JP4716741B2 - Sugar chain-deficient HGF α chain partial protein - Google Patents

Description

  The present invention relates to a variant of a partial protein of HGF (hepatocyte growth factor) for preventing or treating cancer or diseases caused by excessive angiogenesis. More specifically, the present invention relates to a partial protein of HGF modified by deleting a sugar chain.

  For the treatment of cancer, surgical therapy, chemotherapy, radiation therapy, or multidisciplinary therapy combining these, etc. are performed, but the establishment of a revolutionary anticancer method has not yet been established. The primary tumor can be removed by surgical therapy, but in cancers that are prone to metastasis, such as pancreatic cancer and lung cancer, even if the primary tumor is small, it is already visible to the eye. They often form invisible small metastatic cancers that are difficult to remove surgically. In addition, with chemotherapy and radiation therapy using anticancer agents, it is difficult to prevent recurrence of resistant cancer and metastasis of surviving cancer even if the primary tumor becomes temporarily small. Moreover, anticancer agents and radiotherapy kill not only cancer cells but also normal cells, resulting in severe side effects, thereby reducing the patient's quality of life and immunity. Thus, there is no method for effectively preventing cancer metastasis, and there is an urgent need for the development of a drug effective in preventing cancer metastasis.

Many studies have been conducted on the mechanism of cancer metastasis in the past. Most cancers occur in epithelial tissues, and cancer cells detach from the primary tumor, break the basement membrane that separates the epithelial tissues, infiltrate surrounding tissues, and invade blood vessels and lymph vessels to flow blood and lymph Is transferred to a distant tissue by invasion / growth into the tissue with angiogenesis from the blood vessels and lymphatic vessels, and metastasis is established.
In order to suppress cancer metastasis, it is considered that any one of these processes should be blocked. Examples of cancer metastasis inhibitors include substances that suppress adhesion to vascular endothelial cells at the metastasis destination (non-patented). Reference 1), angiogenesis inhibitors (see Non-Patent Document 2), substances that suppress invasion of cancer cells (see Patent Document 1), inhibitors of basement membrane degrading enzymes (Patent Document 2 and Non-Patent Documents) 3, etc.).

  Recently, tumor dormancy therapy has attracted attention regarding cancer treatment. Tumor dormancy therapy is to put cancer cells into a dormant state using an angiogenesis inhibitor. Angiogenesis inhibitors do not kill cancer cells directly, but inhibit angiogenesis, thereby breaking the nutrient and oxygen supply pathways necessary for cancer cell growth, inducing apoptosis, and resting cancer cells To. As angiogenesis inhibitors, angiostatin (see non-patent document 2), endostatin (see non-patent document 4) and the like are known.

Angiogenesis occurs by capillary growth of existing blood vessels. Angiogenesis is essential for many physiological processes such as embryogenesis, wound healing, and tissue or organ regeneration, while abnormal angiogenesis occurs at pathologies such as tumor growth and at metastatic tumor sites. The onset of tumor angiogenesis is known to be induced by vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), HGF (HGF; Hepatocyte growth factor) and the like.
In addition to such angiogenic activity, HGF has mitogenic activity, motogenic activity, and morphogen activity, and originally functions as a regenerative factor that supports the natural healing power of the living body. It is known that various actions of HGF are exerted by binding of HGF to c-Met / HGF receptor on target cells. It is known that c-Met / HGF receptor is often overexpressed in cancer cells (see Non-Patent Documents 5 to 7), and HGF induces invasion / metastasis to tumor cells. (Refer nonpatent literature 8.). A cancer cell induces invasion and metastasis while utilizing the action of HGF of a living body by interaction with stromal cells surrounding the cancer cell (see Non-Patent Documents 8 and 9). Therefore, if it is possible to inhibit HGF from binding to the c-Met / HGF receptor of tumor cells, it is considered that tumor invasion and metastasis can be suppressed.

  NK4 is a protein composed of the N-terminal hairpin domain of the α chain of HGF and four kringle domains. NK4 binds to the c-Met / HGF receptor and acts as an antagonist of HGF (see Non-Patent Documents 10 and 11). NK4 is known to suppress tumor invasion and metastasis by HGF antagonist activity. Furthermore, NK4 is known to suppress not only HGF but also angiogenesis by VEGF and bFGF by a mechanism different from HGF antagonist activity (see Non-Patent Document 12). Thus, NK4 is a bifunctional molecule having HGF antagonist activity and angiogenesis inhibitory action at the same time, and NK4 is expected to be used as a new anticancer agent.

  Moreover, NK4 is based on angiogenesis inhibitory action, and various diseases caused by abnormal growth of blood vessels such as rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, macular degeneration in the elderly, excessive scar formation during wound healing. Use as a preventive or therapeutic agent is also expected. As mentioned above, angiogenesis is essential for maintaining tissue metabolism and maintaining the functional homeostasis of the living body under normal conditions, but abnormal angiogenesis is associated with various diseases including inflammatory diseases. It is known that For example, diseases such as proliferative diabetes, psoriasis vulgaris, rheumatoid arthritis, diabetic retinopathy, macular degeneration of the elderly, excessive scar formation during wound healing, and metastasis or recurrence of solid tumors are blood vessels, especially peripheral capillaries It has been reported that this is caused by abnormal growth of the cells (see non-patent documents 13 and 14). NK4 having an angiogenesis inhibitory action is promising as a preventive or therapeutic agent for these diseases.

  Furthermore, NK4 can be expected to be used as a preventive or therapeutic agent for infection of Listeria and malaria. When Listeria monocytogenes infects humans, c-Met / HGF receptor is used as a scaffold for infection (see Non-patent document 15), and in infection with malaria parasite, c-Met / HGF receptor is activated by HGF. It is known that it is essential for the initial mechanism of infection (see Non-Patent Document 16). Therefore, it is considered that NK4, which is an HGF antagonist, exhibits preventive and therapeutic effects on these infectious diseases.

  As described above, in order to use the NK4 protein that exhibits an effect in the prevention and treatment of various diseases as a pharmaceutical preparation, it is necessary to produce it in large quantities using cells by genetic engineering techniques. Conventionally, it is known that NK4 can be produced using animal cells such as Chinese hamster ovary (CHO) cells (see Patent Document 3). However, in general, a method for producing proteins using animal cells such as CHO cells is known. The cost is high, and it can be thought that this will lead to an increase in drug prices.

  As a method for inexpensively producing a protein recombinantly, a method is known in which a target gene is introduced into a prokaryote such as Escherichia coli and expressed (see Non-Patent Document 17). However, there is a problem that sugar chains are not added to recombinant proteins produced by prokaryotes such as E. coli. This is because prokaryotes such as Escherichia coli do not have endoplasmic reticulum and Golgi bodies, which are sugar chain biosynthesis fields.

  Unlike the case of DNA or protein biosynthesis, the addition of a sugar chain to a protein and the modification of a sugar chain in an animal cell are post-translational modifications that do not depend on a template. This post-translational modification is carried out through a complex mechanism mediated by a number of sugar chain biosynthesis-related enzymes located in the intracellular organelles called the endoplasmic reticulum and Golgi apparatus. In other words, monosaccharides are sequentially cut and added according to a complex biosynthetic pathway based on the cooperation of specific monosaccharides and enzymes specific to the binding mode (sugar hydrolase and glycosyltransferase). The sugar chain is elongated so as to obtain the sugar chain structure (see Non-Patent Document 18). It is known that the sugar chain added to the protein in this way is deeply related to the overall life phenomenon of higher organisms (see Non-Patent Documents 19 and 20).

  It is known that more than half of the proteins in the human body exist as glycoproteins with added sugar chains (see Non-Patent Document 21). If there is no state, there is a concern that the activity may be lost. For example, it is known that erythropotin known as an erythropoietic hormone loses its medicinal properties when a sugar chain is removed (see Non-Patent Document 22).

  Yeast is known as a cell capable of producing a protein at a low cost and having a glycosylation ability (see Non-Patent Documents 23 to 25). Since yeast is a eukaryote and has an endoplasmic reticulum and a Golgi apparatus, it has a sugar chain biosynthesis mechanism. However, since the sugar chain biosynthesis mechanism of yeast is greatly different from that of animal cells, when a protein having a glycosylation site is produced in yeast, a yeast-type sugar chain is added. The sugar chain structure of yeast is greatly different from the sugar chain structure of humans and other mammals (see Non-Patent Document 26), and such recombinant proteins are antigenic to humans and other mammals. It cannot be used as a human or animal medicine.

