WO2003102186A1 - Adn induisant l'expression specifique des cellules cancereuses et vecteur d'expression specifique des cellules cancereuses - Google Patents

Adn induisant l'expression specifique des cellules cancereuses et vecteur d'expression specifique des cellules cancereuses Download PDF

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WO2003102186A1
WO2003102186A1 PCT/JP2003/006844 JP0306844W WO03102186A1 WO 2003102186 A1 WO2003102186 A1 WO 2003102186A1 JP 0306844 W JP0306844 W JP 0306844W WO 03102186 A1 WO03102186 A1 WO 03102186A1
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dna
nucleotide sequence
cancer
vector
gene
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Japanese (ja)
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Kohji Egawa
Katsuo Noguchi
Eri Kuwada
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Medinet Co., Ltd.
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Priority to AU2003241985A priority Critical patent/AU2003241985A1/en
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Publication of WO2003102186A1 publication Critical patent/WO2003102186A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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 cancer cell-specific expression-inducing DNA which specifically causes gene expression in cancer cells, a recombinant vector containing this DNA, and particularly, this DNA and a target protein or a target polypeptide or R
  • MAGE peptides identified from melanoma cells in cancer immunology in recent years have been the starting point, and cancer antigens are being identified actively.
  • the name of the antigen is that the abnormal protein produced by cell carcinogenesis appears on the cell surface following the flow of the antigen presenting mechanism. Sex is also specific to individual cancers. In the living body, no detailed studies have been made on antigens that are strictly specific and commonly produced in cancer and on the mechanism by which such substances are expressed specifically in cancer cells.
  • the present inventor studied the intrinsic anti-cancer reactivity of a living body using mice. As a result of the study, it was revealed that there are T cells that respond commonly to various cancer cells in the immune response related to the anticancer response. Therefore, assuming that these T cells recognize antigens commonly present on the surface of various cancer cells, we sought what the antigens are.
  • the Qa-2 antigen is an antigen expressed on the surface of normal lymphocytes of a mouse strain, and is one of the non-classical MHC class I antigen genes on the chromosome 17 QaZT1a region of the mouse. It is known that the gene encodes a Q9 gene having almost the same structure as that of Q7. However, the cancer cell surface molecule detected by the Qa-2 antibody is a different molecule from the Qa-2 antigen, which is the non-classical MHC class I antigen gene on mouse chromosome 17 QaZT1a region. One of the groups, the Q5 antigen gene, was shown to encode. In addition, the mouse experimental cancer cells must express the Q5 antigen, a product of the Q5 gene, in all experimental cancers examined, regardless of the mouse strain, organ, or cause of canceration. It was confirmed.
  • the Q5 antigen partially shares antigenicity with Qa-2 mice (mice that do not express the Qa-2 antigen).
  • Qa-2 + mice can be treated with lymphocytes of Qa-2 + mice (mice expressing the Qa-2 antigen) as antigens.
  • an experiment was performed to transplant Q5 + Qa-2 cancer cells (cancer cells that express the Q5 antigen and do not express the Qa-2 antigen). Resistance to transplantation was induced.
  • anti-Qa-2 immunization was performed after transplantation of cancer cells, and it was revealed that resistance to metastasis of Q5 + Qa-2 ⁇ cancer cells was also induced.
  • the Q5 antigen is not an antigen produced by gene mutation or an abnormal peptide derived therefrom, but an expression of the original Q5 gene which is also present in normal cells. However, the mechanism by which the Q5 antigen gene is specifically expressed in cancer cells was unknown. Disclosure of the invention
  • the present invention focuses on the mechanism by which the Q5 antigen gene is specifically and commonly expressed in cancer cells, and elucidates the DNA that specifically induces the expression of the Q5 antigen gene in cancer cells.
  • the purpose of the present invention is to provide diagnostic methods, immunotherapy methods, prophylactic methods, recurrence prevention methods, cancer gene therapy methods, and drugs useful for these methods.
  • the above-mentioned cancer includes not only a state of cancer but also a state in which cells mutated due to a virus infection or the like, that is, a “pre-cancerous state”.
  • pre-cancerous state refers to a state in which, for example, a certain gene is mutated, but has not become an infinitely proliferating state.
  • the present inventors have studied the mechanism by which the Q5 antigen gene is specifically expressed in cancer cells. In the meantime, if the Q5 antigen is specifically expressed in cancer cells, the Q5 antigen gene is not transcribed in normal cells, but is transcribed only as the cells become cancerous. The idea was that there should be a regulatory mechanism that governs transcription. If there is expression-inducing DNA in the living body that causes gene expression in a cancer cell-specific and common manner, this DNA can be used to develop gene therapy for cancer, and develop immunotherapy and immunoprevention methods. It is considered to be extremely useful because it can be used for the development of diagnostic drugs for cancer and general cancer.
  • the present invention provides a cancer cell-specific expression-inducing DNA that induces gene expression in a cancer cell-specific manner as described below, a cancer cell-specific expression vector containing the same,
  • the present invention provides a transformant by introduction, and further provides these uses, for example, a method for detecting cancer, a diagnostic agent, a therapeutic method, a preventive method, a method for preventing recurrence, a cancer gene therapeutic method, and the like.
  • a method for detecting cancer for example, a diagnostic agent, a therapeutic method, a preventive method, a method for preventing recurrence, a cancer gene therapeutic method, and the like.
