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WO1999011786A1 - GENE pancpin ET COMPOSITION DE THERAPIE GENIQUE - Google Patents

GENE pancpin ET COMPOSITION DE THERAPIE GENIQUE Download PDF

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Publication number
WO1999011786A1
WO1999011786A1 PCT/JP1998/003841 JP9803841W WO9911786A1 WO 1999011786 A1 WO1999011786 A1 WO 1999011786A1 JP 9803841 W JP9803841 W JP 9803841W WO 9911786 A1 WO9911786 A1 WO 9911786A1
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WIPO (PCT)
Prior art keywords
gene
vector
cells
cancer
cell
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PCT/JP1998/003841
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English (en)
Japanese (ja)
Inventor
Kouichi Ozaki
Masami Nagata
Tsutomu Fujiwara
Hisanobu Hirano
Hiroyuki Kyushiki
Takashi Okamoto
Masashi Niimi
Original Assignee
Otsuka Pharmaceutical Co., Ltd.
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Publication of WO1999011786A1 publication Critical patent/WO1999011786A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a spleen cancer cell-related gene associated with spleen cancer cell growth, division, and metastasis, and more particularly, to serine, a serine proteinase-inhibiting gene.
  • the present invention relates to a novel kidney-specific cancer-related gene (hereinafter, this gene is referred to as "pancpin gene”), a novel protein encoded by the gene, and a specific antibody thereof.
  • the present invention provides a vector for gene transfer for gene therapy, which contains all or a part of the pancpin gene, a cell into which the pancpin gene has been introduced by the vector, Gene Therapy Composition for Gene Therapy of Human Cancer, In particular, Renal Cancer (Cancer Therapeutic Agent to Cancer Metastasis Inhibitor) Using the Same or Cell as an Active Ingredient, Use of the Gene Therapeutic Composition
  • the present invention relates to a method of gene therapy, and to the use of the pancpin gene for producing the composition for gene therapy.
  • Spleen cancer is one of the poorest prognoses among gastrointestinal malignancies, as it ranks fourth and fifth in the ranks of cancer-related deaths in Japan and western countries (Poston, JG, et al. , Gut., 32, 800-812 (1991)) 0
  • the most important goal in cancer research is to identify early genetic changes that lead to cancer. Determining the alterations of this gene has led to the development of genetic tools for the early diagnosis of the deadly disease, cancer, and new therapeutic approaches to treat this cancer more effectively.
  • Serine's proteases play a central role in regulating a wide variety of physiological processes, including cell aggregation, fibrosis, growth, malignant transformation and inflammation This is the power (Potempa, J., et al., J. Biol. Chem., 269. 15957-15960 (1994)) 0
  • One branch of the serine family which belongs to the serine proteinase inhibitor, is a five-protein (ovalbumin) protein. , Maspin, squamous cell carcinoma antigen, plasminogen activator-inhibitor-2 (PA1-2) and monocyte neutrophil elastase inhibitor (Remold_0, Donnel, E., FEBS Lett., 315, 105-108 (1993)) 0
  • TNF tumor necrosis factor
  • IFN interferon
  • Stimulating therapy through the immune system through the introduction of a drug, a method of introducing a drug resistance gene (MDR), a suicide gene (HSV-TK inheritance: cancer cyclovir cytosine after r 'administration) Cells are killed when administered), and the expression of oncogenes, cancer potentiators or their receptor genes, etc., to antisense oligonucleotides. Attempts have been made to reduce the risk by using a reboom.
  • MDR drug resistance gene
  • HSV-TK inheritance cancer cyclovir cytosine after r 'administration
  • ADA deficiency a representative disease, familial hypercholesterolemia, cystic fibrosis, hereditary diseases such as hemophilia, and melanosis. Includes malignant brain tumors, leukemias, neuroblastomas, renal cancer, etc.
  • the gene therapy is useful as a method for enhancing immunity of patients with these various diseases and as a cancer treatment by introducing a drug resistance gene and an antisense gene.
  • Protocols for gene therapy for these diseases were developed by the Recombinant DNA Advisory Committee of the NIH and FDA in the United States, and after they were approved for clinical practice, their Implemented as gene therapy for each disease
  • An object of the present invention is to provide information useful for elucidating the role of various inhibitors belonging to the Serpin family, which is desired in the art, and in particular, to provide serine 'protease' inhibitors.
  • An object of the present invention is to provide a novel homolog of a protein having homology to one serpin, a gene encoding the same, and an antibody that specifically recognizes the protein.
  • Another object of the present invention is to provide a gene therapy technique, particularly a gene transfer vector effective for cancer gene therapy, a cell having the vector, and a gene therapy using the vector and the cell.
  • An object of the present invention is to provide a novel gene therapy composition capable of suppressing tumor formation, tumor progression and cancer metastasis, and a cancer treatment method and a cancer metastasis suppression method using the same.
  • the present inventors succeeded in isolating and identifying a new kidney-specific cancer-related gene that matches the above-mentioned purpose.
  • the present inventor has also succeeded in developing a gene transfer vector containing the above-mentioned kidney-specific cancer-related gene and suitable for gene therapy for the above-mentioned purpose, and a cell carrying the same.
  • the present invention has been completed based on these findings.
  • the present invention contains a nucleotide sequence encoding any of the following proteins (a) and (b):
  • a pancp in gene particularly a human gene, is provided.
  • SEQ ID NO: consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1; A protein that acts to suppress metastasis.
  • the pancpin gene having a base sequence represented by SEQ ID NO: 3, the pancpin protein encoded by the gene, and an antibody that specifically recognizes the protein Is provided.
  • pancpin gene comprising any one of the following polynucleotides (a) and (b), particularly a human gene:
  • SEQ ID NO: a polynucleotide consisting of the nucleotide sequence represented by 2 and a polynucleotide that hybridizes under stringent conditions.
  • a DNA fragment of the above gene used as a specific probe or a specific primer for gene detection.
  • a salt represented by SEQ ID NO: 3 A vector for gene transfer for transfection and therapy containing the pancpin gene containing all or a part of the base sequence, and a cell having the vector are provided.
  • the above-mentioned vector includes a retrovirus vector, an adenovirus vector, an HlV human immunodeficiency virus) vector, and an adeno-associated virus vector.
  • a retrovirus vector Preferably, it is selected from a health information vector, a simple information vector and an Epstein-type information vector.
  • the above-mentioned cells are those having these vectors, and in particular, these vectors are used to form a membrane by fusing liposomes with inactivated Sendai virus by co-precipitation of canoleic acid phosphate.
  • Membrane fusion ribosome method for embedding in fusion ribosome Plasmid DNA is coated with gold, and DNA is physically introduced into cells by high-pressure discharge, Plasmid DNA DNA method directly into organs and tumors in vivo, the cation-ribosome method to introduce genes embedded in multi-layer positively charged ribosomes into cells, and the target cells.
  • a gene transfer method selected from the ligand-DNA complex method in which a ligand that binds to a receptor to be expressed is bound to DNA.
  • a cancer treatment composition and a cancer metastasis suppressing composition which suppress the growth of cancer cells or suppress cancer metastasis by administering the above-described vector for gene transfer and cells having the same to a cancer patient cancer therapy and cancer metastasis suppressing method according to arrangement is provided O 0
  • pancpin gene of the present invention is a spleen-specific cancer-related gene having homology to serpin, which is a serine protease-inhibitor.
  • the pancpin protein encoded by the pancpin gene of the present invention was prepared using the FASTA program [Person WR, et al., Proc. Atl. Acad. Sci., USA, 85, 2444-2448 (1988)) and the results of a search using GenBank / EMBL database.
  • -Inhibitor-2 PAI-2, related to inflammation
  • kidney cancer is a very poor prognosis among cancer types, and most patients with a definitive diagnosis die in a short period of time.
  • An important clinical feature of the spleen cancer is its early metastasis to lymph nodes and distant organs.
  • the spleen cancer has a high ability to invade normal tissues and other tissues (organs) and has a high likelihood of detachment from the primary layer. The molecular mechanism of transferability is not yet well understood.
  • pancpin gene according to the present invention was As a result, the expression is reduced in most spleen cancer specimens, and normal cancer tissue contains normal cells. Therefore, when high expression of pancpin in normal spleen tissue is considered The decrease in expression in this dramatic cancer specimen is considered to indicate that the pancpin gene is also reduced in normal cells around the cancer cells. This suggests that the pancpin gene of the present invention is one of the genes responsible for maintaining the funinotype of normal cells.
  • pancpin gene is mainly expressed in normal spleen and not in spleen cancer cell lines or many spleen cancer specimens, and based on the analysis of SSCP (single strand conformation polymorphism) and DNA sequence,
  • SSCP single strand conformation polymorphism
  • the lack of the pancpin gene plays an important role in the development or progression of spleen cancer, due to the fact that two missense mutations are identified in tumor DNA from 16 spleen cancer specimens. This gene is suggested to have potential value in canceration.
