WO2022057018A1 - 基于免疫佐剂的水凝胶组合物及其应用 - Google Patents

基于免疫佐剂的水凝胶组合物及其应用 Download PDF

Info

Publication number
WO2022057018A1
WO2022057018A1 PCT/CN2020/124668 CN2020124668W WO2022057018A1 WO 2022057018 A1 WO2022057018 A1 WO 2022057018A1 CN 2020124668 W CN2020124668 W CN 2020124668W WO 2022057018 A1 WO2022057018 A1 WO 2022057018A1
Authority
WO
WIPO (PCT)
Prior art keywords
sodium alginate
immune adjuvant
tumor
aptamer
cell death
Prior art date
Application number
PCT/CN2020/124668
Other languages
English (en)
French (fr)
Inventor
刘庄
孙乐乐
Original Assignee
苏州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州大学 filed Critical 苏州大学
Priority to US18/044,565 priority Critical patent/US20240148874A1/en
Publication of WO2022057018A1 publication Critical patent/WO2022057018A1/zh

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/04—Alginic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • 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
    • C12N2310/16—Aptamers

Definitions

  • the present invention relates to the field of tumor treatment preparations, in particular to an immunoadjuvant-based hydrogel composition and its application.
  • Tumor is a malignant disease that threatens human survival. With the aging of the world's population and changes in living environment and daily habits, the number of deaths due to cancer is increasing dramatically every year. Now, chemotherapy and radiotherapy are the main methods of clinical tumor treatment other than surgical resection. In recent years, many studies have found that certain chemotherapeutic drugs (anthracyclines and oxaliplatin) as well as ionizing radiation can induce tumor cell immunogenic death, which is a unique cell death pathway related to apoptosis, the death of cells Release endogenous danger signals.
  • chemotherapeutic drugs anthracyclines and oxaliplatin
  • calreticulin an endoplasmic reticulum calcium-binding protein
  • HMGB1 high mobility group B1
  • ATP adenosine triphosphate
  • chemotherapeutic drugs currently in clinical development or use have many problems, including strong hydrophobicity, low bioavailability, instability, greater toxicity and side effects, lack of specificity, etc., and cannot fully meet the clinical needs of tumor treatment.
  • In situ hydrogel formulations are generally in the state of solution, suspension or semi-solid, and the hydrogel system will undergo a phase transition immediately after being injected into the administration site, from solution or suspension to semi-solid or solid state.
  • the advantages of this system include local and site-specific effects, prolonged drug delivery, reduced drug dosage, improved bioavailability, reduced side effects, and improved patient comfort and compliance.
  • researchers have developed hydrogel systems that gel in response to pH, temperature, ions, sound waves or light.
  • the sodium alginate hydrogel system is an ideal tumor in situ drug delivery system, which belongs to the hydrogel system of ion-mediated gel formation.
  • Sodium alginate is the sodium salt of alginic acid. Alginic acid is linked by aL-mannuronic acid (M unit) and bD-guluronic acid (G unit) by 1,4-glycosidic bonds and is composed of different proportions of GM, A copolymer of MM and GG fragments.
  • M unit L-mannuronic acid
  • G unit bD-guluronic acid
  • the stability, solubility, viscosity and safety of sodium alginate make it a good excipient for pharmaceutical preparations.
  • the carboxyl groups of sodium alginate can coordinate with divalent metal ions such as calcium ions and copper ions to form gels.
  • sodium alginate hydrogel system as a tumor in situ drug delivery system is that it can use divalent metal ions such as calcium ions in the local tissue fluid of the tumor to form a gel, compared with pH, temperature or light-mediated glue, easier to handle.
  • divalent metal ions such as calcium ions in the local tissue fluid of the tumor to form a gel
  • pH, temperature or light-mediated glue easier to handle.
  • the use of sodium alginate hydrogel to directly encapsulate chemotherapeutic drugs and immune adjuvants for local tumor administration has shown good efficacy in animal tumor models.
  • immune adjuvant-encapsulated hydrogels for enhanced local radiotherapy and radioimmunotherapy has not yet been reported, especially the hydrogel system with radiotherapy-responsive immune adjuvant release function remains to be developed.
  • Immune adjuvants can significantly enhance the immunogenicity of antigens by enhancing the immune response by enhancing the antigen processing and presentation efficiency of antigen-presenting cells. It has been demonstrated that the introduction of immune adjuvants into tumors in treatments capable of inducing immunogenic death of tumor cells can effectively enhance anti-tumor immune responses and produce synergistic therapeutic effects. Because systemic administration of immune adjuvants can lead to serious side effects, such as cytokine storm, local injections (eg, by percutaneous puncture) are often employed to administer immune adjuvants directly to tumors. However, in most clinical tumor treatments, low doses of chemotherapy drugs or radiation are used repeatedly to reduce side effects.