Insect cells are also hosts having sugar chain-adding ability and can produce proteins relatively inexpensively, but the sugar chain structure of insect cells is different from that of human type (Non-patent Document 27). ), And may be antigenic to humans and other mammals.
Therefore, remove the sugar chain from the protein produced using yeast or insect cells, or introduce a gene into which the mutation is introduced into the glycosylation site in the protein molecule. Thus, it is conceivable to produce a protein without glycosylation. However, as described above, there is a concern that if a protein originally present in a form to which a sugar chain is added is made free of a sugar chain, it loses its activity.

NK4 is a fragment of HGFα chain and contains 3 sugar chain addition sites (see Non-Patent Documents 28 and 29) of HGFα chain. Is NK4 active when sugar chain is removed from NK4? It's not known at all.
JP-A-3-31214 JP-A-5-194414 JP 2003-250549 A Iwamoto. Y, et al., Science, 1987, p. 1132-1134 Cao Y, et al., The Journal of Clinical Investigation, 1988, Vol. 101, p. 1055-1063 Irimura. T. et al., Biochemistry, 1989, 25, p. 5322-5328 Blezinger. P, et al., Nature biotechnology. 1999, Vol. 17, p. 343-348 Di. Renzo et al., Oncogene, 1991, Vol. 6, p. 1997-2003 Di. Renzo et al., Cancer research, 1995, vol. 55, p. 1129-1138 Nakajima. M et al., Cancer, 1999, vol. 85, p. 1894-1902 (1999) Jiang. WG, et al., Critical reviews in oncology / hematology, 1999, Vol. 29, p. 209-248 Nakamura. T et al., Cancer research, 1997, vol. 57, p. 3305-3313 Date. K. et al., FEBS letters, 1997, vol. 420, p. 1-6 Date. K. et al., Oncogene, 1998, Vol. 17, p. 3045-3054 Kuba. K. et al., Cancer research, 2000, 60, p. 6737-6743 Polverini PJ., Critical reviews in oral biology and medicine, 1995, vol. 6 (3), p. 230-247 Forkman J., Nature medicine, 1995, Volume 1 (No. 1), p. 27-31 Shen, Y. et al., Cell, 2000, 103, p. 501-510 Carrollo, M., et al., Nature Med, 2003, Vol. 9, p. 1363-1369 Swarts, JR, Current opinion in biotechnology, 2001, Vol. 12, p. 195-201 Kornfeld, R. and one other, Annual review of biochemistry, 1985, vol. 54, p. 631-664 Kobata, A., European journal of biochemistry, 1992, 209, p. 483-501 Barki, A, Glycobiology, 1993, Volume 3, p. 97-130 Goochee, C.F. et al., Biotechnology, 1991, Vol. 9, p. 1347-1355 Takeuchi, M. and 1 other, Glycobiology, 1991, Volume 1, p. 337-346 Wiseman A., Endeavor, 1996, Volume 20, p. 130-132 Russell C. et al., Australian journal of biotechnology., 1991, Vol. 5, p. 48-55 Bookholz, RG and one other person, Biotechnology, 1991, Vol. 9, p. 1067-1072 Gemmill, TR, and one other person, Biochimica et biophysica acta, 1999, 1426, p. 227-237 Altmann, F. et al., Glycoconjugate journal, 1999, Vol. 16, p. 109-123 Hara, H., et al., Journal of biochemistry, 1993, 114, p. 76-82 Shimizu, N. et al., Biochemical and biophysical research communications, 1992, 189, p. 1329-1335

  An object of the present invention is to provide a modified product in which a sugar chain of a partial protein of HGF, particularly HGF α chain and α chain intramolecular fragment is deleted, and a method for producing the same.

  As a result of intensive studies on the sugar chain function of the NK4 protein in order to solve the above problems, the present inventors have found that the function of NK4 is maintained even if the sugar chain of the NK4 protein is removed. It was completely unexpected that the NK4 protein, which is a bifunctional molecule having both an HGF antagonist activity and an angiogenesis inhibitory action, retains these functions even when the sugar chain is removed. Based on the above findings, the present inventors have further studied and have completed the present invention.

That is, the present invention
(1) A protein lacking at least one sugar chain of the sugar chain addition site of HGF α chain, and is any one of the following proteins (a) to (c), wherein the protein is an N-terminal hairpin domain followed by 4 A partial protein of sugar chain-deficient HGF, which has two kringle domains, has an antagonist activity against the action of HGF via c-Met / HGF receptor, and has an anti-angiogenic action;
(A) a protein having an amino acid sequence represented by positions 32 to 494 of SEQ ID NO: 1;
(B) a protein having an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, added or inserted in positions 32 to 494 of SEQ ID NO: 1; or (c) position 32 of SEQ ID NO: 1 To a protein having at least 80% homology with the amino acid at position 494,
(2) the partial protein of sugar chain-deficient HGF according to the above (1), wherein an amino acid is substituted so that no sugar chain is added in the amino acid sequence of at least one sugar chain addition site of the protein;
(3) The partial protein of sugar chain-deficient HGF according to the above (2), wherein the amino acid substitution is at least one of the following (a) to (d):
(A) Asn-X-Ser or Asn-X-Thr (wherein X represents an amino acid other than Pro), among the consensus sequences for N-linked glycosylation, Asn of at least one consensus sequence Is replaced with another amino acid;
(B) Among the consensus sequences for N-linked glycosylation wherein the amino acid sequence is Asn-X-Ser or Asn-X-Thr (X represents an amino acid other than Pro), the Ser of one consensus sequence or Thr, or Ser or / and Thr of two or more consensus sequences are replaced with other amino acids;
(C) X of at least one consensus sequence among N-linked glycosylation consensus sequences of which the amino acid sequence is Asn-X-Ser or Asn-X-Thr (X represents an amino acid other than Pro) Is substituted with Pro; or (d) at least one of Ser or Thr undergoing O-linked glycosylation is substituted with another amino acid,
(4) The partial protein of sugar chain-deficient HGF according to the above (2), wherein the amino acid substitution is at least one of the following (a) to (d):
(A) In the amino acid sequence represented by SEQ ID NO: 1, the amino acid at position 294 or / and 296 is replaced with another amino acid, or / and the amino acid at position 295 is replaced with Pro to position 294. No sugar chain added;
(B) in the amino acid sequence represented by SEQ ID NO: 1, the amino acid at position 402 and / or 404 is replaced with another amino acid, or / and the amino acid at position 403 is replaced with Pro to position 402. A sugar chain is not added; or (c) a sugar chain is not added at position 476 by substitution of the amino acid at position 476 of the amino acid sequence shown in SEQ ID NO: 1 with another amino acid,
(5) The sugar chain-deficient type according to any one of (1) to (4) above, wherein the amino acids at positions 162 to 166 of the amino acid sequence represented by SEQ ID NO: 1 are deleted. A partial protein of HGF, and (6) any one of (1) to (5) above, wherein the amino acids at positions 479 to 494 of the amino acid sequence represented by SEQ ID NO: 1 are deleted. A partial protein of the sugar chain-deficient HGF as described,
About.

The present invention also provides:
(7) DNA comprising a base sequence encoding the partial protein of sugar chain-deficient HGF according to any one of (1) to (6) above,
(8) a vector incorporating the DNA according to (7) above,
(9) The vector described in (8) above is introduced into a cell, the cell is cultured, and a sugar chain-deficient HGF partial protein is produced in the cell or in a culture solution of the cell. The method for producing a partial protein of sugar chain-deficient HGF according to any one of the above (1) to (6), wherein a partial protein of sugar chain-deficient HGF is collected and purified from a culture medium of cells,
(10) The method for producing a partial protein of sugar chain-deficient HGF according to (9), wherein the cell is a eukaryotic cell,
(11) The method for producing a sugar chain-deficient HGF partial protein according to the above (10), wherein the eukaryotic cell is a yeast or insect cell,
(12) The vector described in (8) above is introduced into an insect individual, a sugar chain-deficient HGF partial protein is produced in the insect individual, and the sugar chain-deficient HGF partial protein is collected and purified from the insect individual. A method for producing a partial protein of a sugar chain-deficient HGF according to any one of the above (1) to (6),
(13) The sugar chain-containing HGF partial protein is treated with an enzyme to remove all or part of the sugar chain, and the sugar chain-deficient HGF partial protein is collected and purified from the enzyme reaction solution. A method for producing a partial protein of sugar chain-deficient HGF according to any one of (1) to (6),
(14) A vector in which a DNA having a base sequence encoding a partial protein of HGF having a sugar chain or a vector according to (8) above is introduced into a cell having no ability to add a sugar chain, the cell is cultured, Producing a sugar chain-deficient HGF partial protein in the cell or in the culture medium of the cell, and collecting and purifying the sugar chain-deficient HGF partial protein from the cell or in the cell culture medium. A method for producing a partial protein of sugar chain-deficient HGF according to any one of (1) to (6),
(15) A sugar chain-deficient HGF partial protein is synthesized by a cell-free protein synthesis system using a DNA having a base sequence encoding an HGF partial protein having a sugar chain or the DNA of (7) above as a template, A method for producing a sugar chain-deficient HGF partial protein according to any one of (1) to (6) above, wherein a sugar chain-deficient HGF partial protein is collected from the synthesis reaction solution and purified.
(16) HGF lacking a sugar chain at all or at least one of the glycosylation sites of HGF is treated with protease or limitedly decomposed by chemical treatment, and the glycoprotein-deficient HGF partial protein is treated from the treatment solution. The method for producing a partial protein of sugar chain-deficient HGF according to any one of (1) to (6) above, and (17) wherein the protease is elastase The production method according to (16) above,
About.

