WO2008072895A1 - Vecteur d'expression de surface de l'antigène spécifique du cancer du foie, alpha-fétoprotéine et micro-organisme transformé par ce vecteur - Google Patents

Vecteur d'expression de surface de l'antigène spécifique du cancer du foie, alpha-fétoprotéine et micro-organisme transformé par ce vecteur Download PDF

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WO2008072895A1
WO2008072895A1 PCT/KR2007/006471 KR2007006471W WO2008072895A1 WO 2008072895 A1 WO2008072895 A1 WO 2008072895A1 KR 2007006471 W KR2007006471 W KR 2007006471W WO 2008072895 A1 WO2008072895 A1 WO 2008072895A1
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afp
fetoprotein
alpha
liver cancer
bacteria
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Moon-Hee Sung
Chul Joong Kim
Seung-Pyo Hong
Il Han Lee
Ji Yeon Kim
Jun-Han Lee
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Bioleaders Corporation
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    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001181—Alpha-feto protein
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • 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
    • C12N15/02—Preparation of hybrid cells by fusion of two or more cells, e.g. protoplast fusion
    • C12N15/03—Bacteria
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

  • the present invention relates to a surface expression vector for alpha-fetoprotein (AFP), which is a liver cancer-specific antigen, bacteria transformed with the vector so as to express AFT on the surface thereof, and a vaccine and a health supplement for preventing or treating liver cancer, which contain said bacteria having AFT expressed on the surface thereof, or a crude extracted antigen from said bacteria as an active ingredient.
  • AFP alpha-fetoprotein
  • liver cancer therapies such as the utilization of liver transplantation and combination therapy of radiotherapy with anticancer drugs
  • successful results of liver cancer treatment were demonstrated only in a small number of cases in which lesions were removed from liver cancer patients by surgical operations or in which liver transplantation was performed.
  • cancer cells metastasize to other organs or when carcinoma is found in many foci there is no satisfactory treatment method, and thus the development of a novel method for treating cancer is urgently required.
  • Typical examples thereof may include a method of removing cancer cells by introducing a specific gene into the cancer cells, and a method of preventing the proliferation and metastasis of cancer cells by introducing a specific antigen into the cancer cells to induce an in vivo cellular immune response to the antigen as a target.
  • Such methods can be applied in cancer therapies, which more specifically target solid cancers such as liver cancer.
  • cancer is more specifically treated by inducing a direct or in vivo immune response to selectively inhibit or kill malignant cells while allowing healthy cells to remain undamaged, unlike prior cancer treatment methods, which are relatively nonspecific and, in some cases, induce severe toxicity.
  • Alpha-fetoprotein which is a liver cancer-specific antigen, is a tumor- inducing fetal protein and is expressed only in tissues (e.g., yolk sacs, fetal livers and digestive tracts), originated from endothelial cells.
  • the serum level of the AFP antigen is considered to be clinically important, because it is increased in most of liver cancer patients and AFT is found at high levels with the progression of disease.
  • the serum AFT level of patients is considered to be attributable to the expression of AFP in liver cell carcinoma, not in the surrounding normal liver tissue, and thus the AFP antigen is thought to be controlled through liver cancer cell-specific expression.
  • studies focused on the immunological treatment of liver cancer using this liver cancer-specific antigen are being conducted.
  • the cell surface display technology is to express a foreign protein on the cellular surface using the surface protein of microorganisms, such as bacteria or yeasts, as a surface anchoring motif, and is used in a wide range of applications, including the production of recombinant live vaccines, the construction and screening of peptide/antibody libraries, whole cell absorbents and bioconversion catalysts.
  • the application range of this technology is determined depending on what kind of protein is expressed on the cell surface, thus, the industrial application potentiality of the cell surface display technology can be said to be significant.
  • a surface anchoring motif is most important.
  • Cell surface anchoring motifs which have been known and used till now, are broadly classified into four kinds, i.e., outer membrane proteins, lipoproteins, secretory proteins, and surface organ proteins such as flagella proteins.
  • proteins present on the outer cell membrane such as LamB, PhoE (Charbit et al, J. Immunol, 139: 1658, 1987; Agterberg et al, Vaccine, 8:85, 1990) and OmpA; lipoproteins, such as TraT (Felici et al, J. MoI Biol, 222:301 , 1991), PAL (peptidoglycan associated lipoprotein) (Fuchs et al, Bio/Technology, 9: 1369, 1991) and Lpp (Francisco et al, Proc. Natl. Acad. Sci.