  • the object of the present invention is to provide a hydrogel composition based on an immune adjuvant and its application.
  • it is responsive to tumor cell death markers and simultaneously realizes the release of immune adjuvants, and can maintain long-term intratumoral retention of immune adjuvants in the interval of radiotherapy.
  • the first object of the present invention is to disclose the application of the immunoadjuvant-based hydrogel composition in the preparation of surgical radiotherapy sensitization preparations.
  • the immunoadjuvant-based hydrogel composition includes sodium alginate and encapsulated sodium alginate water-soluble immune adjuvants in .
  • the immune adjuvant-based hydrogel composition includes sodium alginate, tumor cell death marker aptamer and a water-soluble immune adjuvant with an extended sequence, sodium alginate and tumor cell death marker aptamer. Covalently linked by peptide bonds, the extended sequence is base-complementary to at least a portion of the sequence of the tumor cell death marker aptamer.
  • the tumor cell death marker aptamer includes an ATP nucleic acid aptamer, and the nucleotide sequence of the ATP nucleic acid aptamer includes the sequence shown in SEQ ID No. 1.
  • Radiotherapy is divided into multiple low-dose treatments, and the radiosensitization preparation based on the immune adjuvant hydrogel composition has a response function to the radiotherapy applied to the tumor, and realizes the release of the immune adjuvant at the synchronization of tumor radiotherapy, In the interval of radiotherapy, the long-term intratumoral retention of immune adjuvant can be maintained.
  • nucleotide sequence of the extended sequence includes the sequence shown in SEQ ID No.2.
  • the water-soluble immune adjuvant includes one or more of polynucleotides, CpG oligodeoxynucleotides, polyinosinic acid, polyICLC, lipopolysaccharide, cell wall peptide, lipid A and cytokines. kind.
  • the water-soluble immune adjuvant comprises CpG oligodeoxynucleotides.
  • the solid tumor is one or more of colon cancer, melanoma, breast cancer, lung cancer, and head and neck.
  • the radiotherapy sensitizing preparation is used under a single irradiation dose of 0.5-10Gray radiation, and the total radiation dose is 5-80Gray.
  • the administration mode of the radiotherapy sensitizing preparation is intravenous injection.
  • the viscosity of sodium alginate is 5-1000Cp.
  • the viscosity of sodium alginate is 50-200Cp.
  • the second object of the present invention is to provide a hydrogel composition based on an immune adjuvant, comprising sodium alginate, a tumor cell death marker aptamer and a water-soluble immune adjuvant with an extended sequence, sodium alginate It is covalently linked to the tumor cell death marker aptamer through a peptide bond, and the extended sequence is base-complementary to at least a part of the sequence of the tumor cell death marker aptamer.
  • the tumor cell death marker aptamer includes an ATP nucleic acid aptamer, and the nucleotide sequence of the ATP nucleic acid aptamer includes the sequence shown in SEQ ID No. 1.
  • nucleotide sequence of the extended sequence includes the sequence shown in SEQ ID No.2.
  • the water-soluble immune adjuvant includes one or more of polynucleotides, CpG oligodeoxynucleotides, polyinosinic acid, polyICLC, lipopolysaccharide, cell wall peptide, lipid A and cytokines. kind.
  • the water-soluble immune adjuvant comprises CpG oligodeoxynucleotides (CpG-ODN).
  • CpG-ODN is an oligodeoxyribonucleic acid (DNA) sequence capable of enhancing the function of antigen-presenting cells.
  • the viscosity of sodium alginate is 5-1000Cp. Its structural formula is as follows:
  • the immune adjuvant-based hydrogel composition of the present invention includes sodium alginate, the carboxyl group of sodium alginate can form a hydrogel mediated by calcium ions in the tumor in situ, and the immune adjuvant is encapsulated in the gel.
  • the use of hydrogels can slow the release of immune adjuvants locally in the tumor, reducing the number of administrations.
  • the optimal antitumor immune response can only be elicited by the presence of higher concentrations of immune adjuvants at the same time as tumor cell death (tumor antigen release).
  • the conventional hydrogel system can release the immune adjuvant slowly, the release is not controlled, and it is difficult to ensure that the immune adjuvant of a suitable concentration exists locally in the tumor at the optimal time point.
