WO2010070746A1 - Canine angiostatin-like polypeptide - Google Patents

Canine angiostatin-like polypeptide 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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French (fr)
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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Publication of WO2010070746A1 publication Critical patent/WO2010070746A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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.

Abstract

It is intended to provide a method for producing an angiostatin-like polypeptide from canine plasminogen having a native sugar chain and a canine angiostatin-like polypeptide obtained by this method.

Description

イヌアンジオスタチン様ポリペプチドCanine angiostatin-like polypeptide
　本発明は、天然型糖鎖を有するイヌプラスミノーゲンからアンジオスタチン様ポリペプチドを製造するための方法、およびこの方法によって得られたイヌアンジオスタチン様ポリペプチドに関する。 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.
　血管新生は、既存の血管から新しい血管が形成される過程であり、血小板や血管内皮細胞から放出される成長因子等と種々の阻害因子によって調節されている。腫瘍における血管新生は腫瘍の増殖に必須であり、それゆえ、血管新生を阻害する物質は、癌の増殖、浸潤および転移を抑制するその能力に基づいて、抗腫瘍剤として有用である可能性がある。 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.
　このような物質の一例としてアンジオスタチンが知られている。アンジオスタチンは、線溶因子であるプラスミノーゲンを分解することにより得られる、プラスミノーゲンのクリングル領域１～４を含むポリペプチドであり、そして動物実験において癌に対する効果を示すことが報告されている（非特許文献１）。 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).
　アンジオスタチンの製造方法としては、ヒト（非特許文献２～３）、イヌ（特許文献１～２）など由来の組換えタンパク質の製造方法が報告されている。これらの方法によって製造されたアンジオスタチンは、天然型の糖鎖構造を有していない。天然型の糖鎖構造を有していなければ投与された対象内で抗原性を示す可能性があり、また糖鎖が活性に影響していれば、活性が失われる可能性もある。そのため、上記のようなアンジオスタチンは抗癌剤としての活性を十分に示さない可能性がある。 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.
　天然型糖鎖を有するアンジオスタチンの生産は、天然型のプラスミノーゲンを精製し、適当な酵素で処理することによって達成され得る。このような方法としては、例えば、プラスミノーゲンをエラスターゼで加水分解する方法が知られている（非特許文献４）。この方法では、エラスターゼの基質特異性の低さのために多くの副生成物が生じるので、プラスミノーゲンからアンジオスタチンを選択的に生成させることが困難である。従って、この方法には、得られるアンジオスタチンの活性が低いという欠点がある。また、すべての市販エラスターゼ標品を使用して活性のあるアンジオスタチンを生産することができるわけではなく、切断またはその後の処理によってアンジオスタチンが変性される可能性も示唆されている（非特許文献４）。 Production of angiostatin having a natural sugar chain can be achieved by purifying natural plasminogen and treating it with an appropriate enzyme. As such a method, for example, 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. In addition, not all commercially available 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).
　本発明者らは、プラスミノーゲンから選択的にアンジオスタチンを生成するために適切な基質特異性を有し、かつ副生成物を生じさせない新たな酵素を探索した。その結果、バチルス・メガテリウム（Bacillus megaterium）Ａ９５４２株が産生するプロテアーゼである「バシロライシンＭＡ」（Bacillolysin MA）を用いて、ヒトプラスミノーゲンを特異的に切断することによってアンジオスタチン様フラグメント（主生成物はＧｌｕ１からＳｅｒ４４１のアミノ酸配列を有するフラグメントである、以下、「ヒトＢＬアンジオスタチン」または「ｈＢＬＡＳ」と記載する）が得られることを見出した（特許文献３）。なお、上記のアミノ酸番号は、ヒトプラスミノーゲンのアミノ酸配列（ＳＷＩＳＳＰＲＯＴアクセッション番号Ｐ００７４７）からシグナルペプチドを除外した成熟ポリペプチド（すなわち、Ｇｌｕ１－Ａｓｎ７９１）のアミノ酸配列に基づく。 The inventors sought new enzymes that have the appropriate substrate specificity to produce angiostatin selectively from plasminogen and do not produce byproducts. As a result, 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. 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).
　さらに、本発明者らは、ヒトＢＬアンジオスタチンが極めて優れた制癌効果を有すること、および水に対する溶解性が高いことから、静脈内投与によっても格別の制癌効果を奏することを見出した（特許文献４）。このように、ヒトプラスミノーゲンにバシロライシンＭＡを作用させることによって得られるアンジオスタチン様フラグメント（ヒトＢＬアンジオスタチン）の有効性は実証されている。 Furthermore, the present inventors have found that 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). Thus, the effectiveness of an angiostatin-like fragment (human BL angiostatin) obtained by allowing bacilolysin MA to act on human plasminogen has been demonstrated.
　癌のような血管新生の阻害が有効な疾患はヒト以外の哺乳動物にも存在する。このような疾患には上記のようなアンジオスタチン様フラグメントが同様の効果を有することが期待される。しかし、例えば、ヒト由来のプラスミノーゲンフラグメントを他の動物に投与すれば、このフラグメントは投与された動物の免疫系によって異種として認識されるので、その効果が発揮されない可能性がある。従って、ヒト以外の動物へ適用のためには、投与しようとする動物由来のプラスミノーゲンを出発材料として使用してアンジオスタチン様フラグメントを得る必要がある。バシロライシンＭＡはヒトプラスミノーゲンに対する作用に基づいて得られた酵素であるので、ヒトプラスミノーゲンのアミノ酸配列とは異なるアミノ酸配列を有するヒト以外の動物由来のプラスミノーゲンにバシロライシンＭＡを作用させた場合に、アンジオスタチン様活性を有するフラグメントが得られるかどうかは不明であった。それゆえ、ヒト以外の哺乳動物への投与に適切な天然型糖鎖を有するアンジオスタチン様フラグメントを得るための方法が望まれていた。
特開２００２－３５５０５６号公報 特表２００２－５２３０３６号公報 特開２００２－２７２４５３号公報 国際公開第２００５／０７９８３５号パンフレット M.S. O'Reilly et al., Cell, 79:315-328 (1994) Shepard, S.R. et al., Protein Expr. Purif., 20:216-227 (2000) Lin, J. et al., J. Ind. Microbiol. Biotechnol., 24:31-35 (2000) M.S. O'Reilly et al., Nature Medicine, 2:689-692 (1996) Diseases that are effective in inhibiting angiogenesis, such as cancer, also exist in mammals other than humans. For such diseases, angiostatin-like fragments as described above are expected to have similar effects. However, for example, if a human-derived plasminogen fragment is administered to other animals, 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. Since 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. 2005/0779835 Pamphlet MS O'Reilly et al., Cell, 79: 315-328 (1994) Shepard, SR et al., Protein Expr. Purif., 20: 216-227 (2000) Lin, J. et al., J. Ind. Microbiol. Biotechnol., 24: 31-35 (2000) MS O'Reilly et al., Nature Medicine, 2: 689-692 (1996)
　本発明の目的は、天然型糖鎖を有するイヌプラスミノーゲンからアンジオスタチン様ポリペプチドを製造するための方法、およびこの方法によって得られたイヌアンジオスタチン様ポリペプチドを提供することである。 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.