  • FimA FimH adhesion of type 1 fimbriae
  • pili protein such as PapA pilu subunit were used in the expression of foreign proteins.
  • an ice nucleation protein Jung et al, Nat. Biotechnol, 16:576, 1998; Jung et al, Enzyme Microb. Technol, 22:348, 1998; Lee et al, Nat.
  • Streptococcus pyogenes Medaglini, D et al, Proc. Natl. Acad. Sci. USA.,
  • S-layer protein EAl of Bacillus anthracis S-layer protein EAl of Bacillus anthracis
  • surface protein of Gram-positive bacteria such as Bacillus subtilis CotB, etc.
  • the present inventors developed a novel vector for effectively expressing an exogenous protein on the cell surface of microorganisms, using a gene encoding poly-gamma glutamic acid synthetase complex (pgsBCA) gene, derived from a Bacillus sp. strain, as a novel surface anchoring motif, and a method for expressing a large amount of an exogenous protein on the surface of microorganisms transformed with said vector (Korean Patent Registration No. 10-0469800). Many studies have been attempted to stably express a pathogenic antigen or an antigen determinant in bacteria, which can produce the antigen or the antigen determinant in large amounts, using surface anchoring motifs through genetic engineering techniques.
  • pgsBCA poly-gamma glutamic acid synthetase complex
  • the oral administration of live nonpathogenic bacteria expressing an exogenous immunogen on the cell surface thereof can induce an immune response, which is more sustained and stronger than that of vaccines containing the conventional attenuated pathogenic bacteria or viruses.
  • the induction of this strong immune response is known to be due to the surface structures of bacteria serving as an adjuvant enhancing the antigenicity of surface-expressed exogenous proteins, and due to an in vivo immune response to live bacteria.
  • the present inventors have made many efforts to develop a vaccine for preventing or treating human liver cancer and, as a result, have constructed bacteria transformed with a recombinant vector for expressing the liver cancer- specific antigen AFP on the surface of the microorganisms using the gene encoding poly-gamma glutamic acid synthetase complex, and have found that liver cancer can be effectively prevented or treated using said bacteria having AFT expressed on the surface thereof, thereby completing the present invention.
  • Another object of the present invention is to provide bacteria transformed with said vector, and a method for producing alpha-fetoprotein (AFP), which comprises culturing said bacteria.
  • AFP alpha-fetoprotein
  • Still another object of the present invention is to provide a vaccine and a health supplement capable of effectively preventing or treating liver cancer.
  • the present invention provides a vector for the surface expression of alpha-fetoprotein (AFP) antigen, which contains a gene encoding liver cancer-specific antigen alpha-fetoprotein (AFP) or a fragment thereof, and one or more poly-gamma glutamic acid synthetase complex- encoding genes selected from the group consisting of pgsA, pgsB and pgsC, in which the fragment of the gene encoding the alpha-fetoprotein (AFP) antigen can induce a humoral immune response to alpha-fetoprotein in mammals.
  • AFP alpha-fetoprotein
  • the present invention also provides recombinant bacteria transformed with said vector and a method for producing alpha-fetoprotein (AFP), the method comprises culturing said recombinant bacteria to express alpha-fetoprotein (AFP) on the surface of the bacteria; and recovering alpha-fetoprotein (AFP).
  • AFP alpha-fetoprotein
  • the present invention also provides a vaccine and a health supplement for preventing or treating liver cancer, which contains either bacteria having alpha- fetoprotein (AFP) expressed on the surface thereof, or a liver cancer-specific alpha-fetoprotein (AFP) antigen crude extracted from said bacteria.
  • AFP alpha- fetoprotein
  • AFP liver cancer-specific alpha-fetoprotein
  • FIG. 1 shows a genetic map of a vector (pBT:pgsA-mAFP) for the surface expression of the liver cancer-specific antigen AFP.
  • FIG. 2 shows the results of Western blot analysis for the expression pattern of a pgsA-mAFP antigen fusion protein in lactic acid bacteria transformed with a pBT:pgsA-mAFP vector (lane 1 : non-transformed Lactobacillus casei; and lane 2: Lactobacillus casei transformed with pBT:pgsA-mAFP).
  • FIG. 3 shows the results of ELISA assay for the antibody titer of mAFP produced in the serum of mice after a Lactobacillus casei strain transformed with the surface expression vector pBT:pgsA-mAFP according to the present invention, was administered orally into mice for a given period of time .
