WO2021047451A1 - 一种dna纳米疫苗及其制备方法和应用 - Google Patents

一种dna纳米疫苗及其制备方法和应用 Download PDF

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WO2021047451A1
WO2021047451A1 PCT/CN2020/113489 CN2020113489W WO2021047451A1 WO 2021047451 A1 WO2021047451 A1 WO 2021047451A1 CN 2020113489 W CN2020113489 W CN 2020113489W WO 2021047451 A1 WO2021047451 A1 WO 2021047451A1
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dna
vaccine
tumor
strand
nano
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French (fr)
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丁宝全
刘少利
蒋乔
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国家纳米科学中心
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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • 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
    • A61K39/00119—Melanoma antigens
    • A61K39/001192—Glycoprotein 100 [Gp100]
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/82—Colon
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/876—Skin, melanoma

Definitions

  • the invention belongs to the technical field of nanomedicine, and relates to a DNA nano vaccine and a preparation method and application thereof, in particular to a tubular DNA nano vaccine loaded with tumor antigens and immune adjuvants, a preparation method thereof, and application in tumor immunotherapy .
  • tumor treatment has the problems of poor prognosis and low five-year survival rate. Improving the effect of tumor treatment is a scientific problem that needs to be solved urgently.
  • the rapid development of tumor immunotherapy methods has brought breakthroughs in tumor treatment.
  • Tumor vaccines especially personalized vaccines for specific patients, are getting more and more attention from researchers, and they are expected to become new tumor treatments in the near future. Program.
  • the effectiveness of tumor vaccines is still limited. For example, free antigens may be quickly eliminated before being internalized by dendritic cells (DC); in the absence of immune adjuvants, tumor vaccines can easily induce immune tolerance in the body, which limits its therapeutic effect.
  • DC dendritic cells
  • CN102614527A discloses an acid-resistant nanoparticle oral DNA anti-tumor vaccine with pH-sensitive characteristics and a preparation method thereof.
  • the oral DNA vaccine is a surface-modified chitosan with alginic acid combined with a tumor-specific antigen Legumain protein DNA plasmid nanoparticles can be efficiently swallowed by dendritic cells and macrophages in the small intestine Pey's lymph nodes, and express the encoded tumor antigens, activate the host's immune killing against tumor cells, have small toxic and side effects, and have strong antigens Presenting effect, but there are problems such as short cycle time and easy to be cleared.
  • DNA origami nanotechnology is a novel and unique DNA self-assembly technology, which has been widely used in bottom-up preparation of nanoscale two-dimensional and three-dimensional DNA nanostructures.
  • DNA nanostructures constructed using DNA nanotechnology have the characteristics of controllable structure and easy modification, and have broad application prospects in drug transport and reversal of drug resistance. Therefore, the development of high-efficiency, low-toxicity, targeting and controllable DNA nanostructures as drug delivery carriers is of great practical significance. Compared with traditional anti-tumor drug delivery carriers, DNA nanostructures have significant advantages in targeted drug delivery and controllable release due to their controllable structure and easy modification.
  • DNA nanostructures are formed by DNA assembly and have good biocompatibility; DNA is assembled according to the principle of base complementary pairing, which has a high degree of structural predictability; internal functional modifications can effectively load a variety of active drugs, including Gene drugs, small molecule chemical drugs, proteins or antibodies, etc.; complex and high-level structures constructed by DNA nanotechnology have good structural stability in cell lysates, and have excellent protection for the drugs loaded inside; the outer surface is short-used Strand DNA hybridization can target modification of functional groups at specific locations, which enhances the targeting of drug delivery systems; DNA nanostructures can also be modified by nanoparticles to controllable opening and closing under specific conditions to achieve controlled drug release the goal of.
  • CN103656662A discloses a method for using a polypeptide-mediated DNA nanostructure as an anti-tumor drug carrier.
  • a polypeptide with a certain function is connected to the DNA nanostructure.
  • the prepared product is a complex of the DNA nanostructure and the polypeptide.
  • the specific functional polypeptide can mediate the DNA nanostructure loaded with the biomolecule into the cell or specifically bind to the receptor on the cell surface, so that the DNA structure can be used as an anti-tumor drug carrier It has potential application value in the development and research of anti-tumor drug carriers and improving the loading efficiency of anti-tumor drug carriers.
  • the DNA nanostructure can only be used as a drug carrier and does not have the effect of tumor treatment.
  • CN109675049A discloses a pH-induced drug sustained-release DNA nanostructure and its preparation method and application.
  • Long single-stranded DNA is synthesized by rolling circle amplification technology, and complementary hybridization with G and C-rich DNA single strands (loading strands) Obtained DNA molecular aggregates with alternating single and double strands, which can load a large amount of Dox; when the pH of the system decreases, the rolling circle amplification product folds to form a triple helix configuration, and the double strands of the DNA molecular aggregates are melted, releasing G and G-rich aggregates.
  • the DNA of C is single-stranded, and the intercalated Dox is released to complete the drug release.
  • the reversible intercalation and release of Dox can be achieved by adjusting the pH; this method uses a DNA nanostructure with good biocompatibility as a drug carrier to increase the loading
  • the drug dosage has the advantages of low cost, simple operation, high sensitivity to pH, and rapid response, but there is a problem of poor targeting of tumors.
  • the present invention provides a DNA nano vaccine and its preparation method and application.
  • the DNA nano vaccine hybridizes tumor antigen polypeptide molecules, double-stranded RNA adjuvants and CpG adjuvants through precise site design.
  • a tubular three-dimensional structure is formed, and a controllable DNA switch is set on the surface of the tubular DNA nanostructure to respond to the acidic environment of endosomes in the antigen-presenting cells, realizing the targets of tumor antigen peptides and immune adjuvants
  • Toward delivery and controllable release a new type of tumor vaccine that is addressable, safe, efficient, controllable, and has high medical value has been developed.
  • the present invention adopts the following technical solutions:
  • the present invention provides a DNA nano vaccine, which includes a DNA nano structure, a tumor antigen polypeptide-DNA complex and an immune adjuvant;
  • the immune adjuvant includes a double-stranded RNA immune adjuvant and/or a CpG immune adjuvant.
  • the DNA nano vaccine has antigen-presenting cell endosome responsiveness, can effectively cause tumor-specific immune response, and effectively inhibit tumor growth.
  • the alkynyl modified tumor antigen polypeptide is connected to the azide modified DNA chain to form a tumor antigen polypeptide-DNA complex through a "click" reaction;
  • the double-stranded RNA immunoadjuvant uses DNA as a template and is transcribed in vitro to form single-stranded RNA, and
  • the two single-stranded RNAs are mixed at a molar ratio of 1:1 and then annealed to obtain double-stranded RNA.
  • the tumor antigen polypeptide and two immune adjuvants are used to play a synergistic effect.
  • the two signal pathways of TLR3 and TLR9 are activated at the same time, which is better than a structure containing only one immune adjuvant.
  • the DNA nanostructure is formed by assembling a DNA template strand, an auxiliary folding DNA strand, and a capture DNA strand.
  • the DNA template strand includes M13mp18 phage genomic DNA and/or lambda phage genomic DNA, more preferably M13mp18 phage genomic DNA.
  • the circular DNA single strand of the M13mp18 bacteriophage is used as the main chain, and the excess short-strand DNA is used as the auxiliary chain.
  • the main chain and the programmable auxiliary chain hybridize and complement each other at a specific position to form a two-dimensional rectangular sheet. Layer DNA nanostructures.
  • genetically modified M13 phage genomic DNA or asymmetric PCR amplification product can also be used as a DNA template chain to construct DNA nanostructures.
  • the nucleotide sequence of the M13mp18 bacteriophage genomic DNA is shown in SEQ ID NO:1;
  • the auxiliary folding DNA chain is designed according to the article "Folding DNA to create nanoscale shapes and patterns. Nature, 2006,440,297-302". Those skilled in the art can select the auxiliary folding DNA chain and capture the design of the DNA chain as needed. It can be increased, decreased or changed according to the needs, and can be designed on the plane of the entire DNA nanostructure.
  • auxiliary folding DNA strand is as follows:
  • the capturing DNA strand includes capturing DNA strand I, capturing DNA strand II, and capturing DNA strand III.
  • the capture DNA strand I is formed by adding a capture sequence I complementary to the DNA sequence of the tumor antigen polypeptide-DNA complex at the 5'end of the auxiliary folded DNA strand, and the nucleotide sequence of the capture sequence I As shown in SEQ ID NO: 16-24;
  • the capture DNA strand II is formed by adding a capture sequence II complementary to the sticky end sequence of a double-stranded RNA immunoadjuvant at the 5'end of the auxiliary folded DNA strand, and the nucleotide sequence of the capture sequence II As shown in SEQ ID NO: 25 ⁇ 33;
  • the capture DNA strand III is formed by adding a capture sequence III complementary to the 5'end extension sequence of the CpG immunoadjuvant at the 5'end of the assisted folding DNA strand, and the core of the capture sequence III
  • the nucleotide sequence is shown in SEQ ID NO: 34-42;
  • each DNA nanostructure has a capture site on the surface.
  • the capture DNA strand at the capture site is complementary to the extension sequence of the tumor antigen polypeptide, double-stranded RNA immunoadjuvant and CpG immunoadjuvant, and hybridizes through DNA annealing.
  • the three are assembled to specific sites on the surface of DNA nanostructures in a certain proportion.
