WO2010070746A1 - Polypeptide de type angiostatine canine - Google Patents

Polypeptide de type angiostatine canine Download PDF

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WO2010070746A1
WO2010070746A1 PCT/JP2008/072974 JP2008072974W WO2010070746A1 WO 2010070746 A1 WO2010070746 A1 WO 2010070746A1 JP 2008072974 W JP2008072974 W JP 2008072974W WO 2010070746 A1 WO2010070746 A1 WO 2010070746A1
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angiostatin
polypeptide
plasminogen
canine
amino acid
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PCT/JP2008/072974
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Japanese (ja)
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惠司 蓮見
幸輔 清水
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株式会社ティムス
国立大学法人東京農工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for producing an angiostatin-like polypeptide from canine plasminogen having a natural sugar chain, and a canine angiostatin-like polypeptide obtained by this method.
  • Angiogenesis is a process in which new blood vessels are formed from existing blood vessels, and is regulated by various inhibitors such as growth factors released from platelets and vascular endothelial cells. Angiogenesis in tumors is essential for tumor growth, and therefore substances that inhibit angiogenesis may be useful as anti-tumor agents based on their ability to suppress cancer growth, invasion and metastasis. is there.
  • Angiostatin is known as an example of such a substance.
  • Angiostatin is a polypeptide containing kringle region 1 to 4 of plasminogen obtained by degrading plasminogen, which is a fibrinolytic factor, and reported to have an effect on cancer in animal experiments. (Non-Patent Document 1).
  • angiostatin As a method for producing angiostatin, methods for producing recombinant proteins derived from humans (Non-patent Documents 2 to 3), dogs (Patent Documents 1 and 2), etc. have been reported. Angiostatin produced by these methods does not have a natural sugar chain structure. If it does not have a natural sugar chain structure, it may be antigenic in the administered subject, and if the sugar chain affects the activity, the activity may be lost. Therefore, angiostatin as described above may not exhibit sufficient activity as an anticancer agent.
  • Non-patent Document 4 a method of hydrolyzing plasminogen with elastase is known (Non-patent Document 4).
  • This method makes it difficult to selectively produce angiostatin from plasminogen because many byproducts are generated due to the low substrate specificity of elastase. This method therefore has the disadvantage that the activity of the resulting angiostatin is low.
  • elastase preparations can be used to produce active angiostatin, and it has been suggested that cleavage or subsequent treatment may modify angiostatin (Non-Patent Documents). 4).
  • angiostatin-like fragment (main product) was obtained by specifically cleaving human plasminogen using “Bacillolysin MA” which is a protease produced by Bacillus megaterium strain A9542.
  • Bacillus megaterium strain A9542 was found to be a fragment having the amino acid sequence of Glu 1 to Ser 441 (hereinafter referred to as “human BL angiostatin” or “hBLAS”) (Patent Document 3).
  • the above amino acid numbers are based on the amino acid sequence of a mature polypeptide (ie, Glu 1 -Asn 791 ) excluding the signal peptide from the amino acid sequence of human plasminogen (SWISSPROT accession number P00747).
  • human BL angiostatin has an extremely excellent anticancer effect and has high solubility in water, and therefore exhibits an exceptional anticancer effect even by intravenous administration ( Patent Document 4).
  • an angiostatin-like fragment human BL angiostatin obtained by allowing bacilolysin MA to act on human plasminogen has been demonstrated.
  • angiostatin-like fragments as described above are expected to have similar effects.
  • the fragment may be recognized as heterologous by the immune system of the administered animal, so that the effect may not be exhibited. Therefore, for application to non-human animals, it is necessary to obtain angiostatin-like fragments using plasminogen from the animal to be administered as a starting material.
  • basilolysin MA is an enzyme obtained based on the action on human plasminogen, basilolysin MA was allowed to act on plasminogen derived from a non-human animal having an amino acid sequence different from that of human plasminogen. In some cases it was unclear whether fragments with angiostatin-like activity could be obtained. Therefore, a method for obtaining an angiostatin-like fragment having a natural sugar chain suitable for administration to mammals other than humans has been desired.
