an inhibitor of cell migration, invasion or angiogenesis that can effectively inhibit angiogenesis, cell migration and cell invasion in vivo by inhibiting the activation of FAK and paxillin.

The present invention to achieve the first object,

Inhibitors of cell migration, infiltration or angiogenesis through inhibition of function of PTK7 protein.

According to another embodiment of the invention, the inhibitor may comprise a soluble PTK7 protein fragment having an extracellular domain of PTK7 protein.

According to one embodiment of the present invention, the soluble PTK7 protein fragment may include the remaining sequence except the 1 to 30th sequence of the amino acid sequence represented by SEQ ID NO: 2 or the amino acid sequence represented by SEQ ID NO: 2, more preferably Preferably, the least active fragment of the soluble PTK7 protein may be amino acid sequence 31 to 409, or amino acid sequence 327 to 409 of the human PTK7 protein of SEQ ID NO: 2.

According to another embodiment of the present invention, the inhibitor may comprise a small scale RNA sequence that mediates specific RNA interference (RNAi) for messenger RNA (mRNA) encoding the PTK7 protein, more preferably The small RNA sequence comprises a small scale inhibitory RNA (siRNA), or the small scale inhibitory RNA (siRNA) sequence, which forms a complementary bond with a messenger RNA (mRNA) sequence encoding a PTK7 protein to inhibit expression of the mRNA sequence. Small hairpin RNA (shRNA).

According to another embodiment of the present invention, the inhibitor may be a PTK7 protein neutralizing antibody that specifically inhibits the activity of PTK7 protein by specifically binding to PTK7 protein, and the neutralizing antibody may recognize the amino acid sequence represented by SEQ ID NO: 2. Can be.

According to another embodiment of the present invention, the cell may be a vascular endothelial cell.

Hereinafter, the terms used in the present specification will be briefly described.

Unless stated otherwise, the term 'nucleic acid' refers to deoxyribonucleotides or ribonucleotides in single- or double-helix form, and inherently nucleotides that bind to nucleic acids in a manner similar to naturally occurring nucleotides.

Known analogs of tide, and unless otherwise indicated, certain nucleic acid sequences include their complementary sequences.

'Operatively linked to' means a functional binding between a nucleic acid expression control sequence (e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, thereby The regulatory sequence will control the transcription and / or translation process of said other nucleic acid sequence.

The term 'recombinant' as used in reference to a cell refers to the cell's replicating heterologous nucleic acid or expressing a peptide or protein encoded by the heterologous nucleic acid. Recombinant cells can also express genes found in the cell's original form, but modified genes can be reintroduced into cells by artificial methods.

'Promoter' means a promoter capable of controlling the expression of a protein in plant cells when the gene of the desired protein is fused downstream of the promoter. In addition, the promoter of the present invention can be further modified by binding to other transcription-translational active sequences.

Neutralizing antibodies are immune antibodies that are involved in neutralizing reactions. When an antigen, such as a virus, is toxic or infectious to an organism, it binds to the antigen and is active. It means an antibody that decays or disappears.

'Soluble protein fragment' is a kind of simple protein that hydrolyzes to produce only amino acids, and means a protein that can be dissolved in water or salt solution.

According to the present invention, it inhibits capillary-like tube formation, cell migration, cell infiltration and cell adhesion by VEGF, and also inhibits the activation of PI3-kinase and Akt. By inhibiting the activation of FAK and paxillin, it is possible to provide an inhibitor that effectively inhibits angiogenesis, cell migration and cell invasion in vivo. Angiogenesis, cell migration and cell infiltration inhibitors according to the present invention can be carried out by a variety of means of inhibiting PTK7 function such as soluble PTK7 protein fragment treatment, PTK7 knockdown and PTK7 neutralizing antibody treatment.

Hereinafter, the present invention will be described in more detail.

As described above, only the extent to which PTK7 protein is overexpressed in a disease state such as a tumor is known, and whether PTK7 protein is directly related to tumor development or related processes, and if so, what mechanism has been affected? Is unknown.

Thus, the present inventors first confirmed that inhibiting the function of human PTK7 protein prevents cell migration, invasion and angiogenesis. Thus, by inhibiting the function of human PTK7 protein, it is possible to prevent cell migration, invasion and angiogenesis and ultimately prevent and treat various diseases caused by cell migration, infiltration and angiogenesis including cancer metastasis.

Hereinafter, to provide various inhibitory means that can inhibit the activity of human PTK7, but the scope of the present invention is not limited thereto.

Specifically, the specific method for inhibiting function of PTK7 protein provided in the present invention may be implemented by various means, for example, by a soluble PTK7 (sPTK7) protein fragment or a part thereof capable of competing with and blocking the function of PTK7 protein. Can be implemented. The soluble PTK7 protein fragment was designed to have an extracellular domain or a portion thereof of PTK7 as shown in FIG. 1, which means capillary-like tube formation of vascular endothelial cells and cells. In addition to inhibiting migration and invasion, they inhibited angiogenesis in vivo, and also inhibited the activation of signaling proteins such as PI3-kinase and Akt. In addition, PTK7-Ig14s fragments designed to have domains from the first Ig loop to the fourth Ig loop of the PTK7 extracellular domain were also identical to sPTK7. It inhibited the migration of vascular endothelial cells. Soluble PTK7 protein fragments according to the present invention are believed to inhibit PTK7 function by binding to ligands of PTK7 that have not yet been identified and acting as decoy receptors that compete with endogenous PTK7 proteins.

Specifically, the soluble PTK7 (sPTK7) protein fragment that can be used in the present invention is not limited in kind as long as it can inhibit the activity of human PTK7 protein, preferably the protein represented by the amino acid sequence of SEQ ID NO: 2 or the Minimal active fragments may be used. As long as the amino acid sequence of SEQ ID NO: 2 can be encoded, the gene nucleotide sequence encoding the amino acid sequence is not limited, but preferably, the gene nucleotide sequence represented by SEQ ID NO: 1 may be used. More preferably, the sPTK7 sequence indicated by SEQ ID NO: 2 includes a His tag, and may include an amino acid sequence except for the ER signal peptide corresponding to Nos. 1 to 30. Meanwhile, as can be seen in Example 9, the minimally active fragment includes 31 to 409 of the sPTK7 amino acid sequence indicated by SEQ ID NO: 2, and more preferably, may include 327 to 409.

Preparation of soluble PTK7 protein fragments of the present invention comprises constructing a vector expressing a operably bound vector (pcDNA3-hPTK7-Ext-His) or a portion thereof to express human sPTK7 with His-tag, and then that the refinement of soluble PTK7 protein using Ni ^{2 +} -NTA column chromatography in HEK293 cells after a nucleic acid transfection (transfection), sPTK7 or remove the HEK293 cells expressing a part of each of the cultures, and the cell culture Glutathione S-transferase (GST) at a contiguous position at the 5'- or 3'-end to facilitate purification of the recombinant peptide or protein expressed from the recombinant expression vector, which may be performed by a method, but is not limited thereto. ), Nucleotide sequences encoding maltose binding protein (MBP), FLAG, 5-7x His (hexahistidine), NusA (N utilization substance A) or Trx (Thioredoxin) Enemies may be included in the.