  • the tumor antigen polypeptide-DNA complex, double-stranded RNA immune adjuvant and CpG immune adjuvant are bound to the DNA nanostructure by capturing DNA strands.
  • the principle of base complementary pairing is used to connect the tumor antigen polypeptide-DNA complex, double-stranded RNA (dsRNA) immune adjuvant and CpG immune adjuvant to the self-assembled two-dimensional sheet DNA nanometer through site design.
  • dsRNA double-stranded RNA
  • CpG immune adjuvant CpG immune adjuvant
  • the quantity and relative position of tumor antigen peptides and immune adjuvants on the surface of the sheet DNA nanostructure are precisely controlled.
  • the number of the tumor antigen polypeptide-DNA complex is 10-30, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, more preferably 15-20.
  • the number of the double-stranded RNA immunoadjuvant is 10-30, for example, it can be 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, more preferably 15-20. and / or
  • the number of the CpG immune adjuvant is 10-30, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, more preferably 15-20.
  • the tumor antigen polypeptide is any tumor antigen polypeptide known to those skilled in the art, and can also be a new tumor antigen polypeptide sequence obtained by screening individualized tumor patients.
  • amino acid sequence of the tumor antigen polypeptide is shown in SEQ ID NO: 11;
  • amino acid sequence shown in SEQ ID NO: 11 is: SIINFEKLRRG.
  • sequence of DNA in the tumor antigen polypeptide-DNA complex is shown in SEQ ID NO: 12;
  • the nucleotide sequence shown in SEQ ID NO: 12 is:
  • the sequence of the tumor antigen polypeptide-DNA complex is:
  • nucleotide sequence of the DNA template used for in vitro transcription and synthesis of double-stranded RNA immunoadjuvant is shown in SEQ ID NO: 13-14;
  • nucleotide sequence of the CpG immune adjuvant is shown in SEQ ID NO: 15;
  • the shape of the DNA nano vaccine includes a rectangular two-dimensional structure and/or a tubular three-dimensional structure.
  • the length of the rectangular two-dimensional structure is 80-100nm, for example, it can be 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, 87nm, 88nm, 89nm, 90nm, 91nm, 92nm, 93nm, 94nm, 95nm. , 96nm, 97nm, 98nm, 99nm or 100nm, more preferably 90-100nm.
  • the width of the rectangular two-dimensional structure is 50 to 70 nm, for example, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm. , 66nm, 67nm, 68nm, 69nm or 70nm, more preferably 50-60nm.
  • the bottom diameter of the tubular three-dimensional structure is 10-25nm, for example, it can be 10nm, 11nm, 12nm, 13nm, 14nm, 15nm, 16nm, 17nm, 18nm, 19nm, 20nm, 21nm, 22nm, 23nm, 24nm or 25nm. , More preferably 19-20 nm. and / or
  • the height of the tubular three-dimensional structure is 80-100nm, such as 80nm, 81nm, 82nm, 83nm, 84nm, 85nm, 86nm, 87nm, 88nm, 89nm, 90nm, 91nm, 92nm, 93nm, 94nm, 95nm, 96nm, 97nm, 98nm, 99nm or 100nm, more preferably 90-100nm.
  • the DNA nanovaccine with a tubular three-dimensional structure has a DNA switch.
  • a DNA switch with acidic environment responsiveness is hybridized on the two long sides of the rectangular sheet DNA nanostructure.
  • the DNA switch is formed by the hybridization of two single-stranded DNAs. The 3'ends of the two single-stranded DNAs are hybridized.
  • the two-dimensional rectangular DNA nanostructure is crimped and closed to form a three-dimensional tubular DNA nano-vaccine, and finally a tubular DNA loaded with tumor antigen peptides and immune adjuvants and a controllable DNA switch is prepared.
  • Nano vaccine, the tubular DNA nano vaccine realizes the loading and transportation of tumor antigen polypeptide and immune adjuvant and the controllable release in lymph nodes, and is a new type of tumor immune vaccine.
  • the DNA switch responds to the acidic environment of the endosome in the antigen presenting cell, controls the opening of the tubular structure, and exposes the tumor antigen polypeptide and two immune adjuvants therein.
  • the number of the DNA switches is 5-10, for example, 5, 6, 7, 8, 9, or 10, and more preferably 8-10. and / or
  • nucleotide sequence of the DNA switch is shown in SEQ ID NO: 43-58;
  • the tubular DNA nano-vaccine used in tumor immunotherapy of the present invention is controllable for the release of loaded tumor antigen polypeptides and immune adjuvants.
  • the tubular DNA nano-vaccine is ingested by antigen-presenting cells and responds to the acid environment of endosomes, so that three This kind of active ingredient is controllably released in endosomes.
  • the present invention provides a method for preparing the DNA nanovaccine as described in the first aspect, and the method includes the following steps:
  • step (1) The annealing product obtained in step (1) is purified by centrifugation, mixed with tumor antigen polypeptide-DNA complex, double-stranded RNA immunoadjuvant and CpG immunoadjuvant in proportion, and then annealed;
  • step (3) Mix the annealing product obtained in step (2) with the DNA switch in proportion and anneal;
  • the annealing conditions in step (1) are: the starting temperature is 95°C to 65°C, the end temperature is 25°C to 4°C, each 1°C is a gradient, each gradient stays for 5 to 10 minutes, and the annealing time is maintained at 2 ⁇ 24h, preferably 7-9h.
  • the starting temperature is 95-65°C, for example 95°C, 93°C, 91°C, 90°C, 87°C, 85°C, 83°C, 81°C, 80°C, 77°C, 75°C, 73°C °C, 71°C, 69°C, 67°C or 65°C.
  • the endpoint temperature is 25 to 4°C, for example, 25°C, 24°C, 23°C, 21°C, 20°C, 19°C, 17°C, 15°C, 13°C, 11°C, 7°C, 5°C °C or 4°C.
  • the temperature of the annealing process is 2-24h, for example, it can be 2h, 4h, 6h, 7h, 8h, 9h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h.
  • the molar ratio of the DNA template strand, the auxiliary folded DNA strand and the captured DNA strand in step (1) is 1:(5-20):(5-20), for example, it can be 1:5:5, 1: 7:7, 1:10:10, 1:15:15 or 1:20:20, preferably 1:(5-10):(5-10).
  • the buffer described in step (1) is 1 ⁇ TAE/Mg 2+ .
  • the annealing conditions in step (2) and step (3) are as follows: the starting temperature is 45 ⁇ 37°C, the ending temperature is 25 ⁇ 16°C, every 1°C is a gradient, each gradient stays for 3 ⁇ 8min, and 3 ⁇ 10 cycles.
  • the starting temperature is 45-37°C, for example, it may be 45°C, 44°C, 43°C, 42°C, 41°C, 40°C, 39°C, 38°C or 37°C.
  • the endpoint temperature is 25-16°C, for example, it may be 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C or 16°C.
  • the number of said cycles is 3-10, for example, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the molar ratio of the annealing product, tumor antigen polypeptide-DNA complex, double-stranded RNA immune adjuvant and CpG immune adjuvant in step (2) is 1:(2-10):(2-10):( 2-10).
  • the molar ratio of the annealing product to the DNA switch in step (3) is 1:(1-2). and / or
  • the steps of centrifugation in step (2) and step (4) are: mixing the obtained annealing product with 1 ⁇ TAE/Mg 2+ buffer and adding it to a 100 kDa spin column to perform centrifugation.
  • the inventor optimized the reaction conditions through a large number of complicated experiments, explored the effects of annealing temperature and reaction time on the nanostructure, and each step and each condition synergized, and finally successfully prepared a tubular DNA nano-vaccine composite structure with excellent performance. .
  • the present invention provides a method for preparing a DNA nanovaccine as described in the first aspect, and the method includes the following steps:
  • the conditions are: from 95°C to 65°C, each 1°C is a gradient, each gradient residence time is 5min; from 65°C to 25°C, every 1°C is a gradient, each temperature gradient residence time is 10min, the whole annealing The process is 7-9 hours to obtain rectangular DNA nanostructures;
  • the annealing product obtained in step (1) is mixed with 1 ⁇ TAE/Mg 2+ buffer and added to a 100kDa spin column, centrifuged, and then combined with tumor antigen polypeptide-DNA complex, double-stranded RNA immunoadjuvant and The molar ratio of CpG immune adjuvant is 1:(2-10):(2-10):(2-10) mixed and annealed.
  • the annealing conditions are: from 45°C to 25°C, with a gradient of 1°C, The residence time of each gradient is 3 ⁇ 5min, and 6 cycles are carried out;
  • step (3) The annealing product obtained in step (2) is mixed with the DNA switch at a molar ratio of 1:(1-2) and annealed.
  • the annealing conditions are: from 45°C to 25°C, with a gradient of 1°C, each The gradient residence time is 3 ⁇ 5min, and 6 cycles are carried out;
  • step (3) The annealing product obtained in step (3) is mixed with 1 ⁇ TAE/Mg 2+ buffer solution and added to a 100 kDa spin column, and centrifuged to obtain a tubular DNA nano-vaccine.
  • the present invention provides a pharmaceutical composition comprising the DNA nano vaccine as described in the first aspect.
  • the pharmaceutical composition further includes any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
  • the present invention provides an application of the DNA nano vaccine as described in the first aspect and/or the pharmaceutical composition as described in the third aspect in the preparation of a tumor immunotherapy vaccine.