  • FIG. 1 is a diagram showing a band obtained by examining the expression of a group of MHC class Ib genes in a mouse tumor cell line by RT-PCR.
  • FIG. 2 is a diagram showing bands obtained as a result of examining a Q5 transcript (mRNA) for each organ of a normal AKR (H-2 k ) mouse.
  • FIG. 3 is a view showing a band as a result of examining a Q5 transcript (mRNA) for each organ of a normal BALBZc (H-2 d ) mouse.
  • Figure 4 is a diagram showing a band of normal C57BL / 6 (H-2 b ) Results of examining the Q 5 transcripts for each organ of the mouse (mRNA A).
  • FIG. 5 is a diagram showing bands as a result of examining the Q5 transcript (mRNA) of each organ of BW5147-carrying AKR mouse.
  • FIG. 6 is a diagram showing a band as a result of examining the Q5 transcript (mRNA) of each organ of the MH134 tumor-carrying C3HZHe mouse.
  • FIG. 7 is a diagram showing bands obtained as a result of examining the expression of CAR in healthy human peripheral blood by the RT-PCR method.
  • FIG. 8 is a diagram showing a restriction map and an adenovirus vector using lacZ for the repo overnight gene.
  • FIG. 9 shows a restriction enzyme map and a plasmid DNA configuration diagram using luciferase as a reporter gene.
  • FIG. 10 is a tissue staining diagram (magnification: 200 times) showing the expression of lacZ in the human cancer cell line HeLa.
  • Figure 11 is a histological staining diagram showing the expression of lacZ in the human cancer cell line DU145 ( ⁇ The rate is 200 times).
  • FIG. 12 is a tissue staining diagram (magnification: 200 times) showing the expression of lacZ in the human cancer cell line MDA-MB-231.
  • FIG. 13 is a tissue staining diagram (magnification: 200 times) showing the expression of lacZ in the human cancer cell line DLD-1.
  • FIG. 14 is a relative activity diagram showing luciferase expression in the human cancer cell line HeLa.
  • FIG. 15 is a relative activity diagram showing luciferase expression in human cancer cell line DU145.
  • FIG. 16 is a relative activity diagram showing the expression of luciferase in the human cancer cell line MDA-MB-231.
  • FIG. 17 is a histostaining (magnification: 200-fold) showing the expression of lacZ in normal cells (PBMC).
  • FIG. 18 is a histological staining diagram (magnification: 200 times) showing the expression of lacZ in normal cells (thymus).
  • FIG. 19 is a histological staining diagram (magnification: 200 times) showing the expression of lacZ in normal cells (testis).
  • FIG. 20 is a histological staining diagram (magnification: 200 times) showing the expression of lacZ in normal cells (liver).
  • FIG. 21 is a graph showing the inhibitory effect of local administration of AdZQ5-H-2D d on the growth of MH134 liver cancer in mice bearing MH134 liver cancer.
  • the present inventors focused on the 3.9 kbp region 5 ′ upstream from the translation initiation codon of the mouse Q5 antigen gene as a region considered to induce cancer cell-specific expression of the Q5 antigen.
  • the function of cancer cell-specific expression induction was confirmed as follows (i) to (vi).
  • the plurality of documents described below are cited and incorporated herein.
  • the G3 PDH gene in FIG. 1 is a housekeeping gene and is also shown as a positive control.
  • the expression control level of the Q5 antigen is basically a transcription level.
  • adenovirus receptor (CAR), which was recently discovered, is not found in lymphocytes, (R P. Leon, T. Hedlund, S J. Meech, S Li, J. Schaack, SP Hunger, R C. Duke and J. DeGregoli, Pro atl. Acad. Sci. USA.vol. 95, pp. 13159 (1998), S. Huang, RI. Endo and G R. Nemerow, J. Virology, vol. 9 , pp. 2257 (1995)) It is known that adenovirus cannot normally infect lymphocytes.
  • CAR adenovirus receptor
  • combining the transcriptional regulatory region of the Q5 antigen gene with adenovirus may provide a complete cancer cell-specific expression induction vector that is not expressed in normal cells but is expressed in cancer cells. it is conceivable that. Therefore, the expression of CAR in healthy human peripheral blood was confirmed by RT_PCR using CAR-specific primers. As a result of examining the peripheral blood of six healthy volunteers, it was confirmed that none of the six volunteers expressed CAR (Fig. 7).
  • the base sequence of the 5 'upstream DNA of the Q5 antigen gene was only identified up to 210 bp [GenomeNet Accession No. XI 6423, (S. Watts, A C. Davis, B Gaut, C Wheeler, L Hill and RS Goodenow, The EMBO J., vol.8, pp.1749 (198 9))]. Therefore, from the AKR-derived BW5147 cells are first H_2 k mice were extracted and purified chromosomal DNA. Then the chromosomal DNA and ⁇ , Q4 k (S. Cc hwemmle, D.Bevec, G.Brem, M B. Urban, P A. Baeuerle and E H.
  • Exo III Exonuclease III
  • the length of the insert DNA of the plasmid in the emerged Escherichia coli colony was confirmed by PCR using primers at both ends of the vector multicloning site, and 17 to 25 clones of different sizes were selected.
  • the nucleotide sequence of the insert portion of each selected clone was decoded by the cycle sequence method. Based on the base sequence decoded from each clone, the base sequence of the entire 4. I kbp region of the insert DNA was joined using the overlapping sequence portion.