On the other hand, the recombinant expression vector of the present invention is a strong promoter (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp) when the prokaryotic cell is a host Promoters, recA promoters, SP6 promoters, trp promoters and T7 promoters, etc.), ribosomal binding sites for initiation of translation, and transcription / detox termination sequences, and genomes of mammalian cells when eukaryotic cells are a host. Promoters derived from mammalian viruses (e.g., metallothionine promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, cytomegalovirus promoters, CMV-IE promoters and HSVs Tk promoter) can be used and polyadenylation as the transcription termination sequence It may have a fever.

On the other hand, the recombinant expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selectable label, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneeti Resistance genes for renal, neomycin, tetracycline and G418 resistance genes.

Preferred expression vectors of the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phages (eg, λgt4? ΛB, λ-Charon, λΔz1 and M13 which are often used in the art). Etc.) or viruses (eg, SV40, etc.), but are not limited thereto.

Host cells capable of stable and continuous cloning and expression of vectors in the methods of the present invention may employ any host cell known in the art, for example E. coli JM109, E. coli BL21 (DE3), E. Bacillus genus strains such as coli DH5α, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Enterobacteria and strains, such as Serratia marcesons and various Pseudomonas species.

In addition, when the vector of the present invention is transformed into eukaryotic cells, as host cells, yeast (Saccharomyce cerevisiae), insect cells and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines) and the like can be used.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl2 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)) when the host cell is a prokaryotic cell. , Hanahan, D., J. Mol. Biol., 166: 557-580 (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); 1983) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, fine injection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) Can be injected into the host cell.

In the present invention, in order to analyze whether sPTK7 inhibits capillary-like tube formation, cell migration and invasion of human vascular endothelial cells, vascular endothelial cells include human umbilical vein. endothelial cells (HUVEC) were used, and vascular endothelial growth factor (VEGF) was used as an activity inducing substance of vascular endothelial cells. In the assay method, various concentrations of soluble PTK7 protein fragments were treated with HUVECs, followed by VEGF treatment after about 30 minutes, and soluble to capillary-like tube formation of HUVECs induced by VEGF. The effect of the PTK7 protein fragment was analyzed. As a result, HUVEC treated with VEGF alone in the absence of soluble PTK7 protein fragments formed reticulated tubes in the form of blood vessels, but when pretreated with soluble PTK7 protein fragments, concentration-dependent capillary of HUVEC induced by VEGF Capillary-like tube formation was inhibited (see FIG. 2).

In addition, in order to analyze whether the cytotoxicity of soluble PTK7 protein fragments in capillary-like tube formation of vascular endothelial cells was affected, the number of HUVEC cells after treatment with soluble PTK7 protein fragments Was investigated by MTT assay, and the results showed that the soluble PTK7 protein fragment did not affect the survival or reproduction of HUVEC (see FIG. 3). Therefore, it was confirmed that capillary-like tube formation inhibiting action of vascular endothelial cells by soluble PTK7 protein fragment was not due to cytotoxicity.

Meanwhile, since cell migration and infiltration are important processes in capillary-like tube formation of vascular endothelial cells, cell migration was measured by a chemotactic motility assay in the Transwell system (FIG. 4). Cell invasion was measured by invasion assay after the growth factor-reduced Matrigel was coated in the Transwell system (see FIG. 5). As a result of quantitative analysis of the number of cells showing migration and infiltration, it was confirmed that the soluble PTK7 protein fragment inhibited the migration and invasion of HUVEC induced by VEGF in a concentration-dependent manner. These results confirm that the soluble PTK7 protein fragment is cytotoxic in vitro and inhibits capillary-like tube formation, cell migration and invasion of vascular endothelial cells. Therefore, the present inventors attempted to confirm whether they have angiogenesis inhibitory activity in vivo. To this end, mouse sPTK7 was prepared by the same method as human sPTK7, and Matrigel itself, Matrigel containing VEGF, and Matrigel containing mouse sPTK7 and VEGF were injected between the abdominal endothelial and the outer shell of the mouse to perform an in vivo Matrigel plug assay. It was. As a result, VEGF induced angiogenesis in the plug, whereas angiogenesis was significantly inhibited in the experimental group treated with VEPT with sPTK7 (see FIGS. 6 and 7).

Meanwhile, in order to determine the minimum active fragment region of sPTK7, Ig loop domains of sPTK7 having a His-tag were expressed and purified, and cell migration was measured by the same method as sPTK7. As a result, it was confirmed that PTK7-Ig14s-His, PTK7-Ig14-His and PTK7-Ig15-His inhibited the migration of HUVEC by VEGF in a concentration-dependent manner (see FIGS. 1 and 10). However, PTK7-Ig13-His showed very weak cell migration inhibition compared to sPTK7. From these results, PTK7-Ig14s-His contains the least active fragment of the human PTK7 extracellular domain portion, in particular the fourth intact Ig loop portion of the human PTK7 extracellular domain portion (amino acid sequence 327-409 of the human PTK7 protein). ) Is essential for the function of PTK7.

In the present invention, angiogenesis through the inhibition of function of PTK7 protein, It is a technical feature to provide inhibitors that can inhibit cell migration and cell invasion, and thus such inhibition of function of PTK7 protein can be achieved through the use of various inhibitors in addition to the method of using soluble PTK7 protein fragment as an inhibitor. For example, a small inhibitory RNA (siRNA) can be used to inhibit the expression of the mRNA sequence by forming a complementary bond with a messenger RNA (mRNA) sequence encoding the PTK7 protein, thereby inhibiting PTK7 protein function, or PTK7 protein using a PTK7 protein neutralizing antibody that inhibits PTK7 protein function using a small hairpin RNA (shRNA) expression vector comprising an (siRNA) sequence, or inhibits the activity of PTK7 protein by specifically binding to the PTK7 protein. It may also interfere with function.

To confirm the inhibitory effect of PTK7 protein function using RNAi, knock down the expression of PTK7 using siRNA and shRNA vectors, and then affect the capillary-like tube formation and Akt phosphorylation of vascular endothelial cells. The impact was analyzed. The degree of knockdown of PTK7 was analyzed by Western blot using PTK7 anti-serum. As a result, PTK7 was expressed after transfection of PTK7 siRNA to HUVEC or nucleic acid transfection of PTK7 shRNA vector to HEK293 cells. Since the expression was observed to be reduced by more than 70%, it was confirmed that PTK7 was knocked down effectively (see FIGS. 11 to 15). Therefore, PTK7 knockdown in vascular endothelial cells has the same effect as sPTK7 treatment, so sPTK7 blocks endogenous PTK7 function, leading to capillary-like tube formation, migration, and It was confirmed that it inhibits infiltration and angiogenesis in vivo.

On the other hand, as another embodiment of the inhibitor can be used PTK7 protein neutralizing antibody that specifically inhibits the activity of PTK7 protein by specifically binding to PTK7 protein, in this case preferably the neutralizing antibody is represented by SEQ ID NO: 2 amino acid sequence Can be recognized (see Example 11).

Inhibitors of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Compositions comprising the protein of the present invention or DNA encoding the same, oral formulations, external preparations, suppositories, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. Formulated in the form of can be used. Carriers, excipients and diluents which may be included in the composition comprising the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Vectors into which the DNA can be inserted to continuously supply the DNA encoding the protein of the present invention to the affected area include an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector. And plasmids that can be expressed in lentivirus vectors, herpes simplex virus vectors, or animal cells.