  • the tumor immunotherapy vaccine is a broad-spectrum anti-tumor vaccine
  • the tumor is selected from one or more of the following: melanoma, breast cancer, colon cancer.
  • the tumor is not limited to a single type of malignant tumor.
  • the anti-tumor immunotherapy effect of the DNA nano vaccine and/or pharmaceutical composition has a broad spectrum, and can be used for the treatment and prevention of a variety of malignant tumors, such as melanoma. , Breast cancer, colon cancer and other malignant tumors.
  • the present invention provides a method for immunotherapy of tumors, the method comprising: administering the DNA nanovaccine according to the first aspect to a subject in need, and the preparation method according to the second aspect The prepared DNA nano vaccine and/or the pharmaceutical composition as described in the third aspect;
  • the tumor is selected from one or more of the following: melanoma, breast cancer, colon cancer.
  • the present invention provides a method for preventing tumors, the method comprising: administering the DNA nanovaccine according to the first aspect to a subject in need, and administering the DNA nanovaccine according to the preparation method according to the second aspect The prepared DNA nano vaccine and/or the pharmaceutical composition according to the third aspect;
  • the tumor is selected from one or more of the following: melanoma, breast cancer, colon cancer.
  • the present invention provides a drug for immunotherapy of tumors, the drug includes the DNA nano vaccine described in the first aspect, the DNA nano vaccine prepared according to the preparation method described in the second aspect, and/ Or the pharmaceutical composition according to the third aspect;
  • the tumor is selected from one or more of the following: melanoma, breast cancer, colon cancer.
  • the present invention provides a drug for preventing tumors, the drug includes the DNA nano vaccine described in the first aspect, the DNA nano vaccine prepared according to the preparation method described in the second aspect, and/or The pharmaceutical composition as described in the third aspect;
  • the tumor is selected from one or more of the following: melanoma, breast cancer, colon cancer.
  • the present invention has the following beneficial effects:
  • the present invention uses DNA nanotechnology, takes the circular DNA single strand of M13mp18 bacteriophage as the main chain, and uses excess short DNA as the auxiliary chain.
  • the main chain and the programmable auxiliary chain hybridize and complement each other at a specific position and fold. Form a two-dimensional rectangular sheet-layer DNA nanostructure with predictable and controllable structure;
  • the present invention uses the capture DNA chain to connect the tumor antigen polypeptide-DNA complex, double-stranded RNA immune adjuvant and CpG immune adjuvant on the surface of the two-dimensional sheet-layer DNA nanostructure formed by self-assembly according to the principle of base complementary pairing. ;
  • the present invention hybridizes a DNA switch with acidic environment responsiveness on the two long sides of a rectangular sliced DNA nanostructure, curls the sliced DNA nanostructure to form a three-dimensional tubular DNA nano vaccine, and prepares a tumor antigen loaded inside.
  • the tubular DNA nano-vaccine of the present invention can respond to the acidic environment in the target cell, realize controllable conformational changes, release tumor antigen molecules and immune adjuvants, and enhance the immune stimulation effect;
  • the present invention precisely controls the number and relative positions of tumor antigen polypeptides and two immune adjuvants on the surface of the sheet DNA nanostructure, and by adjusting the ratio of the three, the effect of simultaneously activating the TLR3 and TLR9 signal pathways is realized, and the synergy is achieved. Synergistic effect
  • the DNA nano-vaccine of the present invention is used as a nano-scale molecular machine for loading tumor antigens and immune adjuvants, and is effectively transported to the lymph nodes to achieve controlled release. It is expected to provide a new nano-vaccine for tumor immunotherapy. Dosage form.
  • Fig. 1 is an atomic force microscope morphology observation diagram of the rectangular sheet-layer DNA nanostructure of Example 1;
  • Fig. 2 is an atomic force microscope morphological observation diagram of the tubular DNA nano-vaccine of Example 2;
  • Figure 3 shows the targeting effect of the tubular DNA nano-vaccine of Example 3 on inguinal lymph nodes after subcutaneous injection
  • Figure 4 is a graph showing the inhibitory effect of the tubular DNA nano vaccine of Example 4 on the lung metastasis of melanoma cells;
  • Figure 5 is a graph showing the inhibitory effect of the tubular DNA nano vaccine of Example 5 on the growth of mouse melanoma;
  • Example 6 is a transmission electron microscope image of the tubular DNA nanostructure of Example 6;
  • FIG. 7 is a transmission electron microscope image of the tubular DNA nanostructure of Example 7.
  • the final concentration of the template strand and assisted folded DNA strand are 20nM and 100nM respectively; the mixture is slowly annealed and annealed using a gradient PCR machine
  • the conditions are: from 95°C to 65°C, each 1°C is a gradient, each gradient residence time is 5min; from 65°C to 25°C, every 1°C is a gradient, each temperature gradient residence time is 10min;
  • the annealing process is 8 hours, and the rectangular sheet-layer DNA nanostructures are obtained;
  • the centrifugation condition is: add 350 ⁇ L 1 ⁇ TAE-Mg to 100 ⁇ L sample Buffer solution, centrifuged at 4800rpm/min for 3min, the volume of the remaining solution in the spin column is about 100 ⁇ L, repeat the centrifugation 4 times; the final collected samples were analyzed by 1% agarose gel electrophoresis and observed under the atomic force microscope.
  • the morphology of the layer structure is: add 350 ⁇ L 1 ⁇ TAE-Mg to 100 ⁇ L sample Buffer solution, centrifuged at 4800rpm/min for 3min, the volume of the remaining solution in the spin column is about 100 ⁇ L, repeat the centrifugation 4 times; the final collected samples were analyzed by 1% agarose gel electrophoresis and observed under the atomic force microscope. The morphology of the layer structure.
  • the results are shown in Figure 1.
  • the constructed DNA nanostructure is a rectangular sheet structure.
  • AFM scanning results show that the rectangular DNA nanostructure is about 90-100nm long and 60-80nm wide, presenting a regular rectangular structure.
  • the sample connected with the tumor antigen polypeptide and the two immunoadjuvants is mixed with the DNA molecule "switch" at a molar ratio of 1:1 and annealed.
  • the annealing conditions are: from 45°C to 25°C, every 1 °C is a gradient, each gradient has a residence time of 5 min, and performs 6 cycles; then the PCR products are separated by centrifugation with a 100kDa spin column, purified and recovered by agarose gel electrophoresis, and purified tubular DNA nano-vaccine containing antigen and adjuvant is obtained.
  • Composite structure is: from 45°C to 25°C, every 1 °C is a gradient, each gradient has a residence time of 5 min, and performs 6 cycles; then the PCR products are separated by centrifugation with a 100kDa spin column, purified and recovered by agarose gel electrophoresis, and purified tubular DNA nano-vaccine containing antigen and adjuvant is obtained.
  • the results are shown in Figure 2.
  • the morphology of the constructed tubular DNA nanostructure was characterized by atomic force microscopy (AFM).
  • the structure was about 90-100 nm in length and 20 nm in width, presenting a regular tubular structure.
  • Cy5 fluorescently labeled tubular DNA nano-vaccine of Example 2 (the tumor antigen polypeptide is SEQ ID NO: 11: SIINFEKLRRG) was inoculated to the base of the tail of C57BL/6 mice. After 24 hours, the mice were anesthetized to death, and the mice were taken. Fluorescence imaging of the inguinal lymph nodes was performed to evaluate the lymph node targeting effect of the tubular DNA nano-vaccine.
  • mice B16-OVA melanoma cells 2.0 ⁇ 10 5 mouse B16-OVA melanoma cells were injected into the tail vein of C57BL/6 mice, and this time was counted as day 0; a certain dose of the tubular DNA nano vaccine of Example 2 (the tumor antigen polypeptide is SEQ ID NO:11: SIINFEKLRRG) was inoculated on the base of the tail of melanoma model mice on the 1st and 7th days. The mice were sacrificed on the 16th day. The lung tissues of the mice were surgically removed and the lung tissues of the mice were observed. The formation of metastases in the middle.
  • the tubular DNA nano vaccine of Example 2 the tumor antigen polypeptide is SEQ ID NO:11: SIINFEKLRRG
  • mice 2.0 ⁇ 10 5 mouse B16-OVA melanoma cells were inoculated on the back of C57BL/6 mice and counted as day 0; on day 4 after inoculation, melanoma basically formed; on day 4 and On the 11th day, mice were inoculated with a certain dose of the tubular DNA nano vaccine of Example 2 (the tumor antigen polypeptide is SEQ ID NO: 11: SIINFEKLRRG) at the base of the tail. The tumor volume was measured every 2 days, and the tumor volume changes were statistically analyzed. happening. The tumor volume is calculated according to the following formula, where d is the smallest diameter of the tumor and D is the largest diameter of the tumor. The mice in the control group were injected with saline.
  • the antigen polypeptide gp100 25-33 (KVPRNQDWL) was selected.
  • KVPRNQDWL antigen polypeptide gp100 25-33
  • the sample connected with the tumor antigen polypeptide and the two immunoadjuvants is mixed with the DNA molecule "switch" at a molar ratio of 1:1 and annealed.
  • the annealing conditions are: from 45°C to 25°C, every 1 °C is a gradient, each gradient has a residence time of 5 min, and performs 6 cycles; then the PCR products are separated by centrifugation with a 100kDa spin column, purified and recovered by agarose gel electrophoresis, and purified tubular DNA nano-vaccine containing antigen and adjuvant is obtained.