  • pancpin protein has homology to serpins suggests that the pancpin gene plays an important role in tumor cell growth and division.
  • serpins can inhibit the growth and division of tumors, for example, mapins are present in epithelial cells of initially normal human breast.
  • mapins are present in epithelial cells of initially normal human breast.
  • mapin cDNA in the fact that mosquitoes defined as expressed mRNA were at reduced or undetectable levels in invasive or metastatic breast cancer.
  • the protein encoded by the pancpin gene is a secretory protein.
  • the protein acts in some growth regulation pathways, possibly in growth inhibition pathways, because it is considered to be a serpin homologue.
  • the pancpin gene according to the present invention can be expected to have an activity of suppressing tumor formation-tumor progression and metastasis of spleen.
  • the pancpin gene and its gene product according to the present invention provide extremely useful information and means, especially for elucidation, understanding, diagnosis, prevention, and treatment of the spleen cancer.
  • the gene of the present invention can be suitably used for developing a new drug or the like that induces the expression of the gene of the present invention, which is used for treatment of the above-mentioned diseases, and the expression of the gene of the present invention in an individual or a tissue or its expression Product detection and mutation (deletion) of the gene And point mutation) or detection of abnormal expression can be suitably used for elucidation and diagnosis of the above diseases.
  • PCR product named “TSA204” (a fragment of pancp in gene, pancp in 5 nucleotides can be deduced from the DNA sequence, and the nucleotide sequence is as shown in SEQ ID NO: 2.
  • the gene of the present invention specifically has a nucleotide sequence encoding a protein having an amino acid sequence consisting of 405 amino acid residues represented by SEQ ID NO: 1 (specific examples of the sequence are as follows).
  • Mosquitoes exemplified as a gene comprising SEQ ID NO: 3 or a gene comprising a polynucleotide comprising all or a part of the nucleotide sequence represented by SEQ ID NO: 2;
  • the gene of the present invention encodes a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1. Also, a gene containing the base sequence described above is included. here, The degree of “deletion, substitution or addition of amino acid” and the position thereof are similar to those of the modified protein, which has the same function as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1. There is no particular limitation as long as it has the same effect. Specific examples include those having an effect of suppressing spleen tumor formation, tumor progression and metastasis.
  • the modification (mutation) of the amino acid sequence may occur in nature, for example, due to mutation or post-translational modification. These can be performed artificially based on the specific example gene).
  • the present invention encompasses all modified genes having the above-mentioned properties regardless of the cause and means of such modification / mutation.
  • Examples of the above-mentioned artificial means include, for example, site specific mutagenesis [Methods in Enzymology,
  • the base sequence represented by SEQ ID NO: 3, which is one embodiment of the gene of the present invention, is one of the codons representing each amino acid residue in the above amino acid sequence (SEQ ID NO: 1). It is also a combination example.
  • the gene of the present invention is not limited to the sequence of this combination example, but it is of course possible to have a base sequence obtained by combining and selecting an arbitrary codon for each amino acid residue. The selection of the codon can be performed according to a conventional method, for example, considering the frequency of use of the codon in the host to be used (Ncleic Acids Res.
  • the gene of the present invention is characterized in that the force represented as the base sequence of single-stranded DNA is based on the base sequence complementary to such a base sequence.
  • Polynucleotides of course, also include components containing both of these. Also, it is not limited to DNA such as cDNA.
  • the gene of the present invention is not limited to a polynucleotide comprising the whole or a part of the nucleotide sequence shown in SEQ ID NO: 2, but may be a certain number of nucleotides. It may be composed of a base sequence having homology. Such genes are at least listed below. Under the strong stringent conditions, the nucleic acid shown in SEQ ID NO: 2 hybridizes with the DNA having the sequence strength, and does not desorb from the DNA even when washed under certain conditions. O
  • DNA having the nucleotide sequence of SEQ ID NO: 2 was hybridized with the DNA under the condition of 65 ° C in 6 SSC at night or in the condition of 37 ° C in 4XSSC containing 50% formamide at night.
  • the gene of the present invention consisting of a base sequence having homology to the above specific DNA, that is, the gene of the present invention having a base sequence which hybridizes with the specific DNA and is not eliminated even by washing under certain conditions, is more preferable.
  • it encodes a protein having an activity similar to that of the protein encoded by the specific DNA, that is, a protein having an effect of suppressing spleen tumor formation, tumor progression and metastasis. Good.
  • the gene of the present invention can be easily produced and obtained by general genetic engineering techniques based on sequence information on specific examples [for example, Molecular Cloning 2nd Ed, Cold Spring Harbor Lab. Press (1989); see, for example, Seisho-Danigaku Experimental Lecture, “Gene Research Methods I, II, III”, edited by the Oral Biochemical Society (1986)].
  • the production of the gene of the present invention is carried out by preparing a cDNA library from an appropriate source having the gene according to a conventional method, and applying a specific application of the gene of the present invention from the library.
  • examples of the origin of the cDNA include various types of cells and tissues capable of expressing the gene of the present invention, and cultured cells derived therefrom, particularly, kidney tissues. Separation of total RNA, separation and purification of mRNA, acquisition of cDNA and cloning thereof can all be carried out in accordance with ordinary methods.
  • cDNA libraries are commercially available, and in the present invention, various commercially available cDNA libraries, for example, various types of commercially available cDNA libraries from Clontech Lab. Inc. A cDNA library can also be used.
  • the method for screening the gene of the present invention from the cDNA library is not particularly limited, and any conventional method can be used. Specifically, for example, immunoscreening using a specific antibody for a protein produced by cDNA How to select the corresponding cDNA clone by using a probe with a probe that selectively binds to the target DNA sequence. — Hybridization, Colony Highbridging — For example, and combinations thereof.
  • Examples of the probe used herein include DNA that can be generally exemplified by DNA chemically synthesized based on information on the nucleotide sequence of the gene of the present invention, and of course, the present invention gene that has already been obtained. And its fragments can be used well.
  • a sense primer and an antisense primer set based on the nucleotide sequence information of the gene of the present invention can be used as a screening probe.
  • the screening method of the gene of the present invention is based on the protein interacting cloning method using the pancpin protein instead of the specific antibody described above.
  • the differential display method [Liand P., et al., Science, 257. 967 971 (1992)] is also used under different conditions. It allows direct comparison and study of mRNA expression between cells or multiple different cell groups.
  • a DNAZRNA amplification method by PCR (Science, 230: 1350 (1985)) is also preferable. It can be used for In particular, when it is difficult to obtain a full-length cDNA in a library, the lace method (RACE: Rapid amplification of cDNA ends; Experimental Medicine, 12 (6): 35 (1994)) 5 '-Race (5, -RACE) 3 ⁇ 4 C Proc. Nat 1. Acad. Sci., USA., 8: 8998 (1988)).
  • the primer used for adopting such a PCR method can be appropriately set based on the sequence information of the gene of the present invention already clarified by the present invention. Can be synthesized.
  • the amplified DNAZNA fragment can be isolated and purified by a conventional method as described above, for example, by gel electrophoresis.
  • the gene of the present invention can also be produced and obtained according to a conventional DNA synthesis technique, for example, a chemical synthesis means such as the above-mentioned phosphoric acid ester method and phosphoric acid amidite method.
  • a chemical synthesis means such as the above-mentioned phosphoric acid ester method and phosphoric acid amidite method.
  • Such chemical synthesis can be performed, for example, using a commercially available automatic oligonucleotide synthesizer.
  • the complementary strand is synthesized and the strands are annealed together under appropriate conditions, or the complementary strand is added using DNA polymerase with an appropriate primer sequence.
  • it can be obtained from a chemically synthesized single-stranded product.
  • the gene of the present invention or various DNA fragments obtained above is Natl. Acad. Sci., USA., 74: 5463 (1977) and maximum guinea-note method C Method in Enzymology, 65: 499 (1980)].
  • the nucleotide sequence can be determined using a commercially available sequence kit or the like.
  • the present invention provides a vector containing the pancpin gene of the present invention (expression vector), a host cell transformed with the vector, and a pancpin protein obtained by culturing the host cell. It also provides a method for producing the.
  • pancpin protein The method for producing the pancpin protein is described in General Genetic Recombination Technology C Science, 22: 1431 (1984); Biochem. Biophys. Res. Comm., 130: 692 (1985); Proc. Natl. Acad. Sci. USA., 80: 5990 (1983) and the above-cited references, etc.].
  • prokaryote and eukaryote can be used as the host cell.
  • prokaryotic hosts include Escherichia coli and Bacillus subtilis.