Dosage forms of the composition containing the protein or DNA encoding the same can be any substrate commonly used in injections. For example, the substrate may be a mixture of distilled water, sodium chloride salt solution, a mixture of sodium chloride and mineral salts or the like, a solution of mannitol, lactose, dextran, glucose, and the like, glycine, arginine, and the like.

Amino acid solutions, organic acid or salt solutions, glucose solutions, and similar solutions. Injectables may also be prepared as solutions, suspensions or colloidal solutions by adding osmotic pressure regulators, pH regulators, vegetable oils such as sesame oil or soybean oil, or surfactants such as lecithin, nonionic surfactants, etc., in the usual manner. Can be. Such injectables may be prepared in powder form, lyophilized form and dissolved immediately before use.

The composition containing the protein or DNA encoding the same according to the present invention as an active ingredient may be dissolved in a pre-sterilized substrate if necessary immediately before gene treatment in the solid phase, or may be used as it is without additional treatment in the liquid phase.

Preferred dosages of the protein of the present invention or DNA encoding the same may vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the amount of 0.0001 to 1000 mg / kg may be administered once to several times per week, or once via a catheter after surgery, locally, at the site of disease caused by angiogenesis. It can be absorbed at the target point. The dosage can be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention. The dosage does not limit the scope of the invention in any aspect.

The protein of the present invention or DNA encoding the same can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

On the other hand, by including the composition of the present invention does not necessarily mean to be approved as a medicine, it can be understood as a concept that includes a conventional functional food or even health supplements.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only to aid the understanding of the present invention, and should not be construed as limiting the scope of the present invention.

< Example  1> cell culture

Human umbilical vein endothelial cells (HUVECs) were isolated by treating collagen degrading enzymes in human umbilical veins. HUVEC contains 20% (w / v) fetal bovine serum (FBS), 100 units / ml penicillin, 100 μg / ml streptomycin, 3 ng / ml basic fibroblast growth factor (bFGF, Cultured from Upstate Biotechnology, USA) and M199 medium (purchased from Invitrogen, USA) containing 5 unit / ml heparin. Meanwhile, HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, 100 unit / ml penicillin and 100 μg / ml streptomycin. All cells were incubated at 37 ° C. with 5% CO ₂ and 95% air.

< Example  2> His - tag Human sPTK7  Preparation of Expression Plasmids

First, a 4.2-kb Eco RI fragment was obtained from human PTK7 full-length cDNA (Park, SK et al., J Biochem (Tokyo) 119: 235-239), and served to the Eco RI site of the pcDNA3 vector (purchased from Invitrogen). By cloning, the human PTK7 expression vector pcDNA3-hPTK7 was produced. The 640-bp cDNA fragment encoding the C-terminal part of the extracellular domain of the human PTK7 protein was amplified by a polymerase chain reaction (PCR) to include His-tag coding sequence and translation end codon. Primer pairs used for PCR were 5'-AAAAGCTCAAGTTCACACCA-3 '(nucleotide sequence 1646-1665 of GenBank U40271) and 5'-GC TCTAGA TCA ATGATGATGATGATGATG CTGGATCATCTTGTAGGG-3' His-tag coding sequence (underlined portion of the sequence), termination codon and Xba I site (Italic portion of the sequence) which is the base sequences 2239-2256 of GenBank U40271. pcDNA3-hPTK7 was digested with Xho I (nucleotide sequence 1829 of GenBank U40271) and Xba I (multi-cloning site of pcDNA3) to remove 1.6 kb of fragments, and then the PCR product was digested with Xho I and Xba I. 0.45 kb fragment was isolated and conjugated to obtain a pcDNA3-hPTK7-Ext-His plasmid, which is a human sPTK7 expression vector. The prepared plasmid was confirmed to have no PCR error through bidirectional sequencing. Since amino acid sequences 1 to 30 of the PTK7 polypeptide were ER signal peptides, when the plasmid was introduced into eukaryotic cells, the entire PTK7 extracellular site and amino acid sequences of amino acid sequences 31 to 703 were expressed.

< Example  3> human and mouse sPTK7 Expression and Purification

Human sPTK7 Expression Vector (pcDNA3-hPTK7-Ext-His) and Mouse sPTK7 Expression Vector (pcDNA3.1-mPtk7-Ext-His) Prepared in Example 2 (Jung, JW et al., Gene 328: 75-84) HEK293 cells were transfected with calcium phosphate (calcium phosphate), respectively, and treated with 1.2 mg / ml of G418 to select G418-resistant cell clones. Selected cell clones were reselected to cell clones stably expressing human sPTK7 and mouse sPTK7 using anti-Penta-His single antibody (Qiagen, Hilden, Germany). The serum-free medium in which the reselected clones were incubated for 7 days was saturated with ammonium sulfate to 70% to precipitate the proteins, and the precipitates were then diluted with 1 mM phenylmethanesulphonyl fluoride (PMSF) and 1 mM. Phosphate buffered saline (PBS) with ethylene diamine tetraacetic acid (EDTA) (137 mM NaCl, 10 mM Na ₂ HPO ₄ , 2.7 mM KCl, 2 mM KH ₂ PO ₄ , pH 7.4 ) And dialyzed with PBS. After the completion of the dialysis samples with Ni ^{2 +} -NTA agarose and loaded on agarose (purchased from Qiagen, Inc.) by imidazole elution, were secured to the mouse and human sPTK7 sPTK7 purified by re-dialyzed with PBS.

Example 4 Analysis of Inhibition of Capillary-like Tube Formation of Vascular Endothelial Cells by sPTK7

The cultured HUVECs were starvated for 6 hours in M199 medium containing 1% FBS, then detached with trypsin and dispersed in the same medium. 2 x 10 ⁵ HUVECs in 0.2 ml medium were pre-reacted with various concentrations of sPTK7 (0, 0.5, 1, 2, 4 ug / ml) for 30 minutes. 0.2 mg of 10 mg / ml growth factor reduction Matrigel ^™ (purchased from BD Biosciences) was polymerized into each well of a 24-well plate, and then each well was treated with sPTK7-treated HUVEC and 20 ng / ml of vascular endothelium. Vascular endothelial growth factor (VEGF) was added. After 24-well plates were incubated at 37 ° C. for 16 hours, the number of blood vessels formed was analyzed by light microscopy (60 ×). The comparison of each group in this analysis and the following analysis was done using Student's t-test, and it was considered that there was no significance level when the P value was less than 0.05. All experimental values were obtained by performing at least three independent experiments and expressed as "mean value ± standard deviation value".

Figure 2 shows the results of analyzing the effect of sPTK7 in the capillary-like tube formation process of HUVEC induced by VEGF. Statistical analysis through Student's t-test showed †† when the P value was less than 0.01 for the control without VEGF, and ** when the P value was less than 0.01 for the control with VEGF. *** when the value is less than 0.001.

As can be seen from the results of FIG. 2, the HUVEC treated with VEGF alone in the absence of sPTK7 increased the number of tubes formed by 45% compared to HUVEC treated with nothing. However, pretreatment with sPTK7 inhibited capillary-like tube formation of HUVEC induced by VEGF in a sPTK7 concentration-dependent manner.