  • Composite structure is: from 45°C to 25°C, every 1 °C is a gradient, each gradient has a residence time of 5 min, and performs 6 cycles; then the PCR products are separated by centrifugation with a 100kDa spin column, purified and recovered by agarose gel electrophoresis, and purified tubular DNA nano-vaccine containing antigen and adjuvant is obtained.
  • the morphology of the constructed DNA nanostructure was characterized by transmission electron microscopy.
  • the structure was about 90-100nm long and about 20nm wide, presenting a regular tubular structure.
  • the antigen Adpgk polypeptide (ASMTNMELM) was selected.
  • ASMTNMELM the antigen Adpgk polypeptide
  • the sample connected with the tumor antigen polypeptide and the two immunoadjuvants is mixed with the DNA molecule "switch" at a molar ratio of 1:1 and annealed.
  • the annealing conditions are: from 45°C to 25°C, every 1 °C is a gradient, each gradient has a residence time of 5 min, and performs 6 cycles; then the PCR products are separated by centrifugation with a 100kDa spin column, purified and recovered by agarose gel electrophoresis, and purified tubular DNA nano-vaccine containing antigen and adjuvant is obtained.
  • Composite structure is: from 45°C to 25°C, every 1 °C is a gradient, each gradient has a residence time of 5 min, and performs 6 cycles; then the PCR products are separated by centrifugation with a 100kDa spin column, purified and recovered by agarose gel electrophoresis, and purified tubular DNA nano-vaccine containing antigen and adjuvant is obtained.
  • the morphology of the constructed DNA nanostructure was characterized by transmission electron microscopy.
  • the structure is about 90-100nm long and 20nm wide, presenting a regular tubular structure.
  • mice 2.0 ⁇ 10 5 mouse B16-F10 melanoma cells were inoculated on the back of C57BL/6 mice and counted as day 0; on day 4 after inoculation, melanoma basically formed; on day 4 and On the 11th day, mice were inoculated with a certain dose of the tubular DNA nano vaccine of Example 6 at the base of the tail, and the tumor volume was measured every 2 days, and the tumor volume was continuously observed for 16 days to statistically analyze the changes in tumor volume.
  • the tumor volume is calculated according to the following formula, where d is the smallest diameter of the tumor and D is the largest diameter of the tumor.
  • the mice in the control group were injected with saline.
  • the tumor size is shown in Table 1 below.
  • the DNA nano-vaccine treatment group can effectively inhibit the proliferation of melanoma in tumor-bearing mice, showing a significant tumor treatment effect.
  • mice 1.0 ⁇ 10 5 mouse MC-38 colorectal cancer cells were inoculated on the back of C57BL/6 mice, which was counted as day 0; on the 4th day after inoculation, colorectal tumors basically formed; On day and day 11, mice were inoculated with 100 nM (100 ⁇ L) of the tubular DNA nano-vaccine of Example 7 at the base of the tail. The tumor volume was measured every 2 days, and the tumor volume was continuously observed for 20 days, and the tumor volume changes were analyzed statistically. The tumor volume is calculated according to the following formula, where d is the smallest diameter of the tumor and D is the largest diameter of the tumor. The mice in the control group were injected with saline.
  • the tumor size is shown in Table 2 below.
  • the DNA nano-vaccine treatment group can effectively inhibit the proliferation of colorectal tumors in tumor-bearing mice, showing a significant tumor treatment effect.
  • the present invention uses the circular DNA single strand of the M13mp18 bacteriophage as the main chain, and the excess short-strand DNA as the auxiliary chain.
  • the main chain and the programmable auxiliary chain hybridize and complement each other at a specific position and fold to form a double One-dimensional rectangular sheet-layer DNA nanostructures, according to the principle of base complementary pairing, use capture DNA strands to connect tumor-specific antigen polypeptides, double-stranded RNA immune adjuvants and CpG immune adjuvants in a self-assembled two-dimensional sheet-layer DNA nanostructure Surface; then, hybridize the acid-responsive DNA "switch" on the two long sides of the rectangular sheet DNA nanostructure, thereby curling the rectangular structure into a tubular structure, and obtaining a tumor antigen and immune adjuvant loaded inside
  • a tubular DNA nanoparticle vaccine with a controllable "switch" to respond to the acidic environment inside the antigen-presenting cell; the bottom diameter of the nanoparticle vaccine is 19nm and

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Abstract

本发明提供了一种DNA纳米疫苗及其制备方法和应用，所述DNA纳米疫苗包括DNA纳米结构、肿瘤抗原多肽-DNA复合物和免疫佐剂；所述免疫佐剂包括双链RNA免疫佐剂和/或CpG免疫佐剂。本发明构建了一种由DNA模板、辅助折叠DNA链和捕获DNA链组装而成的纳米结构，通过捕获DNA链与功能成分杂交，实现了肿瘤抗原分子和免疫佐剂分子在DNA自组装纳米结构表面的精确定位组装；同时，在管状DNA纳米结构的一侧设计可控的DNA分子"开关"，能够在进入抗原递呈细胞后响应内涵体的酸环境，响应性的开启管状结构从而释放肿瘤抗原和免疫佐剂分子，具有肿瘤抗原特异性免疫刺激效果，是一种用于多种恶性肿瘤的免疫治疗与预防的肿瘤疫苗。

Description

一种DNA纳米疫苗及其制备方法和应用
相关申请的交叉引用