The present invention also provides:
(18) A medicament comprising the sugar chain-deficient HGF partial protein according to any one of (1) to (6) as an active ingredient,
(19) The medicament according to the above (18), which is an angiogenesis inhibitor,
(20) The medicament according to (18) above, which is an antagonist for the action of HGF via the c-Met / HGF receptor,
(21) The medicament according to any one of (18) to (20) above, which is a tumor invasion, growth or metastasis inhibitor.
(22) The medicament according to any one of (18) to (20), which is an apoptosis inducer,
(23) The medicament according to (20) above, which is an agent for preventing or / and treating infectious diseases,
(24) The medicament according to (23) above, which is an infection caused by malaria or Listeria protozoa, and (25) a gene therapy drug comprising the DNA according to (7) above,
About.
Furthermore, the present invention uses a sugar chain-deficient HGF partial protein for the production of a pharmaceutical preparation for angiogenesis suppression and the like, and administers a medicament containing the sugar chain-deficient HGF partial protein as an active ingredient to a patient. The present invention relates to a method for preventing and treating angiogenesis. The present invention also includes the use of a DNA encoding a sugar chain-deficient HGF partial protein for the production of a gene therapy drug for angiogenesis suppression, etc., and a DNA encoding a sugar chain-deficient HGF partial protein. The present invention relates to a method for preventing or treating angiogenesis and the like by administering a gene therapy drug to a patient.

Since the sugar chain-deficient HGF partial protein of the present invention (hereinafter abbreviated as a sugar chain-deficient HGF partial protein) can be produced in yeast or insect cells, it is a sugar chain-deficient HGF partial protein. Can be produced at low cost. In particular, it is significant that sugar chain-deficient NK4 can be produced at low cost.
Since the sugar chain-deficient HGF partial protein (eg, sugar chain-deficient NK4) of the present invention has the same activity as the HGF partial protein (eg, NK4) having a sugar chain in HGF antagonist activity and angiogenesis-inhibiting action. It can be a substitute for a partial protein of HGF having a sugar chain (for example, NK4). Therefore, the sugar chain-deficient HGF partial protein of the present invention can be used for tumor invasion suppression, growth suppression, metastasis suppression, apoptosis induction or / and angiogenesis suppression. In addition, a medicament containing DNA encoding the sugar chain-deficient HGF partial protein of the present invention can be used as a gene therapy drug.

Hereinafter, the present invention will be described in detail.
In the present invention, “partial protein of sugar chain-deficient HGF” refers to a partial protein of HGF from which all or at least one sugar chain addition site of HGF α chain, preferably all sugar chains are deleted. The “partial protein of HGF” has an amino acid sequence represented by positions 32 to 494 of SEQ ID NO: 1, for example, in humans and mammals (eg, dogs, cats, cows, horses, pigs, sheep, etc.) An HGF α chain protein having an N-terminal hairpin domain followed by four kringle domain structures, and a protein of an intramolecular fragment of HGF having at least the N-terminal hairpin domain followed by four kringle domains, preferably an intramolecular HGF α chain A protein fragment. The protein of the intramolecular fragment of HGF α chain is 1 to 30, preferably 1 to 25, more preferably 1 to 21 amino acids in the HGF α chain (for example, positions 32 to 494 of SEQ ID NO: 1). A protein in which is deleted. Specifically, for example, the amino acid sequence represented by SEQ ID NO: 1 has been deleted from positions 162 to 166, and a 5-amino acid deletion type HGFα chain represented by positions 32 to 489 of SEQ ID NO: 2 is present. Can be mentioned. Further, for example, NK4 represented by the amino acid sequence from position 32 to position 478 of SEQ ID NO: 1 (the amino acid sequence of positions 479 to 494 of the amino acid sequence represented by SEQ ID NO: 1 has been deleted), and For example, NK4 represented by the amino acid sequence from position 32 to position 473 of SEQ ID NO: 2 (the amino acid sequence of position 474 to position 489 of the amino acid sequence represented by SEQ ID NO: 2 has been deleted) and the like. It is done.
In the sugar chain-deficient HGF partial protein of the present invention, 1-30, preferably 1-25 amino acids of the amino acid sequence of the HGF α chain (for example, positions 32 to 494 of SEQ ID NO: 1) A protein having a deleted, substituted, added or inserted amino acid sequence refers to a protein in which an amino acid has been deleted, substituted, added or inserted by genetic engineering techniques or / and mutation. Specifically, the protein of the above-described intramolecular fragment of the HGF α chain, the protein having an amino acid added to the N-terminal side and / or C-terminal side of the HGF α chain, or / and the sugar chain of the HGF α chain by a genetic engineering technique A protein in which the amino acid of the amino acid sequence of the addition site has been deleted, substituted, added or inserted so that a sugar chain is not added. In addition, a protein in which amino acids have been deleted, substituted, added or inserted by a well-known technical method such as specific mutagenesis, and the number of naturally occurring amino acids (1 to several) are missing. Proteins that have been deleted, substituted, added or inserted are also included.
The partial protein of sugar chain-deficient HGF also includes proteins having homology of 80% or more, preferably 85% or more, more preferably 90% or more with the amino acid sequence of HGF α chain. In the present invention, “homology” means the degree of coincidence of amino acid residues constituting each sequence by comparing the primary structures of proteins.
Further, those having a modified amino acid residue pyroglutamic acid as the N-terminal amino acid of the protein are also included in the sugar chain-deficient HGF partial protein of the present invention.
Moreover, the partial protein of the sugar chain-deficient HGF of the present invention is a protein having an antagonistic activity against the action of HGF via the c-Met / HGF receptor and an angiogenesis-inhibiting action. Examples of the action of HGF via c-Met / HGF receptor include tyrosine phosphorylation of c-Met / HGF receptor, motogenic activity, mitogenic activity, and morphogenic activity. (Matsumoto, K. and Nakamura, T., Biochem. Biophys. Res. Commun., 1997, 239, p. 639-644). The angiogenesis inhibitory action is not limited to the mechanism as long as it is an action that inhibits the neovascularization, but preferably means an action that inhibits angiogenesis involving HGF, FGF, and VEGF.

  Deletion of the sugar chain of the HGF partial protein is preferably mutated to the base sequence so that the amino acid of the amino acid sequence is deleted, substituted or added so that the sugar chain is not added at least at one of the sugar chain addition sites of the HGF α chain. However, it may also be caused by treating a partial protein of HGF having a sugar chain with an enzyme.

As a method for introducing a mutation into a gene of a partial protein of HGF so that the sugar chain is not added at at least one sugar chain addition site of the HGF α chain, a base sequence corresponding to the amino acid sequence of the sugar chain addition site to be deleted It is better to introduce a mutation into When sugar chains are added to proteins, sugar chains include N-linked sugar chains and O-linked sugar chains, and the following mutations are introduced for each.
A consensus sequence [Asn-X-Ser or Asn-X-Thr (X represents an amino acid other than proline)] is known for the N-linked sugar chain. When a consensus sequence is present, a sugar chain binds to Asn (Kobata, A., Eur. J. Biochem., 1992, 209, p. 483-501). Therefore, Asn in the consensus sequence is converted to another amino acid (eg, Gln), or the nucleotide sequence is mutated to convert Ser or Thr to another amino acid (eg, Gly, Ala, etc.). By introducing, the N-linked sugar chain at the corresponding site can be deleted. In this case, the amino acid used for the conversion is preferably appropriately selected from amino acids that do not form a new consensus sequence with the amino acid sequences before and after the consensus sequence. Further, a mutation may be introduced into the base sequence so that proline is introduced into the X site in the consensus sequence.
There is no consensus sequence for O-linked glycans, but in O-linked glycans, sugar chains are added to the hydroxyl groups of Ser or Thr, so that Ser or Thr at the site that undergoes O-linked glycan addition. The sugar chain at the corresponding site can be deleted by introducing a mutation into the base sequence so that is converted to another amino acid (eg, Gly). In this case, as the amino acid used for the conversion, it is preferable to appropriately select an amino acid that does not form the consensus sequence with the amino acid sequence before and after the amino acid.