< Example  5> In vascular endothelial cells sPTK7 Toxic effects analysis by

MTT assay was used to analyze whether cytotoxicity of sPTK7 had an effect on capillary-like tube formation of vascular endothelial cells. 20 μl of 0.1% gelatin solution was added to each well of a 48-well plate and dried. 2.0 x 10 ⁴ HUVECs were added to each well, followed by incubation for 24 hours, followed by starvation for 6 hours in M199 culture medium containing 1% FBS. After incubating for 36 hours with various concentrations of human sPTK7 and 10 ng / ml VEGF in each well, the number of living cells was analyzed by MTT assay (Lee, SJ et al., Biochem Biophys Res Commun 312: 1196). -1201). As can be seen from the results of FIG. 3, the number of cells increased by 40.5% when only VEGF was treated, compared to the sample without VEGF, but in the sample treated with VEGF and the sample treated with sPTK7 together with VEGF, There was no difference. Therefore, sPTK7 did not affect the survival or reproduction of HUVEC. It was confirmed that sPTK7 did not inhibit capillary-like tube formation of vascular endothelial cells by cytotoxicity. there was.

< Example  6> In vascular endothelial cells sPTK7 Cell migration and infiltration inhibition assay by

Since cell migration and infiltration are important processes in capillary-like tube formation of vascular endothelial cells, cell migration was measured by a chemotactic motility assay in a Transwell system (see FIG. 4). Cell invasion was measured by invasion assay after the growth factor reduction Matrigel was coated in the Transwell system (see FIG. 5). Quantitative analysis of cell migration and measurement of cell infiltration were performed as follows.

Specifically, the bottom of the transwell was coated with 10 μl of 0.1% gelatin and dried. HUVECs were starved for 6 hours in an M199 culture medium containing 1% FBS, then detached with trypsin and dispersed in the same medium. Various concentrations of sPTK7 were added to make HUVEC 1 × 10 ⁶ / ml and reacted for 30 minutes. 0.1 mL of HUVEC reacted in the upper chamber of the transwell was added, and the M199 culture medium containing 10 ng / ml of VEGF and 1% FBS was added to the lower chamber and incubated at 37 ° C. for 4 hours. Cells migrated to the bottom of the filter were stained with hematoxylin and eosin.

On the other hand, the method of measuring cell invasion was the same as the method of measuring cell migration, but the difference between the dry and culture time was 24 hours by coating 80 μl of 10 mg / ml growth factor reduction Matrigel ^{TM on the} top of the transwell. After incubation, vascular endothelial cells infiltrated or infiltrated under the transwell were observed under an optical microscope (200-fold).

4 and 5 are the results of analyzing the effect of sPTK7 on cell migration and infiltration of HUVEC induced by VEGF. Cell migration (FIG. 4) and cell infiltration (FIG. 5) of HUVECs were determined using VEGF as a chemoattractant under and without growth factor reduction Matrigel, respectively, in the Transwell system. Statistical analysis by Student's t test, ††† when the P value is less than 0.001 for the control group not treated with VEGF, and *** when the P value is less than 0.001 for the control group treated with VEGF.

As can be seen from Figures 4 and 5, VEGF increased the migration of HUVEC 3.3 times, infiltration 5 times, but sPTK7 decreased the cell migration and infiltration according to the concentration. Therefore, it was confirmed that sPTK7 inhibits both migration and infiltration of HUVEC induced by VEGF in a concentration-dependent manner.

Example 7 Analysis of In vivo Angiogenesis Inhibition by sPTK7

Since sPTK7 has no cytotoxicity against vascular endothelial cells and inhibits capillary-like tube formation, cell migration and invasion, it is analyzed whether sPTK7 inhibits angiogenesis in vivo. It was. For mouse level in vivo analysis, 20 units of 0.6 ml Matrigel ^™ containing nothing, containing 200 ng of mouse VEGF, or 200 ng of mouse VEGF and 20 μg of mouse sPTK7 Heparin was added and injected to the 7-week-old C57BL / 6 female mice between the ventral endothelial and the cortex. After 7 days, the plugs were collected and photographed, and the hemoglobin level of each plug was measured with Drabkin's reagent kit 525 (purchased from Sigma-Aldrich, St. Louis, MO, USA) to quantify angiogenesis. .

6 and 7 show the results of analyzing the effect of sPTK7 on in vivo angiogenesis induced by VEGF. Five mice in each group were injected with Matrigel, Matrigel with mouse VEGF or Matrigel with mouse sPTK7 and VEGF, respectively. FIG. 6 shows the results of angiogenesis in Matrigel plugs taken from each group of mice 7 days after Matrigel injection. FIG. 4B shows the amount of hemoglobin in each Matrigel plug to quantify the degree of angiogenesis. It is measured. Statistical analysis by Student's t test, ††† when the P value is less than 0.001 for the control group not treated with VEGF, marked with ** when the P value is less than 0.01 for the control group treated with VEGF.

As a result of the measurement, the plug containing only VEGF was reddish brown, whereas the plug containing Matrigel ^™ itself or the plug containing sPTK7 and VEGF was pale yellow (see FIG. 6), and the plug containing only VEGF contained almost 7 g / dl of hemoglobin. However, in the plug containing sPTK7 and VEGF, the value was reduced to 1.5 g / dl (see FIG. 7). From the above results, it was confirmed that sPTK7 inhibits angiogenesis in vivo.

< Example  8> In vascular endothelial cells sPTK7 Inhibition Analysis of Signaling Protein Activation

We investigated whether sPTK7 inhibits the signaling process when inhibiting capillary-like tube formation, cell migration and invasion of HUVEC. Subconfluent culture HUVECs were starved for 6 hours in M199 medium containing 1% FBS. After 30 minutes of starvation, 4 μg / ml of sPTK7 was added and cultured. Then, 10 ng / ml of VEGF was added and incubated for 10 minutes (for 5 minutes in case of KDR phosphorylation assay). After incubation, the cells were wiped with PBS and 350 μl of radioimmunoprecipitation assay (RIPA) buffer solution (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-) per 100 mm plate. 100, 0.1% SDS, 0.5% Sodium deoxycholate, 25 mM beta-glycerophosphate, 50 mM NaF, 1 mM Na ₃ VO ₄ , 1 mM PMSF) and dissolved for 20 minutes, centrifuged at 4 ℃, 12,000g for 5 minutes. Isolate to obtain supernatant. In the case of immunoprecipitation, 1 μg of antibody was added to 350 μl of the supernatant, followed by reaction at 4 ° C. for 12 hours. 20 μl of protein-A Sepharose (50% slurry) was added to each reactant and reacted at 4 ° C. for 1 hour, followed by centrifugation to obtain a precipitate. In the case of western blot, SDS gel sample buffer was put in an immunoprecipitate or cell lysate supernatant, and the sample was prepared. Then, the sample was prepared by electrophoresis on SDS-PAG and blotted onto the nitrocellulose membrane, and the antibodies were reacted. PI3-kinase activity was measured with a competitive ELISA kit (purchased from Echelon Biosciences, Salt Lake City, UT, USA) using immunoprecipitates.