本申请要求2019年09月09日提交的第CN201910849707.2号中国发明专利申请的优先权，所述申请以引用的方式整体并入本文。
技术领域
本发明属于纳米医学技术领域，涉及一种DNA纳米疫苗及其制备方法和应用，尤其涉及一种装载有肿瘤抗原和免疫佐剂的管状DNA纳米疫苗及其制备方法和在肿瘤免疫治疗中的应用。
背景技术
目前，肿瘤治疗存在着预后较差、五年生存率低的问题，提高肿瘤的治疗效果是亟待解决的科学问题。近年来快速发展的肿瘤免疫治疗方法为肿瘤治疗带来了突破性成果，肿瘤疫苗，尤其是针对特定患者的个体化疫苗越来越受到研究者的重视，有望在不久的将来成为新的肿瘤治疗方案。然而，肿瘤疫苗的有效性仍然有限。例如，游离抗原在被树突状细胞(dendritic cells，DC)内化之前可能被迅速清除；在没有免疫佐剂存在的条件下，肿瘤疫苗容易诱导机体产生免疫耐受，限制了其治疗效果。如何提高免疫系统的响应能力，进一步提高肿瘤疫苗的治疗效果并减少副作用的发生，是亟待解决的问题。近期研究证实，药物递送系统可以通过调节免疫治疗药物的生物分布、定位、体内稳定性和释放动力学，来增强免疫刺激治疗的功效。因此，利用生物材料设计新型的纳米肿瘤疫苗，具有重要的研究意义和广阔的应用前景。
CN102614527A公开了一种具有pH敏感特性的抗酸纳米颗粒口服DNA抗肿瘤疫苗及制备方法，所述的DNA口服疫苗是一种由海藻酸进行表面修饰的壳聚糖结合编码肿瘤特异性抗原Legumain蛋白DNA质粒的纳米颗粒，能够高效在小肠派氏淋巴结中被树突状细胞及巨噬细胞吞噬，并表达编码的肿瘤抗原，激活宿主对肿瘤细胞的免疫杀伤，毒副作用小，且具有强烈的抗原呈递作用，但是存在循环时间短、易被清除等问题。
DNA折纸纳米技术是一种新颖而独特的DNA自组装技术，已被广泛应用于自下而上(Bottom-up)制备纳米尺度的二维和三维DNA纳米结构。利用DNA纳米技术构建的DNA纳米结构具有结构可控且易于修饰的特点，在药物运输和逆转耐药等方面具有广阔的应用前景。因此，研制高效、低毒、靶向和可控的DNA纳米结构作为药物运输载体，具有重大的实践意义。与传统的抗肿瘤药物运输载体相比，DNA纳米结构由于其结构可控和易于修饰的特点，在药物靶向运输和可控释放等方面具有显著的优势。DNA纳米结 构由DNA组装形成，具有良好的生物相容性；DNA按照碱基互补配对原则进行组装，具有高度的结构预测性；内部进行功能化修饰，能够有效装载具有活性的多种药物，包括基因药物、小分子化学药物、蛋白质或者抗体等；由DNA纳米技术构建的复杂高级结构在细胞裂解液中具有良好的结构稳定性，对于内部装载的药物起到优良的保护作用；外表面使用短链DNA杂交，可以在特定部位靶向修饰功能基团，增强了载药系统的靶向性；DNA纳米结构还可以通过修饰纳米粒子，从而在特定条件下可控开启和关闭，达到控制药物释放的目的。
CN103656662A公开了一种利用多肽介导的DNA纳米结构用作抗肿瘤药物载体的方法，将具备一定功能的多肽连接在DNA纳米结构上，所制备的产物为DNA纳米结构与多肽的复合物，DNA纳米结构表面负载生物分子后与细胞相互作用时，特定功能的多肽能介导负载生物分子的DNA纳米结构进入细胞或特异性地与细胞表面的受体结合，实现DNA结构用作抗肿瘤药物载体的目的，在抗肿瘤药物载体的开发、研究及提高抗肿瘤药物载体的载入效率等方面具有潜在的应用价值，但是所述DNA纳米结构仅能够作为药物载体，不具有肿瘤治疗的效果。
CN109675049A公开了一种pH诱导的药物缓释DNA纳米结构及其制备方法与应用，通过滚环扩增技术合成了长单链DNA，与富含G和C的DNA单链(loading链)互补杂交得到单双链交替的DNA分子聚集体，可负载大量的Dox；当体系的pH降低时，滚环扩增产物折叠形成三螺旋构型，DNA分子聚集体双链解链，释放富含G和C的DNA单链，并且释放出嵌插的Dox，从而完成药物释放，可通过调节pH实现Dox的可逆嵌插和释放；该方法利用生物相容性良好的DNA纳米结构作为药物载体，提高载药量，具有成本低廉、操作简便、对pH响应灵敏度高、反应快速等优点，但是存在对肿瘤的靶向性差的问题。
因此，构建一种新型的肿瘤疫苗，实现疫苗的靶向输送和可控释放，在提高治疗效果的同时减少副作用的发生，在肿瘤免疫治疗领域具有重要意义。
发明内容
针对现有技术的不足，本发明提供了一种DNA纳米疫苗及其制备方法和应用，所述DNA纳米疫苗通过精确的位点设计将肿瘤抗原多肽分子、双链RNA佐剂和CpG佐剂杂交到DNA纳米结构的内部，形成管状三维结构，并在管状DNA纳米结构的表面设置可控DNA开关，以响应抗原递呈细胞中内涵体的酸性环境，实现了肿瘤抗原多肽和免疫佐剂的靶向输送和可控释放，开发得到一种可寻址、安全高效、可控释放、具有较高医用价值的新型肿瘤疫苗。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种DNA纳米疫苗，所述DNA纳米疫苗包括DNA纳米结构、肿瘤抗原多肽-DNA复合物和免疫佐剂；
所述免疫佐剂包括双链RNA免疫佐剂和/或CpG免疫佐剂。
本发明中，DNA纳米疫苗具有抗原递呈细胞内涵体响应性，可有效引起肿瘤特异性免疫反应，有效抑制肿瘤的生长。
本发明中，炔基修饰肿瘤抗原多肽通过“click”反应与叠氮修饰DNA链连接形成肿瘤抗原多肽-DNA复合物；双链RNA免疫佐剂以DNA为模板，体外转录形成单链RNA，并将两条单链RNA按照1:1的摩尔比混合后退火得到双链RNA。
本发明中，采用肿瘤抗原多肽和两种免疫佐剂发挥协同作用，通过精准控制三者的相对位置，同时激活TLR3和TLR9两个信号通路，优于仅包含一种免疫佐剂的结构。
优选地，所述DNA纳米结构由DNA模板链、辅助折叠DNA链和捕获DNA链组装形成。
优选地，所述DNA模板链包括M13mp18噬菌体基因组DNA和/或λ噬菌体基因组DNA，更优选为M13mp18噬菌体基因组DNA。
本发明中，利用M13mp18噬菌体的环状DNA单链作为主链，过量的短链DNA作为辅链，通过主链与可编程的辅链在特定位置的杂交互补，折叠形成一种二维长方形片层DNA纳米结构。
本发明中，经过基因改造的M13噬菌体基因组DNA或非对称PCR扩增产物也可以作为DNA模板链，用于构建DNA纳米结构。
优选地，所述M13mp18噬菌体基因组DNA的核苷酸序列如SEQ ID NO:1所示；
Figure PCTCN2020113489-appb-000001
Figure PCTCN2020113489-appb-000002
Figure PCTCN2020113489-appb-000003
Figure PCTCN2020113489-appb-000004
Figure PCTCN2020113489-appb-000005
本发明中，辅助折叠DNA链根据文章“Folding DNA to create nanoscale shapes and patterns.Nature,2006,440,297-302”进行设计，本领域技术人员可以根据需要挑选辅助折叠DNA链，而捕获DNA链的设计可以根据需要进行增多、减少或位点的变更，在整个DNA纳米结构的平面上均可以进行设计。
示例性地，所述辅助折叠DNA链举例如下：
Figure PCTCN2020113489-appb-000006
优选地，所述捕获DNA链包括捕获DNA链I、捕获DNA链II和捕获DNA链Ⅲ。
优选地，所述捕获DNA链I由在辅助折叠DNA链的5’端加上与肿瘤抗原多肽-DNA复合物的DNA序列互补杂交的捕获序列I形成，所述捕获序列I的核苷酸序列如SEQ ID NO:16～24所示；
Figure PCTCN2020113489-appb-000007
Figure PCTCN2020113489-appb-000008
优选地，所述捕获DNA链II由在辅助折叠DNA链的5’端加上与双链RNA免疫佐剂的粘性末端序列互补杂交的捕获序列II形成，所述捕获序列II的核苷酸序列如SEQ ID NO:25～33所示；
Figure PCTCN2020113489-appb-000009
Figure PCTCN2020113489-appb-000010
和/或优选地，所述捕获DNA链Ⅲ由在辅助折叠DNA链的5’端加上与CpG免疫佐剂的5’端延长序列互补杂交的捕获序列Ⅲ形成，所述捕获序列Ⅲ的核苷酸序列如SEQ ID NO:34～42所示；
Figure PCTCN2020113489-appb-000011
根据上述设计，确保每个DNA纳米结构表面具有捕获位点，捕获位点处的捕获DNA链与肿瘤抗原多肽、双链RNA免疫佐剂和CpG免疫佐剂的延伸序列互补，通过DNA退火杂交，将三者按照一定比例组装至DNA纳米结构表面的特定位点。
优选地，所述肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂通过 捕获DNA链结合在DNA纳米结构上。
本发明中，利用碱基互补配对原则，通过位点设计将肿瘤抗原多肽-DNA复合物、双链RNA(dsRNA)免疫佐剂和CpG免疫佐剂连接在自组装形成的二维片层DNA纳米结构表面，精准控制肿瘤抗原多肽和免疫佐剂在片层DNA纳米结构表面的数量和相对位置。
优选地，所述肿瘤抗原多肽-DNA复合物的个数为10-30个，例如可以是10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个或30个，更优选为15～20个。
优选地，所述双链RNA免疫佐剂的个数为10-30个，例如可以是10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个或30个，更优选为15～20个。和/或
优选地，所述CpG免疫佐剂的个数为10-30个，例如可以是10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个或30个，更优选为15～20个。
本发明中，肿瘤抗原多肽为本领域技术人员知晓的任意肿瘤抗原多肽，也可以是根据个体化肿瘤患者筛选得到的新的肿瘤抗原多肽序列。
优选地，所述肿瘤抗原多肽的氨基酸序列如SEQ ID NO:11所示；
SEQ ID NO:11所示的氨基酸序列为：SIINFEKLRRG。
本发明中，所述肿瘤抗原多肽-DNA复合物中DNA的序列如SEQ ID NO:12所示；
SEQ ID NO:12所示的核苷酸序列为：
Figure PCTCN2020113489-appb-000012
示例性地，所述肿瘤抗原多肽-DNA复合物的序列为：
Figure PCTCN2020113489-appb-000013
本发明中，用于体外转录合成双链RNA免疫佐剂的DNA模板的核苷酸序列如SEQ ID NO:13～14所示；
SEQ ID NO:13：
5’-TAATACGACTCACTATAGGTAAACTCTTTGCGCACATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCGCTGGAAGATGGAACCGCTGGAGAGCAACTGCATAAGGCTATGAAGAGATACGCCCTGGTTCCTGGAACAATTGCTTTTACAGATGCACATAT-3’；
SEQ ID NO:14：
5’-TTAATACGACTCACTATAGGATATGTGCATCTGTAAAAGCAATTGTTCCAGGAACCAGGGCGTATCTCTTCATAGCCTTATGCAGTTGCTCTCCAGCGGTTCCATCTTCCAGCGGATAGAATGGCGCCGGGCCTTTCTTTATGTTTTTGGCGTCTTCCAT-3’.
优选地，所述CpG免疫佐剂的核苷酸序列如SEQ ID NO:15所示；
SEQ ID NO:15：
5’-GTTAGTGTTAGTGTTAGTTTGCAAGCTGTTGGGTTACCACCTTCATTGGAAAACGTTCTTCGGGGCGTTCTTAGGTGGTAACC-3’.