  • Escherichia coli especially Escherichia coli K 1
  • eukaryotic host cells include cells such as vertebrates and yeasts. Examples of the former include COS cells (Cell, 23: 175 (1981)), which are monkey cells, and Chinese. 'Hamster ovary cells and their dihydrofolate reductase-deficient strains, C Proc. Natl. Acad. Sci., USA., 77: 4216 (1980)].
  • Mosquitoes that are preferably used, such as yeast cells of the genus Romyces, are not limited to these.
  • a promoter is used upstream of the gene so that the gene of the present invention can be expressed in the vector using a vector that can be replicated in the host cell.
  • an expression plasmid to which an SD (Shine and Dalgarno) base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis have been added can be suitably used.
  • the above vectors are generally plasmids derived from E. coli, for example, pBR322, pBR325, pUC12,
  • the expression vector usually includes a promoter located upstream of the gene of the present invention to be expressed, a splice site of RNA, and polyadenylation.
  • expression vector examples include, for example, pSV2dhfrCol.Cell.Biol., Which holds the initial promoter of SV40, ⁇ : 854 (1981)]. This can be exemplified. In addition, various known commercial vectors can be used.
  • Examples of commercially available vectors used for expression systems using animal cells include, for example, pEGFP-N, pEGFP-C (above C Iontech), pIND (Invitrogen), pc DNA 3.1 Vectors for animal cells, such as 1 / His (Invitrogen), pFastBacHT (Gibco BRL), pAcGHLT (PharMingen), pAc5 / V5-His And insect cells such as pMTZV5-His, pMT / Bip / V5His (all from Invitrogen).
  • an expression vector when a yeast cell is used as a host for example, PAM82 having a promoter for the acid phosphatase gene (Proc. Natl. Acad. Sci., USA., 80: 1 (1983)].
  • PAM82 having a promoter for the acid phosphatase gene
  • yeast cells include, for example, pPICZ, pPICZ ⁇ (all from Invitrogen).
  • the promoter there is no particular limitation on the promoter.
  • Escherichia is used as a host, for example, a tripphan (trp) promoter, an lpp promoter, a lac promoter, etc.
  • the recA Promoter, the PL / PR motor, etc. can be used appropriately.
  • the host is a bacterium belonging to the genus Bacillus, for example, SP01 promoter, SP02 promoter, penP promoter and the like are preferable.
  • Preferred promoters when yeast is used as a host include, for example, pH05 promoter, PGK promoter, GAP promoter, ADH promoter and the like.
  • a promoter derived from SV40 for example, a promoter derived from SV40, a retrovirus promoter, a metallothionoline promoter, a heat shock promoter, a site promoter, etc.
  • Gallo wireless promoters, SRa motors, and the like can be exemplified as preferred examples.
  • a normal fusion protein expression vector can also be preferably used, and a specific example of the vector is glutathione-S— Examples include pGEX (Promega) for expression as a fusion protein with Lansferase (GST).
  • GST Lansferase
  • Method for Introducing the Desired Recombinant DNA (Expression Vector) into Host Cells ⁇ The transformation method is also not particularly limited, and various general methods can be employed.
  • the obtained transformant can also be cultured according to a conventional method, and the culture expresses and produces the pancpin protein coded by the gene of the present invention, and is transformed. Accumulates or is secreted intracellularly, extracellularly or on the cell membrane of the body.
  • the medium used for the above-mentioned culture various types commonly used depending on the host cell used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cell.
  • the recombinant protein (pancpin protein) thus obtained can be separated and purified, if desired, according to various separation procedures utilizing its physical properties, chemical properties, and the like.
  • Biochemical Data Book II pp. 1175-1259, 1st edition, 1st edition, issued by Tokyo Chemical Dojin on June 23, 1980;
  • Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, ultrasonic crushing, ultrafiltration, Molecular sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, affinity chromatography, high-performance liquid chromatography Rotomog Examples include various liquid chromatographies such as Raphine (HPLC), dialysis, and combinations thereof. Particularly preferred methods include binding a specific antibody to pancpin protein. An example of an affinity chromatograph that uses a column that has been used.
  • the present invention also provides a novel pancpin protein itself obtained, for example, as described above.
  • the protein is characterized by having an action of suppressing spleen tumor formation, tumor progression and metastasis, and is useful in the field of medicine as described above.
  • the pancpin protein can also be used as a specific antibody for the protein, that is, as an immunizing antigen for preparing an anti-pancpin antibody.
  • the components used as antigens here can be, for example, proteins produced in large amounts by the above-described genetic engineering techniques or fragments thereof, and these antigens can be used. By doing so, desired antisera (polyclonal antibodies) and monoclonal antibodies can be obtained.
  • Monoclonal antibodies can also be obtained in the usual manner by producing fusion cells of plasma cells (immunocytes) and morphocytoma cells of animals immunized with the above-mentioned immunizing antigen, and obtaining the desired antibody. This can be carried out by selecting a clone that produces chromosome and culturing the clone.
  • the immunized animal is generally selected in consideration of compatibility with the plasmacytoma cells used for cell fusion, and a mouse or rat is generally advantageously used. Immunization is the same as in the case of the above antiserum, and can be carried out in combination with a normal adjuvant if desired.
  • Fusion of the above immune cells and plasmacytoma cells can be carried out in the presence of a conventional fusion promoter, for example, polyethylene glycol (PEG) or Sendai virus (HVJ) according to a known method. And the separation of desired hybridomas can be performed in the same manner [Meth. In Enzymol., 73: 3 (1981);
  • the search for the antibody-producing strain of interest and the cloning into a single clone are also performed by a conventional method.
  • the search for the antibody-producing strain is carried out by the ELIS method using the above immunizing antigen [Meth. In
  • the antibody of the present invention can be collected from the thus obtained hybridoma by culturing the hybridoma by a conventional method and obtaining the culture supernatant. This method is carried out by, for example, administering a mammal to a mammal that is compatible with the animal, growing the animal, and obtaining the resulting ascites.
  • the former method is suitable for obtaining high-purity antibodies
  • the latter method is suitable for mass production of antibodies.
  • the antibodies obtained in this way can be further subjected to salting out, gel filtration, affinity chromatography, etc. It can be purified by any conventional means.
  • the antibody thus obtained is characterized by having binding properties to the pancpin protein of the present invention, which is determined by the above-mentioned purification of the pancpin protein and measurement by the immunological technique. It can be used to advantage for identification.
  • the present invention also provides such a novel antibody.
  • the expression of the gene of the present invention in various tissues can be detected.
  • Such detection can be performed according to a conventional method, for example, RT-PCR and reverse transcribed-polymerase reaction; ES Kawasaki, et al. Amplincation oi RNA. In PCR Protocol, A Guide to methods and applications, Academic Press , Inc., SanDiego, 21-27 (1991)] for RNA amplification and Northern blot analysis.
  • the primer used is not particularly limited as long as it is specific to the gene of the present invention and can specifically amplify only the gene of the present invention. And the sequence can be set appropriately. Usually, it can have a partial sequence of the order of 20 to 30 nucleotides.
  • the present invention also provides a specific primer for detecting the pancpin gene of the present invention and a DNA fragment to be used as Z or a specific probe.
  • An example of the vector for gene transfer containing the pancpin gene according to the present invention is the expression vector for the pancpin protein described above. Specific examples of such a vector for gene transfer and cells having the vector are described in Example 2 below. These are effective for various types of gene therapy, particularly for the treatment of suppressing spleen tumor formation, tumor progression and metastasis.
  • the vector for introducing the gene of the present invention comprises an appropriate promoter, a 5 ′ mRNA leader sequence, a start site, a nucleic acid sequence containing a gene sequence to be introduced (pancpin gene sequence), a 3 ′ untranslated region, It can be a viral or non-noorious vector in which rear denier signals are functionally connected in a permutation at an appropriate distance.
  • the gene therapy method of the present invention is carried out by infecting and introducing appropriate cells such as spleen cancer cells targeting the vector for gene transfer of the present invention. It can suppress cell growth and cancer metastasis.
  • the gene therapy method using the gene transfer vector containing the pancpin gene according to the present invention can be regarded as a method of supplying a wild-type pancpin function to cells having a mutant pancpin allele. It can be. When such a function is supplied to the cells, the neoplastic growth of the recipient cell Z target cells is suppressed.
  • the wild-type pancpin gene (or a part of the gene) can be introduced into cells using a vector (the present invention vector) that maintains the gene extrachromosomally. In this case, the gene is expressed from outside the chromosome.
  • the gene When the gene is introduced into a cell having a mutated pancpin allele and expressed, the gene should encode a part of the pancpin protein required for non-neoplastic growth of the cell. Is an important requirement.