  • sequences of SEQ ID Nos: 1, 2, 3, and 4 may be abbreviated as Hind, Bst, Pvu, or Nco at the position of each restriction enzyme.
  • Genomnet DDBJ FASTA A homology search of the determined sequence with Genomnet DDBJ FASTA revealed that none of the sequences had a homologous sequence over the entire region.
  • a plasmid DNA having a 5 'upstream region of the Q5 gene upstream thereof was constructed (Fig. 9), and its transcription activity was quantified.
  • Examination using human cancer cell lines HeLa (cervical cancer), DU145 (prostate cancer), and ViDA-MB-231 (breast cancer) revealed expression of the reporter gene in all cancer cells ( Figures 14, 15, 16).
  • the SV40 promoter was compared with pGL3Q5. Hind, the expression was 50% or more (Table 2).
  • normal cells S There are cell lines that can be cultured in / 'or primary cells that are called normal cells S, but there is no guarantee that they are truly normal. Therefore, the inventors used normal cells of the tumor-bearing mouse //? Vivo system for evaluation in normal cells.
  • Recombinant adenovirus comprising a D NA of the structure shown in FIG. 8, AdZHind-lacZ, A d / Bst-lacZ, was purified Ad / Pvu-lacZ ⁇ AdZNco- lacZ, respectively 3 X 1 0 9 pfu / mouse Each group was administered to 2 to 4 animals, and 3 days later, each organ was taken out and frozen sections were prepared.
  • ⁇ lacZ positive
  • X lacZ negative
  • the corresponding DNA is DNA that induces expression in a cancer cell-specific manner. If a vector containing the DNA is introduced into a cell, any foreign gene linked downstream can be used only when the cell is a cancer cell. To express The inventors have found that the present invention can be performed and completed the present invention.
  • the present invention provides a cancer cell-specific expression-inducing DNA that induces gene expression in a cancer cell-specific manner, and a cancer cell-specific expression vector containing the same.
  • Cancer cell-specific expression induction DNA that induces gene expression in a cancer cell-specific manner, and a cancer cell-specific expression vector containing the same.
  • the DNA of the present invention contains the predicted transcription initiation site of the Q5 antigen gene of the mouse non-classical histocompatibility antigen gene (MHC class Ib gene). Is a DNA having
  • A DNA consisting of at least a portion of the DNA (SEQ ID NO: 1) from 2667 bases to 22 bases 5 'upstream from the transcription start point of the Q5 antigen gene of the mouse nonclassical histocompatibility complex gene (MHC class Ib gene) .
  • E DNA consisting of at least a part of DNA (SEQ ID NO: 1) from 3901 bases to 22 bases 5 'upstream of the transcription start point of the Q5 antigen gene of the mouse nonclassical histocompatibility complex gene (MHC class Ib gene) .
  • a ' a DNA in which an arbitrary number of consecutive base sequences of 1 to 1236 bases from 5 to 3,903 bases are added to the DNA of SEQ ID NO: 1;
  • B ' A DNA in which an arbitrary number of consecutive base sequences of 1 to 624 bases from 1 2043 bases to 1666 bases are further added to the DNA of SEQ ID NO: 2 from 5' upstream 12043 bases to 12666 bases.
  • C DNA in which an arbitrary number of consecutive base sequences of 1 to 627 bases from 1,415 bases to 1,204 bases are added to the DNA of SEQ ID NO: 3.
  • D A DNA in which an arbitrary number of consecutive base sequences of 1 to 584 bases from 5 'upstream to 830 bases to 1,413 bases are further added to the DNA of SEQ ID NO: 4 from 830 bases to 1,413 bases.
  • DNA retains its main function even if one, two or less, or three or less, or several or fewer bases in the sequence are added, deleted, substituted or inserted. Often. Therefore, as long as the DNA of the present invention has the activities (1), (2), and (3) described below, one or several bases or less in the sequences (1-1) to (1-3) described above are included. But also includes added, deleted, substituted or inserted sequences.
  • the DNA of the present invention also includes a sequence in which one or several bases or less in the above-mentioned sequences (1-1) to (1-3) are mutated with a point mutation.
  • the DNA of the present invention includes the above-mentioned sequence portions (1-1) to (1-4) as long as it has the activity (1) or (2) described later.
  • the DNA of the present invention specifically hybridizes with the complementary strand of the above-mentioned sequence (1-1) to (1-6) as long as it has the activity of (1) or (2) described later. To include.
  • the term “specifically hybridize” as used herein refers to hybridization in 0.1 XSSC and 0.1% SDS for at least 68 ° C for 2 hours.
  • the DNA of the present invention has an activity of inducing gene expression downstream thereof in a cancer cell-specific manner.
  • the DNA of the present invention has an activity of inducing gene expression downstream thereof in a cancer cell-specific manner regardless of the type of cancer.
  • DNA of another embodiment of the present invention is a nucleotide sequence complementary to the above-mentioned DNA of (1-1) to (1-7).
  • antisense sequences may function to inhibit the expression of genes downstream of cancer cell-specific expression-inducing DNA.
  • nucleotide sequence is a sequence consisting of a nucleotide having a base of a pyrimidine base or a purine base, and includes DNA, RNA and derivatives thereof.