  • FIG. 4 is a graphic diagram showing the results of ELISPOT analysis for the mAFP-specific cytotoxic activity of T-cells in the spleen and thymus of mice orally administered with a Lactobacillus casei strain transformed with the surface expression vector pBT:pgsA-mAFP according to the present invention (A: spleen; and B: thymus).
  • FIG. 5 is a graphic diagram showing the results of intracellular IFN- ⁇ staining for the mAFP-specific cytotoxic activity of T cells in the spleen and thymus of mice orally administered with the inventive Lactobacillus casei strain expressing an mAFP antigen on the surface thereof.
  • FIG. 6 is a graphic diagram showing the results of MTT assay for the mAFP- specific cytotoxic activity of the spleen- and thymus-derived T cells of mice orally administered with the inventive Lactobacillus casei strain expressing an mAFP antigen on the surface thereof (A: spleen; and B: thymus).
  • FIG. 7 is a graphic diagram showing the results of tumor proliferation rate as a function of time, measured after administering a Lactobacillus casei strain (observed to have an antigen determinant expressed on the surface thereof), transformed with the inventive surface expression vector pBT:pgsA-mAFP, orally into mice, and then injecting tumor cells into the mice.
  • FIG. 8 is a photograph showing the size of tumors appearing in mice, measured after administering a Lactobacillus casei strain (observed to have an antigen determinant expressed on the surface thereof), transformed with the inventive surface expression vector pBT:pgsA-mAFP, orally into mice, and then injecting tumor cells into the mice (A: non-transformed Lactobacillus casei, and B: Lactobacillus casei transformed with pBT: pgsA-mAFP).
  • liver cancer-specific antigen alpha-fetoprotein AFP
  • a recombinant expression vector containing one or more poly-gamma glutamic acid synthetase complex-encoding genes, selected from the group consisting of pgsA, pgsB and pgsC, and a gene encoding a liver cancer-specific AFP antigen was constructed.
  • the vector pBT:pgsA-mAFP capable of expressing liver cancer- specific alpha-fetoprotein (AFP) on the surface of microorganisms was constructed, and lactic acid bacteria Lactobacillus casei were transformed with the surface expression vector pBT:pgsA-mAFP to express the liver cancer- specific antigen alpha-fetoprotein on the surface of the lactic acid bacteria.
  • the present invention relates to a vector for the surface expression of alpha-fetoprotein (AFP), which contains a gene encoding liver cancer-specific antigen alpha-fetoprotein (AFP) or a fragment thereof, and one or more poly-gamma glutamic acid synthetase complex-encoding genes selected from the group consisting of pgsA, pgsB and pgsC, in which the fragment of the gene encoding the alpha-fetoprotein (AFP) antigen can induce a humoral immune response to alpha-fetoprotein in mammals.
  • AFP alpha-fetoprotein
  • the present invention relates to a recombinant bacteria transformed with said vector.
  • said vector preferably has a SlpA7 promoter
  • said bacteria are preferably selected from the group consisting of E. coli, Salmonella typhi, Salmonella typhimurium, Vibrio cholerae, Mycobacterium bovis, Shigella, Bacillus, lactic acid bacteria, Staphylococcus, Propionibacterium acnes, Listeria monocytogenes and Streptococcus. More preferably, the bacteria are lactic acid bacteria.
  • the present invention relates to a method for producing alpha- fetoprotein (AFP), which comprises culturing said transformed bacteria to express alpha-fetoprotein (AFP) on the surface of the bacteria.
  • AFP alpha- fetoprotein
  • lactic acid bacteria having alpha-fetoprotein expressed on the surface thereof were administered orally into mice, and after a given time period, an antibody to alpha-fetoprotein (AFP) in the mouse serum was measured.
  • AFP alpha-fetoprotein
  • the hAFP-specific cytotoxicity of T cells in the spleen and thymus of mice was measured by ELISPOT assay, MTT assay and intracellular IFN- ⁇ (gamma) staining.
  • ELISPOT assay ELISPOT assay
  • MTT assay intracellular IFN- ⁇ (gamma) staining.
  • lactic acid bacteria having alpha-fetoprotein expressed on the surface thereof were administered orally into mice, and after a given time period, tumor cells overexpressing alpha-fetoprotein (AFP) were injected into the mice, and tumor proliferation rate in the mice as a function of time was measured.