优选地，所述DNA纳米疫苗的形状包括长方形二维结构和/或管状三维结构。
优选地，所述长方形二维结构的长为80～100nm，例如可以是80nm、81nm、82nm、83nm、84nm、85nm、86nm、87nm、88nm、89nm、90nm、91nm、92nm、93nm、94nm、95nm、96nm、97nm、98nm、99nm或100nm，更优选为90～100nm。
优选地，所述长方形二维结构的宽为50～70nm，例如可以是50nm、51nm、52nm、53nm、54nm、55nm、56nm、57nm、58nm、59nm、60nm、61nm、62nm、63nm、64nm、65nm、66nm、67nm、68nm、69nm或70nm，更优选为50～60nm。
优选地，所述管状三维结构的底部直径为10～25nm，例如可以是10nm、11nm、12nm、13nm、14nm、15nm、16nm、17nm、18nm、19nm、20nm、21nm、22nm、23nm、24nm或25nm，更优选为19～20nm。和/或
优选地，所述管状三维结构的高为80～100nm，例如可以是80nm、81nm、82nm、83nm、84nm、85nm、86nm、87nm、88nm、89nm、90nm、91nm、92nm、93nm、94nm、95nm、96nm、97nm、98nm、99nm或100nm，更优选为90～100nm。
进一步优选地，所述管状三维结构的DNA纳米疫苗上有DNA开关。
本发明中，通过在长方形片层DNA纳米结构的两条长边上杂交具有酸性环境响应能力的DNA开关，所述DNA开关由两条单链DNA杂交形成，两条单链DNA的3’端互补形成酸性响应的双链“锁”，将二维长方形DNA纳米结构卷曲闭合形成三维管状DNA纳米疫苗，最终制备得到内部装载有肿瘤抗原多肽和免疫佐剂、同时具有可控DNA开关的管状DNA纳米疫苗，所述管状DNA纳米疫苗实现了肿瘤抗原多肽和免疫佐剂的装载、运输和在淋巴结中的可控释放，是一种新型肿瘤免疫疫苗。
本发明中，DNA开关响应抗原递呈细胞中内涵体的酸性环境，控制管状结构打开，暴露其中的肿瘤抗原多肽和两种免疫佐剂。
更进一步优选地，所述DNA开关的个数为5～10个，例如可以是5个、6个、7个、8个、9个或10个，再优选为8～10个。和/或
更进一步优选地，所述DNA开关的核苷酸序列如SEQ ID NO:43～58所示；
Figure PCTCN2020113489-appb-000014
Figure PCTCN2020113489-appb-000015
本发明用于肿瘤免疫治疗的管状DNA纳米疫苗，对于装载的肿瘤抗原多肽和免疫佐剂的释放是可控的，管状DNA纳米疫苗被抗原递呈细胞摄取后响应内涵体的酸环境，使 三种有效成分在内涵体中可控释放出来。
第二方面，本发明提供了一种如第一方面所述的DNA纳米疫苗的制备方法，所述方法包括以下步骤：
(1)将DNA模板链、辅助折叠DNA链和捕获DNA链按比例混合于缓冲液中，退火，得到长方形DNA纳米结构；
(2)将步骤(1)得到的退火产物离心纯化，与肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂按比例混合后退火；
(3)将步骤(2)得到的退火产物与DNA开关按比例混合后退火；
(4)将步骤(3)得到的退火产物离心纯化，得到管状DNA纳米疫苗。
优选地，步骤(1)所述退火的条件为：起点温度95℃～65℃，终点温度25℃～4℃，每1℃为一个梯度，每个梯度停留5～10min，保持退火时间为2～24h，优选为7～9h。
优选地，所述起点温度为95～65℃，例如可以是95℃、93℃、91℃、90℃、87℃、85℃、83℃、81℃、80℃、77℃、75℃、73℃、71℃、69℃、67℃或65℃。
优选地，所述终点温度为25～4℃，例如可以是25℃、24℃、23℃、21℃、20℃、19℃、17℃、15℃、13℃、11℃、7℃、5℃或4℃。
优选地，所述退火过程的温度为2～24h，例如可以是2h、4h、6h、7h、8h、9h、10h、12h、14h、16h、18h、20h、22h或24h。
优选地，步骤(1)所述DNA模板链、辅助折叠DNA链和捕获DNA链的摩尔比为1:(5-20):(5-20)，例如可以是1:5:5、1:7:7、1:10:10、1:15:15或1:20:20，优选为1:(5-10):(5-10)。
优选地，步骤(1)所述的缓冲液为1×TAE/Mg 2+。
优选地，步骤(2)和步骤(3)所述退火的条件为：起点温度45～37℃，终点温度25～16℃，每1℃为一个梯度，每个梯度停留3～8min，进行3～10个循环。
优选地，所述起点温度为45～37℃，例如可以是45℃、44℃、43℃、42℃、41℃、40℃、39℃、38℃或37℃。
优选地，所述终点温度为25～16℃，例如可以是25℃、24℃、23℃、22℃、21℃、20℃、19℃、18℃、17℃或16℃。
优选地，所述循环的个数为3～10个，例如可以是3个、4个、5个、6个、7个、8个、9个或10个。
优选地，步骤(2)所述退火产物、肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂的摩尔比为1:(2-10):(2-10):(2-10)。
优选地，步骤(3)所述退火产物与DNA开关的摩尔比为1:(1-2)。和/或
优选地，步骤(2)和步骤(4)所述离心的步骤为：将得到的退火产物与1×TAE/Mg 2+缓冲液混合并加入100kDa的离心柱，进行离心。
本发明中，发明人通过大量复杂的实验摸索，优化反应条件，探究退火温度和反应 时间对纳米结构的影响，各步骤各条件协同增效，最终成功制备得到性能优良的管状DNA纳米疫苗复合结构。
作为优选技术方案，本发明提供了一种如第一方面所述的DNA纳米疫苗的制备方法，所述方法包括以下步骤：
(1)将DNA模板链、辅助折叠DNA链和捕获DNA链按摩尔比为1:(5-20):(5-20)混合于1×TAE/Mg 2+缓冲液中进行退火，退火的条件为：从95℃到65℃，每1℃为一个梯度，每个梯度停留时间为5min；从65℃到25℃，每1℃为一个梯度，每个温度梯度停留时间为10min，整个退火过程为7-9h，得到长方形DNA纳米结构；
(2)将步骤(1)得到的退火产物与1×TAE/Mg 2+缓冲液混合并加入100kDa的离心柱，进行离心，随后与肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂按摩尔比为1:(2-10):(2-10):(2-10)混合并退火，退火的条件为：从45℃到25℃，每1℃为一个梯度，每个梯度停留时间为3～5min，进行6个循环；
(3)将步骤(2)得到的退火产物与DNA开关按摩尔比为1:(1-2)混合并退火，退火的条件为：从45℃到25℃，每1℃为一个梯度，每个梯度停留时间为3～5min，进行6个循环；
(4)将步骤(3)得到的退火产物与1×TAE/Mg 2+缓冲液混合并加入100kDa的离心柱，进行离心，得到管状DNA纳米疫苗。
第三方面，本发明提供了一种药物组合物，所述药物组合物包括如第一方面所述的DNA纳米疫苗。
优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
第四方面，本发明提供了一种如第一方面所述的DNA纳米疫苗和/或如第三方面所述的药物组合物在制备肿瘤免疫治疗疫苗中的应用。
根据第四方面所述的应用，所述肿瘤免疫治疗疫苗为广谱抗肿瘤疫苗；
优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
本发明中，所述肿瘤不限于单一类型的恶性肿瘤，DNA纳米疫苗和/或药物组合物的抗肿瘤免疫治疗效果具有广谱性，可以用于多种恶性肿瘤的治疗与预防，例如黑色素瘤、乳腺癌、结肠癌等恶性肿瘤。
第五方面，本发明提供了一种用于免疫治疗肿瘤的方法，所述方法包括：对有需要的受试者给予第一方面所述的DNA纳米疫苗、按照第二方面所述的制备方法而制得的DNA纳米疫苗和/或如第三方面所述的药物组合物；
优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
第六方面，本发明提供了一种用于预防肿瘤的方法，所述方法包括：对有需要的受试者给予第一方面所述的DNA纳米疫苗、按照第二方面所述的制备方法而制得的DNA 纳米疫苗和/或如第三方面所述的药物组合物；
优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
第七方面，本发明提供了一种用于免疫治疗肿瘤的药物，所述药物包括第一方面所述的DNA纳米疫苗、按照第二方面所述的制备方法而制得的DNA纳米疫苗和/或如第三方面所述的药物组合物；
优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
第八方面，本发明提供了一种用于预防肿瘤的药物，所述药物包括第一方面所述的DNA纳米疫苗、按照第二方面所述的制备方法而制得的DNA纳米疫苗和/或如第三方面所述的药物组合物；
优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
与现有技术相比，本发明具有如下有益效果：
(1)本发明利用DNA纳米技术，以M13mp18噬菌体的环状DNA单链作为主链，以过量的短链DNA作为辅链，通过主链与可编程的辅链在特定位置的杂交互补，折叠形成结构可预测和可控制的二维长方形片层DNA纳米结构；