　本発明は、
［１］イヌプラスミノーゲンをバシロライシンＭＡで消化することを含む、アンジオスタチン様ポリペプチドの製造方法；
［２］イヌプラスミノーゲンをバシロライシンＭＡで消化することによって得られる、アンジオスタチン様ポリペプチド；
［３］Ｎ末端アミノ酸が配列番号１のアミノ酸配列の７８位のアルギニンまたは８１位のロイシンであり、還元条件下のドデシル硫酸ナトリウム－ポリアクリルアミドゲル電気泳動によって決定される分子量が３５～３８ｋＤａである、［２］のアンジオスタチン様ポリペプチド；
［４］クリングル領域１～３を含み、ＮＴＰおよびクリングル領域４～５を含まない、［２］または［３］のアンジオスタチン様ポリペプチド；
［５］イヌプラスミノーゲンが天然型の糖鎖を有する、［２］～［４］のいずれかのアンジオスタチン様ポリペプチド；
［６］［２］～［５］のいずれかのアンジオスタチン様ポリペプチドを含む、血管新生阻害用組成物
に関する。 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 angiogenesis-inhibiting composition comprising the angiostatin-like polypeptide of any one of [2] to [5].
イヌプラスミノーゲン（ｃＰｌｇ）のバシロライシンＭＡ（ＢＬ－ＭＡ）消化物および精製イヌアンジオスタチン様ポリペプチド（ｃＢＬＡＳ）のドデシル硫酸ナトリウムポリアクリルアミド電気泳動（ＳＤＳ－ＰＡＧＥ）の結果を示す図である。It is a figure which shows the result of the sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of the basilolysin MA (BL-MA) digest of canine plasminogen (cPlg) and the refined canine angiostatin-like polypeptide (cBRAS). 各種濃度のイヌアンジオスタチン（ｃＢＬＡＳ）の、血管新生に対する効果を示すグラフである。図中「ＶＥＧＦ」は１０ｎｇ／ｍＬの血管内皮増殖因子（ＶＥＧＦ）を添加した群を示し、横軸はイヌアンジオスタチン（ｃＢＬＡＳ）の濃度を示し、縦軸は全視野に対する抗ＣＤ３１抗体で染色された領域の割合（％）を示す。It is a graph which shows the effect with respect to angiogenesis of canine angiostatin (cBLAS) of various density | concentrations. In the figure, “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.
　本明細書において使用する「イヌプラスミノーゲン」（「ｃＰｌｇ」ともいう）なる用語は、イヌ由来のプラスミノーゲンを指す。プラスミノーゲンは、動物の血漿中に存在する、プラスミンの前駆体であり、プラスミノーゲンアクチベータによって切断されて活性なプラスミンに変換される。プラスミンは５個のクリングル領域（Ｋ１～Ｋ５）を含むＨ鎖と、セリンプロテアーゼ活性中心を含むＬ鎖とからなる。本発明の目的のためには天然型の糖鎖を有するプラスミノーゲンが好適に使用される。「天然型の糖鎖を有する」とは、天然のプラスミノーゲンに結合した糖鎖と実質的に同一の構造を有する糖鎖を同一の部位に有することを意味する。イヌプラスミンのＨ鎖にはＮ結合型糖鎖が結合する可能性があるコンセンサス配列（Ａｓｎ－Ｘａａ－Ｓｅｒ／Ｔｈｒ）が２箇所存在する（配列番号１の１１７～１１９位および２８９～２９１位）。このうち後者はヒトプラスミンのＮ結合型糖鎖結合部位に相当し、イヌプラスミンにおいても実際に糖鎖が結合していると考えられる。天然型糖鎖を有するイヌプラスミノーゲンは、例えば、イヌ血漿から精製して得ることができる。あるいは、公知のイヌプラスミノーゲンのアミノ酸配列（配列番号１）に基づいて、組換えＤＮＡ技術などを使用して産生されたプラスミノーゲンであっても、天然型糖鎖を有するのであれば使用することができる。配列番号１のアミノ酸配列において、プラスミノーゲンアクチベータによる切断部位は５６３位と５６４位との間に存在し、７８から５６３位がＨ鎖に、５６４～７９３位がＬ鎖に相当し、１～７７位の領域はＮ末端ペプチド（N-terminal peptide；ＮＴＰ）と称する。クリングル領域をそれぞれの領域の最もＮ末端側および最もＣ末端側のシステイン（Ｃｙｓ）残基にはさまれた領域と定義すれば、クリングル領域１～５はそれぞれ配列番号１の８４～１６２位、１６６位～２４３位、２５６～３３３位、３６０～４３７位、４６４～５４３位の領域に相当する。 As used herein, the term “dog plasminogen” (also referred to as “cPlg”) 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. For the purpose of the present invention, 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. There are two consensus sequences (Asn-Xaa-Ser / Thr) that can bind N-linked sugar chains to the H chain of canine plasmin (positions 117 to 119 and 289 to 291 in SEQ ID NO: 1). . Among these, the latter corresponds to the N-linked sugar chain binding site of human plasmin, and it is considered that sugar chains are actually bound also in canine plasmin. Canine plasminogen having a natural sugar chain can be obtained, for example, by purification from canine plasma. Alternatively, plasminogen produced using recombinant DNA technology or the like based on the amino acid sequence of known canine plasminogen (SEQ ID NO: 1) is used as long as it has a natural sugar chain. can do. In the amino acid sequence of SEQ ID NO: 1, the cleavage site by plasminogen activator exists between positions 563 and 564, positions 78 to 563 correspond to the H chain, positions 564 to 793 correspond to the L chain, and 1 to The region at position 77 is called an N-terminal peptide (NTP). If 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.