  The sugar chain deficiency site of the sugar chain-deficient HGF partial protein corresponds to the sugar chain addition site of the HGF partial protein, and for example, human HGF having the amino acid sequence shown in positions 32 to 478 of SEQ ID NO: 1. In NK4, which is a partial protein of the α chain intramolecular fragment, Asn (N-linked type) at position 294, Asn (N-linked type) at position 402, Thr at position 476 (O -Bond type). In addition, NK4, which is an intramolecular fragment of the α chain of 5-amino acid-deleted human HGF having the amino acid sequence shown in positions 32 to 473 of SEQ ID NO: 2 (hereinafter abbreviated as 5-amino acid deleted NK4) ) Are Asn (N-linked) at position 289, Asn (N-linked) at position 397, and Thr (O-linked) at position 471 in SEQ ID NO: 2. The consensus sequence of NK4 is present at positions 294 to 296 and positions 402 to 404 of the amino acid sequence represented by SEQ ID NO: 1, and is represented by SEQ ID NO: 2 for the 5-amino acid deletion type NK4. In the amino acid sequence from position 289 to position 291 and position 397 to position 399.

As a method for introducing a mutation into a base sequence, for example, when introducing a mutation into DNA encoding NK4, a mutation primer corresponding to the portion into which the mutation is to be introduced is synthesized, and a known technique such as the Kunkel method is used. Can do. In addition, mutation can be introduced more easily by using a commercially available mutation introduction kit or the like. Alternatively, from the known nucleotide sequence information of NK4, the nucleotide sequence of the portion where mutation is to be introduced can be modified, and DNA can be produced by chemical synthesis using a conventionally known method. Examples of the chemical synthesis method include a method of chemically synthesizing with a DNA synthesizer such as a DNA synthesizer model 392 (manufactured by Perkin Elmer Co., Ltd.) using the phosphoramidite method.
A DNA encoding a partial protein of the HGF α chain, such as NK4, can be obtained, for example, according to the method described in JP-A-2003-250549 or the method described therein. For example, a primer based on a known HGF base sequence is prepared, and DNA is amplified by PCR using a cDNA synthesized from HGF mRNA contained in human tissues or cells or HGF cDNA selected from a cDNA library as a template. And DNA encoding NK4 can be obtained. In addition, cDNA can be amplified from mRNA contained in human tissues or cells by RT-PCR. It can also be obtained by the above chemical synthesis.
A vector suitable for expression of a partial protein of a sugar chain-deficient HGF by excising the DNA from a recombinant vector such as a plasmid or phage containing a DNA encoding the amino acid sequence of the partial protein of the sugar chain-deficient HGF with a restriction enzyme A recombinant expression vector can be prepared by recombining with a restriction enzyme and DNA ligase downstream of the promoter. More specifically, in the downstream direction of transcription, in order, if necessary, (1) a promoter, (2) a ribosome binding site, (3) a start codon, (4) a base encoding a partial protein of the sugar chain-deficient HGF of the present invention. Constructed to contain DNA containing sequence, (5) stop codon, (6) terminator.

  In addition, by incorporating a secretory signal sequence before the base sequence encoding the partial protein of sugar chain-deficient HGF, the partial protein of sugar chain-deficient HGF can be secreted outside the cell. The signal sequence is preferably one that is recognized and processed by the host cell. For example, when the host belongs to the genus Escherichia, the signal sequence of PhoA, the signal sequence of OmpA, etc., and when the host is of the genus Bacillus, the signal sequence of α-amylase, the signal sequence of subtilisin, etc. are yeast. In this case, the signal sequence of MFα, the signal sequence of SUC2, and the like can be used. When the host is a mammalian cell, the signal sequence of HGF, the signal sequence of insulin, the signal sequence of α-interferon, etc. can be used. In the partial protein of human HGF, it is particularly preferable to use the signal sequence of human HGF. Specifically, for example, the base sequence encoding the amino acid sequence shown in the first to 31st positions of SEQ ID NO: 1 corresponds to the signal sequence.

The DNA includes not only a DNA comprising a base sequence encoding a partial protein sequence of a sugar chain-deficient HGF in which a mutation is introduced into the base sequence of the sugar chain addition site, but also (a) the sugar chain-deficient HGF. DNA encoding a protein having a base sequence in which one or several bases of a base sequence encoding a partial protein sequence have been deleted, substituted, added or inserted, and having an HGF antagonist activity and angiogenesis inhibitory action (B) hybridizing under stringent conditions with DNA having a base sequence complementary to DNA having a base sequence encoding a partial protein sequence of the sugar chain-deficient HGF, and inhibiting HGF antagonist activity and angiogenesis DNA comprising a base sequence encoding a protein having an action, or (c) a partial protein sequence of the sugar chain-deficient HGF Having DNA at least 80% or more homology with a nucleotide sequence encoding, and is intended to encompass DNA consisting of a nucleotide sequence encoding a protein having an HGF antagonist activity and neovascularization inhibitory action.
Regarding the above base sequence, when “one or several bases are deleted, substituted, added or inserted”, it is a number that can be naturally generated by a well-known technical method such as site-directed mutagenesis or the like. It means that (1 to several) bases have been deleted, substituted, added or inserted.
DNA that can hybridize under stringent conditions means DNA obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like using the above DNA as a probe.
The stringent conditions include, for example, an SSC solution having a salt concentration of about 0.1 to 2 times (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride, 15 mM sodium citrate), and a temperature of about. Hybridization conditions at about 65 ° C.
DNA having homology is DNA having at least about 80% homology, preferably DNA having about 85% homology, more preferably about 90% or more homology under highly stringent conditions. Refers to DNA having The high stringent conditions are, for example, conditions in which the sodium concentration is about 19 to 40 mM, preferably about 19 to 20 mM, and the temperature is about 50 to 70 ° C., preferably about 60 to 65 ° C. Say. In particular, the most preferable conditions are when the sodium concentration is about 19 mM and the temperature is about 65 ° C.

  As vectors that can be used in the present invention, plasmids such as pBR322, pUC18, and pUC19 (Toyobo) are used when Escherichia coli is the host, and plasmids such as pUB110 (Sigma) are used when Bacillus subtilis is the host. When using as a host, plasmids such as pYES2 (Invitrogen) and pRB15 (ATCC37062) can be used. As expression vectors for animal cells, pCAGGS and pCXN2 (Niwa, H., Yamamura, K. and Miyazaki, J., Gene, 1991, Vol. 108, p.193-200, JP 03-168087), pcDL-SRα (Takebe, Y. et al., Mol. Cell. Biol. 1988, Vol. 8, p.466-472) and the like. In addition, bacteriophage λgt10, λgt11 (Stratagene), virus SV40 (BRL), BPV (ATCC VR-703), vectors derived from retrovirus genes, etc. can be listed, but these vectors can be replicated and amplified in the host. If there is no particular limitation.

  The promoter and terminator are not particularly limited as long as they correspond to the host used for the expression of the base sequence encoding the target partial protein of sugar chain-deficient HGF. Examples of the promoter include trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, etc. when the host is Escherichia coli, and when the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter etc. are mentioned. When animal cells are used as hosts, Rous sarcoma virus (virus RSV), MPSV, poliovirus, fowl head virus, adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), hepatitis B virus, simian Examples include promoters obtained from virus genomes such as virus 40 (SV40) and vaccinia virus, merothionein promoter, heat shock promoter, and the like. In addition, when a higher mammalian host is used, an enhancer is preferably introduced into the vector. Introducing an enhancer increases transcription. Enhancers include SV40 enhancer, cytomegalovirus early promoter / enhancer, polyomer enhancer, adenovirus enhancer, and the like. As the terminator, trp terminator, 1pp terminator, etc. when the host is Escherichia coli, amyF terminator, etc. when the host is Bacillus subtilis, CYC1 terminator, etc. when the host is yeast, and SV40 when the host is an animal cell. A terminator, HSV1TK terminator, etc. can be illustrated. These promoters and terminators are appropriately combined depending on the host to be used.

