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WO2006079291A1 - Désoxynucléotide synthétique artificiel monobrin, préparation pour vaccin issue dudit désoxynucléotide et applications - Google Patents

Désoxynucléotide synthétique artificiel monobrin, préparation pour vaccin issue dudit désoxynucléotide et applications Download PDF

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WO2006079291A1
WO2006079291A1 PCT/CN2006/000183 CN2006000183W WO2006079291A1 WO 2006079291 A1 WO2006079291 A1 WO 2006079291A1 CN 2006000183 W CN2006000183 W CN 2006000183W WO 2006079291 A1 WO2006079291 A1 WO 2006079291A1
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antigen
seq
cancer
peptide
heat shock
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Li-Ying Wang
Mu-Sheng Bao
Yong-Li Yu
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Changchun Huapu Biotechnology Co Ltd
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Changchun Huapu Biotechnology Co Ltd
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    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/117—Nucleic acids having immunomodulatory properties, e.g. containing CpG-motifs
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/17—Immunomodulatory nucleic acids

Definitions

  • the present invention relates to a synthetic single-stranded deoxynucleotide capable of enhancing the immunostimulatory action of an antigen or antigen composition, and a vaccine composition comprising the single-stranded deoxynucleotide and an antigen or antigen composition. Furthermore, the invention relates to the use of synthetic single-stranded deoxynucleotides for the preparation of vaccine compositions, and to the use of said vaccine compositions as medicaments. Background technique
  • CpG is a dinucleotide in which cytosine and guanine are linked by a phosphate, wherein C represents cytosine, G represents guanine, p represents phosphoric acid, and cytosine is located at the 5' end.
  • CpG 0DN artificially synthesized single-stranded DNA
  • CpG 0DN single-stranded DNA
  • enhancing B cells, T cells, NK cells, and antigen presenting cells Such as monocytes, macrophages and dendritic cells
  • neutrophil activity and showed significant clinical application prospects (Weiner GJ, The iramunobiology and clinical potential of immunostimulatory CpG oligodeoxynucleotides. J Leukoc Biol 2000 Oct; 68 (4) : 455-63).
  • synthetic single-stranded oligonucleotides can take a wide variety of forms due to the different sequences.
  • Heat shock protein is a family of molecular companion proteins found in many organisms. In the process of immune response, heat shock proteins can exhibit the following four basic functions: 1. Carrying antigens into antigen-presenting cells including dendritic cells; 2. Directing antigens into antigen-presenting cells into MHC Class I pathway processing and presentation; 3. Stimulation of antigen-presenting cells including dendritic cells to express costimulatory molecules, secreting cytokines; 4. Antigens that bind antigens and heat shock proteins to form complexes or fusion proteins, Obtaining the property of activating antigen-specific cytotoxic T lymphocytes (Tamura Y, Peng P, Liu K, Daou M, Srivastava PK. Immunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Science 1997 Oct 3 ; 278 (5335) : 117-20 ).
  • Cytotoxic T lymphocyte is one of the most powerful individuals to kill tumor cells and kill cells infected with virus-infected cells.
  • Exogenous protein antigens are mainly processed by MHC class II pathway in antigen-presenting cells including dendritic cells after application of the individual, and stimulate humoral immune response (Heikema A, Agsteribbe E, Wilschut J, Huckriede A. Generation of heat shock protein-based vaccines by intracellular loading of gp96 with antigenic peptides. Immunol Lett 1997 Jun 1 ; 57 (1-3) : 69-74), can not effectively induce antigen-specific CTL o
  • Mycobacterial heat shock protein 65 is capable of carrying an antigenic peptide fused or conjugated or coupled thereto into the MHC class I antigen-presenting pathway, activating antigen-peptide-specific cytotoxic T lymphocytes, and killing tumor cells expressing the antigen peptide.
  • Hepatitis C virus is the causative agent of hepatitis C and has infected more than 170 million people worldwide. Liver cancer and cirrhosis are two serious complications caused by HCV infection. Worldwide, the combination of alpha-interferon and ribavirin is a method of treating hepatitis C virus infection with an effective rate of approximately 40% (Jon Cohen. The Scientific Challenge of Hepatitis C. Science, 1999, 285: 26) - 30).
  • the fusion protein formed by the fusion of Mycobacterium tuberculosis heat shock protein 65 (HSP65) and the multi-epitope HCV core antigen is a genetically engineered recombinant protein vaccine for preventing and treating hepatitis C (CN02122116. 2, China). '
  • Chlamydia Trachomatis is a non-sports bacterium that specializes in intracellular parasitic microorganisms, which can cause trachoma, inclusion body conjunctivitis, genitourinary diseases, sexually transmitted diseases, lymphogranuloma, endometritis, salpingitis, pelvic cavity in humans. Inflammation, tubal infertility and ectopic pregnancy of the fallopian tube.