As can be seen in Figure 8, sPTK7 inhibited KDR (VEGFR2) phosphorylation induced by VEGF in HUVEC, and not only inhibited Akt phosphorylation but also inhibited the activity of PI3-kinase that activates Akt. PI3-kinase is known to be deeply involved in angiogenesis, cell migration and cell infiltration by increasing the expression of angiogenetic cytokines (Brader, S. and Eccles, SA, Tumori 90: 2-8). In addition, Akt is known to play an important role in angiogenesis by contributing to vascular maturation and permeability as well as survival, reproduction and migration of endothelial cells (Chen, J. et al., Nat Med, 11: 1188-1196; Manning , BD and Cantley, LC, Cell 129: 1261-1274). Taken together, it can be expected that PI3-kinase and Akt activation inhibition plays an important role in the inhibition of endothelial cell migration, invasion and angiogenesis by sPTK7.

Moreover, sPTK7 also inhibited the phosphorylation of Akt induced by bFGF in HUVEC. Therefore, sPTK7 was expected to inhibit angiogenesis, cell migration and invasion by blocking intracellular signaling processes such as PI3-kinase and Akt activated by various signaling inducers such as VEGF and bFGF.

Treatment of endothelial cells with VEGF is known to induce tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin (Waltenberger, J. et al., J Biol Chem 269: 26988-26995; Abedi, H. and Zachary, I., J Biol Chem 272: 15442-15451. In addition, PI3-kinase / Akt signaling mechanisms have been reported to be involved in actin reorganization and endothelial cell migration promoted by VEGF (Morales-Ruiz, M. et al., Circ Res 86: 892- 896; Radisavljevic, Z. et al., J Biol Chem 275: 20770-20774).

In order to confirm the effect of sPTK7 on the activation of FAK and paxillin, after treatment with sPTK7 and VEGF in HUVEC in the same manner as in the above example, immunoprecipitation of FAK and paxillin with respective antibodies and then Western phosphotyrosine antibody Blots were performed to analyze the phosphorylation of FAK and paxillin. Specifically, for immuno-fluorescence microscopy, hunger-treated HUVECs were treated with sPTK7 for 30 minutes, and then cultured for 1 hour after VEGF was added. The cultured cells were treated with 3.7% paraformaldehyde for 10 minutes to fix the cells, treated with 0.5% Triton X-100 for 5 minutes, and blocked with 3% BSA for 1 hour. Subsequently, the reaction was performed with an antibody of paxillin for 2 hours, and then reacted for 1 hour with Phodoidin-FITC and a Rhodamine-conjugated secondary antibody. As a result, as shown in Figure 9, the effect of sPTK7 on the activation of FAK and paxillin observed, sPTK7 in HUVEC was observed to inhibit tyrosine phosphorylation of FAK and paxillin induced by VEGF.

  Thus, sPTK7 could be expected to inhibit the adhesion and migration of vascular endothelial cells by inhibiting local adhesion formation and actin reconstitution. These results suggest that inhibition of PTK7 function may inhibit cell migration, cell infiltration, and cell adhesion in vascular endothelial cells as well as other types of cells expressing PTK7.

< Example  9: human PTK7 of Extracellular  Part of the domain Minimum activity  Decision>

A deletion mutant of sPTK7 was constructed to determine the minimal activity of human PTK7 extracellular domain. Specifically, as shown in FIG. 1, pcDNA3.1-hPTK7-Ig13-His (the first Ig loop to the third Ig loop and the fourth Ig loop portion (amino acid sequences 31 to 344 of SEQ ID NO: 2) and His tag Expression], pcDNA3.1-hPTK7-Ig14-His [expression of the first Ig loop to the fourth Ig loop and part of the fifth Ig loop (amino acid sequence 31 to 436 of SEQ ID NO: 2) and His tag] and pcDNA3 .1-hPTK7-Ig15-His (expressing the first Ig loop to the fifth Ig loop and part of the sixth Ig loop (amino acid sequences 31 to 528 of SEQ ID NO: 2) and His tag) respectively after the last codon The polymerase chain reaction (PCR) method was mutated to have his tag codon and translation end codon.

Among them, pcDNA3.1-hPTK7-Ig14-His was prepared as follows. In the previously prepared human sPTK7 expression vector (pcDNA3-hPTK7-Ext-His), the PCR was linked to the 436 amino acid of the 5th Ig loop entry of the extracellular domain of the human PTK7 protein to include His-tag coding sequence and translation end codon. Amplified through. Primer pairs used for PCR were 5'-TAATACGACTCACTATAGGG-3 '(T7 promoter sequences 863-882 of the pcDNA3 vector) and 5'-GC TCTAGA TCA ATGATGATGATGATGATG CTGGGTCAGGCAATCCAA-3' (SEQ ID NO: 1). Nucleotide sequence of 1453-1470, His-tag coding sequence (underlined portion of the sequence), termination codon and XbaI site (italic portion of the sequence). Thereafter, the PCR product and the pcDNA3.1 vector were cleaved with EcoRI and XbaI to conjugate fragments as in Example 2, so that a part of the first Ig loop to the fourth Ig loop and the fifth Ig loop of the human PTK7 extracellular domain (sequences) A pcDNA3.1-hPTK7-Ig14-His plasmid expressing the amino acid sequence Nos. 1 to 436 of No. 2 and His tag was obtained. The remaining plasmids were also obtained in this manner.

pcDNA3.1-hPTK7-Ig14s-His [expression of His tag and part from the first Ig loop to the fourth Ig loop (except amino acid sequence 31 of SEQ ID NO: 2) except for the fifth Ig loop entry part] In the case of pcDNA3.1-hPTK7-Ig14-His, nucleotide sequences 1390 to 1470 of SEQ ID NO: 1 were deleted by PCR. Primer pairs used for PCR were 5'-CATCACTGTGGCC CATCATCATCATCATCAT TGA TCTAGA GGGCC-3 '[SEQ ID NOS: 1377-1389, His-tag coding sequence (underlined portion of the sequence), termination codon and XbaI site (above) The italic part of the sequence) and the base sequence 997-1001 (the gothic part of the sequence) of the pcDNA3.1 vector] and 5'- GATGATGATGATG GGCCACAGTGATGTTGACATCCTGTCTC-3 '(part of the His-tag coding sequence (underlined) SEQ ID NOs: 1362-1389. The PCR product was treated with DpnI, transformed into XL1-Blue Escherichia coli, and colonies were selected and cultured to obtain a deleted plasmid. The prepared plasmids were confirmed to have no PCR error through bidirectional sequencing.

   Expression plasmids of the human PTK7 extracellular domain portions prepared in this example (pcDNA3.1-hPTK7-Ig13-His, pcDNA3.1-hPTK7-Ig14s-His, pcDNA3.1-hPTK7-Ig14-His and pcDNA3.1 -hPTK7-Ig15-His) was expressed and purified as in Example 3, PTK7-Ig13-His (amino acid sequence Nos. 31 to 344 of human PTK7 protein), PTK7-Ig14s-His (human PTK7 protein) as shown in FIG. Amino acid sequences 31 to 409), PTK7-Ig14-His (amino acid sequences 31 to 436 of human PTK7 protein) and PTK7-Ig15-His (amino acid sequences 31 to 528 of human PTK7 protein) were obtained.