  • DNA refers to adenine, guanine, cytosine, and thymine as bases, as well as small amounts of methylated bases such as 5-methylcytosine and 6-methylaminopurine. And so on.
  • the DNA of the present invention may be one obtained by screening and isolating a chromosomal DNA library, one obtained by the PCR method, or one synthesized.
  • the vector used in the method for producing DNA of the present invention is not particularly limited as long as it can introduce a gene into a known host such as Escherichia coli.
  • a shuttle vector having a cloning site for a restriction enzyme having no recognition site is selected, and the DNA of the present invention is ligated at this cloning site. This is introduced into an appropriate host such as Escherichia coli, a desired clone is selected, and vector DNA is extracted and purified from the obtained clone.
  • the vector into which the DNA of the present invention is incorporated and specifically expressed in cancer cells is not particularly limited as long as it is a vector used for gene transfer into animal cells.
  • a plasmid vector, a retrovirus vector, an adenovirus Vectors, adeno-associated virus vectors, simple herpes virus vectors, HIV vectors, ribosome vectors and the like can be used. After insertion into the virus vector production shuttle vector, it can be incorporated into the virus vector.
  • adenovirus vectors are preferred in that they are introduced into cancer cells. If the vector contains a promoter sequence for expression of the vector itself, it is preferable to delete that region.
  • the nucleotide sequence encoding the target protein or polypeptide sequence to be incorporated and expressed in the cancer cell-specific expression vector of the present invention may be any nucleotide sequence that can be expressed by the vector (2-1).
  • a nucleotide sequence encoding at least a part of a non-self MHC class I molecule a nucleotide sequence encoding at least a part of a non-self MHC class II molecule, a site force Nucleotide sequence encoding ins, nucleotide sequence encoding site force receptor, nucleotide sequence encoding a polypeptide that induces cell death, nucleotide sequence encoding costimulatory molecules (CD80, CD86, etc.), cancer antigen Nucleotide sequence encoding a tumor suppressor gene Preferred is an antisense nucleotide sequence of an oncogene.
  • RNA sequence encoding the RNA sequence a nucleotide sequence encoding lipozyme (RNA having enzymatic activity) or RNAi (RNA interference) against an oncogene or a mutant gene is preferable.
  • nucleotide sequences Since all of the above nucleotide sequences are known, they can be obtained by cloning from a DNA library by a general method. These nucleotide sequences are chemically synthesized using known methods such as the phosphotriester method and the amidite method. You can also get. It can also be obtained from GeneBank.
  • the cancer cell-specific expression-inducing DNA and the nucleotide sequence encoding the desired protein, polypeptide or RNA are integrated into the vector's cloning site.
  • the method for constructing a cancer cell-specific expression vector using a commercially available adenovirus vector is described below.
  • a shuttle vector having a restriction enzyme cloning site where no recognition site is present in the cancer cell-specific expression induction DNA is selected, and the cancer cell-specific expression induction DNA is ligated at the cloning site. This is introduced into E. coli, a desired clone is selected, and vector DNA is extracted and purified from the obtained clone.
  • a nucleotide encoding the protein, polypeptide or RNA of interest was ligated to the cloning site downstream of the cancer cell-specific expression-inducing DNA, and this was introduced into Escherichia coli, and the desired clone was selected and obtained. Extract and purify vector DNA from clones. From the obtained vector DNA, a region containing the cancer cell-specific expression induction DNA and the above-mentioned nucleotide sequence is cut out with a restriction enzyme (a cut surface having a blunt end) and taken out. Thus, the desired DNA was amplified in E. coli.
  • This fragment is inserted into a cosmid vector Yuichi pAxcw, which is an adenovirus vector dephosphorylated after digestion with a restriction enzyme (Swal), and packaged using a packaging kit (GigapacklllXL (Stratagene)). .
  • This is infected to VCS-257 E. coli or the like, and cosmid DNA is purified from the resulting colonies according to a conventional method, and a desired vector is selected.
  • the obtained vector was used together with DNA-TPC for 293 cells by the calcium phosphate method.
  • the cells are infected and cultured for 7 to 15 days in a 96-well plate. Collect the dead cells and their medium from the wells in which 293 cells have died, freeze-thaw six times, and centrifuge. The supernatant is used as the primary virus solution.
  • the virus fluid contains "recombinant adenovirus particles" consisting of cancer cell-specific expression-inducing DNA + adenovirus vector DNA incorporating DNA encoding the desired protein / polypeptide or RNA. .
  • the primary virus solution is added to the culture supernatant of 293 cells and HeLa cells, and 293 cells are completely killed and HeLa cells are selected not to die. In this step, those contaminated with wild-type adenovirus are eliminated, and the target recombinant adenovirus amplification product is selected. Dead cells and their medium are collected, freeze-thawed six times, and then centrifuged as in the preparation of the primary virus solution, and the resulting supernatant is used as the secondary virus solution. . On the other hand, DNA is prepared from dead cells, and it is confirmed whether or not the desired vector DNA is obtained. If the DNA is confirmed, a tertiary virus solution and a quaternary virus solution are further prepared.
  • the tertiary virus solution is used to infect 293 cells, and the vector whose desired structure can be confirmed is designated as a cancer cell-specific expression vector. If a desired amount of virus particles is obtained, it is not necessary to prepare a quaternary virus solution, and if necessary, a higher-order virus solution may be prepared. After confirming that no natural virus is contaminated, the quaternary virus solution is purified by density gradient centrifugation or the like, and the titer is determined. The virus solution prepared as described above is applied to gene therapy.