  • JP 2002-355056 A Japanese translation of PCT publication No. 2002-523036 JP 2002-272453 A International Publication No.
  • An object of the present invention is to provide a method for producing an angiostatin-like polypeptide from canine plasminogen having a natural sugar chain, and a canine angiostatin-like polypeptide obtained by this method.
  • the present invention [1] A method for producing an angiostatin-like polypeptide, comprising digesting canine plasminogen with basilolysin MA; [2] Angiostatin-like polypeptide obtained by digesting canine plasminogen with basilolysin MA; [3] The N-terminal amino acid is arginine at position 78 or leucine at position 81 of the amino acid sequence of SEQ ID NO: 1, and the molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions is 35 to 38 kDa Angiostatin-like polypeptide of [2]; [4] Angiostatin-like polypeptide of [2] or [3] comprising kringle regions 1 to 3 and not including NTP and kringle regions 4 to 5; [5] The angiostatin-like polypeptide according to any one of [2] to [4], wherein canine plasminogen has a natural sugar chain; [6] An an
  • VEGF represents a group to which 10 ng / mL vascular endothelial growth factor (VEGF) was added, the horizontal axis represents the concentration of canine angiostatin (cBLAS), and the vertical axis was stained with anti-CD31 antibody for the entire visual field. The ratio (%) of the remaining area.
  • dog plasminogen refers to dog-derived plasminogen.
  • Plasminogen is a precursor of plasmin, present in animal plasma, which is cleaved by plasminogen activator and converted to active plasmin. Plasmin consists of an H chain containing five kringle regions (K1 to K5) and an L chain containing a serine protease active center.
  • plasminogen having a natural sugar chain is preferably used. “Having a natural sugar chain” means having a sugar chain having substantially the same structure as a sugar chain bound to natural plasminogen at the same site.
  • Canine plasminogen having a natural sugar chain can be obtained, for example, by purification from canine plasma.
  • plasminogen produced using recombinant DNA technology or the like based on the amino acid sequence of known canine plasminogen is used as long as it has a natural sugar chain. can do.
  • NTP N-terminal peptide
  • the kringle region is defined as a region sandwiched between the most N-terminal and C-terminal cysteine (Cys) residues of each region
  • the kringle regions 1 to 5 are positions 84 to 162 of SEQ ID NO: 1, respectively. This corresponds to the regions of the 166th to 243rd, 256th to 333th, 360th to 437th, and 464th to 543th positions.
  • Bacillolysin MA refers to a protease produced by Bacillus megaterium strain A9542. Basilolysin MA specifically cleaves human plasminogen to produce an angiostatin-like fragment (the main product is a fragment having the amino acid sequence of Glu 1 to Ser 441 ; the amino acid number is the amino acid sequence of human plasminogen (SWISSPROT The activity to generate a mature polypeptide (ie, based on the amino acid sequence of Glu 1 -Asn 791 ) excluding the signal peptide from session number P00747). Bacillus megaterium A9542 strain was established in Tsukuba Center, Central 1-1-1 Tsukuba Center, Ibaraki 305-8586, Japan, under the patent number FERM P-18268 in 2001. Deposited on March 21.
  • angiostatin refers to a polypeptide that is derived from plasminogen and exhibits angiogenesis inhibitory activity, including plasminogen kringle regions 1 to 4.
  • Angiostatin-like polypeptide refers to a plasminogen-derived polypeptide that exhibits angiogenesis inhibitory activity similar to Angiostatin. According to the present invention, an angiostatin-like polypeptide is obtained by digesting canine plasminogen with basilolysin MA.
  • the angiostatin-like polypeptide of the present invention is not necessarily composed of a single molecular species, and may be a mixture of a plurality of molecular species having different N-terminal amino acids or sugar chain structures.
  • an angiostatin-like polypeptide obtained by digesting canine plasminogen with basilolysin MA is also referred to as “cBLAS”.
  • the polypeptide portion other than the sugar chain is an amino acid sequence derived from canine plasminogen.