  • the tumor cell death marker aptamer is used in the composition of the present invention to connect the water-soluble immune adjuvant through base pairing, when radiotherapy is applied to the tumor, the radiotherapy causes the tumor cell to die and releases the tumor cell death marker (such as ATP), the binding force between the tumor cell death marker and the tumor cell death marker aptamer is stronger, so that the water-soluble immune adjuvant is released from the hydrogel.
  • the immune adjuvant-based hydrogel composition can be applied to prepare a radiosensitizing preparation to assist in enhancing the antitumor immune response induced by multiple low-dose radiotherapy.
  • the third object of the present invention is to provide a preparation method of the above-mentioned immunoadjuvant-based hydrogel composition, comprising the following steps:
  • tumor cell death marker aptamer Reacting sodium alginate and amino-modified tumor cell death marker aptamer in solution to obtain a sodium alginate-adenosine triphosphate nucleic acid aptamer conjugate; preferably, the tumor cell death marker aptamer is ATP A nucleic acid aptamer, the nucleotide sequence of which includes the sequence shown in SEQ ID No.1;
  • DNA hybridization reaction is carried out with sodium alginate-ATP nucleic acid aptamer conjugate and water-soluble immune adjuvant with extended sequence in buffer to obtain a hydrogel composition based on immune adjuvant, wherein, The extended sequence is base-complementary to at least a portion of the sequence of the tumor cell death marker aptamer.
  • the molar ratio of the carboxyl group in the sodium alginate and the amino group in the aptamer of the tumor cell death marker modified by the amino group is 100-5000:1.
  • the molar ratio of the carboxyl group in the sodium alginate and the amino group in the aptamer of the tumor cell death marker modified by the amino group is 1000-1100:1.
  • step (1) the sodium alginate is activated and then reacted with the amino-modified tumor cell death marker aptamer.
  • step (1) includes the following steps:
  • step (S2) adding sodium acetate solution to the product of step (S1), adding ethanol after mixing, and then reacting at -80°C to obtain a sodium alginate-adenosine triphosphate nucleic acid aptamer conjugate.
  • step (S1) the concentration of the aqueous solution of sodium alginate is 0.1-0.2 mg/mL.
  • step (S1) the concentration of the aqueous solution of the amino-modified tumor cell death marker aptamer is 0.1 mmol/L.
  • the amino group on the amino-modified tumor cell death marker aptamer and the carboxyl group on the sodium alginate form a covalent bond to connect to the sodium alginate , the reaction is mediated by an activator.
  • Water-soluble immune adjuvants are assembled to sodium alginate by DNA hybridization of extended sequences with tumor cell death marker aptamers. The release of water-soluble immune adjuvants from sodium alginate is due to the specific binding of tumor cell death markers and tumor cell death marker aptamers, thereby opening the DNA formed by water-soluble immune adjuvants and tumor cell death marker aptamers Double chain.
  • the amino group is connected to the 5' end of the tumor cell death marker aptamer.
  • the amino group-modified tumor cell death marker aptamer includes a tumor cell death marker aptamer and a single amino group at one end thereof, and the tumor cell death marker aptamer is a section of the tumor cell death marker aptamer obtained through exponential enrichment.
  • the oligodeoxyribonucleic acid (DNA) sequence screened by the phylogenetic evolution of ligands (SELEX) technology, the specific sequence is 5'-acctgggggagtattgcggaggaaggt-3' (SEQ ID No. 1), which can specifically interact with ATP. combine to form specific three-dimensional structures.
  • the water-soluble immune adjuvant with extended sequence is preferably CpG-ODN with extended sequence, wherein CpG-ODN is a kind of oligodeoxyribonucleic acid (DNA) that can improve the function of antigen presenting cells Sequence, its sequence is: 5'-tccatgacgttcctgacgtt-3' (SEQ ID No.3), its 3' end is extended a section of extension sequence, and this extension sequence consists of a sequence that is partially complementary to ATP nucleic acid aptamer: 5'-accttcctccgcaa -3' (SEQ ID No. 2) composition.
  • CpG-ODN is a kind of oligodeoxyribonucleic acid (DNA) that can improve the function of antigen presenting cells Sequence
  • its sequence is: 5'-tccatgacgttcctgacgtt-3' (SEQ ID No.3)
  • this extension sequence consists of a sequence that is
  • the CpG-ODN with an extended sequence forms a aptamer with adenosine triphosphate (ATP) through DNA hybridization with the sodium alginate-ATP nucleic acid aptamer.
