WO2009116764A2 - Cell migration, cell infiltration, or neovascularization inhibitor through degradation of function of ptk7 protein - Google Patents
Cell migration, cell infiltration, or neovascularization inhibitor through degradation of function of ptk7 protein Download PDFInfo
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- WO2009116764A2 WO2009116764A2 PCT/KR2009/001298 KR2009001298W WO2009116764A2 WO 2009116764 A2 WO2009116764 A2 WO 2009116764A2 KR 2009001298 W KR2009001298 W KR 2009001298W WO 2009116764 A2 WO2009116764 A2 WO 2009116764A2
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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.
- 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.
- 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).
- 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).
- the first problem to be solved by the present invention is to inhibit angiogenesis, cell migration, cell invasion and cell adhesion by VEGF, inhibit the activation of PI3-kinase and Akt, and also FAK and Park By also inhibiting the activation of silin, to provide an inhibitor of cell migration, infiltration or angiogenesis that can effectively inhibit angiogenesis, cell migration and cell invasion in vivo.
- the present invention to achieve the first object,
- Inhibitors of cell migration, infiltration or angiogenesis through inhibition of function of PTK7 protein are inhibitors of cell migration, infiltration or angiogenesis through inhibition of function of PTK7 protein.
- the inhibitor may comprise a soluble PTK7 protein fragment having an extracellular domain of PTK7 protein.
- 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.
- the inhibitor may comprise a small RNA sequence that mediates specific RNA interference (RNAi) for messenger RNA (mRNA) encoding the PTK7 protein, more preferably the Small RNA sequences comprise small inhibitory RNA (siRNA), or small inhibitory RNA (siRNA) sequences, that form complementary bonds with messenger RNA (mRNA) sequences encoding PTK7 protein to inhibit expression of the mRNA sequence. Hairpin RNA (shRNA).
- siRNA small inhibitory RNA
- shRNA Hairpin RNA
- 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.
- the neutralizing antibody may recognize the amino acid sequence represented by SEQ ID NO: 2.
- the cell may be a vascular endothelial cell.
- 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.
- '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.
- a nucleic acid expression control sequence e.g., a promoter, signal sequence, or array of transcriptional regulator binding sites
- the term 'recombinant' as used in connection with a cell refers to the cell's replicating a 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.
- 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.
- Inhibitors by inhibiting angiogenesis, cell migration, cell infiltration and cell adhesion by VEGF, by inhibiting the activation of PI3-kinase and Akt, and also inhibit the activation of FAK and paxillin, Inhibitors can be provided that effectively inhibit angiogenesis, cell migration and cell infiltration 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.
- 1 is a schematic diagram showing the structure of PTK7 polypeptide and all or part of the extracellular domain of PTK7 secreted from cells.
- Figure 2 is a graph showing the results of analyzing the effect of sPTK7 on the angiogenesis 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.
- FIG. 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.
- FIG. 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.
- 15 is a graph showing the results of analyzing the effect of knockdown of PTK7 on VEGF-induced angiogenesis 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.
- the present inventors first confirmed that inhibiting the function of human PTK7 protein prevents cell migration, invasion and angiogenesis.
- 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.
- the specific method for inhibiting function of PTK7 protein provided by the present invention can be implemented by various means, for example, by soluble PTK7 (sPTK7) protein fragment or part thereof that can compete with PTK7 protein and block its function.
- soluble PTK7 sPTK7
- the soluble PTK7 protein fragment was designed to have an extracellular domain or portion thereof of PTK7 as shown in FIG. 1, which inhibits angiogenesis, cell migration and invasion of vascular endothelial cells, while in vivo Inhibition of angiogenesis and inhibition of the activation of signaling proteins such as PI3-kinase and Akt was also confirmed.
- 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.
- 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.
- the gene base sequence encoding the amino acid sequence is not limited, but preferably, the gene base sequence represented by SEQ ID NO: 1 may be used.
- 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.