  • the wild-type pancpin gene or a portion thereof is preferably introduced into a mutant cell so that recombination with the endogenous mutant pancpin gene present in the cell occurs. Such recombination requires the occurrence of double recombination in which the pancpin gene mutation is corrected.
  • a vector for introducing a desired gene (source vector) for both recombination and extrachromosomal maintenance is already known in the art as described later.
  • the pancpin gene of the present invention can be introduced into such a known vector for gene introduction and used.
  • the vector for introducing the gene of the present invention can be introduced into an appropriate cell such as a cell having the above mutant allele or a patient's cancer cell, for example, by electroporation, coprecipitation of phosphoric acid phosphate, viral traits, or the like. It can be carried out according to various methods known in the art for introducing DNA into cells, including introduction.
  • the cells transformed with the wild-type pancpin gene can be used as a drug for suppressing cancer or suppressing cancer metastasis or as a model system for therapeutic research. Can be used.
  • the gene therapy method of the present invention using the gene transfer vector of the present invention includes a copy of the pancpin gene linked to an expression control element, and further comprises a tumor cell of a patient.
  • a virus or a plasmid vector capable of expressing the gene product in the cell is prepared and introduced into a tumor cell of a patient. You.
  • the suitable source vector used for preparing the vector for introducing the gene of the present invention is not particularly limited.
  • US Pat. No. 5,252,479 The vectors disclosed in the specification and PCT International Publication W093 / 07282 can be used.
  • the desired vector can be prepared according to a conventional method, for example, as described in the above-mentioned patent specification and published specification.
  • the vector for transfection of the present invention containing the pancpin gene or a fragment thereof can be introduced into a patient's tumor cell by local or systemic injection administration to the patient's tumor site. You. According to the above systemic administration, any tumor cells that can metastasize to other sites can be reached. If the transduced gene is not permanently incorporated into the chromosome of each target tumor cell, the administration should be repeated periodically. You just have to return it.
  • the gene therapy method according to the present invention includes, as described above, an in vivo method in which a vector or cell for gene transfer is directly administered into the body, and a method in which a cell once targeted from the patient's body is used. Both ex vivo and ex vivo methods include removing the gene and introducing the gene outside the body, and then returning the cells to the body.
  • the specific gene of the present invention not all of the specific gene, that is, a gene consisting of the entire sequence is necessarily required. As long as it retains substantially the same function as the function, the above-mentioned modified or partially composed gene can also be used.
  • the gene transfer vector containing the pancpin gene of the present invention and the gene therapy composition containing, as an active ingredient, the cell into which the pancpin gene has been introduced by using the vector are particularly applicable to cancer.
  • the targets of gene therapy include, besides the cancer, genetic markers, hereditary diseases, and viral diseases such as AIDS.
  • the target cells for gene transfer can be appropriately selected depending on the target of gene therapy. This includes cancer cells and tumor tissues as well as secretory cells such as lymphocytes, fibroblasts, hepatocytes, hematopoietic stem cells, spleen, and thyroid. Cells are included.
  • Methods for introducing the pancpin gene into cells for the gene therapy of the present invention include a viral method and a non-viral method.
  • a viral method in consideration of the fact that the pancpin gene is a foreign gene expressed in normal cells, for example, a retrovirus vector in which the pancpin gene of the present invention is introduced as a vector is used.
  • retrovirus vectors such as immunoreceptors, such as adenovirus vectors, HIV vectors, and adenoviruses: AAV (adeno associated virus) Norwegian I Norres Vector
  • Vectors can also be used as vectors for introducing the present gene into cells.
  • Non-viral gene transfer methods include phosphoric acid co-precipitation; DNA-encapsulated ribosomes are fused with inactivated Sendai virus whose genes have been disrupted by ultraviolet light beforehand.
  • Membrane fusion ribosome method to create fusion ribosomes and directly fuse with cell membrane to introduce DNA into cells
  • the ligand-DNA complex method described above includes, for example, a method in which a glycoprotein receptor expressed by hepatocytes is used as a target and a glycoprotein is used as a ligand [Wu, et al., J. Biol. Chem., 26i. 14338 (1991); Ferkol, et al., FASEB J., 7, 1081-1091 (1993)], and Transferrin receptor, which strongly expresses tumor cells. And a method using transbrin as a ligand with a target [Wagner et al., Proc. Natl. Acad. Sci., USA., 87, 3410 (1990)]. included.
  • the gene transfer method for gene therapy according to the present invention may be a combination of various biological and physical gene transfer methods as described above.
  • Examples of the method by the combination include a method in which a certain size of plasmid DNA is combined with a polylysine-conjugated antibody specific to adenovirus hexon protein.
  • the gene of the present invention can be introduced by binding the obtained complex to adenovirus vector and infecting the cell with the thus obtained trimolecular complex. . In this way, efficient binding, internalization and endosomal degradation can be achieved before the DNA coupled to the adenovirus vector is damaged.
  • the ribosome ZDNA complex can mediate gene transfer directly in vivo.
  • the retro-Winores vector system in the above method comprises a virus vector and a helper cell (packaging cell).
  • the helper cells pre-express genes such as the retrovirus structural protein gag (the structural protein in the viral particle), po1 (reverse transcriptase), and enV (the coat protein). A cell that does not generate virus particles.
  • the Winores vector has a packaging symbol, nanore, and LTR (long terminal repeats), but has structural genes such as gag, po1, and env necessary for Winores replication. Absent.
  • the packaging signal is a sequence that becomes a tag during assembly of viral particles, and includes a selection gene (neo, hyg) and a desired transgene (pancpin gene) integrated into the cloning site. ) Is inserted instead of the viral gene.
  • the insert should be as short as possible, the packaging signal should be widened to include a part of the gag gene, and the ATG of the gag gene should be used. It is important not to leave it.
  • the helper cells produce the vector genomic RNA due to the viral structural proteins. Are packaged to form viral particles and are secreted. After the virus particles as recombinant viruses infect target cells, DNA reverse transcribed from viral genomic RNA is incorporated into the cell nucleus, and the gene inserted in the vector is expressed.
  • a method for increasing the efficiency of introduction of a desired gene a method using a fragment containing a cell binding domain of fibronectin, a halin binding site and a conjugation segment is used.
  • the method of using the adenovirus vector will be described in detail.
  • the production of the adenovirus vector can be performed by the method described in Berckner C Berkner, KL, Curr. Topics Microbiol. Immunol., 1583966 (1992)), Setoguchi, Y. et al., Blood, 84, 2946-2953 (1994), Kanekae Yumi, et al. [Experimental Medicine, 12, 28-34 (1994) 3, and Kenaichi, et al., Ketner, G. Natl. Acad. Sci., USA., 91. 6186-6190 (1994)).
  • the E1 and / or E3 gene region of the adenovirus early gene is first removed.
  • the desired desired exogenous gene expression unit (the desired transgene, ie, a pancpin gene, a promoter for transcribing the gene, a polyA that confers stability of the transcribed gene) and a plasmid vector containing a part of adenovirus genome DNA;
  • the plasmid containing the adenovirus genome is simultaneously transfected into, for example, 293 cells.
  • a non-proliferative adeno which is a vector for introducing the gene of the present invention containing the desired gene.
  • the adenovirus gene DNA can be incorporated into a cosmid vector to produce a 3 'adenovirus vector to which a terminal protein has been added.
  • YAC vectors Table 8-8
  • the adeno-associated virus (AAV) vector as described in, for example, Keiya Ozawa, History of Medicine, Vol. L75, No. 9, 619-622 (1995). It was discovered as a small virus that gets mixed into the culture of Denovirus. This confirmed the presence of ParvoWinores, which grows autonomously in host cells without the need for helper virus for virus replication, and the genus of Virdevirus, which requires helper Vinoles. Kotin, RM and Berns, KI, Virology, 170, 460-467 (1989)] 0 The AAV is an abundant host that spreads a wide range of cells and infects various cells, and the viral genome is 468 in size.
  • ITR inverted terminal repeat
  • Recombinant AAV can be prepared by utilizing the property of AAV to be integrated into chromosomal DNA, and thus the gene transfer vector of the present invention can be prepared.
  • This method is described, for example, in the literature C Nienhuis, AW, et al., Viruses and Bone Marrow (ed.by Young, NS), Marcelekker, New York, 1993, pp.351-414: Ferrari, FK , et al., J, Viol, 70, 3227-3234 (1996), etc.].
  • plasmid AAV vector plasmid
  • ITRs at both ends of 5 ′ and 3 ′ of wild-type AAV were left and a desired transgene (pancpin gene) was inserted between them.
  • the viral proteins required for virus replication and virus particle formation are supplied by another helper plasmid. It is necessary to ensure that there is no common nucleotide sequence between the two and that no wild-type virus is produced by genetic recombination. Thereafter, both plasmids were introduced, for example, by transfection into 2993 cells, and then Herno.