  • AFP alpha-fetoprotein
  • the present invention relates to a vaccine and a health supplement for preventing or treating liver cancer, which contains either bacteria having alpha-fetoprotein (AFP) expressed on the surface thereof, or a liver cancer-specific alpha-fetoprotein (AFP) antigen crude extracted from said bacteria as an active ingredient.
  • AFP alpha-fetoprotein
  • AFP liver cancer-specific alpha-fetoprotein
  • said vaccine can be administered orally or with food, and injected subcutaneously, intraperitoneally or intranasally, but the scope of the present invention is not limited thereto.
  • a mouse AFP gene (1818 bp) was used as a liver cancer-specific antigen gene, but it will obvious to those skilled in the art that any gene fragment can be used without limitation, as long as it can induce a humoral immune response to AFP in mammals.
  • bacteria for use as host cells for transformation any bacteria selected from gram-negative bacteria E. coll., Salmonella typhi, Salmonella typhimurium, Vibrio cholerae, Mycobacterium bovis and Shigella, and gram-positive bacteria Bacillus, lactic acid bacteria, Staphylococcus, Propionibacterium acnes, Listeria monocytogenes and Streptococcus may be used in the following examples, as long as they are attenuated or not toxic or attenuated, when they are administered in vivo.
  • edible microorganisms e.g., lactic acid bacteria, Bacillus subtilis, etc.
  • lactic acid bacteria lactic acid bacteria, Bacillus subtilis, etc.
  • Example 1 Construction of surface expression vector pBT:pgsA-mAFP for mAFP
  • pgsA outer membrane protein genes
  • pgsB and pgsC outer membrane protein genes
  • pgsA was used to construct vector pBT:pgsA-mAFP, which can use gram-negative microorganisms and gram-positive microorganisms as host cells and can express liver cancer-specific antigen alpha- fetoprotein (AFP) on the surface of microorganisms.
  • SlpA7 SEQ ID NO: 1
  • the SlpA7 promoter has a 214-bp base sequence (SEQ ID NO: 1) in which a 124-bp base sequence derived from the HCE promoter is continuously linked to a 90-bp domain selected based on a conserved sequence appearing in the promoter o ⁇ slpA gene encoding the S-layer protein of lactic acid bacteria.
  • the pAT vector in the present invention is a general purpose vector which uses gram-positive microorganisms as host cells (Trieu-Cuot, et al, Gene, 29:99, 1991 ; Zhou and Johnson, Biotech. Lett., 29: 121 , 1993). However, in the art to which the present invention pertains, it is also used as a backbone vector for constructing a recombinant vector, which can be used to construct a transformant using gram-negative microorganisms (US 2002/309560, US 2002/217613, US 2002/197153, Jung CM., et al, J. Bacteriology, 181 :2816, 1999).
  • an mAFP gene was obtained by PCR using, as a template, the mAFP of a pGEM T vector containing about 1818-bp AFP gene (SEQ ID NO: 2), with primers of SEQ ID NOS: 3 and 4 (underlines in SEQ ID NOS: 3 and 4 indicate BamUl and Xba ⁇ digestion sites, respectively.
  • SEQ ID NO: 3 5'- cgc ggatcc aag tgg ate aca ccc get -3'
  • SEQ ID NO: 4 5'- gga tctaga tta tta aac gcc caa age ate -3'
  • Vector pBT:pgsA-mAFP capable of expressing mAFP on the surface of gram- negative microorganisms and gram-positive microorganisms as host cells was constructed by treating the mAFP gene with restriction enzymes BamHl and Xbal and linking it to the 3 '-terminal end of the outer membrane protein gene pgsA of surface expression vector pBT:pgsA, which is involved in the synthesis of poly- gamma glutamic acid, inaccordance whti a translation codon (FIG. 1).
  • Example 2 Cell surface expression of mAFP (alpha-fetoprotein)
  • the surface expression vector pBT:pgsA-mAFP constructed in Example 1, was introduced into Lactobacillus casei to construct transformants expressing mAFP on the surface thereof.
  • the transformants were cultured in MRS medium ⁇ Lactobacillus MRS, Becton Dickinson and Company Sparks, USA) at 37 ° C to induce the expression of cancer-specific protein mAFP on the surface of the transformants.
  • the cultured transformants were adjusted to the same cell concentration, and a given amount of protein was extracted from the transformants and denatured, thus preparing a sample.