- the sPTK7 amino acid sequence shown in SEQ ID NO: 2 includes 31 to 409, 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 After transfection of the HEK293 cells to HEK293 cells, HEK293 cells expressing sPTK7 or a part thereof are isolated and cultured, and the soluble PTK7 proteins are purified in the cell culture using Ni 2+ -NTA column chromatography.
- pcDNA3-hPTK7-Ext-His operably bound vector
- Glutathione S-transferase 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.
- MBP maltose binding protein
- FLAG FLAG
- 5-7x His hexahistidine
- NusA N utilization substance A
- Trx Thioredoxin
- the recombinant expression vector of the present invention is a strong promoter (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ 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.
- a strong promoter e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp
- Promoters derived from mammalian viruses e.g., metallothionine promoters
- 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
- adenovirus late promoters e.g., vaccinia virus 7.5K promoters, SV40 promoters, cytomegalovirus promoters, CMV-IE promoters and HSVs Tk promoter
- 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.
- 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, ⁇ gt4? ⁇ B, ⁇ -Charon, ⁇ z1 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 DH5 ⁇ , 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.
- E. coli JM109 E. coli BL21 (DE3)
- E. Bacillus genus strains such as coli DH5 ⁇ , E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuring
- yeast Sacharomyce cerevisiae
- insect cells e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines
- CHO cell line Choinese hamster ovary
- W138 BHK, COS-7, 293
- HepG2, 3T3, RIN and MDCK cell lines e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293)
- HepG2, 3T3, RIN and MDCK cell lines e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) , HepG2, 3T3, RIN and MDCK cell lines
- 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.
- 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 Neuroporation
- liposome-mediated transfection Wong, TK et al., Gene, 10:87 (1980)
- DEAE- Dextran treatment Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)
- gene balm Yamaang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)
- vascular endothelial growth factor was used as an inducer of endothelial activity.
- various concentrations of soluble PTK7 protein fragments were treated with HUVECs, followed by VEGF treatment after about 30 minutes, and the effects of soluble PTK7 protein fragments on angiogenesis of HUVECs induced by VEGF were analyzed.
- HUVEC treated with VEGF alone in the absence of soluble PTK7 protein fragments formed reticulated vessels, but in the case of pretreatment with soluble PTK7 protein fragments, HUVEC induced concentration-dependently. Formation was inhibited (see FIG. 2).
- 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).
- 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 to 409 of the human PTK7 protein). ) Is essential for the function of PTK7.
- angiogenesis through the inhibition of function of PTK7 protein
- a small inhibitory RNA is used to inhibit the expression of the mRNA sequence by forming a complementary bond with a messenger RNA (mRNA) sequence encoding the PTK7 protein to inhibit PTK7 protein function or to inhibit such small inhibitory RNA.
- mRNA messenger RNA
- PTK7 protein using PTK7 protein neutralizing antibody that inhibits PTK7 protein function using small hairpin RNA (shRNA) expression vectors comprising the (siRNA) sequence, or inhibits the activity of PTK7 protein by specifically binding to PTK7 protein. It may impair function.
- shRNA small hairpin RNA
- siRNA and shRNA vectors were used to knock down PTK7 expression, and the effects on vascular endothelial cell formation and Akt phosphorylation were analyzed.
- the degree of knockdown of PTK7 was analyzed by Western blot using PTK7 anti-serum.
- 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).
- PTK7 knockdown in vascular endothelial cells has the same effect as sPTK7 treatment, suggesting that sPTK7 blocks the function of endogenous PTK7 to inhibit vascular endothelial cell formation, migration, invasion and in vivo angiogenesis. I could confirm it.
- 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 that 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- injectables may be prepared in powder form, lyophilized form and dissolved immediately before use.
- 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.
- 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. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
- 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 can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
- 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.
- HUVECs Human umbilical vein endothelial cells
- HUVEC contains 20% (w / v) fetal bovine serum (FBS), 100 units / ml penicillin, 100 ⁇ g / 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.