  • the major outer membrane protein (M0MP) of Chlamydia trachomatis stimulates protective immunity.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein epitope antigen peptide is a recombinant protein which has preventive and therapeutic effects on human Chlamydia trachomatis infection and related diseases (CN02141977. 9) , China).
  • Human Prostate specific antigen is a tissue-specific antigen expressed in prostate cancer cells.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and PSA epitope antigen peptide is a recombinant protein for preventing and treating human prostate cancer (CN01134935. 2, China).
  • the MUC1 protein is a type of mucins that are expressed at high levels in breast cancer, ovarian cancer, lung cancer, prostate cancer, and colorectal cancer cells, and are targets for immunotherapy.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and MUC1 antigen peptide is a tumor that expresses MUC1 protein (including but not limited to breast) Cancer) Recombinant protein with preventive and therapeutic effects (CN01.102614 6, China).
  • HER - 2 protein (HER - 2) is an epidermal growth factor receptor (Tadashi Yamamoto, et al. Similarity of protein encoded by the human c - erbB-2 gene to epidermal growth factor receptor. Nature vol 319 16 January 1986 , 230-234) Transmembrane proteins with high homology, overexpressed in 30% of human breast tumor cells, are targets for breast cancer immunotherapy.
  • the fusion protein formed by fusion of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide is a recombinant protein that has preventive and therapeutic effects on human breast cancer (CN01136347. 9, China).
  • One of the objects of the present invention is to provide a synthetic single-stranded deoxynucleotide capable of enhancing the immunostimulatory effect of an antigen or antigen composition, which can be used as an adjuvant for an antigen or antigen composition to enhance an antigen or antigen composition.
  • Immune stimulation consist of single-stranded DNA molecules containing one or more synthetic oligonucleotides, which may be partially sulfurized, fully vulcanized, or unvulcanized.
  • the artificially synthesized single-stranded deoxynucleotide of the present invention comprises at least one sequence selected from any one of the following formulas (1) to (5):
  • the artificially synthesized single-stranded deoxynucleotide of formula (2) has a sequence selected from any one of the following: 5, -ggggACgATACgTCggggggg-3 ' (SEQ ID N0: 1)
  • Ggggggggggggg)g (Qg : IDO1073SE NATCACACATTT- --- ggggggggggg)g (Q63 NO :10TSE IDTTCcccAA- -- 5, -gggggCgTCgTTTTCgTCgACgAATT-3 ' (SEQ ID NO: 143)
  • the synthetic single-stranded deoxynucleotide has a sequence selected from any one of the following:
  • the artificially synthesized single-stranded deoxynucleotide of formula (4) has a sequence selected from any one of the following -
  • the artificially synthesized single-stranded deoxynucleotide of formula (5) has a sequence selected from any one of the following: 5' - TTCgTCgTTTgATCgATgTTCgTTgggggg- 3, (SEQ ID NO: 25) '
  • the artificially synthesized single-stranded deoxynucleotide of the present invention further comprises a modification of each group in the base of the single-stranded deoxynucleotide, wherein the modification includes a non-thio modification, a thio modification, a partial thio Modifications, rare base modifications (dl, dU, etc.), methylation modifications, thiol groups, Aminol inker C6s or Thiol-C6 S-S, etc., are used in conjunction with other materials. Such modifications are well known to those of ordinary skill in the art.
  • Another object of the present invention is to provide a vaccine composition
  • a vaccine composition comprising the above-described single-stranded deoxynucleotide and an antigen or antigen composition, wherein the antigen or antigen composition is preferably formed of mycobacterial heat shock protein 65 and an antigen peptide A complex or fusion protein that is a preparation that induces antigen-specific CTL.
  • the antigenic peptide refers to a peptide segment having immunogenicity or a peptide having immunogenicity in a state of forming a complex, a conjugate or a fusion protein with other proteins, for example: a multi-table in 122116.
  • Hepatitis C virus core antigen antigen peptide which has preventive and therapeutic effects on diseases caused by hepatitis C virus infection and hepatitis C virus infection, and the specific sequence is as follows:
  • Glu lie Asp Asn Glu (SEQ ID NO: 181 )
  • the Chlamydia trachomatis in 9 is mainly a multi-epitope antigen polypeptide, which has therapeutic and preventive effects on related diseases caused by infection of Chlamydia and Chlamydia, and the specific sequences are as follows: Glu Phe Pro Ala Tyr Gly Arg His Met Gin Asp Ala Glu Met Phe Thr Asn Ala Ala Cys Met Ala Leu Asn lie Trp Asp Glu Leu Asn Val Leu Cys Asn Ala Ala Glu Phe Thr lie Asn Lys Pro Lys Gly Tyr Val Gly Lys Glu Phe Pro Leu Ala Leu Asp Ala Ala Thr Gly Thr .