  • the angiostatin-like polypeptide of the present invention has a natural sugar chain.
  • the angiostatin-like polypeptide may be further purified by subjecting this digest to affinity chromatography (eg, Lys-Sepharose).
  • Angiogenesis inhibitory activity can be measured in vitro, for example, by observing the effect of a test substance on angiogenesis by endothelial cells using a commercially available kit or the like.
  • angiogenesis inhibitory activity can be measured in vivo by observing the effect of a test substance on the growth of Lewis lung cancer transplanted subcutaneously into mice, for example as described in US Pat.
  • the angiostatin-like polypeptide of the present invention is, for example, 34-40 kDa, preferably 35-38 kDa, more preferably 36-36 by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) under reducing conditions.
  • the molecular weight is 37 kDa.
  • the N-terminal amino acid of the angiostatin-like polypeptide of the present invention is arginine at position 78 (Arg 78 ) or leucine at position 81 (Leu 81 ) of the amino acid sequence of SEQ ID NO: 1.
  • the C-terminal amino acid of the angiostatin-like polypeptide of the invention is tyrosine (Tyr 342 ) at position 342 of the amino acid sequence of SEQ ID NO: 1.
  • the angiostatin-like polypeptide of the present invention has an amino acid sequence of Arg 78 or Leu 81 to Tyr 342 of the amino acid sequence of SEQ ID NO: 1 except that the polypeptide portion (polypeptide backbone) other than its sugar chain Consists of.
  • the amino acid sequences of Arg 78 -Tyr 342 and Leu 81 -Tyr 342 of the amino acid sequence of SEQ ID NO: 1 are shown in SEQ ID NOs: 2 and 3, respectively.
  • This region includes the kringle regions 1 to 3, but does not include the NTP and the kringle regions 4 to 5.
  • the main product of angiostatin-like fragment obtained by digesting human plasminogen with basilolysin MA has the amino acid sequence of human plasminogen Glu 1 to Ser 441 . This sequence includes NTP and kringle regions 1-4.
  • the angiostatin-like polypeptide derived from canine plasminogen of the present invention has a structure very different from the angiostatin-like fragment derived from human.
  • the recombinant canine angiostatins described in Patent Documents 1 and 2 each contain kringle regions 1 to 4 of canine plasminogen and have a structure different from that of the angiostatin-like polypeptide of the present invention. ing.
  • the conditions for digestion of plasminogen with basilolysin MA are appropriately selected so as to obtain a polypeptide exhibiting angiogenesis inhibitory activity. Since basilolysin MA requires Ca 2+ for its enzymatic reaction, digestion is performed in the presence of Ca 2+ .
  • the reaction temperature is selected in consideration of the optimum temperature of basilolysin MA (about 50 to 60 ° C.), the stability of the substrate (plasminogen), etc., for example, 0 to 60 ° C., preferably 20 to 50 ° C., more preferably Is 30 to 40 ° C., more preferably 37 ° C.
  • Other conditions such as reaction time, enzyme and substrate concentration in the reaction solution are also appropriately determined by those skilled in the art.
  • the reaction time is, for example, 0.5 to 2.5 hours, preferably 1 to 2 hours.
  • concentration of enzyme (basilolysin MA) (concentration calculated based on a molecular weight of 34 kDa) is, for example, 50 to 500 nM, preferably 100 to 300 nM, more preferably 150 nM.
  • concentration of the substrate (dog plasminogen) is, for example, 0.1 to 2 mg / mL, preferably 0.2 to 1.0 mg / mL, more preferably 0.5 mg / mL.
  • the angiostatin-like polypeptide obtained by the production method of the present invention exhibits angiogenesis inhibitory activity. Since basilolysin MA is an enzyme obtained based on the action on human plasminogen, basilolysin MA is added to plasminogen derived from dogs, which are non-human animals having an amino acid sequence different from the amino acid sequence of human plasminogen. It was unclear whether a fragment having angiostatin-like angiogenesis inhibitory activity would be obtained when allowed to act. For example, the cleavage site of human plasminogen by basilolysin MA is between serine (Ser) at position 441 and valine (Val) at position 442.