　本明細書において使用する「バシロライシンＭＡ」（「ＢＬ－ＭＡ」ともいう）なる用語は、バチルス・メガテリウム（Bacillus megaterium）Ａ９５４２株が産生するプロテアーゼを指す。バシロライシンＭＡは、ヒトプラスミノーゲンを特異的に切断して、アンジオスタチン様フラグメント（主生成物はＧｌｕ１からＳｅｒ４４１のアミノ酸配列を有するフラグメント；アミノ酸番号はヒトプラスミノーゲンのアミノ酸配列（ＳＷＩＳＳＰＲＯＴアクセッション番号Ｐ００７４７）からシグナルペプチドを除外した成熟ポリペプチド（すなわち、Ｇｌｕ１－Ａｓｎ７９１）のアミノ酸配列に基づく）を生成する活性を示す。バチルス・メガテリウム Ａ９５４２株は、〒３０５－８５６６茨城県つくば市東１－１－１　つくばセンター　中央第６、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ Ｐ－１８２６８の下、２００１年３月２１日に寄託されている。 As used herein, the term “bacilolysin MA” (also referred to as “BL-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.
　本明細書において使用する「アンジオスタチン」なる用語は、プラスミノーゲンに由来し、プラスミノーゲンのクリングル領域１～４を含む、血管新生阻害活性を示すポリペプチドを指す。本明細書において使用する「アンジオスタチン様ポリペプチド」なる用語は、アンジオスタチンと同様の血管新生阻害活性を示すプラスミノーゲン由来のポリペプチドを指す。本発明によれば、アンジオスタチン様ポリペプチドは、イヌプラスミノーゲンをバシロライシンＭＡで消化することによって得られる。なお、本発明のアンジオスタチン様ポリペプチドは必ずしも単一の分子種からなるものではなく、Ｎ末端アミノ酸や糖鎖構造の異なる複数の分子種の混合物であってもよい。本明細書において、イヌプラスミノーゲンをバシロライシンＭＡで消化して得られるアンジオスタチン様ポリペプチドを「ｃＢＬＡＳ」ともいう。本発明によれば天然型の糖鎖を有するイヌプラスミノーゲンが出発材料として使用されるので、その糖鎖以外のポリペプチド部分（「ポリペプチド骨格」という）はイヌプラスミノーゲン由来のアミノ酸配列からなり、そして本発明のアンジオスタチン様ポリペプチドは天然型糖鎖を有する。この消化物をアフィニティクロマトグラフィー（例えば、Ｌｙｓ－Ｓｅｐｈａｒｏｓｅ）に供することによってアンジオスタチン様ポリペプチドをさらに精製してもよい。血管新生阻害活性は、例えば、市販のキットなどを使用して、内皮細胞による血管新生に対する試験物質の効果を観察することによってインビトロで測定することができる。あるいは、血管新生阻害活性は、例えば特許文献４に記載されるように、マウスに皮下移植したルイス肺癌の成長に対する試験物質の効果を観察することによってインビボで測定することができる。 As used herein, the term “angiostatin” refers to a polypeptide that is derived from plasminogen and exhibits angiogenesis inhibitory activity, including plasminogen kringle regions 1 to 4. As used herein, the term “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. In the present specification, an angiostatin-like polypeptide obtained by digesting canine plasminogen with basilolysin MA is also referred to as “cBLAS”. According to the present invention, since canine plasminogen having a natural sugar chain is used as a starting material, the polypeptide portion other than the sugar chain (referred to as “polypeptide skeleton”) is an amino acid sequence derived from canine plasminogen. And 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. Alternatively, 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.
　１つの実施態様において、本発明のアンジオスタチン様ポリペプチドは、還元条件下のドデシル硫酸ナトリウム－ポリアクリルアミド電気泳動（ＳＤＳ－ＰＡＧＥ）で例えば３４～４０ｋＤａ、好ましくは３５～３８ｋＤａ、より好ましくは３６～３７ｋＤａの分子量を示す。１つの実施態様において、本発明のアンジオスタチン様ポリペプチドのＮ末端アミノ酸は、配列番号１のアミノ酸配列の７８位のアルギニン（Ａｒｇ７８）または８１位のロイシン（Ｌｅｕ８１）である。１つの実施態様において、本発明のアンジオスタチン様ポリペプチドのＣ末端アミノ酸は、配列番号１のアミノ酸配列の３４２位のチロシン（Ｔｙｒ３４２）である。１つの実施態様において、本発明のアンジオスタチン様ポリペプチドは、その糖鎖以外のポリペプチド部分（ポリペプチド骨格）が、配列番号１のアミノ酸配列のＡｒｇ７８またはＬｅｕ８１からＴｙｒ３４２までのアミノ酸配列からなる。配列番号１のアミノ酸配列のＡｒｇ７８－Ｔｙｒ３４２およびＬｅｕ８１－Ｔｙｒ３４２のアミノ酸配列をそれぞれ配列番号２および３に示す。この領域にはクリングル領域１～３は含まれるが、ＮＴＰおよびクリングル領域４～５は含まれない。上記のヒトプラスミノーゲンをバシロライシンＭＡで消化して得られるアンジオスタチン様フラグメントの主生成物はヒトプラスミノーゲンのＧｌｕ１からＳｅｒ４４１のアミノ酸配列を有する。この配列にはＮＴＰおよびクリングル領域１～４が含まれる。このように、本発明のイヌプラスミノーゲン由来のアンジオスタチン様ポリペプチドは、ヒト由来のアンジオスタチン様フラグメントと非常に異なった構造を有している。また、特許文献１および２に記載される組換えイヌアンジオスタチンはいずれもイヌプラスミノーゲンのクリングル領域１～４を含むものであり、本発明のアンジオスタチン様ポリペプチドとは異なる構造を有している。 In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. Thus, 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. In addition, 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.