  • Lys Asp Ala Ser lie Asp Tyr His Glu Trp Gin Ala Ser Leu Ala Leu Ser Tyr Arg Leu Asn Met Phe Thr Pro Tyr lie Gly Val Lys Trp Ser Arg Ala Ser Phe Asp Ala Asp Thr Tyr Lys Leu (SEQ ID NO : 182)
  • Human prostate specific antigen cytotoxicity T lymphocyte multi-epitope single copy antigen polypeptide which has preventive and therapeutic effects on human prostate cancer, and the specific sequence is as follows:
  • MUC1 antigen cytotoxic T lymphocyte epitope antigen peptide in CN011026146 which has preventive and therapeutic effects on tumors expressing MUC1 including, but not limited to, breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer cells, etc., specific sequence as follows:
  • Another object of the present invention is to provide a method of producing a vaccine composition comprising mixing an effective amount of the above-described synthetic single-stranded deoxynucleotide with an effective amount of an antigen or antigen composition.
  • an effective amount of the synthetic single-stranded deoxynucleotide is mixed with an effective amount of a complex or fusion protein formed by the mycobacterial heat shock protein 65 and the antigenic peptide.
  • Another aspect of the invention provides a method of enhancing the immunostimulatory effect of an antigen or antigen composition by using a synthetic single-stranded deoxynucleotide in combination with an antigen or antigen composition.
  • the synthetic single-stranded deoxynucleotide can significantly enhance the activity of the mycobacterial heat shock protein 65 antigen peptide fusion protein to induce antigenic peptide-specific CTL, which is significantly enhanced.
  • Mycobacterial heat shock protein 65 antigen peptide fusion protein stimulates mouse to inhibit the growth of antigenic peptide-expressing tumor cells, and significantly enhances the anti-tumor, anti-virus and anti-tumor specificity of mycobacterial heat shock protein 65 antigen peptide fusion protein-induced antigen peptide Anti-Chlamydia infection activity.
  • a further aspect of the invention is the use of a vaccine composition for the preparation of a vaccine for the treatment of a viral infection, a bacterial infection, a parasitic infection, an allergy or cancer.
  • This includes administering an effective amount of an immunological composition consisting of a synthetic single-stranded deoxynucleotide to an antigen or antigen composition to a human or mammal in need of treatment.
  • the viral infection, bacterial infection, parasitic infection, allergy or cancer includes, but is not limited to, related diseases caused by hepatitis C virus infection, related diseases caused by infection of chlamydia and chlamydia, prostate cancer, breast cancer, Ovarian cancer, lung cancer, gastric cancer, endometrial cancer, salivary gland cancer, adenocarcinoma, colon and rectal cancer, non-small cell lung cancer, lung adenocarcinoma, and the like.
  • the design sequence is as follows - ( 1 ) (G) n (L) n X 1 X 2 CGY 1 Y 2 (M) n (G) n
  • 3 ⁇ 4 A, T or G;
  • L, M A, T, C or G;
  • n is 0-6
  • n is 0-6; , -TCgTAACgTTgTTTTTAACgTT-3 ' (SEQ ID NO: 9) Ggggggggg)Q ( :72TccTTc3S ID NOccET- - ggggggggg) (Q1cEO :7cTcTTS I NTcTDT - ggggggg)ggg (Q0 NO :7cCATSE IDcTcccTClT— - )gggggggggggg (QD NO :69S IcTEccAccAcTcTcTcT3T- - ggggg) (QO :67TTCTCT3SE ID NcAACAAATI - g )gggggg (Q058ccccSED N:cTcTccc3 ITcT— - ggggg) (QO57ccccc3SE IN :TcTDcTcT- -- gggggs (Q"c
  • Gggg ()gggQS7E ID N0:1cccT3ACATAcTTcTcTTC- - gggg)g (QN IDO: 15TCT3SEcTcTcTTcTT_ - gg)gg (QN IDO :14cSECTTTTTTTCTTcTI - gggQ)gg (gS ID N :2T3EO1cTTTcTTcTTTcT— — ggg)g (Qg IED NO10TT3S :CAcATCTCTATAcT- I , -TCgTCgACgTCgTTgggCggg-3 ' (SEQ ID NO:73)
  • DNA synthesis uses a solid phase phosphoramidite triester method to synthesize DNA fragments. This method has the advantages of efficacious and rapid coupling, and has been widely used in DNA chemical synthesis.