  • a gene therapy method for cancer and a cancer vaccine can be developed by using the cancer cell-specific expression vector of the present invention.
  • the cancer cell-specific vector of the present invention is introduced into a cancer cell, and the integrated target nucleotide is expressed.
  • the cancer cells are not particularly limited as long as they are eukaryotic cells, but mammalian cells such as humans and mice are preferable from the viewpoint of usefulness.
  • the cancer cells may be present in the living body or may be taken out of the living body.
  • cancer cells are prepared from cancer tissue directly collected from a living body, it is highly possible that the cells are contaminated with bacteria or the like. Therefore, it is preferable to add an antibiotic to the culture solution.
  • Examples of a method for introducing the polar cell-specific expression vector of the present invention into cancer cells ex'ra include a calcium phosphate method, an electroporation method (electroporation method), a liposomal method, a DEAE dextran method, a lipofection method, and a microporous method.
  • a general method such as an injection method can be used. If it is a viral vector, it can be introduced by infecting cancer cells by including it in a medium.
  • In vivo methods for introducing into cancer cells include intravenous injection, arterial injection, intramuscular injection, and local injection of a cancer cell-specific expression vector suspended in physiological saline or the like to obtain an appropriate titer. Examples of the method include introduction by oral administration, transoral administration, and transdermal administration.
  • Culture of the cancer cells into which the cancer cell-specific expression vector has been introduced ex vivo is performed according to a conventional cell culture method.
  • RNA expression of the target protein / polypeptide or RNA can be confirmed by a known method for analyzing gene products.
  • a fluorescent antibody method, an enzyme antibody method, a method using flow cytometry, a Western blotting method, an immunoprecipitation method, an RT-PCR method, a cDNA base sequencing method and the like can be mentioned.
  • CTL cytotoxic T cells
  • Cytotoxic ⁇ cells can be produced by using a cancer cell-specific expression vector according to the present invention and using a processed cell in which a cancer cell has expressed an antigen molecule such as a non-self MHC class I molecule.
  • CTL can be induced.
  • a processed cell which is a transformant into which the cancer cell-specific expression vector of the present invention has been introduced and can express a desired protein or polypeptide
  • CTL cancer cell Cytotoxic T cells
  • the obtained cytotoxic T cells can be returned to a living body and used for treating cancer.
  • the cancer cell-specific expression vector of the present invention is introduced into a cell of a subject such as a human tissue, organ, or body fluid, and a desired protein or polypeptide downstream of the cancer cell-specific expression DNA of the present invention is introduced. If the nucleotide sequence encoding the peptide expresses a desired protein or the like, the cell of the subject is known to be a cancer cell, and the presence of the cancer cell can be determined.
  • the present invention provides a test method in which a cancer cell-specific expression vector is introduced into a subject to determine the presence of cancer cells based on the presence or absence of expression, and a diagnostic agent for cancer containing cancer cell-specific expression DNA. it can.
  • cancer can be diagnosed if cancer cells are present, and not only can the cancer be diagnosed, but also cells that have not been sick but have been infected by a virus (hepatitis C, etc.) Is present, and so-called precancerous conditions that gradually progress to cancer can also be diagnosed.
  • the nucleotide sequence introduced into the vector together with the cancer cell-specific expression DNA of the present invention includes, in addition to the nucleotide sequence encoding the desired protein or polypeptide, LacZ, luciferase, GFP ( A reporter gene such as Green Fluorescent Protein) can be used.
  • Cancer cells are mutations of their own cells, and the immune system cannot be recognized as non-self in vivo. When it grows abnormally, the disease progresses.
  • a desired protein, polypeptide or RNA can be expressed in a cancer cell-specific manner, so that the immune system expresses a protein or polypeptide that can be recognized as non-self. This makes it possible to recognize cancer cells as non-self and eliminate the cancer cells, thus enabling effective cancer treatment.
  • cancer cells are present, not only treatment for clinically developing cancer disease, but also mutant cells that gradually move to cancer, such as viral infections (hepatitis C, etc.)
  • the condition exists that is, a so-called precancerous condition
  • the term includes treatment and prevention and prevention of recurrence.
  • a fusion protein may be produced by some mutation, but even if it is not caused by an oncogene, it may cause cancer.
  • Cancer can be treated by using the vector of the present invention containing RNAi capable of causing RNA interference with a mutant gene or DNA encoding lipozyme.
  • the present invention provides a method for immunologically treating cancer, a method for preventing immunity, and a method for preventing recurrence using a vector containing a cancer cell-specific expression-inducing DNA.
  • cytotoxic T cells CTL
  • cancer cell-specific expression induction vector of the present invention may be used for studies on cancer cell-specific transcription factors and studies on inhibition of the transcription factors.
  • mice normal BALB / c mice and normal C57BL / 6 mice, each 10 weeks of age, were used by two males.
  • Heart blood was collected from each mouse under ether anesthesia to obtain about 1 ml of peripheral blood.
  • This peripheral blood was diluted to 7.5 ml each with physiological saline (saline), layered on 3 ml of lymphoprep (manufactured by NYC0MED), centrifuged at room temperature at 2,500 rpm for 30 minutes, and centrifuged.
  • a blood mononuclear cell (PBMC) fraction was obtained.