  The cell that can be used as a host is not particularly limited, and examples thereof include eukaryotic cells such as animals, plants, insects, and eukaryotic microorganisms, and prokaryotic cells such as prokaryotic microorganisms. These cells may form an individual, and an animal individual, a plant individual, or an insect individual may be used as a host. As eukaryotic cells, both adherent cells and suspension cells can be used. For example, eukaryotic cells that produce and accumulate HGF partial proteins in the cells may be used, or eukaryotic cells that secrete and produce HGF partial proteins. It may be a cell. Examples of animal cells include CHO cells (Chinese hamster ovary cells), COS cells, BHK cells, mouse C127 cells, and Hela cells. Examples of plant cells include rice, tobacco, and Arabidopsis. Examples of insect cells include cells such as Sf9 and Sf21. Insect individuals can include, for example, silkworms. Examples of eukaryotic microorganisms include yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida bodiniii, Pichia pastoris, and Aspergillus spp., Trichoderma spp. Examples of prokaryotic microorganisms include Escherichia coli and Bacillus subtilis. Among these, prokaryotic cells do not have the ability to add glycosylation. Therefore, even if an expression vector encoding a glycosylated HGF partial protein is introduced. Good. Yeast, insect cells or insect individuals are preferred.

  Sugar chain-deficient HGF partial protein expression vectors include competent cell method (J. Mol. Biol., 1970, Vol. 53, p. 154), protoplast method (Proc. Natl. Acad. Sci. USA, 1978). Year 75, p. 1929), calcium phosphate method (Science, 1983, Vol. 221, p. 551), DEAE dextran method (Science, 1982, Vol. 215, p. 166), electric pulse method ( Proc. Natl. Acad. Sci. USA, 1984, Vol. 81, p. 7161), in vitro packaging method (Proc. Nat. Acad. Sci. USA, 1975, Vol. 72, p. 581), virus Vector method (Cell, 1984, 37, p. 1053) or microin Ekushon method (Exp.Cell.Res., 1984 years, 153 vol, P.347) is introduced into a host by etc., transformants are prepared.

  The obtained transformant is cultured in an appropriate medium according to the host in order to produce the target sugar chain-deficient HGF partial protein. The medium contains a carbon source, a nitrogen source, inorganic substances, vitamins, serum, drugs and the like necessary for the growth of the transformant. Examples of the medium include LB medium (Nissui Pharmaceutical), M9 medium (J. Exp. Mol. Genet., Cold Spring Laboratory, New York, 1972, p. 431) when the transformant host is E. coli. In the case where the host is yeast, YEPD medium (Genetic Engineering, vol. 1, Plenum Press, New York, 1979, p. 117) can be used. When the host is an animal cell, examples include MEM medium, DMEM medium, RPMI 1640 medium (Nissui Pharmaceutical) containing 20% or less fetal bovine serum. The transformant is usually cultured in the range of 20 ° C to 45 ° C and pH of 5 to 8, and aeration and agitation are performed as necessary. When the host is an adherent animal cell or the like, a carrier such as glass beads, collagen beads, or acetylcellulose follow fiber is used. It can be carried out if the transformant grows even under other medium composition or culture conditions, and is not limited thereto.

  In culture, when a protease inhibitor is added to the culture solution, the degradation of the sugar chain-deficient HGF partial protein can be suppressed, leading to an improvement in the productivity of the sugar chain-deficient HGF partial protein. Examples of protease inhibitors to be added include benzamidine, aprotinin, leupeptin, antipain, chymostatin, pepstatin A, phenylmethanesulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), and the like.

  The sugar chain-deficient HGF partial protein thus produced in the culture supernatant of the transformant or in the transformant is obtained by known salting-out method, solvent precipitation method, dialysis method, ultrafiltration method, gel electricity It can be separated, collected and purified by a combination of electrophoresis, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, affinity chromatography and the like. In particular, a combination of salting out with ammonium sulfate, S-sepharose ion chromatography, heparin sepharose affinity chromatography, and phenyl sepharose reverse phase chromatography, or a combination of salting out with ammonium sulfate, S-sepharose ion chromatography, and anti-HGF antibody sepharose affinity chromatography Etc. are preferable and effective purification methods.

  In addition, the partial protein of sugar chain-deficient HGF of the present invention is obtained by obtaining a partial protein of HGF to which a sugar chain has been added by a known method, and then treating the partial protein of HGF with an enzyme that removes the sugar chain. Can also be obtained. As an enzyme for removing a sugar chain, glycopeptidase F, glycopeptidase A, etc. can be used for the purpose of removing an N-linked sugar chain. Removal of the O-linked sugar chain is achieved by a combination of sialidase, fucosidase, O-glycanase and the like. At this time, it is desirable to add a protease inhibitor to the reaction solution. Examples of protease inhibitors to be added include benzamidine, aprotinin, leupeptin, antipain, chymostatin, pepstatin A, phenylmethanesulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), and the like. The HGF partial protein from which the sugar chain has been removed by enzymatic treatment can be collected as the partial protein of the sugar chain-deficient HGF of the present invention and purified by the purification method described above.

Furthermore, the sugar chain-deficient HGF partial protein of the present invention can also be obtained by using a cell-free protein synthesis system. A cell-free protein synthesis system uses a cell extract prepared from Escherichia coli, rabbit reticulocyte, wheat germ, etc., or encodes a target protein using a protein synthesis factor group contained in the cell extract It refers to a method of protein synthesis using DNA or mRNA as a template without using living cells. Cell extracts contain molecular groups necessary for protein synthesis such as ribosomes, tRNAs, translation factors, etc. When an amino acid serving as an energy source and substrate such as ATP and GTP is added to this, protein is synthesized. . Instead of the cell extract, a protein synthesis factor group contained in the cell extract may be mixed and used. Since the cell-free protein synthesis system does not include the endoplasmic reticulum or the Golgi apparatus, a sugar chain-deficient HGF part lacking a sugar chain using a DNA or mRNA encoding a partial protein of HGF having a glycosylation site as a template Can produce protein. Alternatively, DNA or mRNA having a mutation introduced at the glycosylation site can be used.
A protease inhibitor can also be added to the reaction solution. Examples of protease inhibitors to be added include benzamidine, aprotinin, leupeptin, antipain, chymostatin, pepstatin A, phenylmethanesulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), and the like. The sugar chain-deficient HGF partial protein synthesized in the cell-free protein synthesis reaction solution can be collected and purified by the purification method described above.

  The partial protein of sugar chain-deficient HGF of the present invention can also be produced by digesting HGF lacking a sugar chain with a protease or specifically cleaving it by chemical treatment. As the HGF used for this purpose, one having a mutation introduced at the glycosylation site based on the already known gene sequence of HGF can be used. Enzymatic degradation of HGF can be carried out by digesting HGF using a specific protease such as elastase, HGF activator, urokinase-type plasminogen activator, matriptase and the like. The resulting sugar chain-deficient HGF partial protein can be collected and purified by the purification method described above.

  The sugar chain-deficient HGF partial protein of the present invention obtained as described above has an antagonistic activity against the action of HGF via the c-Met / HGF receptor and also has an anti-angiogenic action of glycosylated HGF. It has the same activity as a partial protein.

  The partial protein of sugar chain-deficient HGF of the present invention is effective as a medicine and is used in the form of a general medical preparation. The pharmaceutical preparation containing the sugar chain-deficient HGF partial protein of the present invention as an active ingredient can take various preparation forms (eg, liquid, solid, capsule, etc.), but is generally an active ingredient. An injection, an inhalant, a suppository, or an oral preparation is used together with only a partial protein of sugar chain-deficient HGF or a pharmaceutically acceptable carrier, and an injection is preferred. The injection can be prepared by a conventional method, for example, a sugar chain-deficient HGF partial protein and a pharmaceutically acceptable carrier in an appropriate solvent (for example, sterile purified water, buffer, physiological saline, etc. ), And then sterilized by filtration with a filter or the like, and then filled into an aseptic container. The partial protein content of sugar chain-deficient HGF in the injection is usually adjusted to about 0.0002 to 3 (W / V%), preferably about 0.001 to 2 (W / V%). Oral drugs are formulated into dosage forms such as tablets, granules, fine granules, powders, soft or hard capsules, liquids, emulsions, suspensions, syrups, and the like. It can be prepared according to the conventional method of chemical conversion. Suppositories can also be prepared by a conventional method using a conventional base (for example, cacao butter, lauric butter, glycerogelatin, macrogol, witepsol). Inhalants can also be prepared according to conventional means on pharmaceutical preparations. The partial protein content of the sugar chain-deficient HGF in the preparation can be appropriately adjusted according to the dosage form, applicable disease and the like.