  • DNA chemical synthesis differs from the enzymatic DNA synthesis process in that it extends from the 5'-3' direction, but starts at the 3' end.
  • the specific reaction steps are as follows:
  • the protective group DMT (dimethoxytrityl) of the nucleotide attached to CpG (Controlled Pore Glass) was removed with trichloroacetic acid to obtain a free 5'-hydroxyl end for the next condensation reaction. .
  • the phosphoramidite-protected nucleomonomer is mixed with the tetrazole activator and introduced into the synthesis column to form a phosphoramidite tetrazole active intermediate (the 3'-end has been activated, but the 5'-end is still affected by DMT Protection), this intermediate will undergo a condensation reaction with a deprotected nucleotide on GpG.
  • the terminal hydroxyl group is often blocked by acetylation, and the general acetylation reagent is acetic anhydride and N-methyl. Mixture Formed together.
  • the nucleoside monomer is linked to the oligonucleotide attached to CpG through a phosphorous ester bond, and the phosphorous ester bond is unstable, and is easily hydrolyzed by an acid or a base.
  • a tetrahydrofuran solution of iodine is usually used.
  • the phosphoryl group is converted to a phosphate triester to obtain a stable oligonucleotide.
  • a deoxynucleotide is attached to the nucleotide of C P G, and the deprotected nucleotide of the newly attached deoxynucleotide 5'" is also removed by trichloroacetic acid.
  • the above activation, ligation, blocking, and oxidation processes are repeated to obtain a crude DNA fragment.
  • the solid phase synthesis oligonucleotide is carried out on a DNA synthesizer. After the deprotection group is removed from the oligonucleotide synthesized by the above method, the purity of the target oligonucleotide is extremely low, and contains a large amount of impurities, and the main impurities are
  • the removed protecting group is ammonia and benzoic acid ammonia and isobutyric acid ammonia, the nitrile ethyl group removed on the nitrile phosphorus group, and the short chain generated during synthesis, so that the amount of the oligonucleotide in the crude product is only About 15%.
  • the efficiency of each step in the synthesis is between 97% and 98%, the cumulative efficiency is not high. Taking the chain lengths of 20mer and 50mer as examples, (97.5%) 20 ⁇ 60%, (97.5%) 50 ⁇ 28%, it can be seen that the target oligonucleotide content in the crude product is very low, even 10%. To. These impurities, especially the large amounts of salts and short chains present in the crude product, cause quantitative inaccuracy and affect the next reaction, so the oligonucleotide must be purified. Purification by polyacrylamide gel electrophoresis (PAGE) is recommended. The purified product is highly purified and can be used in most molecular biology experiments to avoid many unexpected problems. If cost savings are considered, for less demanding experiments, such as simple PCR reactions, desalting purification can be used.
  • PAGE polyacrylamide gel electrophoresis
  • the oligonucleotide DNA is 0D 26 .
  • the value is measured.
  • Absorbance oligonucleotide solution as defined in 1 0D 26 1, 1cm light path under the standard 1ml quartz cuvette 260nm wavelength. .
  • the base composition is not identical for each particular oligonucleotide, the 1260 0D oligonucleotide DNA weighs approximately 33 g.
  • Example 3 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on heat shock protein hepatitis C virus antigen peptide fusion protein
  • HCV65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-HCV multi-epitope HCV core antigen fusion protein
  • Oligo synthetic deoxyoligonucleotide
  • B16 cells HCV+B16 cells
  • Oligo 1 SEQ ID N07
  • Oligo 2 SEQ ID NO106
  • Oligo 3 SEQ ID NO 103
  • Oligo 8 SEQ ID NO 123
  • Oligo 10 SEQ ID NO 159
  • mice 2 ⁇ ⁇ HSP- HCV
  • Oligo group injection 50 ⁇ ⁇ Oligo
  • HSP-HCV + 01igo group injection 20 ⁇ ⁇ HSP- HCV, 50 ⁇ ⁇ 01igo
  • HSP-HCV and Oligo were prepared in PBS at the required concentrations, and HSP-HCV+Oligo was injected with HSP-HCV and Oligo.
  • mice in each group were injected with physiological saline, HSP-HCV application solution, Oligo application solution and HSP-HCV+Oligo mixture.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • the cells were resuspended in IMM medium containing 10% FBS, and the mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP-HCV (20 (g/ml). 37 ° C, 5% C0 2 culture 7 Days.
  • Mouse spleen cells were harvested for effector cells.
  • HCV+B16 cells were cultured in 10% FBS in IMDM medium. The cells were incubated with Cr-labeled HCV+B16 cells (1 hour at 37 ° C, 5% C0 2 ). The cells were washed three times with IMDM containing 10% FBS.