　プラスミノーゲンのバシロライシンＭＡでの消化の条件は、血管新生阻害活性を示すポリペプチドが得られるように適宜選択される。バシロライシンＭＡはその酵素反応にＣａ２＋を必要とするので、消化はＣａ２＋の存在下行われる。反応温度は、バシロライシンＭＡの至適温度（約５０～６０℃）、基質（プラスミノーゲン）の安定性などを考慮して選択され、例えば０～６０℃、好ましくは２０～５０℃、より好ましくは３０～４０℃、さらに好ましくは３７℃である。反応時間、酵素および基質の反応液中の濃度などの他の条件も当業者によって適宜決定される。反応時間は、例えば０．５～２．５時間、好ましくは１～２時間である。酵素（バシロライシンＭＡ）の濃度（分子量３４ｋＤａに基づいて算出される濃度）は例えば５０～５００ｎＭ、好ましくは１００～３００ｎＭ、より好ましくは１５０ｎＭである。基質（イヌプラスミノーゲン）の濃度は例えば０．１～２ｍｇ／ｍＬ、好ましくは０．２～１．０ｍｇ／ｍＬ、より好ましくは０．５ｍｇ／ｍＬである。 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. The 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. The 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.
　本発明の製造方法によって得られるアンジオスタチン様ポリペプチドは、血管新生阻害活性を示す。バシロライシンＭＡはヒトプラスミノーゲンに対する作用に基づいて得られた酵素であるので、ヒトプラスミノーゲンのアミノ酸配列とは異なるアミノ酸配列を有するヒト以外の動物であるイヌ由来のプラスミノーゲンにバシロライシンＭＡを作用させた場合に、アンジオスタチン様の血管新生阻害活性を有するフラグメントが得られるかどうかは不明であった。例えば、バシロライシンＭＡによるヒトプラスミノーゲンの切断部位は４４１位のセリン（Ｓｅｒ）と４４２位のバリン（Ｖａｌ）との間である。一方、これらのアミノ酸に相当するイヌプラスミノーゲンのアミノ酸は配列番号１の４４３位のセリン（Ｓｅｒ）および４４４位のＡｌａである。このような基質のアミノ酸配列の相違に加えて、バシロライシンＭＡの切断部位を一次アミノ酸配列のみから予測することはできないという理由から、イヌプラスミノーゲンにバシロライシンＭＡを作用させて得られるポリペプチドの構造を予測することはできなかった。従って、上記の知見は予想外であった。また、本発明の製造方法によって得られるアンジオスタチン様ポリペプチドは、天然型糖鎖を有するプラスミノーゲンを出発材料として得られるので、天然型糖鎖を有する。従って、天然型糖鎖を有していない場合に生じる可能性のある抗原性や失活などの問題を考慮する必要がないという点で本発明は有利である。 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. On the other hand, 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. In addition to such differences in the amino acid sequence of the substrate, 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.
　本発明により、上記のアンジオスタチン様ポリペプチドを含む組成物が提供される。この組成物は、血管新生を阻害するために使用することができる。例えば、本発明のアンジオスタチン様ポリペプチドを含有する組成物を対象に投与することによって、血管新生の阻害が所望される疾患を処置することができる。疾患としては癌が例示される。投与対象は処置を必要とする任意の哺乳動物であり得るが、好ましくはイヌである。組成物には、他の血管新生阻害剤、薬学的に許容される賦形剤など、任意の成分をさらに含めることができる。製剤の形態および投与経路は、公知のものから任意に選択することができる。例えば、注射用製剤を静脈注射によって対象に投与することができる。 The present invention provides a composition comprising the above-mentioned angiostatin-like polypeptide. This composition can be used to inhibit angiogenesis. For example, by administering to a subject a composition containing an angiostatin-like polypeptide of the invention, 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.
　以下、実施例により本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.
実施例１：バシロライシンＭＡ（ＢＬ－ＭＡ）の精製
　グルコース１％、コーンスターチ ３％、大豆ミール１％、ペプトン ０．５％、イーストエキス０．５％、ＣａＣＯ3 ０．２％、ＣＢ４４２ ０．０１％を含む液体培地（ｐＨ７．０）１００ｍＬを含む５００ｍＬ容三角フラスコ中で、バチルス・メガテリウム Ａ９５４２株（〒３０５－８５６６茨城県つくば市東１－１－１　つくばセンター　中央第６、独立行政法人産業技術総合研究所特許生物寄託センターに受託番号ＦＥＲＭ Ｐ－１８２６８の下、２００１年３月２１日に寄託）を２８℃、６日間振とう培養した。培養後、培養液３Ｌをセライト（Celite（登録商標））を通してろ過し、そのろ液１ＬをＨ2Ｏで希釈して５Ｌにし、イソプロピルアルコールを最終濃度５％（ｖ／ｖ）となるように添加した。この混合物を、２０ｍＭ　ＭＥＳ（２‐［Ｎ‐モルホリノ］エタンスルホン酸）－ＮａＯＨ（ｐＨ６．５）／５％イソプロピルアルコールで平衡化したカルボキシルメチルセルロース（生化学工業株式会社）カラム（４００ｍＬのゲル）に流速１５ｍＬ／分でアプライした。同じ緩衝液６００ｍＬで洗浄した後、２０ｍＭ　ＭＥＳ－ＮａＯＨ（ｐＨ６．５）／５％イソプロピルアルコール／０．２Ｍ ＮａＣｌで溶出した。その溶出画分を６０ｍＬずつ分画し、活性の認められた画分を集めた。その純度をＳＤＳ－ＰＡＧＥで確認し、精製品９０ｍｇを得た。なお、以下では、ＢＬ－ＭＡの濃度を、その分子量（３４ｋＤａ）に基づいて算出される「ｎＭ」、「μＭ」などの単位で表示する。 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. After culturing, 3 L of the culture broth is filtered through Celite (Celite (registered trademark)), 1 L of the filtrate is diluted with H 2 O to 5 L, and isopropyl alcohol is adjusted to a final concentration of 5% (v / v). Added. This mixture was applied to a carboxymethylcellulose (Seikagaku Corporation) column (400 mL gel) equilibrated with 20 mM MES (2- [N-morpholino] ethanesulfonic acid) -NaOH (pH 6.5) / 5% isopropyl alcohol. It was applied at a flow rate of 15 mL / min. After washing with 600 mL of the same buffer, elution was performed with 20 mM MES-NaOH (pH 6.5) / 5% isopropyl alcohol / 0.2 M NaCl. The eluted fractions were fractionated 60 mL each, and fractions in which activity was recognized were collected. The purity was confirmed by SDS-PAGE, and 90 mg of purified product was obtained. In the following, the concentration of BL-MA is displayed in units such as “nM” and “μM” calculated based on the molecular weight (34 kDa).