It is preferable to add a stabilizer when the pharmaceutical preparation of the sugar chain-deficient HGF partial protein of the present invention is formulated. Examples of the stabilizer include albumin, globulin, gelatin, alanine, glycine, mannitol, glucose, dextran, sorbitol, ethylene glycol and the like. In addition, the pharmaceutical preparation of the present invention may contain other additives necessary for formulation, such as a solvent (eg, physiological saline, sterile purified water, water for injection), excipient (eg, fructose, D-sorbitol, glucose). Starch, crystalline cellulose, dextrin, etc.), binder (eg, gelatin, corn starch, tragacanth, gum arabic, etc.), solubilizer (eg, lauromacrogol, polysorbate 80, polyoxyethylene hydrogenated castor oil 60, gum arabic, Sodium benzoate, etc.), antioxidants (eg, L-ascorbic acid, tocopherol, sodium edetate, etc.), soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), isotonic agents (eg, sodium chloride) , Glucose, D-mannitol, glycerin, etc.), buffer (eg citric acid, sodium citrate) Lithium, acetic acid, sodium acetate, lactic acid, sodium hydrogenphosphate, etc.), thickeners (gum arabic, carmellose, popidone, methylcellulose, etc.), preservatives (for example, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate) Chlorobutanol, benzyl alcohol, benzalkonium chloride, etc.), pH adjusters (hydrochloric acid, sodium hydroxide, citric acid, acetic acid, etc.) and the like.
In the case of a liquid preparation, it is desirable to store it after removing moisture by freeze storage or freeze drying. The freeze-dried preparation is used by adding distilled water for injection at the time of use and re-dissolving it.
In the case of an oral preparation, granules, tablets and the like are coated with an enteric coating agent (for example, cellulose acetate phthalate, methacrylic acid copolymer, hydroxypropyl cellulose phthalate, carboxymethyl ethyl cellulose, etc.). It is preferable that the capsule is an enteric capsule.

  The preparation of the present invention can be administered by an appropriate administration route according to the preparation form. For example, it can be administered into a vein, artery, subcutaneous, intramuscular, etc. in the form of an injection. The dose is appropriately adjusted depending on the disease, symptoms, age, weight, etc. of the patient. For example, for adults, 0.01 mg of a normal sugar chain-deficient HGF partial protein (eg, sugar chain-deficient NK4) is usually used. It is appropriate to administer this once a day or several times a day.

The sugar chain-deficient HGF partial protein of the present invention can be used for tumor invasion suppression, growth suppression, metastasis suppression, apoptosis induction or / and angiogenesis suppression. Therefore, the medicament containing the partial protein of sugar chain-deficient HGF of the present invention can be used as a prophylactic / therapeutic agent for cancer or / and a disease caused by angiogenesis, and a cancer metastasis preventive agent.
Examples of target cancers include lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, gallbladder cancer, kidney cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, oral cancer, colon cancer, colon cancer, uterine cancer, bile duct cancer , Pancreatic islet cell carcinoma, adrenal cortex cancer, bladder cancer, testicular cancer, testicular tumor, thyroid cancer, skin cancer, malignant carcinoid tumor, malignant melanoma, osteosarcoma, soft tissue sarcoma, neuroblastoma, Wilms tumor, retinoblast Tumors, melanoma, glioma, etc., among others, ovarian cancer, pancreatic cancer, stomach cancer, gallbladder cancer, kidney cancer, prostate cancer, breast cancer, esophageal cancer, liver cancer, oral cancer, colon cancer, colon cancer, sarcoma, melanoma Or glioma is preferable, and ovarian cancer, pancreatic cancer, gastric cancer, and gallbladder cancer are particularly preferable.

  In addition, the medicament containing the sugar chain-deficient HGF partial protein of the present invention can be used in combination with other antitumor agents and the like. Other anti-tumor agents include, for example, alkylating agents (eg, nitrogen mustard, melphalan, cyclophosphamide, chlorambucil, thiotepa, dibromomannitol, dibromodarcitol, carmustine, lomustine, semustine, nimustine hydro Chloride, chlorozotocin, ranimustine, busulfan, procarbazine, etc.), various antimetabolites (for example, 6-mercaptopurine, azathioprine, 6-thioguanine, thioinosine, fluorouracil, tegafur, carmofur, doxyfluridine, broxuridine, cytarabine, enotitabine, methotrexate, methotrexate, methotrexate Trexate, etc.), antitumor antibiotics (eg daunorubicin, aclarubicin, doxorubicin, actinomycin D, mitomycin C, And other antitumor agents (eg, cisplatin, carboplatin, tamoxifen, L-asparaginase, acebraton, schizophyllan, picibanil, ubenimex, krestin, etc.), antitumor plant components (camptothecin, vindesine, vincristine, vinblastine, etc.) ), BRM (biological response control substance; for example, tumor necrosis factor, indomethacin, etc.), cell adhesion inhibitor (for example, substance having RGD sequence, etc.), matrix metalloprotease inhibitor (for example, marimastat, batimastat, etc.) Hormones such as hydrocortisone, dexamethasone, methylprednisolone, prednisolone, plasterone, betamethasone, triamcinolone, oxymetholone, nandrolone, methenolone, Festrol, ethinyl estradiol, chlormadinone, medroxyprogesterone, etc.), vitamins (eg, tocopherol, etc.), antibiotics (eg, ampicillin, sulbenicillin, cephalexin, cephalodine, tobramycin, chlorralphenicol, tetracycline hydrochloride, kitacemycin, lincomycin hydrochloride, etc.) Chemotherapeutic agents (for example, sulfamethizole, sulfamonomethoxine, enoxacin, norfloxacin, ofloxacin, quinoxacin, etc.) and the like can be mentioned. Furthermore, the medicament containing the sugar chain-deficient HGF partial protein of the present invention can also be used in combination with surgical therapy or radiation therapy. Therefore, the medicament containing the partial protein of the sugar chain-deficient HGF of the present invention can suppress tumor invasion, growth suppression, metastasis suppression, apoptosis induction in combination with other antitumor agents and / or surgical therapy or radiotherapy. Or / and can be used to suppress angiogenesis.

  Examples of the disease caused by angiogenesis when the medicament containing the partial protein of sugar chain-deficient HGF of the present invention is used as a prophylactic / therapeutic agent for diseases caused by angiogenesis include, for example, rheumatoid arthritis, psoriasis, Osler-Webber ( Osler-Webber syndrome, myocardial angiogenesis, peripheral vasodilatation, hemophilic arthritis, ocular angiogenic diseases (eg diabetic retinopathy, retinopathy of prematurity, aging macular degeneration, corneal transplantation) Hematopoietic organs including rejection, angiogenic glaucoma, post-lens fibroproliferation, or perosis), angiofibroma, benign tumor (eg, hemangioma, acoustic neuroma, neurofibroma, trachoma, purulent granuloma, etc.), leukemia Tumor, solid tumor, tumor metastasis, wound granulation and the like.

In addition, the medicament containing the sugar chain-deficient HGF partial protein of the present invention can be widely applied to the prevention or treatment of diseases caused by excessive or abnormal stimulation of endothelial cells. Although there is no restriction | limiting in particular as this disease, For example, intestinal adhesion, Crohn's disease, atherosclerosis, scleroderma, for example, excessive scar formation, such as a keloid, etc. can be mentioned. Furthermore, the sugar chain-deficient HGF partial protein of the present invention, in particular, a drug containing sugar chain-deficient NK4 is useful as a fertility regulator based on preventing angiogenesis necessary for embryo implantation. As a result, it is useful as a prophylactic or therapeutic agent for diseases involving angiogenesis such as cat scratch disease and ulcers.
Furthermore, the sugar chain-deficient HGF partial protein can be used as a prophylactic / therapeutic agent for infectious diseases such as Listeria and malaria.
The base sequence encoding the partial protein of the sugar chain-deficient HGF of the present invention can also be used as a gene therapy drug. Examples of the disease targeted for gene therapy include the above-mentioned diseases.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited at all by these Examples.
In addition, the meaning of each abbreviation used in an Example is as follows.
HGF: hepatocyte growth factor bFGF: basic fibroblast growth factor LB: Luria-Bertani
Amp: ampicillin SDS-PAGE: SDS-polyacrylamide gel electrophoresis Tween 80: polyoxyethylene (20) sorbitan monooleate

The base sequence encoding the 5-amino acid deleted NK4 shown in SEQ ID NO: 3 was incorporated into the pCAGGS vector. The resulting vector is called pCAGGS-NK4.
In order to introduce mutations at three glycosylation sites (positions 289, 397, and 471 of SEQ ID NO: 2) present in the NK4 protein, three types of mutation primers (5′-phosphorylation) shown in Table 1 were used. Synthesis and mutagenesis were performed using the pCAGGS-NK4 vector as a template. As a result of this mutation, Asn289 and Asn397 are replaced with Gln and Thr471 is replaced with Gly in the amino acid sequence of NK4 of the 5-amino acid deletion type. SEQ ID NO: 4 shows the total amino acid sequence after mutagenesis. For mutation introduction, QuikChange Multi Kit manufactured by STRATAGENE was used.