  • 51 Cr-labeled HCV+B16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, each well ⁇ containing 1 ⁇ 10 4 cells. Three effector cells ( ⁇ ) were added at a ratio of 100:1 to the target, and three replicate wells were set. Incubate for 4-6 hours at 37 ° C, 5% C0 2 . The 96-well culture plate was centrifuged for 5 minutes (3000 rpm). The ⁇ supernatant was aspirated from each well and the radioactivity was measured. The cytotoxic activity of effector cells was calculated according to the following formula, expressed as specific killing rate.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the activity of the heat shock protein hepatitis C virus antigen peptide fusion protein by immunologically inducing HCV core antigen-specific cytotoxic T lymphocytes (p ⁇ 0.05). This CTL kills HCV-infected cells in vivo.
  • synthetic single-stranded deoxynucleotides significantly enhance the antiviral (including but not limited to hepatitis C virus) activity of the heat shock protein hepatitis C virus antigen peptide fusion protein.
  • B16 cells transfected with the HCV core antigen multi-epitope antigen peptide gene can be used as a cell model for hepatitis C virus infection (CN02122116. 2).
  • CN02122116. 2 hepatitis C virus infection
  • the injection of synthetic single-stranded deoxynucleotides (Oligo) and Mycobacterium tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein can stimulate mouse CTL to kill HCV in vivo. Cell activity.
  • HCV65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-HCV multi-epitope HCV core antigen fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • HC B16 cells transfection of HCV core antigen B16 cells
  • mice Forty male C57/BL6 mice, 6-8 weeks, were divided into normal saline group (injected saline), HSP-HCV group (20 ⁇ ⁇ HSP-HCV), and Oligo group (injected 50 ⁇ ⁇ Oligo) and HSP-HCV+Oligo group (20 ⁇ ⁇ HSP-HCV, 50 ⁇ ⁇ Oligo).
  • HSP-HCV and Oligo were prepared in PBS at the required concentrations, and HSP-HCV+Oligo was injected with HSP-HCV and Oligo. Each group of mice was injected with normal saline, HSP-HCVs Oligo on days 1, 14, and 21 And HSP- HCV+01igo. At day 24 HCV + B16 cells were seeded (1105 cells / animal), the injection site was the right dorsal skin.
  • mice On the 15th day after inoculation of the tumor, the mice were sacrificed to take the tumor and weigh the tumor.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of M. tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein to stimulate mouse CTL to kill HCV cells in vivo (p ⁇ 0.05), The performance is as follows: The combined use of synthetic single-stranded oligonucleotides and Mycobacterium tuberculosis heat shock protein 65 multi-epitope HCV core antigen fusion protein inhibits the growth ability of tumor cells expressing HCV core antigen significantly.
  • Example 4 Synthetic single-stranded deoxynucleotides enhance the specificity of CTL activity induced by the heat shock protein Chlamydia trachomatis major outer membrane protein epitope antigen peptide fusion protein
  • Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein HSP65-Chla
  • HSP65-Chla Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • B16 cells Chola 16 cells of the major outer membrane protein antigen peptide gene of Chlamydia trachomatis (ATCC, see CN02141977. 9).
  • mice 40 female C57/BL6 mice of 6-8 weeks, 10 rats in each group, divided into normal saline group (injected saline), HSP65-Chla group (injected 20 ⁇ ⁇ HSP65-Chla), Oligo group (injected 50 ⁇ ) Oligo), HSP65-Chla +01igo group (injection 2 ( ⁇ g HSP65-Chla, 50 ⁇ ⁇ 01igo).
  • HSP65-Chla and Oligo were prepared in PBS at the required concentrations, and HSP65-Chla+Oligo was injected with HSP65-Chla and Oligo. Groups of mice were injected with saline, HSP65-Chla, Oligo and HSP65_Chla+01igo on days 1, 14, and 21, respectively. Four subcutaneous injections (SC) were performed on the limbs of the mice near the lymph nodes.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in the usual manner, and the following procedure was as described in Example 1.
  • Chla+B16 cells were cultured in 10% FBS in IMDM medium. Target cells were seeded with 51 Cr-labeled Chla + B16 cells, and the following procedure was performed as described in Example 1.
  • Synthetic single-stranded deoxynucleotides significantly enhance Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis
  • the outer membrane protein antigen peptide fusion protein was induced to induce the activity of Chlamydia-specific CTL (p ⁇ 0.05). This CTL kills Chlamydia-infected cells in vivo. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein 65 and Chlamydia trachomatis major outer membrane protein antigen peptide fusion protein in the treatment and prevention of Chlamydia infection and its related diseases.