- FBS fetal bovine serum
- bFGF basic fibroblast growth factor
- M199 medium purchasedd from Invitrogen, USA
- HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS, 100 unit / ml penicillin and 100 ⁇ g / ml streptomycin. All cells were incubated at 37 ° C. with 5% CO 2 and 95% air.
- DMEM Dulbecco's Modified Eagle Medium
- Primer pairs used for PCR were 5'-AAAAGCTCAAGTTCACACCA-3 '(nucleotide sequences 1646-1665 of GenBank U40271) and 5'-GC.
- TCTAGA TCA ATGATGATGATGATGATG CTGGATCATCTTGTAGGG-3 ' [His-tag coding sequence (underlined portion of the sequence), nucleotide codons 2239-2256 of GenBank U40271, termination codon and Xba I site (italic part of the sequence).
- pcDNA3-hPTK7 Xho I nucleotide sequence 1829 of GenBank U40271
- Xba I was cut into I (multi-cloning site of pcDNA3) to remove the 1.6 kb fragment, and then the PCR result was removed.
- Xho I and Xba By cutting with I, a 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 that there is no PCR error through bidirectional sequencing.
- amino acid sequences 1 to 30 of the PTK7 polypeptide are 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.
- 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.
- PMSF phenylmethanesulphonyl fluoride
- PBS Phosphate buffered saline
- EDTA ethylene diamine tetraacetic acid
- samples were loaded on Ni 2+ -NTA agarose (purchased from Qiagen), eluted with imidazole, and repurified with PBS to obtain purified human sPTK7 and mouse sPTK7.
- the cultured HUVECs were starvated for 6 hours in M199 medium containing 1% FBS, then separated 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 TM (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.
- Figure 2 shows the results of analyzing the effect of sPTK7 in the vascularization 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.
- 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.
- pretreatment with sPTK7 inhibited angiogenesis of VEGF-induced HUVECs depending on sPTK7 concentration.
- MTT assay was used to analyze whether the cytotoxicity of sPTK7 influenced the vascular endothelial cell formation.
- 20 ⁇ l 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.
- 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.
- 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, it was found that sPTK7 did not affect the survival or reproduction of HUVEC, and it was confirmed that the vascularization inhibitory effect of vascular endothelial cells by sPTK7 was not caused by cytotoxicity.
- cell migration was measured by a chemotactic motility assay in the Transwell system (see FIG. 4), and growth factor reduction Matrigel coated in the Transwell system.
- Cell invasion was measured by an invasion assay (see FIG. 5). Quantitative analysis of cell migration and measurement of cell infiltration were performed as follows.
- the bottom of the transwell was coated with 10 ⁇ l 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.
- 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 ⁇ l 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 ⁇ ).
- FIG. 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.
- 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.
- sPTK7 Since sPTK7 has no cytotoxicity against vascular endothelial cells and inhibits angiogenesis, cell migration and invasion, sPTK7 can be expected to inhibit angiogenesis in vivo.
- a 7-week-old C57BL containing 20 units of heparin was added to 0.6 ml Matrigel TM containing nothing, 200 ng of mouse VEGF, or 200 ng of mouse VEGF and 20 ⁇ g of mouse sPTK7. / 6 female mice were injected between the ventral endothelial and the outer shell. 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. .
- FIG. 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.
- the plug containing only VEGF was reddish brown, whereas the plug containing Matrigel TM 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.
- 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.
- sPTK7 inhibits the signaling process in inhibiting HUVEC angiogenesis, cell migration and invasion.
- Subconfluent culture HUVECs were starved for 6 hours in M199 medium containing 1% FBS. After 30 minutes of starvation, 4 ⁇ g / 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).
- 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 °C, 12,000g for 5 minutes. Isolate to obtain supernatant.
- RIPA radioimmunoprecipitation assay
- 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).
- 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.
- PI3-kinase and Akt activation inhibition plays an important role in the inhibition of endothelial cell migration, invasion and angiogenesis by sPTK7.
- 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.