  The vector having the mutation introduced therein is E. coli. E. coli XL10 Gold competent cells were transformed and Amp resistant colonies were picked up on LB / Amp plates. A plasmid was extracted from each of the obtained clones, and the target clone was screened by analyzing the nucleotide sequence of the coding part of NK4. A vector in which the target mutation was introduced at three positions of the nucleotide sequence encoding NK4 and that no other mutation was confirmed was selected and used in the subsequent experiments. The resulting mutant vector is referred to as pCAGGS-NK4-NG.

  PCAGGS-NK4-NG was then transfected into COS-7 cells. COS-7 cells were cultured by adding 10% fetal calf serum (FCS) to Dulbecco's modified Eagle medium (DMEM). Cells were replaced with serum-free DMEM medium immediately prior to transfection. Transfection was performed by the lipofection method using LIPOFECTAMINE 2000 (Invitrogen Corporation). Six hours after transfection, the medium was changed to DMEM containing 1% FCS, 5 mM benzamidine, and heparin was added at 1 μg / mL. After 24 hours, the culture supernatant was collected by filtration through a 0.22 μm filter, and then replaced with a fresh medium (1% FCS, 1 μg / mL heparin, DMEM containing 5 mM benzamidine) and further cultured for 24 hours. After 24 hours, the culture supernatant was again collected in the same manner. The collected culture supernatant was collected and stored at −80 ° C. until purification. The concentration of sugar chain-deficient NK4 secreted into the culture solution was analyzed by ELISA.

The above medium was thawed, mixed, filtered again through a 0.22 μm filter, and HiTrap heparin (Bed) equilibrated with 50 mM Tris-HCl (pH 7.5), 0.01% Tween 80, 0.3 M sodium chloride. volume: 5 mL) (Amersham Biosciences) was added at a flow rate of 1 mL / min. The column was washed with 50 mM Tris-hydrochloric acid (pH 7.5), 0.01% Tween 80, 0.3 M sodium chloride, and then the NaCl-deficient NK4 was eluted by increasing the NaCl concentration to 2 M. Elution was performed at a flow rate of 1 mL / min and fractionated at 2.5 mL / tube. Fractions containing sugar chain-deficient NK4 were collected, and buffer exchanged to 50 mM Tris-HCl (pH 7.5), 0.01% Tween 80, 0.3 M sodium chloride by ultrafiltration. This was added to a Mini S column (Bed volume: 0.8 mL) (Amersham Biosciences) equilibrated with the same buffer at a flow rate of 0.4 mL / min. The column was washed with 50 mM Tris-hydrochloric acid (pH 7.5), 0.01% Tween 80, 0.3 M sodium chloride, and then the NaCl-deficient NK4 was eluted by increasing the NaCl concentration to 1 M. Elution was performed at a flow rate of 0.4 mL / min, and fractionation was performed at 0.4 mL / tube. Fractions containing sugar chain-deficient NK4 were collected, collected, and the purified state was confirmed by SDS-PAGE.
Further, NK4 protein was prepared by CHO cells according to the method described in JP-A-2003-250549 (referred to as glycosylated NK4).
FIG. 1 shows the result of silver-staining the gel after reducing the glycosylated NK4 and sugar-deficient NK4 proteins and performing electrophoresis by SDS-PAGE. A band of glycosylated NK4 was observed at a position corresponding to a molecular weight of 67 kDa. In sugar chain-deficient NK4, the band was shifted to a position corresponding to the molecular weight lacking the sugar chain (molecular weight of NK4 protein main body 51 kDa).

  MDCK-3B cells were suspended in DMEM medium (containing 10% FCS), seeded on a 96-well plate at 5000 cells / well, and HGF was added to a concentration of 30 pM. At this time, glycosylated NK4 or glycosylated NK4 was added at a concentration of 3 nM, 30 nM or 85 nM, and cultured at 37 ° C. for 20 hours, and then the degree of scatter was observed with a microscope (FIG. 2). ). The normal form of MDCK-3B cells is as shown in FIG. 2A. However, when HGF is added and cultured, the cells are dispersed (FIG. 2B). When glycosylated NK4 was added simultaneously with HGF, cell dispersion was suppressed, and the cell dispersion was suppressed as the concentration of glycosylated NK4 increased (FIGS. 2C, D, and E). Furthermore, sugar chain-deficient NK4 suppressed the dispersion of MDCK-3B cells to the same extent as sugar chain-added NK4 (FIGS. 2G, H, I). That is, sugar chain-deficient NK4 had the same level of HGF antagonist activity as sugar chain-added NK4. When HGF is not added, there is no change in MDCK-3B cells even when sugar chain-added NK4 or sugar chain-deficient NK4 is added (FIG. 2F, J).

  Human umbilical vein vascular endothelial cells (HUVEC) were suspended in MDCB131 medium (containing 5% FCS), seeded on a 96-well plate at 2000 cells / well, and cultured for 24 hours without adding bFGF. Thereafter, the medium was replaced with fresh MDCB131 medium (containing 5% FCS), and at the same time, bFGF was added to 3 ng / mL, followed by culturing for 72 hours. At this time, at the same time as bFGF was added, glycosylated NK4 or glycosylated NK4 was added to a concentration of 30 nM or 85 nM. The number of HUVEC cells after 72 hours of culture was counted, and the effect of inhibiting the growth of HUVEC by glycosylated NK4 or glycosylated NK4 was compared (FIG. 3). When the glycosylated NK4 or the glycosylated NK4 was not added, HUVEC proliferated by the action of bFGF, and the number of cells increased. When glycosylated NK4 was added simultaneously with bFGF, the growth of HUVEC was suppressed, and the growth of HUVEC was suppressed as the concentration of glycosylated NK4 increased. In addition, sugar chain-deficient NK4 suppressed the growth of HUVEC to the same extent as sugar chain-added NK4. That is, sugar chain-deficient NK4 had an angiogenesis inhibitory effect similar to that of sugar chain-added NK4.

  In order to examine the invasion inhibitory action of glycosylated NK4 and glycosylated NK4 on cancer cells, human gallbladder cancer cells (GB-d1) were suspended in DMEM medium (containing 10% FCS), and Matrigel chamber (BD BIOCOAT Matrigel chamber, BD Biosciences) (15000 cells / chamber). The chamber was set on a 24-well plate, and DMEM medium (containing 10% FCS) was added to the outside of the chamber. Further, HGF was added to the DMEM medium outside the chamber to a concentration of 110 pM. At this time, sugar chain-added NK4 or sugar chain-deficient NK4 was added to the DMEM medium outside the chamber at a concentration of 1 nM, 11 nM, or 85 nM and cultured at 37 ° C. for 24 hours. Thereafter, the number of Gb-d1 cells infiltrating the outside of the chamber was counted. The number of infiltrating cells when only HGF was added was taken as 100, and the number of cancer cells invading outside the chamber was shown as a relative value (FIG. 4). Matrigel mimics the basement membrane, and cancer cells in the chamber infiltrate outside the chamber by the action of HGF added outside the chamber. When the glycosylated NK4 or the glycosylated NK4 was not added, a large number of cancer cells infiltrated the outside of the chamber due to the infiltration-inducing action of HGF. When glycosylated NK4 was added simultaneously with HGF, the invasion of cancer cells was suppressed, and the invasion was suppressed as the concentration of glycosylated NK4 increased. In addition, sugar chain-deficient NK4 suppressed invasion of GB-d1 cells to the same extent as sugar chain-added NK4.