  • the sample was fractioned by SDS-PAGE, and the resulting protein was transferred to a polyvinylidene-difluoride membrane (PVDF, Bio-Rad).
  • PVDF polyvinylidene-difluoride membrane
  • the PVDF membrane to which the protein has been transferred, was blocked in a blocking buffer (50 mM Tris-HCl, 5% skim milk, pH 8.0) for 1 hour with stirring, and then a rabbit-derived polyclonal primary antibody to pgsA, diluted 1000 fold in a blocking buffer, was added to the PVDF membrane, which was then allowed to react for 4 hours.
  • the membrane was washed with buffer and allowed to react with an avidin-biotin reagent for 1 hour, followed by washing. Then, a substrate (H 2 O 2 ) and a color-developing reagent (DAB) were added to the membrane to induce a color development reaction, thus observing the expression of the fusion protein (pgsA-hAFP) in the transformants.
  • a substrate H 2 O 2
  • DAB color-developing reagent
  • the linker-containing pgsA has a molecular weight of about 43.6 kDa and mAFP has a molecular weight of about 66.6 kDa, and thus the fusion protein pgsA-mAFP expressed in the transformants has a molecular weight of about 110 kDa.
  • Example 3 Anticancer effect of microorganisms having AFP expressed on the surface thereof
  • Example 2 The immune response of animal cells to the Lactobacillus casei strain expressing AFP on the surface thereof, produced in Example 2, and the anticancer effect of the Lactobacillus casei strain expressing AFP on the surface thereof, were analyzed.
  • the Lactobacillus casei strain having AFP expressed on the surface thereof was administered orally into mice for a given period of time, and then a humoral immune response to AFP in the mouse blood was examined, before liver cancer cells overexpressing AFP were injected subcutaneously into the mice.
  • mice Male, 5-week-old, Korea Biolink Co., Korea
  • test group and control group each consisting of
  • the Lactobacillus casei strain having AFP expressed on the surface thereof was administered orally into the prepared test group mice at a dose of about 1 x 10 10 /day for a total of 10 days (5 days at the first week and 5 days at the second week), and a booster dose was administered orally into the mice for 5 days at each of the fourth week and the sixth week.
  • mice Before the Lactobacillus casei strain having AFP expressed on the surface thereof was administered into the mice, preimmune serum was collected from the mice. Also, from the mice, sera were collected at the third week (first serum), the fifth week (second serum) and seventh week (third week) and were comparatively analyzed for humoral immunity. To the mice of the control group, a Lactobacillus casei strain not expressing AFP on the surface thereof was administered at a dose of about 1 x 10 10 /day for the same period as that of the test group according to the same method as that of the test group.
  • AFP antigen (Fitzgerald, USA) was coated on a 96-well Nunc-immune plate. The antigen coating was performed by diluting the antigen in a 0.1 M bicarbonate coating buffer (pH 9.6) to a concentration of 100 ng/100 ⁇ i per well, and incubating the plate at 4 ° C overnight. The AFP antigen-coated plate was washed three times with PBST, and then blocked with 10% skim milk in PBS (blocking solution) for 2 hours.
  • PBS blocking solution
  • the blocked plate was allowed to react with a primary antibody (mouse serum) at 37 ° C for 2 hours and then react with a peroxidase-conjugated secondary antibody for 1 hour. After completion of each of the reactions, the plate was washed five times with PBST. Finally, the plate was allowed to react with an OPD substrate solution at room temperature for 20 minutes, and then a 0.2 N H 2 SO 4 reaction stopper solution was added thereto to stop the enzymatic reaction. Then, the plate was measured for absorbance at 450 nm using an ELISA reader, thus measuring the specific IgG antibody titer to AFP in the mice serum. As a result, as it can be seen in FIG.
  • the Lactobacillus casei strain having AFP expressed on the surface thereof was administered orally into mice for a given period of time, and then a cellular immune response to AFP in T cells in the spleen and thymus of the mice was measured.
  • mice were selected from 15 mice of the mouse group described in Example 3 (1), and the spleen and the thymus were cut out from each of the selected mice and placed in the respective tubes containing 15 ml of RPMI medium (Gibco BRL, USA)).
  • the spleen and thymus tissues were transferred into sterilized Petri dishes and ground using two sheets of sterilized slide glass, and cells were extracted from each of the tissue membranes. AU the contents in the Petri dishes were transferred into 50-ml tubes, which were filled with RPMI medium.