実施例２：イヌプラスミノーゲン（ｃＰｌｇ）の精製
　ｃＰｌｇの精製を以下の１）～８）に記載する手順に従って行った。クロマトグラフィーの流速は特に指定がない限り、８ｍＬ／分であった。 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.
　１）４５ｍＬのＬｙｓ－Ｓｅｐｈａｒｏｓｅ（GE healthcare、17-0960-01）を、平衡化緩衝液（０．１Ｍ　ＮＨ４ＨＣＯ３（ｐＨ　８．３））を用いてＸＫ２６／２０カラム（GE healthcare、18-1000-72）に充填した（φ２．６×１１ｃｍ）。 1) 45 mL of Lys-Sepharose (GE healthcare, 17-0960-01) was added to an XK26 / 20 column (GE healthcare, 18) using an equilibration buffer (0.1 M NH 4 HCO 3 (pH 8.3)). -1000-72) (φ2.6 × 11 cm).
　２）カラムを２２５ｍＬの平衡化緩衝液で平衡化した。 2) The column was equilibrated with 225 mL of equilibration buffer.
　３）３８０μＬの１０５　ＫＩＵ／ｍＬのアプロチニン（和光純薬、016-11826）を、３８０ｍＬのイヌ血漿（オリエンタル酵母）に添加した（血漿は、低温で、泡立たせないように取り扱った）。 3) 380 μL of 10 5 KIU / mL aprotinin (Wako Pure Chemicals, 016-11826) was added to 380 mL of canine plasma (Oriental yeast) (the plasma was handled at low temperature to prevent foaming).
　４）この血漿を上記Ｌｙｓ－Ｓｅｐｈａｒｏｓｅにアプライし、その後、１３５０　ｍＬの洗浄緩衝液（０．１Ｍ　ＮＨ４ＨＣＯ３（ｐＨ　８．３）／０．５Ｍ　ＮａＣｌ）で洗浄した（流速１５ｍＬ／分）。 4) The plasma was applied to the Lys-Sepharose and then washed with 1350 mL of washing buffer (0.1 M NH 4 HCO 3 (pH 8.3) /0.5 M NaCl) (flow rate: 15 mL / min) .
　５）カラムの３倍容量の溶出緩衝液（０．１Ｍ　ＮＨ４ＨＣＯ３（ｐＨ　８．３）／０．２Ｍ　ε－アミノカプロン酸（ＥＡＣＡ））を用いて溶出した。２８０ｎｍの吸光を観察し、検出されるピークに相当する画分の溶出液を回収した。 5) Elution was performed using 3 column volumes of elution buffer (0.1 M NH 4 HCO 3 (pH 8.3) /0.2 M ε-aminocaproic acid (EACA)). Absorption at 280 nm was observed, and the eluate of the fraction corresponding to the detected peak was collected.
　６）溶出液の一部をＳＤＳ－ＰＡＧＥにより評価し、ｃＰｌｇの純度、収量を確認した。残りの溶出液は以下のように硫安沈殿により濃縮した。溶出液１００ｍＬに対して、乳鉢で十分に細かくした硫酸アンモニウムを、氷上で溶出液を攪拌しながら、１０ｇを５分程度の添加速度で３９ｇ添加した（６０％飽和度）。全ての硫安を添加後、さらに１５分間攪拌し、１６，０００×ｇ、４℃で３０分間遠心分離し、上清を別のチューブにデカンテーションにより移し、沈殿のあるチューブを再度１６，０００×ｇ、４℃で５分間遠心分離し、上清を、ピペットを用いて先程のチューブに移した。得られた沈殿と上清はそれぞれ、－３０℃、４℃で保存した。 6) A part of the eluate was evaluated by SDS-PAGE to confirm the purity and yield of cPlg. The remaining eluate was concentrated by ammonium sulfate precipitation as follows. To 100 mL of the eluate, 39 g of ammonium sulfate, which was sufficiently fined in a mortar, was added at an addition rate of about 5 minutes while stirring the eluate on ice (60% saturation). After all ammonium sulfate has been added, the mixture is further stirred for 15 minutes, centrifuged at 16,000 × g for 30 minutes at 4 ° C., the supernatant is transferred to another tube by decantation, and the tube with the precipitate is again 16,000 × g. g Centrifugation at 4 ° C. for 5 minutes, and the supernatant was transferred to the previous tube using a pipette. The resulting precipitate and supernatant were stored at −30 ° C. and 4 ° C., respectively.
　７）凍結（－３０℃）したｃＰｌｇに、数ｍＬのＴＢＳ／Ｔ（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ　７．４（２５℃））／１００ｍＭ　ＮａＣｌ／０．０１％　Ｔｗｅｅｎ８０）を添加して沈殿を溶解し、この溶液を他の凍結ｃＰｌｇを含むチューブに順次移し、同様の操作を繰り返し、高濃度のｃＰｌｇ溶液を調製した。 7) Add several mL of TBS / T (50 mM Tris-HCl (pH 7.4 (25 ° C.)) / 100 mM NaCl / 0.01% Tween 80) to cPlg frozen (−30 ° C.) to dissolve the precipitate Then, this solution was sequentially transferred to another tube containing frozen cPlg, and the same operation was repeated to prepare a high-concentration cPlg solution.
　８）上述で調製したｃＰｌｇ溶液を透析膜セルロースチューブ２０／３２（三光純薬）に移し、２０倍量のＴＢＳ／Ｔに対して透析を行った。２時間ごとにＴＢＳ／Ｔを５回交換し、ｃＰｌｇの精製物とした。 8) The cPlg solution prepared above was transferred to a dialysis membrane cellulose tube 20/32 (Sanko Junyaku) and dialyzed against 20 times the amount of TBS / T. The TBS / T was changed 5 times every 2 hours to obtain a purified product of cPlg.