- VEGF focal adhesion kinase
- paxillin focal adhesion kinase
- FAK focal adhesion kinase
- paxillin paxillin
- 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).
- 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, the reaction was carried out for 1 hour after the reaction with Rhodamine-conjugated secondary antibody and Phalloidin-FITC, followed by observation with a confocal microscope.
- 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.
- sPTK7 could be expected to inhibit the adhesion and migration of vascular endothelial cells by inhibiting local adhesion formation and actin reconstitution.
- 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 It was mutated by polymerase chain reaction (PCR) to have His tag codon and translation end codon.
- PCR poly
- pcDNA3.1-hPTK7-Ig14-His was prepared as follows.
- 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 pcDNA3 vector) and 5'-GC TCTAGA TCA ATGATGATGATGATGATG CTGGGTCAGGCAATCCAA-3' for the fifth Ig loop entry (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).
- 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]
- nucleotide sequences 1390 to 1470 of SEQ ID NO: 1 were deleted by PCR.
- Primer pairs used for PCR were 5'-CATCACTGTGGCC CATCATCATCATCAT 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.
- PTK7-Ig13-His PTK7-Ig14s-His
- PTK7-Ig14-His PTK7-Ig14-His
- PTK7-Ig15-His influence the cell migration.
- 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.
- 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.
- 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.
- RNA Interference (RNAi) Analysis of Angiogenesis and Akt Phosphorylation of Vascular Endothelial Cells During PTK7 Knockdown by RNA Interference (RNAi) Method
- 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 vascular endothelial angiogenesis and Akt phosphorylation were analyzed. Knockdown of PTK7 using PTK7 siRNA in HUVEC was performed by the following method.
- siRNA small interfering RNA
- shRNA small hairpin RNA
- 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.
- Opti-MEM I serum-reduced medium
- siRNA Dharmacon's ON-TARGET plus family J- 003167-13, J-003167-14, J-003167-15 and J-003167-16
- Lipofectamine purchased from Invitrogen
- 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.
- M199 medium purchased from Invitrogen
- 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).
- 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).
- Mission TRC shRNA vectors calcium phosphate
- the nucleic acid transfected HEK293 cells were changed to DMEM medium containing 10% FBS, 100 unit / ml penicillin and 100 ⁇ g / 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).
- PTK7 siRNA mixture was used to analyze the effect of PTK7 knockdown on cellular function.
- 15 shows the results of analyzing the effect of knockdown of PTK7 on VEGF-induced angiogenesis in HUVEC.
- PTK7 knockdown HUVECs were placed on growth factor-reducing Matrigel and incubated for 16 hours to determine the number of blood vessels produced, and statistically analyzed by Student's t test, with a P value of less than 0.01 for mock transfection without VEGF.
- ⁇ P value is less than 0.001 ⁇
- VEGF-treated control mock + VEGF
- Akt phosphorylation was observed at levels similar to, but VEGF-induced Akt phosphorylation was inhibited in HUVEC 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.
- sPTK7 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.
- sPTK7 antiserum 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 ⁇ l of immunoprecipitation assay buffer per 60 mm plate. Western blots were then performed on the cell lysate supernatant.
- PTK7 neutralizing monoclonal antibodies as well as PTK7 neutralizing polyclonal antibodies may be a method of inhibiting cell migration, invasion and angiogenesis by inhibiting the function of PTK7.
- the sPTK7 expression and PTK7 neutralizing aptamer treatment may also inhibit cell migration and invasion. It can block angiogenesis by blocking the normal function of vascular endothelial cells.
- 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.
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Abstract
Description
Claims (9)
- PTK7 단백질의 기능을 저해하는 세포의 이동, 침윤 또는 혈관신생 억제제.Inhibitor of cell migration, infiltration or angiogenesis that inhibits the function of PTK7 protein.