  • Each PBMC fraction was diluted to 1 Om1 with a saline solution, centrifuged at 1500 rpm at 4 ° C for 5 minutes, and washed by sedimenting PBMC. After this washing was performed twice, the precipitate was recovered, dissolved in 1 ml of Isogen (Nippongene: Nippongene), and immediately stored at 180 ° C. Separately, each organ of each mouse was excised, thoroughly washed with a saline solution, homogenized in lm1 Isogen, and immediately stored at -80 ° C.
  • RNA sample 50 ng was used for the reverse transcription reaction. It was evaluated by PCR method using -3 '. At this time, G3PDH-specific primers, 5'-ACCACAGTCCATGCCATCAC-3 ', and 5'-TCCACCACCCTGTTGCTGTA-3' were used as positive controls. From each RNA sample, PCR was performed using a Q5-specific primer without a reverse transcription reaction, and it was confirmed that DNA amplification did not occur. The results are shown in Figures 2, 3, and 4. As shown in Figure 2, it was only very slight transcripts slightly To thymus peripheral blood lymphocytes were detected in normal AKR (H-2 k) mice.
  • BW5147 lymphoma that had been passaged in the abdominal cavity of AKR mice was collected and After washing twice, it was transplanted into the abdominal skin of another AKR mouse at 2 ⁇ 10 5 cells / 501 / mouse.
  • each organ was collected in the same manner as in a normal mouse.
  • MH134 J3 dried cancer was transplanted into C3HZHe mice, and each organ was recovered.
  • RNA was extracted and purified from each organ collected as in normal mice, and analyzed by RT-PCR.
  • transcripts were detected only in peripheral blood mononuclear cells (Fig. 5).
  • MH134 tumor-bearing C3H / He mice it was not detected in any organ (Fig. 6).
  • Example 4 Ku Q5 k gene 5, the cloning of the upstream DNA> - from AKR-derived BW5147 cells initially H- 2 3 ⁇ 4 mice were extracted and purified chromosomal DNA according to a conventional method. Next, nested PCR was performed using this chromosomal DNA as type I. The base sequence of the primers used was S. Cchwe mardle, D. Bevec, G. Brem, MB Urban, PA Baeuerle and E. Refer to the nucleotide sequence of chromosomal DNA reported in H.
  • the primer sequence for 1st—PCR is 5′-ATCCT CCAAAGGCACATGTGACATG-3 ′ and 5′-CCACTGCTTGTTGTTTTTGGCGATCTT-3 ′
  • the second primer sequence for PCR is 5′-TCCACTGTCTCCMCATGGCGAACG-
  • the nested PCR yielded a PCR product containing a DNA fragment of about 8.3 kbp. 3, 5, and 5'-CGCGCACGGCTTCGCACTTGCATT-3 '.
  • the above nested PCR product was treated with T4 DNA polymerase to make the DNA ends blunt and purified, and then phosphorylated at the ends by T4 polynucleotide kinase treatment.
  • pUC118 was digested with SmaI, the terminal phosphate was removed by alkaline phosphatase treatment, and purified vector DNA was prepared.
  • 8.3 kb pDNA fragment and vector DNA were ligated by a ligation reaction, and introduced into E. coli JM-109 by electroporation. The size of the insert DNA of the emerging colonies was examined, and the desired clone having 8.3 kbp was selected.
  • pUC 118 ZQ5-5 '(Sac-E2) was used, and the nucleotide sequence was determined as follows by the Size-fractionated Uni-directional deletion (SUD) method.
  • pUC118 / Q5-5 '(Sac-E2) was digested with Apa I and Xba I, followed by Exonuclease III (Exoffl) digestion for 5, 10, 15, 20 or 25 minutes.
  • the ends of these ⁇ ⁇ ⁇ ⁇ -digested DNAs were blunted with Mung Bean nuclease and Klenow fragmaent, and after agarose gel electrophoresis, DNA fragments of each size were recovered and purified.
  • the DNA thus obtained was circularized by self-ligation and introduced into E.
  • the size of the insert DNA of the plasmid in the emerging Escherichia coli colony was confirmed by PCR reaction using universal primers at both ends of the vector cloning site, and 17 to 25 clones having different sizes were selected.
  • the nucleotide sequence of the insert portion of each selected clone was decoded by the cycle sequence method. Base sequence decoded from each clone Based on the sequence, the nucleotide sequence of the entire 4.1 kb region of the insert DNA was joined using the overlapping sequence portion.
  • the transcription inducing activity could be maintained high if the region was 14 'bases to 1422 bases (Pvu II site) to +22 bases or more, 5' upstream from the expected transcription start site. It is unclear whether the region matches the region required for transcriptional repression in E. coli, and it is necessary to examine transcriptional regulation in normal cells. However, there are some cell lines that can be cultured in ⁇ 0 and primary cultured cells, but there is no guarantee that they are truly normal at the time of examination. Therefore, the present inventors carried out in an in vivo system using tumor-bearing mice.
  • AdZHind-lacZ, Ad / Bst-lacZ, Ad / Pvu-lacZ, and AdZNco-lacZ did not stain normal cells other than cancer tissues at all (Fig. 17, Fig. 18, Figure 19, Figure 20, Table 3).
  • a cancer cell-specific expression induction vector can be provided by combining with a vector that does not introduce a gene into PBMC like an adenovirus vector.