  • Example 5 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on Mycobacterium tuberculosis heat shock protein 65 and human prostate specific antigen (PSA) epitope antigen peptide fusion protein 1. Synthetic single Enhancement of inducible specific CTL activity by HSP65-HCV by strand oligonucleotide
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • PSA human prostate specific antigen antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotides
  • B16 cells PSA + B16 cells transfected with the PSA antigen peptide gene (ATCC reference CN01134935. 2).
  • mice Forty male C57/BL6 mice, 6-8 weeks, were divided into normal saline group (injected saline), HSP-PSA group (20 ⁇ ⁇ HSP-PSA), 01 i go group (injection) 50 ⁇ ⁇ 01 i go ), HSP-PSA+Oli go group (20 ⁇ ⁇ HSP-PSA, 50 ⁇ ⁇ 01igo).
  • mice in each group were injected with normal saline, HSP-PSA, Oligo and HSP-PSA+Oli go o in four subcutaneous injections (SC;) in the limbs of the mice near the lymph nodes.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • the cells were resuspended in IMDM medium containing 10% FBS, and mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP_PSA (20 ( ⁇ g/ml).
  • PSA + B16 cells transfected with the PSA antigen peptide gene were cultured in IMDM medium containing 10% FBS.
  • PSA+B16 cells were labeled with 61 Cr (cultured for 1 hour, 37X, 5% C0 2 ).
  • Use IMDM with 10% FBS Wash the cells three times in total.
  • 51 Cr-labeled PSA 16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, 100 ⁇ l per well, containing 1 ⁇ 10 4 cells.
  • the effector cells were added at a ratio of 100:1 to the target, and three replicate wells were set. The following operations and cytotoxicity calculations are as described in relation to Example 1.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of PSK-specific CTLs induced by Mycobacterium tuberculosis heat shock protein 65 and PSA antigen peptide fusion proteins (p ⁇ 0.05). This CTL can kill PSA-expressing tumors. cell. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the heat shock protein PSA antigen peptide fusion protein. Prevention and treatment of the biological activity of prostate cancer. 2. Enhancement of anti-expression of PSA tumor by HSP65-PSA by synthetic single-stranded oligonucleotide
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • PSA human prostate specific antigen antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • B16 cells PSA + B16 cells transfected with the PSA antigen peptide gene.
  • mice Forty male C57/BL6 mice, 6-8 weeks old, were divided into normal saline group (injected with normal saline), HSP-PSA group (20 ⁇ ⁇ HSP-PSA), and Oligo group (injected 50 ⁇ ⁇ Oligo), HSP-PSA+01igo group (20 g HSP-PSA, 50 ⁇ 01igo).
  • HSP-PSA and Oligo were prepared in PBS, and HSP-PSA+Oligo group was injected with HSP-PSA and Oligo. Mice were injected with saline, HSP-PSA, 01igo and HSP_PSA+01igo on days 1, 14, and 21, respectively. PSA+B16 cells (1 ⁇ 10 5 /piece) were inoculated on the 28th day, and the injection site was subcutaneous in the right back of the mouse.
  • mice On the 15th day after the tumor was inoculated, the mice were sacrificed to take the tumor and weighed the tumor.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the growth of Mycobacterium tuberculosis heat shock protein 65 and PSA antigen peptide fusion proteins (P ⁇ 0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of heat shock proteins and PSA antigen peptide fusion proteins in the prevention and treatment of prostate cancer.
  • Example 6 Enhancement of immunostimulatory effect of synthetic single-stranded deoxynucleotides on heat shock protein-MUC1 antigen peptide fusion protein 1. Synthesis of specific CTL activity by HSP65-MUC1 induced by synthetic single-stranded oligonucleotide Enhancement
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-MUC1 antigen peptide fusion protein HSP-MUC1 antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotides
  • B16 cells MUC1+B16 cells transfected with the WJC1 antigen peptide gene (ATCC reference CN01102614.6).
  • mice 40 female C57/BL6 mice of 6-8 weeks, 10 rats in each group were divided into normal saline group (injected saline), HSP-MUC1 group (20 ⁇ ⁇ HSP-MUCl injection), Oligo group (injected 50 ⁇ 6) Oligo), HSP- MUCl+Oligo group (20 ⁇ ⁇ HSP-MUCl, 50 ⁇ ⁇ 01igo).
  • HSP-MUCl and Oligo were prepared in PBS to the desired concentration, and the HSP-MUCl+Oligo group was injected with HSP-MUC1 and Oligo.