   Cell migration was measured as in Example 6 to confirm whether the obtained PTK7-Ig13-His, PTK7-Ig14s-His, PTK7-Ig14-His, and PTK7-Ig15-His influence the cell migration. As can be seen in FIG. 10, the migration of HUVECs was increased 3.3-fold when only VEGF was treated, compared to when VEGF and sPTK7 or fragments thereof were not treated. PTK7-Ig13-His did not reduce cell migration with concentration, but PTK7-Ig14s-His, PTK7-Ig14-His, PTK7-Ig15-His and sPTK7 reduced cell migration to a similar degree with concentration. Statistical analysis using Student's t test showed that the P value was less than 0.001 for the control group without VEGF. PTK7-Ig13-His was less than 0.01 for the VEGF-treated control group, but PTK7-Ig14s-His, PTK7- Ig14-His, PTK7-Ig15-His, sPTK7 had a P value of less than 0.001. Thus, PTK7-Ig14s-His contains the least active fragment of the human PTK7 extracellular domain portion, and the fourth Ig loop portion (amino acid sequence 327-409 of the human PTK7 protein) in the human PTK7 extracellular domain portion is in complete form. It was confirmed that what is present is important for the function of PTK7.

Example 10 Analysis of Capillary-like Tube Formation and Akt Phosphorylation Inhibition of Vascular Endothelial Cells During PTK7 Knockdown by RNA Interference (RNAi) Method

In addition to sPTK7, as an alternative method of blocking PTK7 function, RNA interference was confirmed. Specifically, knocking down the expression of PTK7 by treating small interfering RNA (siRNA) of PTK7 or by inducing RNA interference by treating a small hairpin RNA (shRNA) vector of PTK7 expressing PTK7 siRNA The effects of capillary-like tube formation and Akt phosphorylation on vascular endothelial cells were analyzed. Knockdown of PTK7 using PTK7 siRNA in HUVEC was performed by the following method. HUVECs cultured in 6 cm petri dishes were replaced with serum-reduced medium (Opti-MEM I, purchased from Invitrogen), followed by 100 nM siRNA (Dharmacon's ON-TARGET plus family J- 003167-13, J-003167-14, J-003167-15 and J-003167-16), or mixtures thereof (see FIG. 11), using Lipofectamine (purchased from Invitrogen) for 4 hours. Incubated for transfection. The nucleic acid transfer HUVECs were replaced with M199 medium (purchased from Invitrogen) containing 20% (w / v) FBS, 3 ng / ml of bFGF (Upstate Biotechnology) and 5 unit / ml of heparin and incubated for 48 hours. It was. As a result of analyzing the knockdown of PTK7 in HUVEC, individual PTK7-siRNAs, J-003167-14, J-003167-15, J-003167-16 and PTK7 siRNA mixtures reduced PTK7 expression by more than 70% compared to negative control. It was shown that their knockdown efficacy is high (see FIG. 12).

On the other hand, knockdown of PTK7 using PTK7 shRNA vector in HEK293 was performed by the following method. HEG293 cells cultured in 6 cm culture dishes were treated with 5 ug of Mission TRC shRNA vectors (calcium phosphate) (SIGMA-ALDRICH's Mission TRC shRNA family TRCN0000006431, TRCN0000006432, TRCN0000006433, TRCN0000006434 and TRCN0000006435, see FIG. 13). ) Were each transfected with nucleic acid. The nucleic acid transfected HEK293 cells were changed to DMEM medium containing 10% FBS, 100 unit / ml penicillin and 100 μg / ml streptomycin after 12 hours, and then cultured for 48 hours. Analysis of PTK7 knockdown by each PTK7 shRNA vector in HEK293 revealed that TRCN0000006433 and TRCN0000006434 reduced the expression of PTK7 by more than 70% compared to the negative control, indicating that their PTK7 knockdown effect was high (FIG. 14).

As in the above results, a PTK7 siRNA mixture was used to analyze the effect of PTK7 knockdown on cellular function. 15 is a result of analyzing the effect of knockdown of PTK7 on capillary-like tube formation induced by VEGF in HUVEC. PTK7 knockdown HUVECs were placed on growth factor-reduced Matrigel and incubated for 16 hours to determine the number of blood vessels generated, and statistically analyzed by Student's t test, with a P value of less than 0.01 for mock transfection without control †††, when the P value is less than 0.001, *** when the P value is less than 0.001 for the control group (mock + VEGF) treated with VEGF is indicated as ***. As can be seen in FIG. 15, capillary-like tube formation was increased by 45% when VEGF was treated in mock transfected HUVECs, and the control siRNA was nucleic acid transfected. Capillary-like tube formation by VEGF was observed in HUVECs similar to that of HUVECs infected with false nucleic acid transfer, but capillary-like tube formation induced by VEGF was observed in HUVECs knocked down PTK7. -like tube formation) was inhibited. In addition, similar to capillary-like tube formation, in Akt phosphorylation assay, VEGF treatment of mock transfected HUVECs increased Akt phosphorylation, and control siRNA was delivered to nucleic acid. In infected HUVECs, Akt phosphorylation was observed to be similar to that of HUVECs infected with false nucleic acid transfer, but VEGF-induced Akt phosphorylation was inhibited in HUVECs knocked down PTK7. Since PTK7 knockdown has the same effect as sPTK7 treatment in vascular endothelial cells, it was confirmed that sPTK7 blocks endogenous PTK7 function and inhibits cell migration, invasion and angiogenesis.

< Example  11> PTK7  By antibody PTK7  Inhibition of vascular endothelial cells Akt  Phosphorylation Inhibition Assay

In addition to sPTK7, as an alternative method of blocking PTK7 function, the antibody of PTK7 could be utilized. In previous examples, sPTK7 treatment has been shown to reduce cell migration, invasion, angiogenesis, and Akt phosphorylation when PTK7 function is inhibited. Therefore, after treatment with sPTK7 antibody, the effect on Akt phosphorylation in vascular endothelial cells was analyzed.

      In order to observe the effect of sPTK7 on Akt phosphorylation in HUVEC, 2 μg / ml of sPTK7 antiserum was added 30 minutes before the end of starvation, followed by 10 ng / ml of VEGF and incubated for 10 minutes. PTK7 anti-serum was prepared by injecting rabbits with purified sPTK7 as described in Example 10 and used after confirming specific recognition of PTK7. After culturing, the cells were wiped with PBS, and the supernatant was obtained in the same manner as in Example 8 with 120 μl of immunoprecipitation assay buffer per 60 mm plate. Western blots were then performed on the cell lysate supernatant. 16 shows the results of analyzing the effect of antiserum of PTK7 on phosphorylation of Akt induced by VEGF in HUVEC. Antisera of sPTK7 was observed to inhibit the phosphorylation of Akt induced by VEGF. Since antiserum of sPTK7 has a similar effect to sPTK7 treatment, antisera of sPTK7 contains PTK7 neutralizing polyclonal antibody, indicating that it inhibits PTK7 function. Therefore, PTK7 neutralizing monoclonal antibodies as well as PTK7 neutralizing polyclonal antibodies may be a method of inhibiting cell migration, invasion and angiogenesis by inhibiting PTK7 function.