  • Infecting adenovirus or non-proliferative type when using 293 cells as one virus produces the desired non-proliferative recombinant AAV. Subsequently, since this recombinant AAV is present in the nucleus, cells are recovered by freeze-thawing, and contaminating adenovirus is inactivated by heating at 56 ° C. If necessary, separate and concentrate the recombinant AAV by ultracentrifugation using cesium chloride. As described above, a desired recombinant AAV for introducing a gene can be obtained.
  • HIV vectors can be prepared, for example, according to the method of Shimada et al. C Shiraada, T., et al., J. Clin. Invest., 88, 1043-1047 (1991)) o
  • HIV virus specifically infects helper T cells with CD4 as a receptor
  • the use of the HIV virus allows tissue-specific transfection that can specifically transfect human CD4-positive cells.
  • HIV vector as a gene transfer vector Can be created.
  • the packaging plasmid CGPE is replaced with the structural genes of gag, pol, and env and the regulatory genes required for their expression (tat.rev, etc.). Is constructed so as to be expressed by the promoter of site megalovirus (CMV) and the polyA signal (polyA) of the human globin gene.
  • CMV site megalovirus
  • polyA polyA signal
  • the vector plasmid HXN was inserted between the two LTRs of HIV, and the neomycin resistance gene of Bacteria, which has a thymidine kinase (TK) promoter as a labeling gene ( neoR), and by inserting the SV40 replication origin into the basic plasmid vector, it can be constructed to efficiently grow in COS cells. You. For example, a large number of neomycin resistance genes (neoR ) Can be produced and released into the culture medium.
  • TK thymidine kinase
  • packaging plus mid CGPE converts the structural genes of gag, po1, and env and the regulatory genes (tat, rev) required for their expression with the promoter (CMV) of Site Megalo Winores.
  • CMV promoter
  • Poly A of human globin gene It was created to be expressed by a signal (po1yA).
  • vector plasmid HXN is a plasmid in which a neomycin resistance gene having a thymidine kinase peptide motor (TK) is inserted between both LTRs of HIV as a marker gene. Yes [Koichi Miyake, Takashi Shimada, History of Medicine, Vol.175, No.9, pp.623-624 (1995)].
  • EBV Epstein-Barr virus
  • B ur kitt is a virus belonging to the Rupesu family to have been separated Ri by cell culture-derived lymphoma [Kief f, E. and Liebowi tz, D.:. Virology, 2nd ed Raven
  • EBV Since the EBV has the activity of transforming cells, it is necessary to prepare a virus lacking the activity of the transform in order to use it as a vector for gene transfer. Must. This can be done as follows.
  • the EBV genome near the target DNA for integration of the desired foreign gene is cloned.
  • the DNA fragment of the exogenous gene and the drug-resistant gene are then incorporated into the vector for recombinant virus production.
  • the vector for preparing a recombinant virus cut out with an appropriate restriction enzyme is transfected into EBV-positive Akata cells.
  • Recombinant virus generated by homologous recombination can be recovered together with wild-type A kata EBV by virus production stimulation by treatment with anti-cell surface immunoglobulin antibody. By infecting this with EBV-negative A kata cells and selecting a resistant strain in the presence of the drug, only the desired recombinant virus without wild-type EBV was infected.
  • Akat cells can be obtained. Furthermore, by inducing viral activity in recombinant virus-infected Akat cells, a desired large amount of recombinant viral vector can be produced.
  • Production of a non-viral vector which introduces a desired gene into target cells without using a recombinant virus vector, can be performed, for example, by a transfection method using membrane-fused liposomes. Can be done.
  • This method is a method in which membrane ribosomes (vesicles composed of a lipid bilayer membrane) are given a fusion activity to a cell membrane, thereby directly introducing ribosome contents into cells.
  • the Sendai virus whose gene has been inactivated by ultraviolet light, and the liposome in which a high molecular substance such as a desired gene or protein is encapsulated are fused at 37 ° C.
  • This membrane-fused liposome has a structure called quasi-virus with cavities derived from liposome on the inside and spikes on the outside with the same spikes as the virus envelope. Further, after purification by sucrose density gradient centrifugation, the membrane-fused liposome is adsorbed to the target cultured cells or tissue cells at 4 ° C.
  • the ribosome content is introduced into the cells, and the desired gene can be introduced into the target cells.
  • the lipids used as liposomes are 50% (molar ratio) cholesterol, lecithin, and a synthetic phospholipid with a negative charge. It is preferable to produce and use a single-walled ribosome.
  • a gene transfer method using a cationic ribosome can be mentioned as a method for introducing a gene into a target cell using another ribosome. The method can be performed according to the method of Yagi et al. [Yagi, K., et al., BBRC, 1-96, 1042-1048 (1993)].
  • This method focuses on the fact that both the plasmid and the cell are negatively charged, applies a positive charge to both the inner and outer surfaces of the ribosome membrane, and removes the plasmid by static electricity. And to increase interaction with cells.
  • the liposome used here is a large, multi-layered liposome with a positive charge.
  • the method for preparing plasmid-embedded cationic MLV is as follows.First, the lipid TMAG (N-(-trimethylthyla nio-acetyl; -didodecyl-D-glutamate chloride), D
  • the expression plasmid incorporating the cDNA of the gene targeted for expression may be expressed in the above-described cationic.
  • the amount of DNA is 0.6 / g and the amount of liposome lipid is 30 nmol, and this is 21% phosphate buffered saline.
  • An example is a method of suspending in a liquid and administering to target cells or patient tissues extracted from a patient every other day.
  • Gene therapy is defined by the Ministry of Health and Welfare Guideline as "administering a gene or a cell into which a gene is introduced into a human body for the purpose of treating a disease.”
  • gene therapy in the present invention includes the definition of the guideline, and further includes the use of antisense or oligonucleotides against the above-mentioned target cells or various diseases including cancer. It also includes treatment by introduction, and introduction of a gene as a marker or cells into which a gene as a marker has been introduced into a human body.
  • the method of introducing a desired gene into a target cell or target tissue according to the gene therapy of the present invention typically includes two methods as described above.
  • the first method after the patient cells to be treated are collected, they are cultured outside the body by adding, for example, interleukin-2 (1 L-2), and then retrovirus-free.
  • This is a method (ex vivo method) in which the target gene contained in the cluster is introduced, and the obtained cells are re-transplanted.
  • This method is suitable for treating ADA deficiency, genetic diseases caused by defective genes, cancer, AIDS, and the like.
  • the second method there is a direct gene transfer method (direct method) in which a target gene is directly injected into a target site such as a patient's body or tumor tissue.
  • the first method of the gene therapy is performed in more detail, for example, as follows. That is, mononuclear cells are collected from monocytes using a blood separation device, and the collected cells are cultured for 72 hours in an appropriate medium such as AIM-V medium in the presence of IL-12 to determine the gene to be introduced. Add the containing vector. To increase the efficiency of gene transfer, centrifuge at 250 ° C for 1 hour at 32 ° C in the presence of prominin, and then centrifuge at 37 ° C for 10 hours. Incubate for 24 hours under% CO2.
  • the cells are further cultured in the presence of IL-2 for 48 hours in an AIM-V medium or the like, the cells are washed with saline, the number of viable cells is calculated, and the gene transfer efficiency is increased.
  • the effect of introducing the target gene is confirmed by the in situ PCR or, for example, when the desired target is an enzyme activity, the degree of the activity is measured.
  • safety measures such as bacterial and fungal culture in cultured cells, the presence or absence of mycoplasma infection, and the search for endotoxin are checked, and after confirming safety, gene transfer of the expected effect volume is performed.
  • the cultured cells containing the target gene are returned to the patient by intravenous drip infusion. Gene therapy is performed by repeating such a method at intervals of, for example, weeks to months.
  • the dosage of the virus vector is appropriately selected depending on the target cell to be introduced. In general, it is preferable to use a dose that gives a virus titer in the range of 1.0 x 10 3 cfu to 1.0 x 10 8 cfu for 1 x 10 8 target cells, for example. No.
  • a virus-producing cell containing a retroviral vector containing a target gene and, for example, a patient's cell are co-cultured and transferred to the target cell. It is also possible to adopt a method of introducing children.
  • the second method (direct method) of the gene therapy of the present invention it is determined in advance by a preliminary experiment outside the body whether or not the objective gene is to be introduced by the introduced gene. Search for vector gene cDNA by PCR method, confirmation by in situ PCR method, or increase of specific activity, which is the desired therapeutic effect by targeted gene transfer, and increase or proliferation of target cells It is desirable to confirm the effect of gene transfer such as suppression.