  • the tubes were left to stand on ice for 15-20 minutes, and 40-45 ml of the supernatants were transferred into 50-ml fresh tubes. Each of the tubes was centrifuged at 1600-2000 rpm for 10 minutes, and the supernatant was removed, thus obtaining pellets.
  • 10 ml of TAC buffer [NH 4 Cl, Tris-Cl (pH7.4) 1OmM], previously warmed to 37 ° C , was added to the pellets to suspend the cells, and the pellet-containing tubes were left to stand in a water bath at 37 ° C for 10 minutes.
  • the contents in the tubes were shaken with a glass pipette, such that the dissolved red blood cells were removed through adsorption onto the glass pipette. Then, the tubes were filled with RPMI medium. The cells in the tubes were washed twice with RPMI medium and suspended in RPMI 1640 medium, thus isolating splenocytes and thymocytes.
  • a cell-mediated immune response to AFP was analyzed according to the following three methods.
  • an ELISPOT assay was carried out.
  • An IFN- ⁇ -specif ⁇ c antibody diluted 200-fold in PBS (pH 7.2), was coated on an ELISPOT plate (BD ELISPOT Mouse IFN ⁇ Set, USA) and incubated overnight.
  • the splenocytes and thymocytes isolated from the mice were dispensed into each well of the plate in an amount of 100 ⁇ £/wel ⁇ at a concentration of 2 x 10 6 cells/ml and allowed to react for 2 hours.
  • 7 ⁇ g/ml of AFP was placed into each well and incubated at 37 ° C in 5% CO 2 for 48 hours.
  • each well was washed five times with PBST, and a 250-fold dilution of a biotinylated secondary antibody (BD Biosciences, USA) was dispensed into each well in an amount of 100 ⁇ /well and allowed to react at room temperature for 2 hours. Then, each well was washed five times with PBST, and a 200-fold dilution of an enzyme- conjugated streptoavidin-HRP reagent (BD Biosciences, USA) was placed into each well in an amount of 100 / ⁇ /well and incubated for 1 hour.
  • a biotinylated secondary antibody BD Biosciences, USA
  • each well was washed five times with PBST and PBS, and a substrate solution (BD AEC Substrate Reagent Set, USA) was added thereto to induce color development. Then, the number of spots appearing in each well was measured with an ELISPOT plate reader (BD Biosciences, USA). As a result, as shown in FIG. 4, the number of spots in the test group was significantly larger than that in the control group. This suggests that the T cells in the thymus and spleen in the mice administered with the Lactobacillus casei strain having AFP expressed on the surface thereof were activated against AFP, thus inducing the cell-mediated immune response.
  • a substrate solution BD AEC Substrate Reagent Set, USA
  • splenocytes and thymocytes were dispensed into a 24-well plate at a cell density of 3 x 10 6 cells/well and incubated at 37 ° C in 5% CO 2 for 2 hours. Then, 7 ⁇ g/ml of AFP was dispensed into each well and allowed to react for 2 hours. After completion of the reaction, BD Golgi STOP (USA) was added thereto in an amount of 4 ⁇ /well and allowed to react at 37 ° C in 5% CO 2 for 9 hours.
  • AFP-stimulated cells in each well were centrifuged at 1600 rpm for 5 minutes and mixed with a CD8-PE antibody (Phycoerythrin; BD Biosciences, USA), diluted 200-fold in FACS staining buffer (DPBS, 1% FBS, 0.09% sodium azide), and then, each well was incubated at 4 ° C for 30 minutes. Each well was washed twice with FACS staining buffer, and 200 ⁇ it of a fixation/permeabilization kit (BD Cytofix/Cytoperm, USA) was added to each well and allowed to react at 4 "C for 20 minutes.
  • a fixation/permeabilization kit BD Cytofix/Cytoperm, USA
  • the cells in each well was washed twice with BD perm wash buffer (BD Cytofix/Cytoperm, USA), and an IFN-7-FITC antibody (BD Biosciences, USA), diluted 200-fold in BD perm wash buffer, was added to the cells and allowed to react at 4 ° C for 30 minutes.
  • the cells were washed twice, suspended in 300 ⁇ i of FACS staining buffer, and then analyzed using an FACS analysis system (Becton Dickinson and company).
  • cytokine IFN- ⁇ involved in the anticancer action of CD8+T cells involved in cell-mediated immunity was expressed in the splenocytes and thymocytes of the test group at a higher level than that in the control group.