  • Each group of mice was injected with physiological saline, HSP-MUC1, Oligo and HSP-MUCl+01igo on days 1, 14, and 21.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • the cells were resuspended in IMDM medium containing 10% FBS, and mouse spleen cells were stimulated by adding IMDM medium containing 10% ConA and HSP-MUC1 (200 g/ral). Incubate at 37 ° C, 5% C0 2 for 7 days. Harvest cells for effector cells.
  • MUC1+B16 cells were cultured in 10% FBS in IMDM medium.
  • PSA+B16 cells were labeled with 51 Cr (1 hour, 37 ° C, 5% C0 2 ) o
  • the cells were washed three times with IMDM containing 10% FBS.
  • 51 Cr-labeled UC1+B16 cells were added to wells of a 96-well culture plate in 10% FBS in IMDM medium, 100 ⁇ l per well, containing 1 ⁇ 10 4 cells.
  • the effector cells were added at a ratio of 100:1 to the target, and three replicate wells were set. The following operations and cytotoxicity calculations are as described in relation to Example 1.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein to induce MUC1-specific cytotoxic T lymphocytes (p ⁇ 0.05), this CTL can kill expression Tumor cells of UC1. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the prevention and treatment of MUC1 tumors such as breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, etc. by the Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein. Learning activity.
  • the synthetic single-stranded oligonucleotide enhances the anti-expression of MUC1 tumor activity by HSP65-MUC1.
  • mice Female C57/BL6 mice were used for 6-8 weeks, divided into normal saline group (injected saline), HSP-MUC1 group (20 ⁇ ⁇ HSP-MUC1 injection), Oligo group (injected 50 ⁇ ⁇ Oligo), HSP-MUCl+Oligo group (20 ⁇ ⁇ HSP-MUC1, 50 ⁇ ⁇ Oligo), 10 in each group.
  • MUC1+B16 cells (I x lO 5 /only) were inoculated on the 28th day, and the injection site was subcutaneously in the right hind back of the mouse.
  • mice On the 15th day after inoculation of the tumor, the mice were sacrificed and weighed.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the growth of MUC1 tumor cells inhibited by Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein (p ⁇ 0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock protein 65-MUC1 antigen peptide fusion protein for the prevention and treatment of UC1 tumors such as breast cancer, ovarian cancer, lung cancer, prostate cancer, colorectal cancer, etc. .
  • Example 7 Enhancement of immunostimulatory effects of synthetic single-stranded deoxynucleotides on heat shock protein HER2 antigen peptide fusion protein
  • HSP-HER-2 antigen peptide fusion protein HSP-HER-2
  • Oligo synthetic single-stranded deoxynucleotide
  • B16 cells HER- 2+B16 cells transfected with the HER-2 antigen peptide gene (ATCC, see CN01136347. 9).
  • mice 6-8 weeks of female C57/BL6 mice were divided into normal saline group (injected saline), HSP-HER-2 group (20 ⁇ ⁇ HSP-HER - 2 injection), Oligo group (50 ⁇ Oligo injection), HSP - HER- 2+01 i go group (injected 20 g HSP-HER-2, 50 g Oligo), 10 in each group.
  • HSP-HER-2 and Oligo were formulated in PBS, and HSP-HER-2+01igo was prepared by mixing HSP-HER-2 with Oligo.
  • mice were injected with saline, HSP-HER-2, Oligo, and HSP-HER-2+OligOo at four subcutaneous injections (SC) in the limbs of the mice near the lymph nodes.
  • SC subcutaneous injections
  • mice were sacrificed by necking on the 26th day.
  • Mouse spleen cells or lymph node cells were taken in a conventional manner to prepare a single cell suspension.
  • IMDM medium containing 10% FBS
  • cells were resuspended, IMDM medium containing 10% ConA and HSP- HER-2 (200 ⁇ ⁇ ) stimulation of mouse spleen cells was added.
  • Incubate at 37 ° C, 5% C0 2 for 7 days. Harvest cells for effector cells.
  • B16 cells (HER-216 cells) transfected with the HER-2 antigen peptide gene were cultured in 10% FBS in IMDM medium.
  • HER-2+B16 cells were labeled with 51 Cr (cultured for 1 hour, 37 ° C, 5% C0 2 ). The cells were washed three times with IMDM containing 10% FBS.
  • 51 Cr-labeled 1 ⁇ -2 16 cells were added to 10% FBS in IMDM medium to wells of a 96-well culture plate, 100 ⁇ l per well, containing 1 ⁇ 10 4 cells.
  • the effector cells were added at a target ratio of 100:1, and three replicate wells were set.