  • An example of applying the gene therapy method of the present invention to a malignant tumor is, for example, after ingesting cancer cells from a patient, performing enzymatic treatment and the like to establish cultured cells, for example, retrospectively. After introducing a desired gene into a cancer cell by using a virus and selecting it by G418 treatment, measuring the expression level of IL-112 and the like (in vivo), and then performing radiation treatment It can be used to illustrate how to administer and inoculate the patient's tumor in or near the tumor.
  • a promoter specific to each tissue can be preferably used.
  • Specific examples include, for liver, albumin, -phytoprotein, a! -Antitrypsin, trans-ferrin, and trans-stille.
  • examples thereof include carboxylate hydrolase I, and carcinoembrogen antigen.
  • estrogens, aroma chromosome cytochrome P450, cholesterol side chain cleavage P450, 17 phanolefar hydroxylase P450 Examples are shown below.
  • Examples of prostate include prostate antigen, gp91-fox gene, prostate-specific culle, and the like.
  • breasts examples include erb-B2, erb- ⁇ 3, ⁇ -casein, ⁇ -lactoglobin, whey protein, and the like.
  • the activator protein C-loglobulin can be exemplified.
  • skin there may be mentioned, for example, 14-14 keratin, human keratin 1 or 6, and leucrin.
  • thyroid gland examples include thyroglobulin, calcitonin, and the like.
  • steocalcin For the kidney, Renin, liver, bone, kidney, phosphatase, erythropoietin, etc.For the kidney, amylase, PAP1, etc. Can be illustrated.
  • HSV Herpes simplex inores — thymidine kinase
  • TK nucleotide analog gancyclovir
  • Another gene therapy approach is to bind to the target cell surface
  • An example of the method is to prepare an immunoliposome containing the antibody for binding to the pancpin gene of the present invention and to selectively and efficiently introduce the embedded cDNA into target cells.
  • combined gene therapy in which the above-mentioned virus vector containing the site force gene and the adenovirus containing the suicide gene are simultaneously administered is also possible. These methods are at the level of those skilled in the art.
  • a pharmaceutical composition or pharmaceutical preparation comprising a pharmaceutically effective amount of the gene-introducing vector of the present invention or a cell into which the target gene has been introduced, together with a suitable pharmaceutical carrier or diluent.
  • Gene therapy agents comprising a pharmaceutically effective amount of the gene-introducing vector of the present invention or a cell into which the target gene has been introduced, together with a suitable pharmaceutical carrier or diluent.
  • Pharmaceutical carriers that can be used in the above pharmaceutical composition include fillers, bulking agents, binders, humectants, disintegrants, and surface active agents that are usually used according to the use form of the formulation. And diluents and excipients such as active ingredients and lubricants. These can be selected and used as appropriate according to the dosage unit form of the obtained preparation.
  • various forms can be selected according to the purpose of treatment, and typical examples are tablets, pills, powders, powders, granules, capsules It includes solid dosage forms such as agents and liquid dosage forms such as solutions, suspensions, emulsions, syrups and elixirs. These are updated Oral, non-transdermal (intratubular or injectable), nasal, vaginal, suppository, sublingual, ointment, etc. According to the method, compounding, molding or preparation can be performed.
  • the pharmaceutical preparation containing the vector for transfection of the present invention includes a retrovirus vector in which the vector is embedded in ribosomes or a desired gene is included.
  • a retrovirus vector in which the vector is embedded in ribosomes or a desired gene is included.
  • Prepared in the form of cultured cells infected with the virus (these are contained in phosphate buffered saline (pH 7.4), Ringer's solution, and injections for intracellular compositions) It can also be prepared in such a form as to be administered, or it can be prepared in such a form that it is administered together with a substance that enhances the efficiency of gene transfer such as protamine.
  • the preparation of the present invention is formed into a tablet form, for example, an excipient, a binder, a disintegrant-surfactant, a disintegration inhibitor, an absorption enhancer, and a humectant which are commonly used as a carrier for the preparation are used.
  • Adsorbents and lubricants can be used.
  • Capsules are prepared by mixing the active ingredient of the present invention with various conventional pharmaceutical carriers and filling the mixture into a hard gelatin capsule or a soft capsule according to a conventional method.
  • Liquid dosage forms for oral administration include conventional inert diluents such as pharmaceutically acceptable solutions containing water, and the like. They can also contain adjuvants such as wetting agents, emulsions and suspending agents. These are prepared according to a conventional method.
  • liquid dosage forms for parenteral administration such as sterile aqueous to non-aqueous solutions, emulsions, suspensions, etc.
  • diluents include, for example, water, ethyl alcohol, propylene glycol Recohol, polyethylene glycol, ethoxylate, isosteolinol alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester and olive oil Vegetable oils such as oil can be used.
  • injectable organic esters for example, ethyl oleate and the like can be blended.
  • solubilizing agents such as buffers, wetting agents, emulsifying agents, suspending agents, preservatives, and dispersing agents.
  • Sterilization can be performed by, for example, filtration through a bacterial retention filter, blending of a bactericide, irradiation treatment, and heat treatment. They can also be prepared in the form of sterile solid compositions, which can be dissolved in sterile water or a suitable sterilizable medium immediately before use.
  • the carrier for the preparation may be, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and semi-synthetic gels. You can use Celide etc.
  • diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, and propylen Vegetable oils such as lenglycol, polyethylene glycol, silicone, bentonite and olive oil can be used.
  • composition for nasal or sublingual administration can be prepared according to a conventional method using well-known standard excipients.
  • the drug of the present invention may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.
  • the administration method of the above pharmaceutical preparation is not particularly limited, and is determined according to various dosage forms, patient age, gender and other conditions, degree of disease, and the like.
  • tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered, injections are used alone or mixed with normal replacement fluids such as glucose and amino acids to be intravenous Administered directly, if necessary alone.
  • Transtubular intramuscular, intramuscular, intradermal, subcutaneous or intraperitoneal administration; suppositories administered rectally; vaginal agents administered vaginal Nasal preparations are administered intranasally, sublingual preparations are administered buccally, and ointments are topically administered transdermally.
  • the amount of the active ingredient of the present invention to be contained in the pharmaceutical preparation and the dose thereof are not particularly limited, and the desired therapeutic effect, It is selected from a wide range according to the administration method, treatment period, patient age, gender and other conditions.
  • the dosage of a retroviral vector containing the desired gene as a pharmaceutical preparation is 1 kg of adult body weight per day, for example, as the titer of retrovirus.
  • the pressure is about lxl O 3 pfu, and about 1 ⁇ 10 15 pfu.
  • the preparation may be administered once or several times a day, or may be administered intermittently at intervals of one to several weeks. Preferably, it can be co-administered with a substance such as protamine, which enhances gene transfer efficiency, or a preparation containing the substance.
  • a substance such as protamine, which enhances gene transfer efficiency, or a preparation containing the substance.
  • the gene therapy according to the present invention When the gene therapy according to the present invention is applied to the treatment of cancer, the above-described various gene therapies can be appropriately combined (combined gene therapy). Combination therapy, immunotherapy, etc. can be used. Furthermore, the gene therapy of the present invention, including its safety, can be performed with reference to the NIH guideline [Recombinant DNA Advisory
  • Figure 1 shows the fragmentation of the amplified cDNA according to Example 1 (Part 2).
  • FIG. 2 is a diagram comparing the homology in the amino acid sequence between the expressed protein of the pancpin gene of the present invention and a known protein.
  • FIG. 3 is a photograph showing the result of examining the position of the gene of the present invention on the chromosome.
  • Figure 4 shows three types of normals according to (1) of Example 1.
  • FIG. 5 is a photograph showing the result of analyzing the pancpin gene mutation in spleen cancer according to (1) of Example 1.
  • FIG. 6 is a schematic diagram of the construction of a vector for introducing a gene of the present invention according to Example 2.
  • FIG. 7 is a photograph showing the expression of TSA204 by RT-PCR.
  • Figure 8 shows the effect of TSA204 on cell growth.
  • FIG. 9 is a photograph showing the results of SDS-PAGE of the recombinant pancpin protein prepared in Example 3.
  • FIG. 10 is a photograph showing the specific reactivity of the monoclonal antibody obtained in Example 3.
  • FIG. 11 is a photograph showing the result of immunostaining of a human spleen section performed using the above monoclonal antibody.
  • Example 1 shows a preparation example of the cells to be transformed
  • Example 3 shows a preparation example of a monoclonal antibody having specific binding property to the pancpin protein of the present invention.
  • the scope of the present invention is not limited to these examples.
  • c DNA is [a - 33 P] labeled by CTP (manufactured by Cane catcher arm Ltd.) 3, in the presence of Ichia anchors de 'primer one
  • PCR amplification of this cDNA was performed as follows. That is, each of the 201 PCR mixes The combined solution is 2 ⁇ 1 RT reaction mixture, 2 1 10 X
  • PCR buffer manufactured by Takara
  • anchors de Oh Li GORE - d T ply Ma first and of 2 5 pmol 5' - Pla i Mar (No. 2 0, 5, -GATCTGACAC-3 'with a 10'-mer dexoxy lignonucleotide (primer) having an arbitrary sequence of 3'.