  • ATP adenosine triphosphate
  • Smart sodium alginate hydrogel preparation with slow-release function of responsive immune adjuvant the preparation is injected into the tumor, mediated by calcium ions in the tumor to form a hydrogel, the immune adjuvant is encapsulated in it, and the tumor is treated with chemotherapy or After radiotherapy, a large amount of ATP is released on the one hand, and tumor vaccines are formed on the other hand.
  • the ATP released by the tumor binds to the ATP nucleic acid aptamer, thereby releasing the immune adjuvant.
  • Dendritic cells in the tumor mature after ingesting tumor vaccines and immune adjuvants, and then migrate to lymph nodes to stimulate the activation of antigen-specific T cells, which in turn kill the tumor.
  • the present invention has at least the following advantages:
  • the present invention provides a hydrogel composition with slow release function of immune adjuvant, which can rapidly form a gel in the aqueous phase mediated by calcium ions, and slowly release water-soluble immunity in response to tumor cell death markers adjuvant.
  • the immune adjuvant-based hydrogel composition of the present invention can be used to prepare a radiotherapy sensitizing preparation.
  • the use of hydrogel for local tumor immune adjuvant sustained release can prolong the existence time of the adjuvant at the tumor site.
  • Reduce the amount of adjuvant improve the bioavailability of the adjuvant, and reduce side effects, so as to better enhance the anti-tumor immune response induced by local radiotherapy.
  • the release of immune adjuvants is controlled by the signal molecules released by tumor cell death, so that a higher concentration of immune adjuvants can be locally produced while tumor antigens are produced.
  • Antigen plus immune adjuvants can better stimulate antigen-presenting cells, thereby Stimulate a stronger anti-tumor immune response to achieve the effect of 1+1 greater than 2.
  • Figure 1 is a schematic diagram of the working principle of a smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant.
  • ATP adenosine triphosphate
  • Figure 2 is a graph showing the results of chemotherapy sensitization results of the sodium alginate hydrogel hydrogel radiotherapy/chemosensitization preparation encapsulating the immune adjuvant CpG-ODN in Example 1 for use in a mouse colon cancer subcutaneous tumor model;
  • lane 1 is adenosine triphosphate aptamer
  • lanes 2-7 are sodium alginate-ATP aptamer conjugates
  • Figure 4 is the result of electrophoresis analysis of the smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant in Example 2:
  • swimming lane 1 is adenosine triphosphate nucleic acid aptamer (A-Apt)
  • lane 2 is CpG-ODN
  • lane 3 is the duplex (A-Apt/CpG) formed by adenosine triphosphate aptamer and CpG-ODN
  • lane 7 is sodium al
  • Figure 5 is a graph of the results of the test for the release of ATP-responsive immune adjuvant CpG-ODN in Example 2;
  • FIG. 6 is a graph showing the results of chemotherapy sensitization of the smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant in a mouse colon cancer subcutaneous tumor model in Example 3;
  • ATP adenosine triphosphate
  • Example 7 is a graph showing the results of immune response evaluation in the chemotherapy sensitization of the smart sodium alginate hydrogel preparation with adenosine triphosphate (ATP)-responsive immune adjuvant slow-release function in Example 4 for use in a mouse colon cancer subcutaneous tumor model;
  • ATP adenosine triphosphate
  • Figure 8 shows the immune memory function evaluation of the cured mice in the chemotherapy sensitization of the mouse colon cancer subcutaneous tumor model with the smart sodium alginate hydrogel preparation with the slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant in Example 5 result graph;
  • ATP adenosine triphosphate
  • FIG. 9 is a graph showing the radiosensitization results of the smart sodium alginate hydrogel preparation with the slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant in a mouse colon cancer subcutaneous tumor model in Example 6;
  • ATP adenosine triphosphate
  • Figure 10 is a graph of the radiosensitization results of the smart sodium alginate hydrogel preparation with the slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant in a mouse melanoma model in Example 7;
  • ATP adenosine triphosphate
  • Figure 11 is a graph showing the results of the distal tumor inhibition effect of the smart sodium alginate hydrogel preparation with adenosine triphosphate (ATP)-responsive immune adjuvant slow-release function combined with immune checkpoint inhibitors used to enhance external radiation therapy in Example 8;
  • ATP adenosine triphosphate
  • Figure 12 is a graph showing the results of the smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant combined with immune checkpoint inhibitors in Example 9 for enhancing the effect of radiotherapy on breast cancer in situ tumors.