  • the thymus was excised from the BALBXc (H-2D d ) mouse and ground with a sterilized slide glass to collect thymocytes.
  • the separated cells 1 X 1 0 6 cel ls / m
  • the suspension was suspended in RPMI-1640 medium containing 10% FBS, 50 gZm1 streptomycin and 50 ii g / ml kanamycin so as to give a value of 1, and seeded on a petri dish.
  • the cell suspension in the I FN- ⁇ added to a 50 n gZm 1, were cultured overnight in C_ ⁇ 2 I Nkyube Isseki scratch. After the culture, the cells were collected, washed with PBS, and lysed with Isogen-LS (Futtsubon Gene). Total RNA was obtained according to the protocol attached to Isogen-LS.
  • RNA was treated with oligodT as a primer to prepare cDNA.
  • a PCR reaction was carried out using this cDNA as type II, with the H-2D d- specific primer—5′-ACTAAGCTTAAAATGGGGGCGATGGCTCCGC-3 ′ and 5′-AGTTCTAGACTTCACACTTTA CMTCTGGGAG-3 ′.
  • H-2D d-specific primer 5′-ACTAAGCTTAAAAAATGGGGGCGATGGCTCCGC-3 ′
  • 5′-AGTTCTAGACTTCACACTTTA CMTCTGGGAG-3 ′ was examined by agarose gel electrophoresis, a band was observed at the expected position of 1.I kbp.
  • the remaining PCR product was digested with restriction enzymes HindIII and XbaI, and pUC19 was inserted into the DNA fragment digested with HindIII and XbaI by a ligation reaction.
  • E. coli JM109 was transformed with this ligation product, and a clone having an insert DNA of 1.1 kbp was selected from the colonies that appeared. Plasmids were extracted and purified from the selected clones, and their nucleotide sequences were confirmed. A plasmid (pUC 19 / H-2D d ) completely matching the nucleotide sequence of GenomeNet DDBJ Accession No. U47326 was selected.
  • pUC 19 / H-2D d was digested with 13 ⁇ 411 (with 1111) 3 ⁇ 4 & 1, and the insert DNA of 1.1 kbp was recovered and purified.
  • This 1.1 kbp DNA fragment was ligated to the pRc-CMV vector digested with Hind III and Xba I.
  • the ligation product was used to transform E. coli JM109 with this ligation product.
  • a clone having 1.1 kbp DNA was selected from the appeared colonies. Extraction of plasmids from selected clones, purified, confirms the nucleotide sequence, 11 Nai black Ichin problems (; - 0] ⁇ / / 11-20 was selected as 4.
  • pRc - CMV / H - a 2D d digested with Nru I and Hind III, H - and the 2D d c DNA was recovered including DNA fragments. This was blunt-ended by T4 DNA polymerase treatment.
  • pUC118 ZQ5-5 '(Sac-E2) was digested with PvuII and BamHI, and the: ⁇ -BamHIDNA fragment of Q5-5' DNA was recovered. This was blunt-ended by T4 DNA polymerase treatment, and a kinase reaction was performed to add a phosphate group to the end.
  • T4 DNA polymerase treatment and a kinase reaction was performed to add a phosphate group to the end.
  • Plasmid DNA was extracted from the emerged colonies, and a clone (pRc-CMVZQ5-H-2D d ) in which PvuII-BamHIDNA of Q5-5 ′ was ligated in the correct direction upstream of H-2D d cDNA was selected.
  • pAxcw was digested with Swal to prepare a cosmid vector treated with CIAP.
  • pRc-CMVZQ5-H-2D d is digested with Bglll and XhoI to recover a DNA fragment containing 0: ⁇ 8 sites of ⁇ -0 ⁇ 1 ⁇ from the PvuII site of Q5-5 ' did.
  • the end of this DNA was flattened and slid by T4 DNA polymerase treatment, and a phosphate reaction was further performed to add a phosphate group to the end.
  • This DNA fragment was subjected to a ligation reaction between the cosmid vector prepared above and a packaging reaction, followed by infection of Escherichia coli VCS-257.
  • Cosmid DNA was extracted from the colonies that appeared, and clones containing the insert DNA were selected. Inn After the cosmid DNA containing the desert DNA was removed from the adeno, the nucleotide sequence of the insert DNA was confirmed. The clones which did not have problems were transfected into HeLa cells, and the cells were collected two days later. The recovered cells were reacted with an anti-H-2D d monoclonal antibody (CEDA LANE) as a primary antibody and FITC-labeled anti-mouse IgG as a secondary antibody, and analyzed using a fluorescence microscope and a flow cytometer. A clone whose fluorescence was confirmed was selected.
  • CEDA LANE anti-H-2D d monoclonal antibody
  • the cosmid DNA (pAxcw / Q5-H-2D d ) selected in the analysis with the adeno-dropping DNA was transfected into 293, and AdZQ5-H-2D d was prepared and purified using a conventional method. Q5-H-2D d was obtained.
  • C3HZHe male mice were purchased at the age of 7 weeks, and after cultivation for one week, 210 5 ] ⁇ 1 H134 hepatoma cells were transplanted into the abdominal skin. After 5 days, at Toko filtration tumor diameter became approximately 7 mm, divided into three groups each group for 5-7 mice, purified group Ad / Q5 - H - a 2D d 3 X 1 0 9 pfu / 50 / l The other group received 3 ⁇ 10 9 pfu / 50 II 1 of AdZAxcw similarly prepared as a negative control, and the other group received saline at the site of 501 tumors. Two days later, the administration was repeated once again. Subsequently, the tumor diameter was measured and the survival or death was confirmed.