　Ｌｏｗｒｙ法によりタンパク質を定量した。１．５ｍＬのチューブ中で、８）で得られた透析後ｃＰｌｇ　２０μＬを４０μＬの０．１　Ｍの水酸化ナトリウムで希釈した。更に、２％（ｗ／ｖ）Ｎａ２ＣＯ３／０．１Ｍ水酸化ナトリウムと０．５％ＣｕＳＯ４・５Ｈ２Ｏ／１％クエン酸ナトリウムを５０：１の割合で混合した溶液３００μＬを先のチューブに添加し、十分混和し、１５分間室温で反応させた。反応後、３０μＬのフェノール試薬を添加し、十分混和し、さらに３０分間室温で反応させた。この反応液の２００μＬを９６ウェルプレートに移し、７５０ｎｍの吸光度を測定した。２０～１００μｇ／ｍＬのＢＳＡを用いて同様の操作を行って吸光度を測定することによって検量線を作成した。この検量線に基づいて試料のタンパク濃度を算出した。 Protein was quantified by the Lowry method. In a 1.5 mL tube, 20 μL of cPlg obtained in 8) after dialysis was diluted with 40 μL of 0.1 M sodium hydroxide. Furthermore, 300 μL 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 μL of a phenol reagent was added, mixed well, and further reacted at room temperature for 30 minutes. 200 μL of this reaction solution was transferred to a 96-well plate and the absorbance at 750 nm was measured. A calibration curve was prepared by measuring the absorbance by performing the same operation using 20 to 100 μg / mL of BSA. The protein concentration of the sample was calculated based on this calibration curve.
　ｃＰｌｇの精製は計１１回行った。透析後のタンパク定量結果から、４４８ｍｇのｃＰｌｇが得られたことが分かった。また、溶出液１μＬをドデシル硫酸ナトリウムポリアクリルアミド電気泳動（ＳＤＳ－ＰＡＧＥ、１０％、還元条件）に供した際に、ｃＰｌｇ以外のバンドが観察されなかったため、純度は９５％以上であると推定した。使用したイヌ血漿の総量は４０３０ｍＬであるため、収率は１１１ｍｇ／Ｌ血漿であった。 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 μL 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.
実施例４：ｃＰｌｇ開裂条件の検討
　ＴＢＳ／Ｔ／Ｃａ（５０ｍＭ　Ｔｒｉｓ－ＨＣｌ（ｐＨ７．４（２５℃））／１００ｍＭ　ＮａＣｌ／０．０１％Ｔｗｅｅｎ８０／１ｍＭ　ＣａＣｌ２）４６．４μＬを１．５ｍＬプラスチックチューブにとり、このチューブに１．６ｍｇ／ｍＬ　ｃＰｌｇ　２５．６μＬ（原液をＴＢＳ／Ｔ／Ｃａで希釈して調製）を添加し、均一に混和した。このチューブに１．５μＭ　ＢＬ－ＭＡ　８μＬ（原液をＴＢＳ／Ｔ／Ｃａで希釈して調製）を添加し、均一に混和した。このチューブを３７℃のウォーターバスで加温し、０．５、１．０、１．５、２．０、２．５時間目にチューブから１０μＬの反応液を取り、別の１．５ｍＬプラスチックチューブに移した。このチューブを直ぐに氷上に移し，６０ｍＭ　ＥＤＴＡ　２μＬを添加して酵素反応を停止させた。続いてこのチューブにＥｚＡｐｐｌｙ（AE-1430、ATTO）１２μＬを添加し、全量（２４μＬ）を電気泳動で分画した。 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 μL in 1.5 mL plastic In a tube, 25.6 μL of 1.6 mg / mL cPlg (prepared by diluting the stock solution with TBS / T / Ca) was added to this tube and mixed uniformly. 8 μL of 1.5 μM BL-MA (prepared by diluting the stock solution with TBS / T / Ca) was added to this tube and mixed uniformly. Heat the tube in a 37 ° C water bath, take 10 μL of the reaction solution from the tube at 0.5, 1.0, 1.5, 2.0, and 2.5 hours, and add another 1.5 mL plastic. Transfer to tube. The tube was immediately transferred to ice, and 2 μL of 60 mM EDTA was added to stop the enzyme reaction. Subsequently, 12 μL of EzApply (AE-1430, ATTO) was added to the tube, and the entire amount (24 μL) was fractionated by electrophoresis.
　その結果、１．０～２．０時間の試料について、ｃＢＬＡＳへの変換を示す２本のバンドが観察された。さらに加温すると、上記２本のバンドのうち、下のバンドの強度がさらに増加し、ｃＰｌｇ由来のｃＢＬＡＳ以外の部分に相当する約５０ｋＤａおよび約５３ｋＤａのバンドが時間経過とともに減少した。ｃＢＬＡＳの下のバンドの強度の増強がこれらの分解物に起因する可能性も考えられた。そこで、ｃＰｌｇの開裂に最適な反応時間は１～２時間とした。 As a result, two bands showing conversion to cBRAS were observed for the samples of 1.0 to 2.0 hours. When the temperature was further increased, the intensity of the lower band of the two bands further increased, and the bands of about 50 kDa and about 53 kDa corresponding to portions other than cPlg-derived cBLAS decreased with time. It was also possible that the enhancement of the intensity of the band under cBRAS was due to these degradation products. Therefore, the optimum reaction time for cPlg cleavage was set to 1 to 2 hours.
　３７℃の反応温度を使用した。ＢＬ－ＭＡの至適温度はより高い温度（５０～６０℃）であるが、基質タンパク質（ｃＰｌｇ）の変性を回避するためにこの温度を選択した。 A reaction temperature of 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).
実施例５：イヌＢＬアンジオスタチン（ｃＢＬＡＳ）の調製
　０．５ｍｇ／ｍＬのｃＰｌｇと１５０ｎＭのＢＬ－ＭＡをＴＢＳ／Ｔ／Ｃａ中、３７℃で２時間インキュベートした。その後、最終濃度が１０ｍＭとなるようにＥＤＴＡ－２Ｎａを添加し、反応を停止した。得られた反応液は直ちにＬｙｓ－Ｓｅｐｈａｒｏｓｅを用いた精製に供した。 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を精製するためのクロマトグラフィーは１ｍＬ／分で実施した。ｃＰｌｇ量２２４ｍｇに相当する上述の反応液を６０ｍＬのＬｙｓ－Ｓｅｐｈａｒｏｓｅ（カラムの５倍量の平衡化緩衝液で平衡しておいた）へアプライした。その後、カラムの１０倍量の平衡化緩衝液で洗浄した。溶出は、グラジエント溶出（カラムの５倍量；平衡化緩衝液中のＥＡＣＡ濃度を０ｍＭから８ｍＭへ増加させた）、８ｍＭ　ＥＡＣＡを含む平衡化緩衝液での溶出（カラムの３倍量）によって行った。溶出画分は１０ｍＬずつ分取した。 Chromatography for purifying cBRAS was performed at 1 mL / min. The above reaction solution corresponding to 224 mg of cPlg was applied to 60 mL of Lys-Sepharose (equilibrated with 5 times the amount of equilibration buffer in the column). Thereafter, the column was washed with an equilibration buffer 10 times the amount of the column. Elution is performed by gradient elution (5 times the amount of the column; the EACA concentration in the equilibration buffer was increased from 0 mM to 8 mM) and elution with the equilibration buffer containing 8 mM EACA (3 times the amount of the column). It was. The elution fraction was collected in 10 mL portions.