  • Synthetic single-stranded deoxynucleotides can significantly enhance the activity of Mycobacterium tuberculosis heat shock protein and HER-2 antigen peptide fusion protein to induce HER-2 specific cytotoxic T lymphocytes (p ⁇ 0.05). CTL can kill tumor cells expressing HER-2. Therefore, synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide fusion proteins for the prevention and treatment of HER-2 tumors such as breast cancer and ovarian cancer. Second, the synthetic single-stranded oligonucleotide enhances the anti-expression of HSP65-HER-2 by HER-2 tumor activity
  • HSP65 Mycobacterium tuberculosis heat shock protein 65
  • HSP-HER-2 HER-2 antigen peptide fusion protein
  • Oligo synthetic single-stranded deoxynucleotide
  • HER-2+B16 cells transfected HER-2 B16 cells of the antigenic peptide gene.
  • HSP-HER-2 injected with 20 ⁇ 8 HSP-HER-2
  • Oligo group injected with 50 ⁇ ⁇ Oligo.
  • HSP-HER- 2+01 i go group (20 ⁇ ⁇ HSP-HER- 2, 50 ⁇ Oligo), 10 in each group.
  • mice were injected with saline, HSP-HER-2, Oligo and HSP-HER-2+01igo on days 1, 14, and 21, respectively.
  • HER-2+B16 cells (1 ⁇ 10 5 /piece) were inoculated, and the injection site was subcutaneous in the right back of the mouse.
  • mice On the 15th day after inoculation of the tumor, the mice were sacrificed and weighed.
  • Synthetic single-stranded deoxynucleotides significantly enhanced the growth of Mycobacterium tuberculosis heat shock protein 65 and HER-2 antigen peptide fusion proteins (P ⁇ 0.05). Synthetic single-stranded deoxynucleotides can significantly enhance the biological activity of Mycobacterium tuberculosis heat shock egg S 65 and HER-2 antigen peptide fusion proteins for the prevention and treatment of HER-2 tumors such as breast cancer and ovarian cancer.

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Abstract

La présente invention concerne un type de désoxynucléotide monobrin contenant des CpG et susceptible de renforcer l'effet immunostimulant d'un antigène ou d'une préparation d'antigène. La présente invention concerne également une préparation pour vaccin constituée dudit désoxynucléotide monobrin ainsi que d'un antigène ou d'une préparation d'antigène, et une méthode d'élaboration dudit vaccin. Ledit désoxynucléotide monobrin contenant des CpG améliore de façon significative l'activité des protéines hybrides protéine de choc thermique/peptide d'antigène pour ce qui est de l'induction de lymphocytes T cytotoxiques spécifiques de l'antigène, ainsi que l'activité antitumorale, antivirale et anti-infectieuse vis-à-vis de Chlamydia spécifique d'un antigène. De plus, la présente invention concerne l'emploi dudit désoxynucléotide monobrin contenant des CpG dans l'élaboration d’une préparation pour vaccin ainsi qu'une méthode d'amplification de l'effet immunostimulant, ladite préparation pour vaccin pouvant être employée en tant que médicament dans le traitement d'infections virales, d'infections bactériennes, d'infections parasitaires, d'allergies ou de cancers.
PCT/CN2006/000183 2005-01-27 2006-01-27 Désoxynucléotide synthétique artificiel monobrin, préparation pour vaccin issue dudit désoxynucléotide et applications Ceased WO2006079291A1 (fr)

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WO2012160183A1 (fr) * 2011-05-26 2012-11-29 Intervet International B.V. Oligodésoxynucléotides immunostimulatoires
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US7615539B2 (en) * 2003-09-25 2009-11-10 Coley Pharmaceutical Group, Inc. Nucleic acid-lipophilic conjugates
US9364531B2 (en) 2010-12-30 2016-06-14 Intervet Inc. Immunostimulatory oligodeoxynucleotides
WO2012160183A1 (fr) * 2011-05-26 2012-11-29 Intervet International B.V. Oligodésoxynucléotides immunostimulatoires
JP2014516533A (ja) * 2011-05-26 2014-07-17 インターベット インターナショナル ベー． フェー． 免疫刺激性オリゴデオキシヌクレオチド
JP2014516534A (ja) * 2011-05-26 2014-07-17 インターベット インターナショナル ベー． フェー． 免疫刺激性オリゴデオキシヌクレオチド
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US20160158347A1 (en) * 2013-07-26 2016-06-09 Intervet Inc. Acceleration of Vector Virus Induced Immune Response in Avians
US9757449B2 (en) * 2013-07-26 2017-09-12 Intervet Inc. Acceleration of vector virus induced immune response in avians
WO2022162177A1 (fr) * 2021-01-29 2022-08-04 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptides antigéniques de chlamydia trachomatis et leurs utilisations à des fins vaccinales

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