  • the amino acids of canine plasminogen corresponding to these amino acids are serine (Ser) at position 443 and Ala at position 444 of SEQ ID NO: 1.
  • the cleavage site of basilolysin MA cannot be predicted from the primary amino acid sequence alone, so that the structure of a polypeptide obtained by reacting basilolysin MA with canine plasminogen could not be predicted. Therefore, the above findings were unexpected.
  • the angiostatin-like polypeptide obtained by the production method of the present invention has a natural sugar chain because it can be obtained using plasminogen having a natural sugar chain as a starting material. Therefore, the present invention is advantageous in that it is not necessary to consider problems such as antigenicity and deactivation that may occur when a natural sugar chain is not present.
  • the present invention provides a composition comprising the above-mentioned angiostatin-like polypeptide.
  • This composition can be used to inhibit angiogenesis.
  • a disease in which inhibition of angiogenesis is desired can be treated.
  • a cancer is illustrated as a disease.
  • the subject to be administered can be any mammal in need of treatment, but is preferably a dog.
  • the composition can further include optional ingredients such as other angiogenesis inhibitors, pharmaceutically acceptable excipients, and the like.
  • the form and route of administration of the preparation can be arbitrarily selected from known ones. For example, an injectable formulation can be administered to a subject by intravenous injection.
  • Example 1 Purification of basilolysin MA (BL-MA) Glucose 1%, corn starch 3%, soybean meal 1%, peptone 0.5%, yeast extract 0.5%, CaCO 3 0.2%, CB442 0.01 In a 500 mL Erlenmeyer flask containing 100 mL of a liquid medium (pH 7.0) containing 1%, Bacillus megaterium A9542 strain (1-1-1 Higashi Tsukuba City, Ibaraki 305-8856 Tsukuba Center Chuo No. 6, Industrial Technology (Deposited on March 21, 2001) under the accession number FERM P-18268 at the Research Center for Patent Biological Deposits) and cultured with shaking at 28 ° C. for 6 days.
  • BL-MA basilolysin MA
  • BL-MA concentration of BL-MA is displayed in units such as “nM” and “ ⁇ M” calculated based on the molecular weight (34 kDa).
  • Example 2 Purification of canine plasminogen (cPlg) Purification of cPlg was performed according to the procedure described in 1) to 8) below. The chromatographic flow rate was 8 mL / min unless otherwise specified.
  • Protein was quantified by the Lowry method.
  • 20 ⁇ L of cPlg obtained in 8) after dialysis was diluted with 40 ⁇ L of 0.1 M sodium hydroxide.
  • 300 ⁇ L of a solution in which 2% (w / v) Na 2 CO 3 /0.1 M sodium hydroxide and 0.5% CuSO 4 .5H 2 O / 1% sodium citrate were mixed at a ratio of 50: 1 was first added. Were mixed well and allowed to react for 15 minutes at room temperature. After the reaction, 30 ⁇ L of a phenol reagent was added, mixed well, and further reacted at room temperature for 30 minutes.
  • CPlg was purified 11 times in total. From the protein quantification results after dialysis, it was found that 448 mg of cPlg was obtained. In addition, when 1 ⁇ L of the eluate was subjected to sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE, 10%, reducing conditions), no band other than cPlg was observed, so the purity was estimated to be 95% or more. . Since the total amount of canine plasma used was 4030 mL, the yield was 111 mg / L plasma.
  • Example 4 Examination of cPlg cleavage conditions TBS / T / Ca (50 mM Tris-HCl (pH 7.4 (25 ° C.)) / 100 mM NaCl / 0.01% Tween 80/1 mM CaCl 2 ) 46.4 ⁇ L in 1.5 mL plastic
  • 25.6 ⁇ L of 1.6 mg / mL cPlg prepared by diluting the stock solution with TBS / T / Ca
  • 8 ⁇ L of 1.5 ⁇ M BL-MA (prepared by diluting the stock solution with TBS / T / Ca) was added to this tube and mixed uniformly.