　ＳＤＳ－ＰＡＧＥにおいてｃＢＬＡＳのバンドが確認された画分を、超純水に対して、超純水を用いた以外は実施例２と同様にして透析し、凍結乾燥し、生理食塩水に溶解後、０．２μｍのディスクフィルターを用いてろ過滅菌した。試料の一部を、タンパク定量（実施例２に記載のＬｏｗｒｙ法による）およびＳＤＳ－ＰＡＧＥによる純度検定に供した。 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 μm 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.
　４４８ｍｇのｃＰｌｇから約１４０ｍｇのｃＢＬＡＳが得られた（３５．７　ｍｇ／ｍＬのｃＢＬＡＳ溶液約４ｍＬ）。また、溶出液１μＬをＳＤＳ－ＰＡＧＥ（１０％、還元条件）に供した際に、ｃＢＬＡＳ以外のバンドが観察されなかったため、純度は９５％以上であると推定した。ｃＢＬＡＳ溶液は、－８０℃で保存した。ｃＰｌｇおよびｃＢＬＡＳのＳＤＳ－ＰＡＧＥでの分子量（それぞれ約１００ｋＤａおよび約３７ｋＤａ）に基づいて、回収率は約８４％と算出された（１４０／（４４８×３７／１００））。 About 140 mg of cBLAS was obtained from 448 mg of cPlg (about 3 mL of 35.7 mg / mL cBRAS solution). Further, when 1 μL 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)).
実施例６：部分アミノ酸配列の決定
　実施例１で得られたｃＢＬＡＳをＳＤＳ－ＰＡＧＥに供した。２本のｃＢＬＡＳに相当する３６～３７ｋＤａのバンドが確認された（図１、左レーン）。ゲル中のタンパク質をＰＶＤＦ膜（フルオロトランス（登録商標）、日本ジェネティクス株式会社）に転写した後、目的のバンドを切り出し、４７６Ａ　Ｐｒｏｔｅｉｎ　Ｓｅｑｕｅｎｃｅｒ（Applied Biosystems）を使用してＮ末端アミノ酸配列を解析した。ｃＰｌｇのＢＬ－ＭＡ消化物（未精製）をＳＤＳ－ＰＡＧＥに供すると、さらにいくつかのバンドが見られた（図１、右レーン）。この中で最も優勢な約５３ｋＤａのバンドも同様に配列解析に供した。 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). After 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). . When 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.
　ｃＢＬＡＳの上下のバンドについて決定されたＮ末端配列はそれぞれＡｒｇ－Ｉｌｅ－Ｔｙｒ－Ｌｅｕ、およびＬｅｕ－Ｓｅｒ－Ｇｌｕ－ＸＸＸ－Ｌｙｓであった。この配列はそれぞれｃＰｌｇのＡｒｇ７８、Ｌｅｕ８１から開始する配列に一致した。またｃＰｌｇのＢＬ－ＭＡ消化物において優勢な約５３ｋＤａのバンドのＮ末端配列はＬｅｕ－Ａｓｐ－Ａｌａ－Ｐｒｏ－Ａｌａであった。この配列はｃＰｌｇのＬｅｕ３４３から開始する配列に一致した。以上の結果から、ｃＢＬＡＳのＣ末端アミノ酸配列はＴｙｒ３４２であると推測した。なお、実施例４において見られた約５０ｋＤａのバンドに対応するポリペプチドのＮ末端はＶａｌ３５６であると決定され、このポリペプチドは上記の約５３ｋＤａのバンドに対応するポリペプチドがさらに分解されて生じたことが示唆された。 The 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. In addition, 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. These results, C-terminal, the amino acid sequence of cBLAS was estimated to be Tyr 342. 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.
実施例７：ｃＢＬＡＳの活性の評価
　血管内皮増殖因子（ＶＥＧＦ）により誘導されるヒト内皮細胞の血管新生に対するイヌＢＬアンジオスタチン（ｃＢＬＡＳ）の作用について、血管新生キット（Angiogenesis kit；倉敷紡績株式会社）を用いて検討した。 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.
　試験群として「生理食塩水群」（水１０ｍＬ中塩化ナトリウム８５ｍｇ）、「ＶＥＧＦ群」（ＶＥＧＦ　１０ｎｇ／ｍＬ）、ならびに「ｃＢＬＡＳ群」（ＶＥＧＦ　１０ｎｇ／ｍＬ＋ｃＢＬＡＳ　１、１０、１００μｇ／ｍＬ、以降、「ｃＢＬＡＳ　１μｇ／ｍＬ群」、「ｃＢＬＡＳ　１０μｇ／ｍＬ群」、「ｃＢＬＡＳ　１００μｇ／ｍＬ群」と称する）の計５群を使用して実験を行った。血管新生キット到着日（１日目）に、培地を、ｃＢＬＡＳ群についてはＶＥＧＦを含まないキット添付の培地（ＶＥＧＦ（－）培地）に試験サンプル（所定の濃度のｃＢＬＡＳ）を添加した培地に交換し、生理食塩水群およびＶＥＧＦ群については試験サンプルと同量の生理食塩水を添加したＶＥＧＦ（－）培地に交換した。２、４、７、９日目に、培地を、ｃＢＬＡＳ群についてはＶＥＧＦを含むキット添付の培地（ＶＥＧＦ（＋）培地）に試験サンプル（所定の濃度のｃＢＬＡＳ）を添加した培地に交換し、生理食塩水群についてはＶＥＧＦ（－）培地に交換し、ＶＥＧＦ群についてはＶＥＧＦ（＋）培地に試験サンプルと同量の生理食塩水を添加した培地に交換した。１１日目に血管新生染色キットの説明書に従って抗ＣＤ３１（ＰＥＣＡＭ－１）抗体での染色を行った。画像解析システムＷｉｎＲＯＯＦ（Visual System）を使用して染色領域（染色された領域の全視野に対する割合）を測定し、得られた数値の統計学的解析をエクセル統計２００６（Ｖｅｒ．１．４１）（社会情報サービス（SSRI））を使用してＤｕｎｎｅｔｔの多重比較により行った。 As a test group, a “saline group” (sodium chloride 85 mg in 10 mL water), a “VEGF group” (VEGF 10 ng / mL), and a “cBRAS group” (VEGF 10 ng / mL + cBRAS 1, 10, 100 μg / mL, hereinafter “ Experiments were performed using a total of 5 groups, “cBLAS 1 μg / mL group”, “cBRAS 10 μg / mL group”, and “cBRAS 100 μg / mL group”. 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. On the second, fourth, seventh, and ninth days, 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, and 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. On day 11, staining with an anti-CD31 (PECAM-1) antibody was performed according to the instructions of the angiogenesis staining kit. Using the image analysis system WinROOF (Visual System), the stained area (the ratio of the stained area to the entire visual field) was measured, and the statistical analysis of the obtained numerical values was performed with Excel Statistics 2006 (Ver. 1.41) ( This was done by Dunnett's multiple comparison using Social Information Service (SSRI).