- 제1항에 있어서, The method of claim 1,상기 억제제는 PTK7 단백질의 세포외 도메인을 갖는 가용성 PTK7 단백질 단편을 포함하는 것을 특징으로 하는 세포의 이동, 침윤 또는 혈관신생 억제제. The inhibitor comprises a soluble PTK7 protein fragment having an extracellular domain of PTK7 protein. Inhibitors of cell migration, infiltration or angiogenesis.
- 제2항에 있어서, The method of claim 2,상기 가용성 PTK7 단백질 단편은 서열번호 2로 표시되는 아미노산 서열 또는 서열번호 2로 표시되는 아미노산 서열 중 1 ~ 30번째 서열을 제외한 나머지 서열을 포함하는 것을 특징으로 하는 세포의 이동, 침윤 또는 혈관신생 억제제. The soluble PTK7 protein fragment comprises an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence represented by SEQ ID NO: 2, except for the 1-30 sequence, cell migration, infiltration or angiogenesis inhibitor.
- 제2항에 있어서, The method of claim 2,상기 가용성 PTK7 단백질의 최소활성단편은 서열번호 2의 인간 PTK7 단백질의 31번 내지 409번 아미노산 서열 또는 327번 내지 409번 아미노산 서열인 것을 특징으로 하는 세포의 이동, 침윤 또는 혈관신생 억제제. The least active fragment of the soluble PTK7 protein is a cell migration, infiltration or angiogenesis inhibitor, characterized in that the amino acid sequence of amino acids 31 to 409 or amino acids 327 to 409 of the human PTK7 protein of SEQ ID NO: 2.
- 제1항에 있어서, The method of claim 1,상기 억제제는 상기 PTK7 단백질을 암호화하는 메신저 RNA (mRNA)에 대하여 특이적 RNA 간섭(RNAi)을 매개하는 소규모 RNA 서열을 포함하는 것을 특징으로 하는 세포의 이동, 침윤 또는 혈관신생 억제제. The inhibitor comprises a small RNA sequence that mediates specific RNA interference (RNAi) to the messenger RNA (mRNA) encoding the PTK7 protein, cell migration, infiltration or angiogenesis inhibitor.
- 제5항에 있어서, The method of claim 5,상기 소규모 RNA 서열은 PTK7 단백질을 암호화하는 메신저 RNA (mRNA) 서열과 상보적 결합을 형성하여 상기 mRNA 서열의 발현을 억제하는 소규모 억제 RNA (siRNA), 또는 상기 소규모 억제 RNA (siRNA) 서열을 포함하는 소규모 헤어핀 RNA (shRNA)인 것을 특징으로 하는 세포의 이동, 침윤 또는 혈관신생 억제제. 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. Inhibitors of migration, infiltration or angiogenesis of cells characterized in that they are small scale hairpin RNA (shRNA).
- 제1항에 있어서, 상기 억제제는 PTK7 단백질과 특이적으로 결합함으로써 PTK7 단백질의 활성을 억제하는 PTK7 단백질 중화항체인 것을 특징으로 하는 세포의 이동, 침윤 또는 혈관신생 억제제. The inhibitor of claim 1, wherein the inhibitor is a PTK7 protein neutralizing antibody that specifically inhibits the activity of the PTK7 protein by specifically binding to the PTK7 protein.
- 제7항에 있어서, 상기 중화항체는 서열번호 2로 표시되는 아미노산 서열을 인식하는 것을 특징으로 하는 세포의 이동, 침윤 또는 혈관신생 억제제. 8. The cell migration, infiltration or angiogenesis inhibitor according to claim 7, wherein the neutralizing antibody recognizes an amino acid sequence represented by SEQ ID NO.
- 제1항에 있어서, The method of claim 1,상기 세포는 혈관내피세포인 것을 특징으로 세포의 이동, 침윤 또는 혈관신생 억제제. The cell is an vascular endothelial cell, characterized in that the movement, infiltration or angiogenesis inhibitor.
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US9777070B2 (en) | 2014-04-30 | 2017-10-03 | Pfizer Inc | Anti-PTK7 antibody-drug conjugates |
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