(2)本发明根据碱基互补配对原则，利用捕获DNA链将肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂连接在自组装形成的二维片层DNA纳米结构表面；
(3)本发明在长方形片层DNA纳米结构的两条长边上杂交具有酸性环境响应能力的DNA开关，将片层DNA纳米结构卷曲闭合形成三维管状DNA纳米疫苗，制备得到内部装载有肿瘤抗原多肽和免疫佐剂、同时具有可控DNA开关的管状DNA纳米疫苗；
(4)本发明的管状DNA纳米疫苗能够在靶细胞内响应酸性环境，实现了可控的构象变化，释放肿瘤抗原分子和免疫佐剂，增强了免疫刺激效果；
(5)本发明精准控制肿瘤抗原多肽和两种免疫佐剂在片层DNA纳米结构表面的数量和相对位置，通过调整三者的比例，实现了同时激活TLR3和TLR9信号通路的效果，发挥协同增效作用；
(6)本发明的DNA纳米疫苗作为一种纳米尺度的分子机器用于肿瘤抗原和免疫佐剂的装载，并有效运输到淋巴结中实现可控释放，有望为肿瘤免疫治疗提供一种纳米疫苗新剂型。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1为实施例1的长方形片层DNA纳米结构的原子力显微镜形貌观察图；
图2为实施例2的管状DNA纳米疫苗的原子力显微镜形貌观察图；
图3为实施例3的管状DNA纳米疫苗经皮下注射后对腹股沟淋巴结的靶向效果；
图4为实施例4的管状DNA纳米疫苗对黑色素瘤细胞肺转移的抑制效果图；
图5为实施例5的管状DNA纳米疫苗对小鼠黑色素瘤生长的抑制效果图；
图6为实施例6的管状DNA纳米结构的透射电镜图；
图7为实施例7的管状DNA纳米结构的透射电镜图。
实施发明的最佳方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1 长方形片层DNA纳米结构的制备
将M13mp18模板链、辅助折叠DNA链(“订书钉”链)和捕获DNA链混合，模板链和辅助折叠DNA链的终浓度分别为20nM和100nM；利用梯度PCR仪对混合物进行缓慢退火，退火的条件为：从95℃到65℃，每1℃为一个梯度，每个梯度停留时间为5min；从65℃到25℃，每1℃为一个梯度，每个温度梯度停留时间为10min；整个退火过程为8h，得到长方形片层DNA纳米结构；
退火程序完毕后，将长方形DNA纳米结构样品取出，用100kDa离心柱(MWCO)离心分离，去除过量的订书钉链和捕获DNA链；离心条件为：向100μL样品中加入350μL 1×TAE-Mg缓冲液，于4800rpm/min的条件下离心3min，离心柱内剩余溶液的体积大约为100μL，重复离心4遍；最终收集的样品分别采用1％琼脂糖凝胶电泳分析和在原子力显微镜下观察片层结构的形貌。
结果如图1所示，构建的DNA纳米结构呈长方形片层结构，原子力显微镜扫描结果显示该长方形DNA纳米结构长约90-100nm，宽约60-80nm，呈现规则的长方形结构。
实施例2 装载肿瘤抗原和免疫佐剂的管状DNA纳米疫苗的制备
将纯化后的长方形片层DNA纳米结构溶液和肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂、CpG免疫佐剂按照1:5:5:5的摩尔比混合均匀，放入梯度PCR仪中，缓慢从45℃降至25℃，每1℃为一个梯度，每个梯度停留时间为5min；进行6个循环；
退火程序结束后，将连接了肿瘤抗原多肽和两种免疫佐剂的样品与DNA分子“开关”按照1:1的摩尔比混合并退火，退火的条件为：从45℃到25℃，每1℃为一个梯度，每个梯度停留时间为5min，进行6个循环；然后用100kDa离心柱离心分离PCR产物，琼脂糖凝胶电泳纯化回收，得到提纯的包载有抗原和佐剂的管状DNA纳米疫苗复合结构。
结果如图2所示，利用原子力显微镜(AFM)对构建的管状DNA纳米结构进行形貌表征，该结构长约90-100nm，宽约20nm，呈现规则的管状结构。
实施例3 管状DNA纳米疫苗的淋巴结靶向效果评价
将一定剂量的Cy5荧光标记的实施例2的管状DNA纳米疫苗(肿瘤抗原多肽为SEQ ID NO:11：SIINFEKLRRG)接种于C57BL/6小鼠尾基部，24h后将小鼠麻醉处死，取小鼠腹股沟淋巴结进行荧光成像，评价管状DNA纳米疫苗的淋巴结靶向效果。
结果如图3所示，与对照组(荧光标记DNA链和荧光标记长方形DNA纳米结构)相比，管状DNA纳米结构在小鼠的腹股沟淋巴结富集效果明显，说明该管状DNA纳米结构作为疫苗的载体具有明显的优势。
实施例4 管状DNA纳米疫苗的抗肿瘤转移效果评价
将2.0×10 5个小鼠B16-OVA黑色素瘤细胞尾静脉注射到C57BL/6小鼠体内，此时计为第0天；将一定剂量的实施例2的管状DNA纳米疫苗(肿瘤抗原多肽为SEQ ID NO:11：SIINFEKLRRG)在第1天和第7天给接种于黑色素瘤模型小鼠的尾基部，在第16天将小鼠处死，手术取下小鼠肺组织，观察小鼠肺组织中转移灶的形成情况。
结果如图4所示，与对照组(生理盐水组)相比，实验组(管状DNA纳米疫苗组)的肺组织中转移灶的数量明显较少，说明管状DNA纳米疫苗对肿瘤转移具有明显的抑制作用。
实施例5 管状DNA纳米疫苗的抗肿瘤效果评价
将2.0×10 5个小鼠B16-OVA黑色素瘤细胞接种于C57BL/6小鼠的背部，此时计为第0天；在接种后的第4天，黑色素瘤基本形成；在第4天和第11天分别在小鼠的尾基部接种一定剂量的实施例2的管状DNA纳米疫苗(肿瘤抗原多肽为SEQ ID NO:11：SIINFEKLRRG)，每隔2天测量肿瘤体积大小，统计分析肿瘤体积变化情况。肿瘤体积按照以下公式计算，其中d为肿瘤的最小直径，D为肿瘤的最大直径，对照组小鼠注射生理盐水。
体积＝(d 2×D)/2
结果如图5(A)和图5(B)所示，相比于生理盐水组，实验组(管状DNA纳米疫苗组)能够有效抑制荷瘤小鼠黑色素瘤的增殖，其中有4只小鼠的肿瘤完全消退，展示出显著的肿瘤治疗效果。
实施例6 装载肿瘤抗原gp100和免疫佐剂的管状DNA纳米疫苗的制备
针对黑色素瘤B16F10，选取抗原多肽gp100 25-33(KVPRNQDWL)。将纯化后的长方形片层DNA纳米结构溶液和肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂、CpG免疫佐剂按照1:5:5:5的摩尔比混合均匀，放入梯度PCR仪中，缓慢从45℃降至25℃，每1℃为一个梯度，每个梯度停留时间为5min；进行6个循环；
退火程序结束后，将连接了肿瘤抗原多肽和两种免疫佐剂的样品与DNA分子“开关”按照1:1的摩尔比混合并退火，退火的条件为：从45℃到25℃，每1℃为一个梯度，每 个梯度停留时间为5min，进行6个循环；然后用100kDa离心柱离心分离PCR产物，琼脂糖凝胶电泳纯化回收，得到提纯的包载有抗原和佐剂的管状DNA纳米疫苗复合结构。
如图6所示，利用透射电镜对构建的DNA纳米结构进行形貌表征，该结构长约90-100nm，宽约20nm，呈现规则的管状结构。
实施例7 装载肿瘤抗原Adpgk和免疫佐剂的管状DNA纳米疫苗的制备
针对结直肠肿瘤，选取抗原Adpgk多肽(ASMTNMELM)。将纯化后的长方形片层DNA纳米结构溶液和肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂、CpG免疫佐剂按照1:5:5:5的摩尔比混合均匀，放入梯度PCR仪中，缓慢从45℃降至25℃，每1℃为一个梯度，每个梯度停留时间为5min；进行6个循环；
退火程序结束后，将连接了肿瘤抗原多肽和两种免疫佐剂的样品与DNA分子“开关”按照1:1的摩尔比混合并退火，退火的条件为：从45℃到25℃，每1℃为一个梯度，每个梯度停留时间为5min，进行6个循环；然后用100kDa离心柱离心分离PCR产物，琼脂糖凝胶电泳纯化回收，得到提纯的包载有抗原和佐剂的管状DNA纳米疫苗复合结构。
如图7所示，利用透射电镜对构建的DNA纳米结构进行形貌表征，该结构长约90-100nm，宽约20nm，呈现规则的管状结构。
实施例8 管状DNA纳米疫苗的抗黑色素瘤效果评价
将2.0×10 5个小鼠B16-F10黑色素瘤细胞接种于C57BL/6小鼠的背部，此时计为第0天；在接种后的第4天，黑色素瘤基本形成；在第4天和第11天分别在小鼠的尾基部接种一定剂量的实施例6的管状DNA纳米疫苗，每隔2天测量肿瘤体积大小，连续观测16天，统计分析肿瘤体积变化情况。肿瘤体积按照以下公式计算，其中d为肿瘤的最小直径，D为肿瘤的最大直径，对照组小鼠注射生理盐水。
体积＝(d 2×D)/2
肿瘤尺寸如下表1所示。相比于对照组，DNA纳米疫苗处理组能够有效抑制荷瘤小鼠黑色素瘤的增殖，展示出显著的肿瘤治疗效果。
表1 管状DNA纳米疫苗的抗黑色素瘤效果评价结果
Figure PCTCN2020113489-appb-000016
实施例9 管状DNA纳米疫苗的抗结直肠肿瘤效果评价
将1.0×10 5个小鼠MC-38结直肠癌细胞接种于C57BL/6小鼠的背部，此时计为第0天；在接种后的第4天，结直肠肿瘤基本形成；在第4天和第11天分别在小鼠的尾基部接种100nM(100μL)的实施例7的管状DNA纳米疫苗，每隔2天测量肿瘤体积大小，连续观测20天，统计分析肿瘤体积变化情况。肿瘤体积按照以下公式计算，其中d为肿瘤的最小直径，D为肿瘤的最大直径，对照组小鼠注射生理盐水。
体积＝(d 2×D)/2
肿瘤尺寸如下表2所示。相比于对照组，DNA纳米疫苗处理组能够有效抑制荷瘤小鼠结直肠肿瘤的增殖，展示出显著的肿瘤治疗效果。
表2 管状DNA纳米疫苗的抗结直肠肿瘤效果评价结果
Figure PCTCN2020113489-appb-000017