As a result, in addition to sPTK7 treatment, PTK7 knockdown, and PTK7 neutralizing antibody treatment, the sPTK7 expression and PTK7 neutralizing aptamer treatment may also inhibit cell migration and invasion. In addition, by blocking the normal function of vascular endothelial cells can inhibit angiogenesis (angiogenesis).

Inhibitors of cell migration, cell infiltration and angiogenesis according to the present invention can be used for the treatment of various diseases including metastatic carcinoma associated with cell migration, infiltration and angiogenesis, subject cells also limited to vascular endothelial cells Rather, it can be applied to various kinds of cells expressing PTK7 and can be utilized to inhibit cell migration, invasion, and adhesion.

1 is a schematic diagram showing the structure of PTK7 polypeptide and all or part of the extracellular domain of PTK7 secreted from cells.

2 is a graph showing the results of analyzing the effect of sPTK7 on capillary-like tube formation of HUVEC induced by VEGF.

3 is a graph showing the results of analyzing the effect of sPTK7 on the increase in the number of cells induced by VEGF in HUVEC.

4 and 5 are graphs showing the results of analyzing the effect of sPTK7 on the cell migration and invasion of HUVEC induced by VEGF.

6 and 7 are photographs and graphs showing the results of analyzing the effect of sPTK7 on in vivo angiogenesis induced by VEGF.

8 is an electrophoresis result and a graph showing the results of analyzing the effect of sPTK7 on the activation of VEGF-induced signaling substances in HUVEC.

      9 is an electrophoresis and confocal fluorescence micrograph showing the results of analyzing the effect of sPTK7 on VEGF-induced FAK, paxillin activation, paxillin local attachment formation and actin filament formation in HUVEC.

FIG. 10 is a graphical representation of the results of analysis to determine the site of minimally active fragments of sPTK7 for cell migration of HUVECs induced by VEGF.

Figure 11 shows the sequence of siRNA (ON-TARGET plus from Dharmacon) used for PTK7 knockdown analysis.

Figure 12 is an electrophoresis picture showing the knockdown result of PTK7 by each siRNA and mixtures thereof.

Figure 13 is the information and target sequence (MISSION TRC shRNA from SIGMA-ALDRICH) of the shRNA vector used for PTK7 knockdown analysis.

14 is an electrophoresis picture showing the knockdown result of PTK7 by shRNA vector.

15 is a graph showing the results of analyzing the effect of knockdown of PTK7 on capillary-like tube formation induced by VEGF in HUVEC.