  • FIG. 21 shows the results of measuring the tumor diameter. Compared with the other two groups, a significant tumor growth inhibitory effect was observed in the AdZQ5-H-2D d- administered group (P ⁇ 0.01).
  • This therapeutic effect is due to the fact that non-self MHC expressed specifically in cancer cells induces an immune response in the mouth in a cancer cell-specific manner, thereby eliminating cancer cells.
  • cancer cells are generally not recognized as non-self in vivo and are immune from exclusion by immune function.
  • even cancer cells express the ara antigen, which the immune system can recognize very strongly. It was thought that cancer cells would be eliminated because the immune system could recognize it as non-self.
  • the use of the Q5 gene upstream transcriptional regulatory region can provide a gene therapy agent and a treatment method in which non-self MHC molecules are expressed in a cancer cell-specific manner. The method was expected to produce high therapeutic effects with few side effects.
  • the Q5 gene upstream transcription regulatory region can express various genes in a cancer cell-specific manner, it was considered to be applicable to various cancer gene therapy / prevention agents.
  • the cancer cell-specific expression-inducing DNA of the present invention is a novel cancer cell-specific expression-inducing DNA that exhibits an expression-inducing activity only in undifferentiated and abnormal cells such as cancer cells.
  • the first vector is a cancer cell-specific expression induction vector having a function of expressing a gene linked downstream in a cancer cell.
  • a desired protein, polypeptide or RNA can be expressed in a cancer cell-specific manner.
  • the polypeptide it is possible to recognize cancer cells as non-self and to eliminate the cancer cells, thereby enabling effective cancer treatment.
  • the cancer cell-specific expression-inducing DNA and the vector according to the present invention can be suitably used for cancer treatment.
  • this vector can be used to develop novel cancer gene therapy and cancer vaccines.

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Abstract

L'invention porte sur la production d'un ADN induisant l'expression spécifique des cellules cancéreuses et qui génère l'expression des gènes spécifiquement dans des cellules cancéreuses. L'invention porte aussi sur un vecteur de recombinaison contenant cet ADN, et plus spécifiquement un ADN contenant au moins une partie d'un ADN possédant + 22 bases à partir de la base 5'-supérieure 2667 démarrant au point d'initiation de transcription du gène de l'antigène Q5 d'un gène d'antigène de souris non classique, histocompatible (gène Ib de la classe du complexe majeur d'histocompatibilité) (SEQ ID NO : 1), et un vecteur le contenant. L'invention porte également sur un procédé consistant à transférer un vecteur contenant l'ADN précité avec une séquence nucléotidique codant une protéine cible, un polypeptide cible ou un ARN dans des cellules cancéreuses et à exprimer le polypeptide cible ou l'ARN spécifiquement dans les cellules cancéreuses. Le vecteur précité peut être utilisé dans la prévention, le traitement et le diagnostic du cancer. Il est également possible de développer un nouveau procédé de thérapie génique pour le cancer et un vaccin contre le cancer.
PCT/JP2003/006844 2002-05-31 2003-05-30 Adn induisant l'expression specifique des cellules cancereuses et vecteur d'expression specifique des cellules cancereuses WO2003102186A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013507A1 (fr) * 1996-09-25 1998-04-02 Mcgill University Promoteur hex ii specifique des tumeurs et utilisation dudit promoteur dans la therapie du cancer
WO1999018195A2 (fr) * 1997-10-03 1999-04-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procedes et compositions permettant d'induire une cytotoxicite specifique aux tumeurs
WO2001023004A1 (fr) * 1999-09-30 2001-04-05 The Trustees Of The University Of Pennsylvania Adenovirus selectifs de synthese utilises en therapie anticancereuse
WO2001090344A1 (fr) * 2000-05-25 2001-11-29 University Of Virginia Patent Foundation $g(b)-hcg promoter based tumor-restrictive gene expression for cancer gene therapy

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013507A1 (fr) * 1996-09-25 1998-04-02 Mcgill University Promoteur hex ii specifique des tumeurs et utilisation dudit promoteur dans la therapie du cancer
WO1999018195A2 (fr) * 1997-10-03 1999-04-15 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procedes et compositions permettant d'induire une cytotoxicite specifique aux tumeurs
WO2001023004A1 (fr) * 1999-09-30 2001-04-05 The Trustees Of The University Of Pennsylvania Adenovirus selectifs de synthese utilises en therapie anticancereuse
WO2001090344A1 (fr) * 2000-05-25 2001-11-29 University Of Virginia Patent Foundation $g(b)-hcg promoter based tumor-restrictive gene expression for cancer gene therapy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EGAWA K. ET AL.: "Protection against metastasis by immunization with an allogeneic lymphocyte antigen", CANCER IMMUNOL. IMMUNOTHER., vol. 41, no. 6, 1995, pages 384 - 388, XP002972316 *
WATTS S. ET AL.: "Organization and structure of the Qa genes of the major histocompability complex of the C3H mouse: implications for a Qa function and class I evolution", EMBO J., vol. 8, no. 6, 1989, pages 1749 - 1759, XP002972315 *

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