综上所述，本发明利用M13mp18噬菌体的环状DNA单链作为主链，过量的短链DNA作为辅链，通过主链与可编程的辅链在特定位置的杂交互补，折叠形成一种二维长方形片层DNA纳米结构，根据碱基互补配对原则，利用捕获DNA链将肿瘤特异性抗原多肽、双链RNA免疫佐剂和CpG免疫佐剂连接在自组装形成的二维片层DNA纳米结构表面；随后，在长方形片层DNA纳米结构的两条长边上杂交具有酸响应能力的DNA“开关”，从而将长方形结构卷曲闭合形成管状结构，得到一种内部装载有肿瘤抗原和免疫佐剂、同时具有可控“开关”以响应抗原递呈细胞内涵体内酸性环境的管状DNA纳米颗粒疫苗；所述纳米颗粒疫苗的底部直径为19nm，高为90nm，可以作为一种纳米尺度的分子机器用于肿瘤抗原和免疫佐剂的装载，有效运输到淋巴结中实现可控释放，有望为肿瘤免疫治疗法提供一种纳米疫苗新剂型。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。

Claims (15)

  1. 一种DNA纳米疫苗，其特征在于，所述DNA纳米疫苗包括DNA纳米结构、肿瘤抗原多肽-DNA复合物和免疫佐剂；
    所述免疫佐剂包括双链RNA免疫佐剂和/或CpG免疫佐剂。
  2. 根据权利要求1所述的DNA纳米疫苗，其特征在于，所述DNA纳米结构由DNA模板链、辅助折叠DNA链和捕获DNA链组装形成。
  3. 根据权利要求1或2所述的DNA纳米疫苗，其特征在于，所述DNA模板链包括M13mp18噬菌体基因组DNA和/或λ噬菌体基因组DNA，优选为M13mp18噬菌体基因组DNA；
    更优选地，所述M13mp18噬菌体基因组DNA的核苷酸序列如SEQ ID NO:1所示。
  4. 根据权利要求1-3任一项所述的DNA纳米疫苗，其特征在于，所述捕获DNA链包括捕获DNA链I、捕获DNA链II和捕获DNA链Ⅲ；
    优选地，所述捕获DNA链I由在辅助折叠DNA链的5’端加上与肿瘤抗原多肽-DNA复合物的DNA序列互补杂交的捕获序列I形成，所述捕获序列I的核苷酸序列如SEQ ID NO:16～24所示；
    优选地，所述捕获DNA链II由在辅助折叠DNA链的5’端加上与双链RNA免疫佐剂的粘性末端序列互补杂交的捕获序列II形成，所述捕获序列II的核苷酸序列如SEQ ID NO:25～33所示；和/或
    优选地，所述捕获DNA链Ⅲ由在辅助折叠DNA链的5’端加上与CpG免疫佐剂的5’端延长序列互补杂交的捕获序列Ⅲ形成，所述捕获序列Ⅲ的核苷酸序列如SEQ ID NO:34～42所示。
  5. 根据权利要求1-4任一项所述的DNA纳米疫苗，其特征在于，所述肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂通过捕获DNA链结合在DNA纳米结构上；
    优选地，所述肿瘤抗原多肽-DNA复合物的个数为10-30个，更优选为15～20个；
    优选地，所述双链RNA免疫佐剂的个数为10-30个，更优选为15～20个；
    优选地，所述CpG免疫佐剂的个数为10-30个，更优选为15～20个；
    优选地，所述肿瘤抗原多肽的氨基酸序列如SEQ ID NO:11所示；
    优选地，所述双链RNA免疫佐剂的DNA模板的核苷酸序列如SEQ ID NO:13～14所 示；和/或
    优选地，所述CpG免疫佐剂的核苷酸序列如SEQ ID NO:15所示。
  6. 根据权利要求1-5任一项所述的DNA纳米疫苗，其特征在于，所述DNA纳米疫苗的形状包括长方形二维结构和/或管状三维结构；
    优选地，所述长方形二维结构的长为80～100nm，更优选为90～100nm；
    优选地，所述长方形二维结构的宽为50～70nm，更优选为50～60nm；
    优选地，所述管状三维结构的底部直径为10～25nm，更优选为19～20nm；和/或
    优选地，所述管状三维结构的高为80～100nm，更优选为90～100nm；
    进一步优选地，所述管状三维结构的DNA纳米疫苗上有DNA开关；更进一步优选地，所述DNA开关的个数为5～10个，再优选为8～10个；和/或
    更进一步优选地，所述DNA开关的核苷酸序列如SEQ ID NO:43～58所示。
  7. 一种如权利要求1-6任一项所述的DNA纳米疫苗的制备方法，其特征在于，所述方法包括以下步骤：
    (1)将DNA模板链、辅助折叠DNA链和捕获DNA链按比例混合于缓冲液中，退火，得到长方形DNA纳米结构；
    (2)将步骤(1)得到的退火产物离心纯化，与肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂按比例混合后退火；
    (3)将步骤(2)得到的退火产物与DNA开关按比例混合后退火；
    (4)将步骤(3)得到的退火产物离心纯化，得到管状DNA纳米疫苗；
    优选地，步骤(1)所述退火的条件为：起点温度95℃～65℃，终点温度25℃～4℃，每1℃为一个梯度，每个梯度停留5～10min，保持退火时间为2～24h，优选为7～9h；
    优选地，步骤(1)所述DNA模板链、辅助折叠DNA链和捕获DNA链的摩尔比为1:(5-20):(5-20)，优选为1:(5-10):(5-10)；
    优选地，步骤(1)所述的缓冲液为1×TAE/Mg 2+；
    优选地，步骤(2)和步骤(3)所述退火的条件为：起点温度45～37℃，终点温度25～16℃，每1℃为一个梯度，每个梯度停留3～8min，进行3～10个循环；
    优选地，步骤(2)所述退火产物、肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂的摩尔比为1:(2-10):(2-10):(2-10)；
    优选地，步骤(3)所述退火产物与DNA开关的摩尔比为1:(1-2)；和/或
    优选地，步骤(2)和步骤(4)所述离心的步骤为：将得到的退火产物与1×TAE/Mg 2+缓冲液混合并加入100kDa的离心柱，进行离心。
  8. 根据权利要求7所述的方法，其特征在于，所述方法包括以下步骤：
    (1)将DNA模板链、辅助折叠DNA链和捕获DNA链按摩尔比为1:(5-20):(5-20)混合于1×TAE/Mg 2+缓冲液中进行退火，退火的条件为：从95℃到65℃，每1℃为一个梯度，每个梯度停留时间为5min；从65℃到25℃，每1℃为一个梯度，每个温度梯度停留时间为10min，整个退火过程为7-9h，得到长方形DNA纳米结构；
    (2)将步骤(1)得到的退火产物与1×TAE/Mg 2+缓冲液混合并加入100kDa的离心柱，进行离心，随后与肿瘤抗原多肽-DNA复合物、双链RNA免疫佐剂和CpG免疫佐剂按摩尔比为1:(2-10):(2-10):(2-10)混合并退火，退火的条件为：从45℃到25℃，每1℃为一个梯度，每个梯度停留时间为3～5min，进行6个循环；
    (3)将步骤(2)得到的退火产物与DNA开关按摩尔比为1:(1-2)混合并退火，退火的条件为：从45℃到25℃，每1℃为一个梯度，每个梯度停留时间为3～5min，进行6个循环；
    (4)将步骤(3)得到的退火产物与1×TAE/Mg 2+缓冲液混合并加入100kDa的离心柱，进行离心，得到管状DNA纳米疫苗。
  9. 一种药物组合物，其特征在于，所述药物组合物包括如权利要求1-6任一项所述的DNA纳米疫苗；
    优选地，所述药物组合物还包括药学上可接受的载体、赋形剂或稀释剂中的任意一种或至少两种的组合。
  10. 一种如权利要求1-6任一项所述的DNA纳米疫苗和/或如权利要求9所述的药物组合物在制备肿瘤免疫治疗疫苗中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述肿瘤免疫治疗疫苗为广谱抗肿瘤疫苗；
    优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
  12. 一种用于免疫治疗肿瘤的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1-6任一项所述的DNA纳米疫苗、按照权利要求7或8所述的制备方法而制得的DNA纳米疫苗和/或如权利要求9所述的药物组合物；
    优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
  13. 一种用于预防肿瘤的方法，其特征在于，所述方法包括：对有需要的受试者给予权利要求1-6任一项所述的DNA纳米疫苗、按照权利要求7或8所述的制备方法而制得的DNA纳米疫苗和/或如权利要求9所述的药物组合物；
    优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
  14. 一种用于免疫治疗肿瘤的药物，其特征在于，所述药物包括权利要求1-6任一项所述的DNA纳米疫苗、按照权利要求7或8所述的制备方法而制得的DNA纳米疫苗和/或如权利要求9所述的药物组合物；
    优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
  15. 一种用于预防肿瘤的药物，其特征在于，所述药物包括权利要求1-6任一项所述的DNA纳米疫苗、按照权利要求7或8所述的制备方法而制得的DNA纳米疫苗和/或如权利要求9所述的药物组合物；
    优选地，所述肿瘤选自以下一种或多种：黑色素瘤、乳腺癌、结肠癌。
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