  • the isolated splenocytes and thymocytes were dispensed into each well of a 96- well Nunc-tissue plate in an amount of 100 ⁇ /well at a cell density of 2 x 10 6 cells/ml and incubated for 2 hours. Then, 7 ⁇ g/ml of AFP was dispensed into each well and allowed to react at 37 ° C in 5% CO 2 for 72 hours. 10 ⁇ l of an MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was added to each well at a final concentration of 0.5 mg/ml and allowed to react at 37 ° C in 5% CO 2 for 4 hours.
  • MTT reagent 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide
  • solubilization solution (Roche, Germany) was dispensed into each well and allowed to react overnight in the same conditions as described above. At the next day, the purple formazan crystals were measured for absorbance at 570 nm with an ELISA reader.
  • Hepa 1-6 cells (ATCC CRL-1830) overexpressing AFP were injected subcutaneously into the left femoral regions of 10 mice of each of the test group mice and control group described in Example 3 (1) using a 30-gauge needle. For 7 weeks after the injection, whether cancer would be produced in the test group mice and the control group mice, was examined. As a result, as shown in FIGS. 7 and 8, in the left femoral regions of the control group mice, cancer cells proliferated to a size of 1500 mm 3 or larger at 6 weeks after the injection of the cancer cells. However, in the test group mice administered with the Lactobacillus casei strain having AFP expressed on the surface thereof, the cancer cells proliferated to a size of only 127 mm . This demonstrates that the proliferation of liver cancer cells in live mice was inhibited by the lactic acid bacteria expressing AFP on the surface thereof.
  • the present invention has an effect to provide a vector for the surface expression of liver cancer-specific antigen AFP and bacteria transformed with said vector using a poly-gamma glutamic acid synthetase complex-encoding gene.
  • inventive bacteria having AFP expressed on the surface thereof are administered into patients, an immune response stronger than that in the case of administration of AFP alone is induced.
  • inventive bacteria having AFP expressed on the surface thereof, and a liver cancer-specific AFP antigen crude extracted from said bacteria are useful for the prevention or treatment of liver cancer.

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Abstract

L'invention concerne un vecteur d'expression de surface de l'alpha-fétoprotéine (AFP) qui est un antigène spécifique du cancer du foie, et des bactéries transformées au moyen de ce vecteur pour exprimer l'AFP à leur surface. Cette invention concerne de manière plus spécifique un vecteur d'expression de surface comprenant un gène codant un complexe d'acide poly-gamma-glutamique synthétase ainsi qu'un gène codant l'antigène spécifique du cancer du foie (AFP), des bactéries transformées au moyen dudit vecteur, et un vaccin ainsi qu'un complément prophylactique ou thérapeutique destiné à prévenir ou traiter un cancer du foie contenant soit les bactéries à la surface desquelles l'AFP est exprimée, soit un antigène extrait brut issu desdites bactéries en tant que principe actif.
PCT/KR2007/006471 2006-12-13 2007-12-12 Vecteur d'expression de surface de l'antigène spécifique du cancer du foie, alpha-fétoprotéine et micro-organisme transformé par ce vecteur WO2008072895A1 (fr)

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KR10-2006-0127297 2006-12-13

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Cited By (3)

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WO2015192120A1 (fr) * 2014-06-13 2015-12-17 Aldar Bourinbaiar Composition orale et procédés d'immunothérapie
CN112877353A (zh) * 2021-01-19 2021-06-01 东北林业大学 一种表达载体及其制备方法和应用
WO2022175951A1 (fr) * 2021-02-18 2022-08-25 Yeda Research And Development Co. Ltd. Bactéries génétiquement modifiées pour générer des vaccins

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KR102389169B1 (ko) * 2015-10-22 2022-04-21 고려대학교 산학협력단 암 특이적 에피토프와 연결된 단백질 나노입자 및 이를 포함하는 암 면역치료용 조성물

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015192120A1 (fr) * 2014-06-13 2015-12-17 Aldar Bourinbaiar Composition orale et procédés d'immunothérapie
CN112877353A (zh) * 2021-01-19 2021-06-01 东北林业大学 一种表达载体及其制备方法和应用
WO2022175951A1 (fr) * 2021-02-18 2022-08-25 Yeda Research And Development Co. Ltd. Bactéries génétiquement modifiées pour générer des vaccins

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