  • the PCR reaction was performed under the following conditions.
  • a radioactive ink is marked on a 3 MM filter paper on which dried gel has been placed in advance, and the band containing the desired cDNA can be obtained by combining this with the autoradiogram. After cutting the included gel together with 3 MM filter paper,
  • the PCR product was converted to pUC118 (Yukakara).
  • Fetal brain Fetal brain
  • adult brain A.
  • PCR amplification was achieved with one primer combination.
  • TSA204 This cloned and sequenced PCR product was named TSA204. This product consisted of 235 nucleotides. F A S T A Program
  • the normal human spleen cDNA library was constructed using Oligo (dT) -primed human normal spleen cDNA and Uni ZAP TM XR (Stratagene). did. 1 X 1 0 6 single off ⁇ one, Ri by di click loans, [shed one 3 2 P] - the scan click Li scratch c DNA fragments labeled by d CTP and used as a probe The wing was performed. Positive clones were selected, and their inserted cDNA portions were subcloned into pBluescript II SK (-) by in vivo excision. I did it.
  • the present inventors confirmed about 200 positive plaques against TSA204. From this result, the amount of transcription between total RNA was calculated to be approximately 0.02%.
  • the TSA204 (pancpin) phenolic length cDNA sequence contains a 125 amino acid protein that encodes a 450 amino acid protein with a calculated molecular weight of 416 179 Da. It contained 1,465 nucleotides, including the open reading frame.
  • pancpin protein sequence contained a signal sequence localized near the amino terminus, which is believed to promote secretion.
  • the pancpin protein contained three cysteine residues (Cys21, Cysl26 and Cys265) and three potential N-glycosylation sites (Asn202, Asn207 and Asn306).
  • FIG. 2 shows the results of comparisons with known proteins in public databases using the FASTA program.
  • Figure 2 shows the amino acids of each protein in one letter and their sequences compared. In the figure, the same amino acids are highlighted.
  • pancpin protein was found to be plasminogen activator-inhibitor-2 (PAI-2) [Ye RD, et al., J. Biol. Chem., 262, 3718]. -3725 (1987) 3, squamous cell carcinoma antigen (SCCA-1) [Schneider S, S., et al., Proc. Natl. Acad. Sci., USA, 92, 3147-3151. (1995)], Monocyte Neutrophil Elastase Inhibitor (EI) C Remold-0 'Donnel, E., Proc. Natl. Acad.
  • PAI-2 plasminogen activator-inhibitor-2
  • SCCA-1 squamous cell carcinoma antigen
  • EI Monocyte Neutrophil Elastase Inhibitor
  • pancpin protein is a new member of the serpin family (Oboanorebmin • Family).
  • the human tissues used were heart (heart), brain (brain), placenta (placenta), lung (lung), and liver.
  • pancpin gene expression is different between human spleen cancer cell lines and squamous carcinoma tissues, four cell lines (BXPC-3 (adenocarcinoma 'undifferentiated, Cancer Invest., 4, 15-23 ( 1986)), ASPC — 1 (Metastatic adenocarcinoma, J. Natl. Cancer Inst., 67, 563-569 (1981)), PANC — 1 (Epithelioid, mast duct carcinoma, Int. J, Cancer, 15, 741-747 (1975)) and MIAP a Ca-2 (adenocarcinoma, Int. J.
  • RNAs were isolated from cell lines and spleen cancer tissues using ISOGEN (manufactured by Wako). All of 1 0 ⁇ 1
  • RNA Treat the RNA with 10 units of RNase-free DNase I (Belinger-Meinheim) for 15 minutes, extract twice with phenol-chloroform and ethanol twice. And precipitated.
  • Single-stranded cDNA was synthesized using Superscript II TM reverse transcriptase (manufactured by Life Technology Co., Ltd.) using oligo d (T) and random primers. 2 ⁇ l of each product was used for PCR amplification.
  • Primers ⁇ 1 and ⁇ 2 of the nucleotide sequence shown as SEQ ID NO: 4 and SEQ ID NO: 5 in the sequence listing were synthesized using an automatic oligonucleotide synthesizer, and these were synthesized for 25 nucleotides. It was used for PCR amplification of DNA.
  • the PCR reaction is 100 ng DNA, 1 // M each primer-, 2.5 mm dNTP and 0.25 U ExTaq
  • the test was performed in a 51 solution containing DNA polymerase (Yukara).
  • the PCR products were separated on a 1.5% agarose gel.
  • pancpin expression was not found in all cell lines and most cancer tissues.
  • pancpin gene in spleen cancer was PCR-mutated in the pancpin gene (exons 1, 2, 3, and 4) by PCR—SSCP. Cleaned.
  • the PCR primers used had a sequence designed in the intron to amplify each exon, and their base sequences were those for exon 1 (SEQ ID NOs: 6 and 7). For exon 2, SEQ ID NOs: 8 and 9; for exon 3, SEQ ID NOs: 10 and 11; and for exon 4, SEQ ID NOs: 12 and 13; Each is shown. Each of these primers was synthesized using an automatic oligonucleotide synthesizer.
  • PCR products in the band indicated by SSCP analysis were submitted to TA Cloning Vector (Novagen).
  • the clones were cloned and the 10 clones were sequenced using an ABI 377 automatic sequencer.
  • PCR products showing the above-mentioned two possibly mutated bands were subcloned into a TA cloning vector (Nonogen) and sequenced 10 clones each.
  • a gene encoding the full length of TSA204 was incorporated into a pcDNA3.1 vector (manufactured by Invitrogen), and the vector for introducing the pancpin gene according to the present invention was used.
  • -Was constructed as follows. An outline of the construction is shown in Figure 6.
  • the pcDNA3.1 vector used in this example has a promoter of the cytomegalovirus, and has resistance to the neomycin resistance gene and ampicillin resistance as drug resistance genes.
  • This is a 5.5 kb vector having a gene, and one of the vectors has a recognition site such as restriction enzymes BamHI, XhoI, and SeaI. Its features are shown in Figure 6 above.
  • P CMV shows site menu Garoui Noresu early promoter Roh Enha capacitors scratch.
  • BGHpA shows Bovine Growth Hormone polyadenylation signal.
  • ⁇ ori indicates the f1 fuzzy replica-short origin.
  • SV40 ori indicates the SV40 initial promoter and origin.
  • Neomycin indicates a neomycin resistance gene.
  • SV40 pA shows the SV40 polyadenylate-shion signal.
  • ColEl indicates a high copy number replication origin in E. coli.
  • Ampicilin indicates an ampicillin resistance gene.
  • T7 indicates a ⁇ 7 promoter ⁇ priming site.
  • ATG indicates the start codon (methionine).
  • HIS 6 indicates an epitope tag (histidine repeated 6 times).
  • Anti-Xpress epitope indicates an epitope tag (Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys).
  • EK site indicates the enterokinase cleavage site.
  • Asp718I, Kpnl, BamHI, XhoI, XbaI and ApaI indicate restriction enzymes, respectively.
  • two primers A and B having the base sequences shown in SEQ ID NO: 14 and SEQ ID NO: 15 were automatically transformed into oligonucleotides. These were synthesized using a reotide synthesizer, and these were used as amplification primers.
  • the RF region of the nucleotides of the full length of 1252 nucleotides (TSA2O04 in the figure) gene (1218 bp), which further contains a termination codon, whose base sequence is shown in SEQ ID NO: 3) was subcloned as follows.
  • the A primer contains a BamHI site (GGATCC), and the B primer contains an XhoI site (CTCGAG).
  • PCR is performed using the above primers and cDNA derived from normal human spleen (pancreas marathon ready cDNA: manufactured by Clontech) to amplify the cDNA.
  • cDNA derived from normal human spleen (pancreas marathon ready cDNA: manufactured by Clontech)
  • a product of about 1200 bases in length was obtained, which was then treated with a restriction enzyme and purified (this is shown as “BamHI-XhoI fragment” in FIG. 6).
  • pancreatic cancer cell line PANC-1 epithelial ductal carcinoma: Int. J. Cancer, 15, 741-747 (1975)
  • the ampicillin-resistant gene sequence site of the pc vector containing the full-length gene of the present invention was cleaved with Sea I at a single site to obtain linear DNA, and the cationic ribosome Cell FECTIN TM
  • TM—TPS N, N ', N'', N-tetramethyl-N, N, N, N''-tetrapalmityl spermine: DOPE (dioleyl phosphatidylethanol amine) molar ratio 1: Transfected in the presence of 1/5 ribosomes.