　生理食塩水群について測定された染色領域は２．７９±０．１３％（平均値±標準偏差）であった。ＶＥＧＦ群、ｃＢＬＡＳ　１μｇ／ｍＬ群、ｃＢＬＡＳ　１０μｇ／ｍＬ群、ｃＢＬＡＳ　１００μｇ／ｍＬ群について測定された染色領域はそれぞれ、３．０６±０．１１％、３．００±０．１１％、２．０８±０．１９％、１．２５±０．１１％であった（図２）。ＶＥＧＦ群と比較して、ｃＢＬＡＳ　１０μｇ／ｍＬ群、ｃＢＬＡＳ　１００μｇ／ｍＬ群では，内皮細胞の増殖が有意に阻害された（Ｐ＜０．０１）。なお、ＶＥＧＦ群では生理食塩水群と比較して有意な血管新生が観察されなかった。生理食塩水のみでは血管新生は起こらないと考えられるので、この結果は、上記条件ではＶＥＧＦによる血管新生効果が観察されなかったことを示す。従って、上記結果は、ｃＢＬＡＳが既存の内皮細胞の生存を阻害している可能性を示唆する。以上のように、本試験条件下においてｃＢＬＡＳはヒト内皮細胞の増殖を有意に阻害することが確認された。 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 μg / mL group, cBRAS 10 μg / mL group, and cBRAS 100 μg / 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). Compared with the VEGF group, endothelial cell proliferation was significantly inhibited in the cBRAS 10 μg / mL group and the cBRAS 100 μg / mL group (P <0.01). In the VEGF group, significant angiogenesis was not observed as compared with the physiological saline group. Since it is considered that angiogenesis does not occur only with physiological saline, this result indicates that the angiogenesis effect by VEGF was not observed under the above conditions. Thus, the above results suggest that cBRAS may inhibit the survival of existing endothelial cells. As described above, it was confirmed that cBLAS significantly inhibited the proliferation of human endothelial cells under the test conditions.
　本発明により、天然型糖鎖を有するイヌプラスミノーゲンからアンジオスタチン様ポリペプチドを製造するための方法、およびこの方法によって得られたイヌアンジオスタチン様ポリペプチドが提供される。 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.

Claims (6)

  1. 　イヌプラスミノーゲンをバシロライシンＭＡで消化することを含む、アンジオスタチン様ポリペプチドの製造方法。 A method for producing an angiostatin-like polypeptide, comprising digesting canine plasminogen with basilolysin MA.
  2. 　イヌプラスミノーゲンをバシロライシンＭＡで消化することによって得られる、アンジオスタチン様ポリペプチド。 Angiostatin-like polypeptide obtained by digesting canine plasminogen with basilolysin MA.
  3. 　Ｎ末端アミノ酸が配列番号１のアミノ酸配列の７８位のアルギニンまたは８１位のロイシンであり、還元条件下のドデシル硫酸ナトリウム－ポリアクリルアミドゲル電気泳動によって決定される分子量が３５～３８ｋＤａである、請求項２記載のアンジオスタチン様ポリペプチド。 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. 2. The angiostatin-like polypeptide according to 2.
  4. 　クリングル領域１～３を含み、ＮＴＰおよびクリングル領域４～５を含まない、請求項２または３記載のアンジオスタチン様ポリペプチド。 The angiostatin-like polypeptide according to claim 2 or 3, comprising kringle regions 1 to 3 and not including NTP and kringle regions 4 to 5.
  5. 　イヌプラスミノーゲンが天然型の糖鎖を有する、請求項２～４のいずれか１項記載のアンジオスタチン様ポリペプチド。 The angiostatin-like polypeptide according to any one of claims 2 to 4, wherein canine plasminogen has a natural sugar chain.
  6. 　請求項２～５のいずれか１項記載のアンジオスタチン様ポリペプチドを含む、血管新生阻害用組成物。 A composition for inhibiting angiogenesis, comprising the angiostatin-like polypeptide according to any one of claims 2 to 5.
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Citations (4)

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Publication number Priority date Publication date Assignee Title
JP2001151691A (en) * 1995-04-26 2001-06-05 Childrens Medical Center Corp Angiostatin fragment, angiostatin aggregate and method for using the same
JP2002355056A (en) * 2000-08-25 2002-12-10 Pfizer Prod Inc Composition and method for diagnosis and therapy of disorder accompanied by angiogenesis
WO2004065595A1 (en) * 2003-01-17 2004-08-05 Ttc Co., Ltd. Affinity trap reactor and single-step process for purifying angiostatin-like fragment from human plasma using the same
WO2005079835A1 (en) * 2004-02-24 2005-09-01 Ttc Co., Ltd. Anticancer agent containing bl angiostatin

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001151691A (en) * 1995-04-26 2001-06-05 Childrens Medical Center Corp Angiostatin fragment, angiostatin aggregate and method for using the same
JP2002355056A (en) * 2000-08-25 2002-12-10 Pfizer Prod Inc Composition and method for diagnosis and therapy of disorder accompanied by angiogenesis
WO2004065595A1 (en) * 2003-01-17 2004-08-05 Ttc Co., Ltd. Affinity trap reactor and single-step process for purifying angiostatin-like fragment from human plasma using the same
WO2005079835A1 (en) * 2004-02-24 2005-09-01 Ttc Co., Ltd. Anticancer agent containing bl angiostatin

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Title
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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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