  A DNA fragment encoding the mature polypeptide of sugar chain-deficient NK4 (5-amino acid deletion type) shown at positions 32 to 473 of SEQ ID NO: 4 is obtained using pCAGGS-NK4-NG described in Example 1 as a template. Used and prepared by PCR. At this time, sequences containing NotI restriction enzyme sites were added to both ends of the DNA sequence. The DNA fragment was digested with NotI and then inserted into the NotI restriction enzyme site of the yeast expression pPIC9K vector (Invitrogen). In the pPIC9K vector, the foreign gene is driven by the AOX1 promoter and the gene product is secreted from the host cell by an α-factor secretion signal. The constructed vector is called pPIC9K-NK4-NG. Next, the vector was linearized by digestion with SacI, and then introduced into the spheroplast of the methanol-assimilating yeast Pichia pastoris KM71 (Pichia pastoris KM 71; manufactured by Invitrogen) by electroporation. Transformed cells were selected by culturing on agar medium without histidine. The Pichia pastoris KM71 strain has a mutation in the his4 gene encoding histidinol dehydrogenase involved in histidine synthesis and cannot grow without the addition of histidine. Therefore, only transformed cells into which the pPIC9K vector containing the His4 gene has been introduced can grow on a plate medium containing no histidine. In this way, 200 His + transformants were selected. Geneticin resistant clones were then selected from His + transformed cells. The pPIC9K vector contains a kanamycin resistance gene that confers geneticin resistance to Pichia yeast. The degree of geneticin resistance depends on the number of integrated kanamycin resistance genes. Therefore, transformed cells that are resistant to high concentrations of geneticin incorporate multiple copies of the vector, thereby highly expressing the recombinant protein. For production of sugar chain-deficient NK4, a clone (KM71-4-48) showing resistance to 4 mg / mL geneticin was used. The KM71-4-48 clone is 30% in 5 mg of BMGY medium [1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6), 1.34% yeast nitrogen base, 0.00004% biotin, 1% glycerol]. The cells were cultured with shaking at 1 ° C. for 1 day, and the cultured cells were collected by centrifugation. The collected cells were then used to induce recombinant expression in BMMY medium (1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6), 1.34% yeast nitrogen base, 0.00004% biotin. , 0.5% methanol) was inoculated and cultured at 30 ° C for 5 days with shaking. Methanol was replenished to a final concentration of 0.5% every 24 hours. Five days later, sugar chain-deficient NK4 secreted into the medium was analyzed. In Western blot using an anti-human HGF polyclonal antibody, a sugar chain-deficient NK4 band in the culture medium was detected at a molecular weight of 51 kDa corresponding to the molecular weight lacking the sugar chain (FIG. 5). The concentration of sugar chain-deficient NK4 in the culture medium was 2 μg / mL as a result of ELISA.

  The sugar chain-deficient HGF partial protein of the present invention is useful as a substitute HGF partial protein for the HGF partial protein to which a sugar chain is added.

FIG. 1 shows the results of SDS-PAGE analysis of glycosylated NK4 and glycosylated NK4. FIG. 2 is a diagram comparing the action of NK4 to inhibit cell migration by HGF between a glycosylated NK4 and a glycosylated NK4. FIG. 3 is a diagram comparing the action of NK4 to suppress the proliferation of vascular endothelial cells by bFGF between sugar chain-added NK4 and sugar chain-deficient NK4. FIG. 4 is a diagram comparing the effect of NK4 on the invasion of cancer cells between sugar chain-added NK4 and sugar chain-deficient NK4. FIG. 5 shows the results of Western blot analysis of glycosylated NK4 and glycosylated NK4 produced by Pichia pastoris.

Claims (20)

Next (A) to (C)
(A) a protein comprising an amino acid sequence represented by positions 32 to 494 of SEQ ID NO: 1;
(B) a protein comprising an amino acid sequence in which one to several amino acids are deleted, substituted, added or inserted in positions 32 to 494 of SEQ ID NO: 1; or
(C) from # 32 of SEQ ID NO: 1 of the 494 of the amino acid at least 90% of any protein in the protein <br/> the amino acid residue matches, at least one position of glycosylation sites acids A protein in which at least one sugar chain at the glycosylation site is deleted by substitution of an amino acid so that a sugar chain is not added in the sequence, and the protein is followed by an N-terminal hairpin domain. it has four kringle domains, and partially the protein carbohydrate-deficient HGF characterized by having angiogenesis inhibitory action and having antagonist activity against the action of HGF which via a c-Met / HGF receptor. The amino acid substitution, partial protein glycosylation-deficient HGF according to claim 1, wherein the at least one of the the following (a) (d);
(A) Asn-X-Ser or Asn-X-Thr (wherein X represents an amino acid other than Pro), among the consensus sequences for N-linked glycosylation, Asn of at least one consensus sequence Is replaced with another amino acid;
(B) Among the consensus sequences for N-linked glycosylation wherein the amino acid sequence is Asn-X-Ser or Asn-X-Thr (X represents an amino acid other than Pro), the Ser of one consensus sequence or Thr, or Ser or / and Thr of two or more consensus sequences are replaced with other amino acids;
(C) X of at least one consensus sequence among N-linked glycosylation consensus sequences of which the amino acid sequence is Asn-X-Ser or Asn-X-Thr (X represents an amino acid other than Pro) Is substituted with Pro; or (d) Of Ser or Thr that undergoes O-linked glycosylation, at least one Ser or Thr is substituted with another amino acid. The amino acid substitution, partial protein glycosylation-deficient HGF according to claim 1, wherein the at least one of the the following (a) (c);
(A) In the amino acid sequence represented by SEQ ID NO: 1, the amino acid at position 294 or / and 296 is replaced with another amino acid, or / and the amino acid at position 295 is replaced with Pro to position 294. No sugar chain added;
(B) in the amino acid sequence represented by SEQ ID NO: 1, the amino acid at position 402 and / or 404 is replaced with another amino acid, or / and the amino acid at position 403 is replaced with Pro to position 402. No sugar chain is added; or (c) the sugar chain is not added to position 476 because the amino acid at position 476 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with another amino acid. The partial protein of sugar chain-deficient HGF according to any one of claims 1 to 3 , wherein the amino acids at positions 162 to 166 of the amino acid sequence represented by SEQ ID NO: 1 are deleted. The partial protein of sugar chain-deficient HGF according to any one of claims 1 to 4 , wherein the amino acids at positions 479 to 494 of the amino acid sequence represented by SEQ ID NO: 1 are deleted. DNA comprising a base sequence encoding the partial protein of sugar chain-deficient HGF according to any one of claims 1 to 5 . A vector incorporating the DNA according to claim 6 . A vector according to claim 7 is introduced into a cell, the cell is cultured, a partial protein of sugar chain-deficient HGF is produced in the cell or in a culture solution of the cell, and the culture solution of the cell or the cell The method for producing a partial protein of sugar chain-deficient HGF according to any one of claims 1 to 5 , wherein a partial protein of sugar chain-deficient HGF is collected and purified. The method for producing a partial protein of sugar chain-deficient HGF according to claim 8 , wherein the cell is a eukaryotic cell. 10. The method for producing a sugar chain-deficient HGF partial protein according to claim 9 , wherein the eukaryotic cell is a yeast or insect cell. A vector according to claim 7 is introduced into an insect individual, a sugar chain-deficient HGF partial protein is produced in the insect individual, and a sugar chain-deficient HGF partial protein is collected from the insect individual and purified. A method for producing a partial protein of sugar chain-deficient HGF according to any one of claims 1 to 5 . The vector 請 Motomeko 7 according introduced into cells without glycosylation ability, culturing the cells, allowed to produce the partial protein of sugar-deficient HGF in the culture fluid of the intracellular or the cells, the cells The method for producing a partial protein of sugar chain-deficient HGF according to any one of claims 1 to 5 , wherein a partial protein of sugar chain-deficient HGF is collected and purified from the culture medium of the cell or the cell. A pharmaceutical comprising the partial protein of sugar chain-deficient HGF according to any one of claims 1 to 5 as an active ingredient. The medicament according to claim 13 , which is an angiogenesis inhibitor. The medicament according to claim 13, which is an antagonist for the action of HGF via c-Met / HGF receptor. The medicament according to any one of claims 13 to 15 , which is an inhibitor of tumor invasion, growth or metastasis. The medicament according to any one of claims 13 to 15 , which is an apoptosis inducer. The medicament according to claim 13 , which is an agent for preventing or / and treating infectious diseases. The pharmaceutical according to claim 18 wherein the infection caused Therefore malaria or Listeria A. A gene therapy drug comprising the DNA of claim 6 .