16 is an electrophoretic photograph showing the results of analyzing the effect of VEGF-induced phosphorylation of Akt by antiserum treatment of PTK7 in HUVEC.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Inhibitors of cell migration, invasion, or angiogenesis by          blocking the function of PTK 7 protein <130> HP-0841 <150> KR10-2008-24599 <151> 2008-03-17 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 2298 <212> DNA <213> Homo sapiens <400> 1 gaattccgga actcccgcct cgggacgcct cggggtcggg ctccggctgc ggctgctgct 60 gcggcgcccg cgctccggtg cgctccgcct cctgtgcccg ccgcggagcg cagtctgcgc 120 gcccgccgtg cgccctcagc tccttttcct gagcccgccg cgatgggagc tgcgcgggga 180 tccccggcca gaccccgccg gttgcctctg ctcagcgtcc tgctgctgcc gctgctgggc 240 ggtacccaga cagccattgt cttcatcaag cagccgtcct cccaggatgc actgcagggg 300 cgccgggcgc tgcttcgctg tgaggttgag gctccgggcc cggtacatgt gtactggctg 360 ctcgatgggg cccctgtcca ggacacggag cggcgtttcg cccagggcag cagcctgagc 420 tttgcagctg tggaccggct gcaggactct ggcaccttcc agtgtgtggc tcgggatgat 480 gtcactggag aagaagcccg cagtgccaac gcctccttca acatcaaatg gattgaggca 540 ggtcctgtgg tcctgaagca tccagcctcg gaagctgaga tccagccaca gacccaggtc 600 acacttcgtt gccacattga tgggcaccct cggcccacct accaatggtt ccgagatggg 660 accccccttt ctgatggtca gagcaaccac acagtcagca gcaaggagcg gaacctgacg 720 ctccggccag ctggtcctga gcatagtggg ctgtattcct gctgcgccca cagtgctttt 780 ggccaggctt gcagcagcca gaacttcacc ttgagcattg ctgatgaaag ctttgccagg 840 gtggtgctgg caccccagga cgtggtagta gcgaggtatg aggaggccat gttccattgc 900 cagttctcag cccagccacc cccgagcctg cagtggctct ttgaggatga gactcccatc 960 actaaccgca gtcgcccccc acacctccgc agagccacag tgtttgccaa cgggtctctg 1020 ctgctgaccc aggtccggcc acgcaatgca gggatctacc gctgcattgg ccaggggcag 1080 aggggcccac ccatcatcct ggaagccaca cttcacctag cagagattga agacatgccg 1140 ctatttgagc cacgggtgtt tacagctggc agcgaggagc gtgtgacctg ccttcccccc 1200 aagggtctgc cagagcccag cgtgtggtgg gagcacgcgg gagtccggct gcccacccat 1260 ggcagggtct accagaaggg ccacgagctg gtgttggcca atattgctga aagtgatgct 1320 ggtgtctaca cctgccacgc ggccaacctg gctggtcagc ggagacagga tgtcaacatc 1380 actgtggcca ctgtgccctc ctggctgaag aagccccaag acagccagct ggaggagggc 1440 aaacccggct acttggattg cctgacccag gccacaccaa aacctacagt tgtctggtac 1500 agaaaccaga tgctcatctc agaggactca cggttcgagg tcttcaagaa tgggaccttg 1560 cgcatcaaca gcgtggaggt gtatgatggg acatggtacc gttgtatgag cagcacccca 1620 gccggcagca tcgaggcgca agcccgtgtc caagtgctgg aaaagctcaa gttcacacca 1680 ccaccccagc cacagcagtg catggagttt gacaaggagg ccacggtgcc ctgttcagcc 1740 acaggccgag agaagcccac tattaagtgg gaacgggcag atgggagcag cctcccagag 1800 tgggtgacag acaacgctgg gaccctgcat tttgcccggg tgactcgaga tgacgctggc 1860 aactacactt gcattgcctc caacgggccg cagggccaga ttcgtgccca tgtccagctc 1920 actgtggcag tttttatcac cttcaaagtg gaaccagagc gtacgactgt gtaccagggc 1980 cacacagccc tactgcagtg cgaggcccag ggggacccca agccgctgat tcagtggaaa 2040 ggcaaggacc gcatcctgga ccccaccaag ctgggaccca ggatgcacat cttccagaat 2100 ggctccctgg tgatccatga cgtggcccct gaggactcag gccgctacac ctgcattgca 2160 ggcaacagct gcaacatcaa gcacacggag gcccccctct atgtcgtgga caagcctgtg 2220 ccggaggagt cggagggccc tggcagccct cccccctaca agatgatcca gcatcatcat 2280 catcatcatt gatctaga 2298 <210> 2 <211> 709 <212> PRT <213> Homo sapiens <400> 2 Met Gly Ala Ala Arg Gly Ser Pro Ala Arg Pro Arg Arg Leu Pro Leu   1 5 10 15 Leu Ser Val Leu Leu Leu Pro Leu Leu Gly Gly Thr Gln Thr Ala Ile              20 25 30 Val Phe Ile Lys Gln Pro Ser Ser Gln Asp Ala Leu Gln Gly Arg Arg          35 40 45 Ala Leu Leu Arg Cys Glu Val Glu Ala Pro Gly Pro Val His Val Tyr      50 55 60 Trp Leu Leu Asp Gly Ala Pro Val Gln Asp Thr Glu Arg Arg Phe Ala  65 70 75 80 Gln Gly Ser Ser Leu Ser Phe Ala Ala Val Asp Arg Leu Gln Asp Ser                  85 90 95 Gly Thr Phe Gln Cys Val Ala Arg Asp Asp Val Thr Gly Glu Glu Ala             100 105 110 Arg Ser Ala Asn Ala Ser Phe Asn Ile Lys Trp Ile Glu Ala Gly Pro         115 120 125 Val Val Leu Lys His Pro Ala Ser Glu Ala Glu Ile Gln Pro Gln Thr     130 135 140 Gln Val Thr Leu Arg Cys His Ile Asp Gly His Pro Arg Pro Thr Tyr 145 150 155 160 Gln Trp Phe Arg Asp Gly Thr Pro Leu Ser Asp Gly Gln Ser Asn His                 165 170 175 Thr Val Ser Ser Lys Glu Arg Asn Leu Thr Leu Arg Pro Ala Gly Pro             180 185 190 Glu His Ser Gly Leu Tyr Ser Cys Cys Ala His Ser Ala Phe Gly Gln         195 200 205 Ala Cys Ser Ser Gln Asn Phe Thr Leu Ser Ile Ala Asp Glu Ser Phe     210 215 220 Ala Arg Val Val Leu Ala Pro Gln Asp Val Val Val Ala Arg Tyr Glu 225 230 235 240 Glu Ala Met Phe His Cys Gln Phe Ser Ala Gln Pro Pro Pro Ser Leu                 245 250 255 Gln Trp Leu Phe Glu Asp Glu Thr Pro Ile Thr Asn Arg Ser Arg Pro             260 265 270 Pro His Leu Arg Arg Ala Thr Val Phe Ala Asn Gly Ser Leu Leu Leu         275 280 285 Thr Gln Val Arg Pro Arg Asn Ala Gly Ile Tyr Arg Cys Ile Gly Gln     290 295 300 Gly Gln Arg Gly Pro Pro Ile Ile Leu Glu Ala Thr Leu His Leu Ala 305 310 315 320 Glu Ile Glu Asp Met Pro Leu Phe Glu Pro Arg Val Phe Thr Ala Gly                 325 330 335 Ser Glu Glu Arg Val Thr Cys Leu Pro Pro Lys Gly Leu Pro Glu Pro             340 345 350 Ser Val Trp Trp Glu His Ala Gly Val Arg Leu Pro Thr His Gly Arg         355 360 365 Val Tyr Gln Lys Gly His Glu Leu Val Leu Ala Asn Ile Ala Glu Ser     370 375 380 Asp Ala Gly Val Tyr Thr Cys His Ala Ala Asn Leu Ala Gly Gln Arg 385 390 395 400 Arg Gln Asp Val Asn Ile Thr Val Ala Thr Val Pro Ser Trp Leu Lys                 405 410 415 Lys Pro Gln Asp Ser Gln Leu Glu Glu Gly Lys Pro Gly Tyr Leu Asp             420 425 430 Cys Leu Thr Gln Ala Thr Pro Lys Pro Thr Val Val Trp Tyr Arg Asn         435 440 445 Gln Met Leu Ile Ser Glu Asp Ser Arg Phe Glu Val Phe Lys Asn Gly     450 455 460 Thr Leu Arg Ile Asn Ser Val Glu Val Tyr Asp Gly Thr Trp Tyr Arg 465 470 475 480 Cys Met Ser Ser Thr Pro Ala Gly Ser Ile Glu Ala Gln Ala Arg Val                 485 490 495 Gln Val Leu Glu Lys Leu Lys Phe Thr Pro Pro Pro Gln Pro Gln Gln             500 505 510 Cys Met Glu Phe Asp Lys Glu Ala Thr Val Pro Cys Ser Ala Thr Gly         515 520 525 Arg Glu Lys Pro Thr Ile Lys Trp Glu Arg Ala Asp Gly Ser Ser Leu     530 535 540 Pro Glu Trp Val Thr Asp Asn Ala Gly Thr Leu His Phe Ala Arg Val 545 550 555 560 Thr Arg Asp Asp Ala Gly Asn Tyr Thr Cys Ile Ala Ser Asn Gly Pro                 565 570 575 Gln Gly Gln Ile Arg Ala His Val Gln Leu Thr Val Ala Val Phe Ile             580 585 590 Thr Phe Lys Val Glu Pro Glu Arg Thr Thr Val Tyr Gln Gly His Thr         595 600 605 Ala Leu Leu Gln Cys Glu Ala Gln Gly Asp Pro Lys Pro Leu Ile Gln     610 615 620 Trp Lys Gly Lys Asp Arg Ile Leu Asp Pro Thr Lys Leu Gly Pro Arg 625 630 635 640 Met His Ile Phe Gln Asn Gly Ser Leu Val Ile His Asp Val Ala Pro                 645 650 655 Glu Asp Ser Gly Arg Tyr Thr Cys Ile Ala Gly Asn Ser Cys Asn Ile             660 665 670 Lys His Thr Glu Ala Pro Leu Tyr Val Val Asp Lys Pro Val Pro Glu         675 680 685 Glu Ser Glu Gly Pro Gly Ser Pro Pro Pro Tyr Lys Met Ile Gln His     690 695 700 His His His His His 705  

Claims (9)

An angiogenesis inhibitor comprising a fourth Ig loop portion of an extracellular domain of a human PTK7 soluble protein that inhibits the function of PTK7 protein as a minimally active fragment. delete delete The method of claim 1, The fourth Ig loop portion is an angiogenesis inhibitor, characterized in that the amino acid sequence 327 to 409 of the human PTK7 soluble protein of SEQ ID NO: 2. The method of claim 1, The inhibitor comprises an angiogenesis inhibitor comprising a small RNA sequence that mediates specific RNA interference (RNAi) for messenger RNA (mRNA) encoding the PTK7 protein. The method of claim 5, The small RNA sequence comprises a small scale inhibitory RNA (siRNA), or the small scale inhibitory RNA (siRNA) sequence, which forms a complementary bond with a messenger RNA (mRNA) sequence encoding a PTK7 protein to inhibit expression of the mRNA sequence. Angiogenesis inhibitor, characterized in that the small scale hairpin RNA (shRNA). The angiogenesis inhibitor according to claim 1, wherein the inhibitor is a PTK7 protein neutralizing antibody that specifically binds PTK7 protein to inhibit the activity of PTK7 protein. The angiogenesis inhibitor according to claim 7, wherein the neutralizing antibody recognizes the amino acid sequence represented by SEQ ID NO: 2. An inhibitor of migration or infiltration of tumor cells comprising as a minimum active fragment a fourth Ig loop portion of the extracellular domain of a human PTK7 soluble protein that inhibits the function of PTK7 protein.

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