  • reaction temperature 37 ° C. was used.
  • the optimal temperature for BL-MA is a higher temperature (50-60 ° C.), but this temperature was chosen to avoid denaturation of the substrate protein (cPlg).
  • Example 5 Preparation of canine BL angiostatin (cBLAS) 0.5 mg / mL cPlg and 150 nM BL-MA were incubated in TBS / T / Ca for 2 hours at 37 ° C. Thereafter, EDTA-2Na was added to a final concentration of 10 mM to stop the reaction. The obtained reaction solution was immediately subjected to purification using Lys-Sepharose.
  • cBLAS canine BL angiostatin
  • the fraction in which cBLAS band was confirmed by SDS-PAGE was dialyzed against ultrapure water in the same manner as in Example 2 except that ultrapure water was used, freeze-dried, and dissolved in physiological saline. The solution was sterilized by filtration using a 0.2 ⁇ m disk filter. A part of the sample was subjected to protein quantification (by the Lowry method described in Example 2) and purity assay by SDS-PAGE.
  • cBLAS was obtained from 448 mg of cPlg (about 3 mL of 35.7 mg / mL cBRAS solution). Further, when 1 ⁇ L of the eluate was subjected to SDS-PAGE (10%, reducing conditions), no band other than cBRAS was observed, so the purity was estimated to be 95% or more.
  • the cBRAS solution was stored at ⁇ 80 ° C. Based on the molecular weights of cPlg and cBRAS on SDS-PAGE (about 100 kDa and about 37 kDa, respectively), the recovery was calculated to be about 84% (140 / (448 ⁇ 37/100)).
  • Example 6 Determination of partial amino acid sequence
  • the cBRAS obtained in Example 1 was subjected to SDS-PAGE. A 36-37 kDa band corresponding to two cBRAS was confirmed (FIG. 1, left lane).
  • the protein in the gel was transferred to a PVDF membrane (Fluorotrans (registered trademark), Nippon Genetics)
  • the target band was cut out and the N-terminal amino acid sequence was analyzed using 476A Protein Sequencer (Applied Biosystems).
  • c-Plg BL-MA digest unpurified
  • was subjected to SDS-PAGE several more bands were seen (FIG. 1, right lane). The most dominant band of about 53 kDa was similarly subjected to sequence analysis.
  • N-terminal sequences determined for the upper and lower bands of cBRAS were Arg-Ile-Tyr-Leu and Leu-Ser-Glu-XXX-Lys, respectively. This sequence matched the sequence starting from ArP 78 and Leu 81 of cPlg, respectively.
  • the N-terminal sequence of the approximately 53 kDa band dominant in the BL-MA digest of cPlg was Leu-Asp-Ala-Pro-Ala. This sequence matched the sequence starting from Leu 343 of cPlg.
  • the N-terminal of the polypeptide corresponding to the band of about 50 kDa found in Example 4 was determined to be Val 356 , and the polypeptide corresponding to the band of about 53 kDa was further decomposed. It was suggested that it occurred.
  • Example 7 Evaluation of cBRAS activity Regarding the effect of canine BL angiostatin (cBRAS) on angiogenesis of human endothelial cells induced by vascular endothelial growth factor (VEGF), angiogenesis kit (Kurashiki Spinning Co., Ltd.) It was examined using.
  • VEGF vascular endothelial growth factor
  • a “saline group” sodium chloride 85 mg in 10 mL water
  • a “VEGF group” VEGF 10 ng / mL
  • a “cBRAS group” VEGF 10 ng / mL + cBRAS 1, 10, 100 ⁇ g / mL, hereinafter “Experiments were performed using a total of 5 groups, “cBLAS 1 ⁇ g / mL group”, “cBRAS 10 ⁇ g / mL group”, and “cBRAS 100 ⁇ g / mL group”.
  • the medium On the day of arrival of the angiogenesis kit (day 1), the medium is replaced with a medium in which the test sample (cBLAS at a predetermined concentration) is added to the medium (VEGF ( ⁇ ) medium) that does not contain VEGF for the cBLAS group.