  • the pcDNA3.1 vector is designed to acquire neomycin resistance in cells into which the gene has been introduced, so that it can be screened by neomycin treatment.
  • eight stable transformants expressing TSA204 were obtained.
  • two stable transformants were obtained by transfecting empty vectors into which nothing was inserted.
  • the expression of the TSA204 gene was expressed by a nucleotide sequence corresponding to a part of the nucleotide sequence of the TSA204 gene shown as SEQ ID NO: 16 and: 17 in the sequence listing. RT-PCR using synthetic primers (C and D) having Synthetic primer C was used for the TSA204 gene.
  • FIG. 7 is a photograph showing the result of electrophoresis of the RT—PCR product, and ⁇ X174 Hae II was used as the marker.
  • PCR product of about 400 bases was observed in the stable transformant of TSA204, but was observed in the empty vector and untreated PANC-1 cells. Clearly not.
  • Figure 8 shows the results.
  • the vertical axis PANC - 1 number of cells (X 1 0 4 Z m 1 )
  • the horizontal axis indicates the number of culture days (Day).
  • the stable transformant of the TSA204 gene (shown as TTSA2004 TransformantsJ in the figure) was untreated (shown as “Wild” in the figure) and the stable transformant of the empty vector (shown as “Wild” in the figure) Compared to "Vector onlyj" in the figure, it was found that proliferation was significantly suppressed.
  • the TSA204 protein functions in the cell to negatively regulate the cell growth ability. Therefore, the TSA204 gene of the present invention
  • a gene transfer vector containing a gene pancpin gene
  • SEQ ID NOs: 18 and: Primers A and B having the sequence shown in 19 were synthesized using an automatic oligonucleotide synthesizer.
  • Primer A contains ECoR1 site (GAATTC) and Primer B contains Xho1 site (CTCGAG).
  • PCR was performed using cDNA (pancreasmarathon ready cDNA: CLONTECH) derived from normal human spleen to amplify the cDNA, and a product of about 860 bases (pancpin protein 120 — Having a DNA sequence encoding amino acid).
  • cDNA pancreasmarathon ready cDNA: CLONTECH
  • Glutathione S-Transferase A fusion protein expressed as a target protein in the cells expressed as a fusion protein was transferred to a buffer (20 mM Tris / HCl pH7.5, ImM EDTA, ImM DTT, Elution was carried out by sonication under 10% sucrose, and the affinity chromatography was performed using Glutathione Sepharose 4B (Amersham pharmacia biotetech) heat. Purified by chromatography.
  • the target protein was eluted from the beads using a (glutathione) solution and confirmed by SDS-PAGE.
  • lane 1 is a molecular weight marker 1 (molecular weight: 6400, 550, 50,000, 350,000), and lane 2 was purified.
  • the target protein pancpin protein (120-406)) is shown.
  • the target protein purified as above is mixed with complete Freund's adjuvant (DIFCO LABORATORIES) to prepare an emulsion, which is made up to a protein mass of 5 g per animal. ,
  • mice Female, 6-8 weeks old, 25-30 g were immunized by subcutaneous injection.
  • spleen cells were collected and fused with mouse myeloma cells P3U1 by the PEG method.
  • G—HOT 1 This hybrid dorma was established on August 21, 1996, by the Ministry of International Trade and Industry of the Ministry of International Trade and Industry of 1-3-3 Higashi, Tsukuba City, Ibaraki Prefecture, Japan (zip code: 305-85-666). Accession number to the Institute of Bioscience and Biotechnology, Japan FERMBP — accepted as 6 4 6 9
  • the desired monoclonal antibody was produced from the hybridoma G-H0T1 obtained above and purified. That is, the BALBZc mice, which had been inoculated with pristane (2,6,10,14—tetramethinolepentadecane, Anolet Dritzi) two to three days before the administration of the hybridoma, Domas were intraperitoneally administered in an amount of 1 ⁇ 10 6 Z animals. Ten to fourteen days after administration, the accumulated ascites was collected, collected, and purified using a kit from noisyorad (BIO-RAD MAPS-II Kit).
  • the subclass of the purified antibody thus obtained was obtained from the Stratagene kit (Iso Detect Isotyping Kit,
  • ES blotting and immunostaining were performed using the purified monoclonal antibody prepared above.
  • FIG. 10 shows the results of the above Western blotting.
  • lane 1 is a control lysate (lysate, lysate) of control E. coli without any transformation
  • lane 2 is an expression vector for the target pancpin protein (120-406).
  • One is a lysate (lysate, lysate) of Escherichia coli that has been transformed and IPTG-induced expression. From the lane 2 in the figure, it is clear that the band corresponding to the pancpin protein (120-406) is specifically stained. This indicates that the monoclonal antibody derived from G-HOT1 specifically reacts with the pancpin protein (120-406) used as an immunogen.
  • Immunostaining was performed as follows. That is, fresh human spleen was fixed in formalin, paraffin sections were prepared, and the sections were avidin-biotinylated enzyme using VECTORAIN's kit (VECTASTAIN ABC Kit, Vector). Staining was performed by the complex method (ABC method). As a primary antibody, ascites fluid of a G-HOT 1 -administered mouse was diluted 500-fold with PBS (containing 0.1% BSA).
  • Fig. 11 shows the results of immunostaining of human slices.
  • pancpin protein which stains black, is observed near the nucleus, which stains blue, indicating that the cytoplasm near the nucleus of the acinar cells is strongly stained.
  • the monoclonal antibody against pancpin protein according to the present invention specifically recognizes pancpin.
  • this antibody can be said to be immunohistochemically effective as follows. That is, the antibody of the present invention is expressed in normal spleen, and its expression is lost in cancer. W
  • pancp inn at the protein level, which has been proven at the 82 mRNA level, and is therefore effective both in diagnosis and in elucidating the mechanism of carcinogenesis.
  • a novel pancpin protein having an effect of suppressing spleen tumor formation, tumor progression and metastasis, a gene encoding the same, and an antibody having specific reactivity with the protein. These are useful for elucidation of cancer such as spleen cancer and canceration, and for diagnosis, prevention and treatment thereof.
  • a vector for gene transfer containing a pancpin gene for gene therapy, a cell having the vector, a gene therapy composition containing the vector or the cell as an active ingredient
  • the present invention provides a gene therapy method utilizing the method, particularly a technique for treating cancer growth such as spleen cancer and inhibiting metastasis5.

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Abstract

Cette invention se rapporte à un gène contenant une séquence de bases codant toute la séquence d'acides aminés représentée par le numéro d'identification de séquence 1 (SEQ ID NO:1) ou une partie de celle-ci; à une protéine obtenue par l'expression de ce gène; à un anticorps spécifique contre cette protéine; à un vecteur de transfert génique contenant ce gène; à des cellules portant ce vecteur; à une composition de thérapie génique contenant ce gène comme principe actif; et à un procédé de thérapie génique. La protéine obtenue par l'expression de ce gène a pour effet d'inhiber la formation, la progression et la métastase de tumeurs dans le pancréas. Elle peut par conséquent servir notamment à mettre en évidence, diagnostiquer, prévenir et traiter les cancers tels que le cancer du pancréas et les transformations malignes de celui-ci. Un tel procédé de thérapie génique a pour effet de supprimer la prolifération des cancers tels que le cancer du pancréas et d'inhiber la métastase de celui-ci.
PCT/JP1998/003841 1997-09-01 1998-08-28 GENE pancpin ET COMPOSITION DE THERAPIE GENIQUE WO1999011786A1 (fr)

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JP9/252770 1997-09-01
JP25277097 1997-09-01
JP4431298 1998-02-10
JP10/44312 1998-02-10

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7820438B2 (en) 2002-06-18 2010-10-26 Eisai R&D Management Co., Ltd. Primary cultured adipocytes for gene therapy

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034957A1 (fr) * 1995-05-02 1996-11-07 Incyte Pharmaceuticals, Inc. Serpine derivee du pancreas
WO1998007735A1 (fr) * 1996-08-16 1998-02-26 Human Genome Sciences, Inc. Inhibiteur de l'activateur du plasminogene derive du pancreas

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996034957A1 (fr) * 1995-05-02 1996-11-07 Incyte Pharmaceuticals, Inc. Serpine derivee du pancreas
WO1998007735A1 (fr) * 1996-08-16 1998-02-26 Human Genome Sciences, Inc. Inhibiteur de l'activateur du plasminogene derive du pancreas

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7820438B2 (en) 2002-06-18 2010-10-26 Eisai R&D Management Co., Ltd. Primary cultured adipocytes for gene therapy
US8071085B2 (en) 2002-06-18 2011-12-06 Eisai Co., Ltd. Primary cultured adipocytes for gene therapy

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