  • the physiological saline group and the VEGF group were replaced with VEGF ( ⁇ ) medium supplemented with the same amount of physiological saline as the test sample.
  • the medium is changed to a medium in which the test sample (cBLAS at a predetermined concentration) is added to the medium (VEGF (+) medium) attached to the kit containing VEGF for the cBLAS group,
  • the physiological saline group was replaced with a VEGF ( ⁇ ) medium
  • the VEGF group was replaced with a medium in which the same amount of physiological saline as the test sample was added to the VEGF (+) medium.
  • staining with an anti-CD31 (PECAM-1) antibody was performed according to the instructions of the angiogenesis staining kit.
  • the staining area measured for the physiological saline group was 2.79 ⁇ 0.13% (mean value ⁇ standard deviation).
  • the stained areas measured for the VEGF group, cBRAS 1 ⁇ g / mL group, cBRAS 10 ⁇ g / mL group, and cBRAS 100 ⁇ g / mL group are 3.06 ⁇ 0.11%, 3.00 ⁇ 0.11%, and 2.08, respectively. They were ⁇ 0.19% and 1.25 ⁇ 0.11% (FIG. 2).
  • endothelial cell proliferation was significantly inhibited in the cBRAS 10 ⁇ g / mL group and the cBRAS 100 ⁇ g / mL group (P ⁇ 0.01).
  • the present invention provides a method for producing an angiostatin-like polypeptide from canine plasminogen having a natural sugar chain, and a canine angiostatin-like polypeptide obtained by this method.

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Abstract

L'invention porte sur un procédé de production d'un polypeptide de type angiostatine à partir de plasminogène canin ayant une chaîne de sucre native et un polypeptide de type angiostatine canine obtenu par ce procédé.
PCT/JP2008/072974 2008-12-17 2008-12-17 Polypeptide de type angiostatine canine WO2010070746A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001151691A (ja) * 1995-04-26 2001-06-05 Childrens Medical Center Corp アンジオスタチンフラグメント、アンジオスタチン凝集体および使用方法
JP2002355056A (ja) * 2000-08-25 2002-12-10 Pfizer Prod Inc 脈管形成を伴う障害の診断及び治療のための組成物及び方法
WO2004065595A1 (fr) * 2003-01-17 2004-08-05 Ttc Co., Ltd. Reacteur a piege par affinite et procede a une etape servant a purifier un fragment de type angiostatine du plasma humain utilisant ce reacteur
WO2005079835A1 (fr) * 2004-02-24 2005-09-01 Ttc Co., Ltd. Agent anticancéreux contenant de la bl-angiostatine

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001151691A (ja) * 1995-04-26 2001-06-05 Childrens Medical Center Corp アンジオスタチンフラグメント、アンジオスタチン凝集体および使用方法
JP2002355056A (ja) * 2000-08-25 2002-12-10 Pfizer Prod Inc 脈管形成を伴う障害の診断及び治療のための組成物及び方法
WO2004065595A1 (fr) * 2003-01-17 2004-08-05 Ttc Co., Ltd. Reacteur a piege par affinite et procede a une etape servant a purifier un fragment de type angiostatine du plasma humain utilisant ce reacteur
WO2005079835A1 (fr) * 2004-02-24 2005-09-01 Ttc Co., Ltd. Agent anticancéreux contenant de la bl-angiostatine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LUCAS, R. ET AL.: "Multiple forms of angiostatin induce apoptosis in endothelial cells", BLOOD, vol. 92, no. 12, 1998, pages 4730 - 4741 *
NARASAKI, R. ET AL.: "Bacillolysin MA, a novel bacterial metalloproteinase that produces angiostatin-like fragments from plasminogen and activates protease zymogens in the coagulation and fibrinolysis systems", J. BIOL. CHEM., vol. 280, no. 14, 2005, pages 14278 - 14287 *
PIRIE-SHEPHERD, S.R. ET AL.: "The role of angiostatin in the spontaneous bone and prostate cancers of pet dogs", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 292, no. 4, 2002, pages 886 - 891 *

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