  • ATP adenosine triphosphate
  • Example 1 Preparation and application of a sodium alginate hydrogel radiotherapy/chemosensitizing preparation encapsulated with immune adjuvant CpG-ODN
  • CpG-ODN CpG oligodeoxynucleotide
  • step (3) mixing the solutions in step (1) and step (2) according to a certain proportion, and adding deionized water to adjust the final concentration of sodium alginate to 10-20 mg/mL, to obtain a CpG-ODN-encapsulated immune adjuvant.
  • Sodium alginate hydrogel radiotherapy/chemosensitizer preparation
  • Sodium alginate hydrogel radiotherapy/chemosensitization preparation encapsulated with immune adjuvant CpG-ODN for chemotherapy sensitization in mouse colon cancer subcutaneous tumor model The mouse CT26 colon cancer tumor was inoculated on day 7 after inoculation.
  • mice The tumor-bearing BALB/c mice were randomly divided into the following 4 groups (6 mice in each group): group 1, control group (untreated, Untreated); group 2, intratumoral injection of sodium alginate, intravenous injection of oxa Liplatin (ALG+OxPt); group 3, intratumoral injection of sodium alginate hydrogel radiotherapy/chemosensitizer preparation (ALG/CpG) coated with immune adjuvant CpG-ODN; group 4, intratumoral injection of coated immune adjuvant Adjuvant CpG-ODN sodium alginate hydrogel radiotherapy/chemosensitizing preparation, intravenous injection of oxaliplatin (ALG/CpG+OxPt); the final concentration of sodium alginate in each treatment group is 10mg/mL, intratumoral injection The volume was 25 ⁇ L, and the dose of CpG-ODN was 15 ⁇ g per mouse. Oxaliplatin was administered intravenously at a dose of 3 mg/kg body weight on days 7, 10, 13 and 16. After
  • Figure 2(a)(b) shows the changes of tumor volume with time and mouse survival rate with time during the experiment, respectively.
  • the results showed (Fig. 2) that compared with the control group, the tumor growth in group 3 was only partially inhibited, the tumor growth in group 2 was significantly inhibited, and the tumor growth in group 4 was more significantly inhibited. 1/6 of the tumor was completely cleared. It shows that the sodium alginate hydrogel radiotherapy/chemosensitizer sensitizing preparation encapsulating the immune adjuvant CpG-ODN can enhance the effect of chemotherapy to a certain extent.
  • step (3) The precipitate obtained by centrifugation is dissolved in deionized water, and step (3) is repeated once, and the obtained precipitate is the sodium alginate-ATP nucleic acid aptamer conjugate, which is dissolved in phosphate buffer.
  • the reaction principle is shown in Fig. 3a.
  • the sodium alginate-ATP nucleic acid aptamer conjugate was analyzed by native polyacrylamide gel electrophoresis, and the results showed (Fig. 3b) that compared with the adenosine triphosphate nucleic acid aptamer, the sodium alginate-ATP aptamer prepared in Example 1
  • the aptamer conjugates showed obvious electrophoretic migration hysteresis, indicating that sodium alginate was coupled with adenosine triphosphate aptamers, and the molecular weight increased, resulting in a slower electrophoretic mobility.
  • the obtained solution was mixed with the sodium alginate-ATP nucleic acid aptamer conjugate, and placed in a refrigerator at 4 °C for more than 2 hours, wherein the molar ratio of the CpG-ODN with the extended sequence to the adenosine triphosphate nucleic acid aptamer was 1: 2.
  • the obtained sample is a smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant.
  • ATP adenosine triphosphate
  • the smart sodium alginate hydrogel preparation was analyzed by non-denaturing polyacrylamide gel electrophoresis, and the results showed (Fig. 4b) that, compared with the sodium alginate-ATP aptamer conjugate
  • the preparation showed obvious electrophoretic migration hysteresis, indicating that the CpG-ODN with the extended sequence hybridized with the adenosine triphosphate aptamer and assembled on the sodium alginate molecule, so that the molecular weight of the smart sodium alginate hydrogel preparation was larger, resulting in Electrophoretic mobility is slowed down.
  • ATP-responsive immune adjuvant CpG-ODN release test In order to be able to monitor the release of CpG-ODN from hydrogels, CpG-ODN modified with a fluorophore at one end was used. According to Examples 1 and 3, adenosine triphosphate was prepared. (ATP)-responsive immune adjuvant sustained-release smart sodium alginate hydrogel formulation. The prepared smart sodium alginate hydrogel preparation is mixed with a sodium alginate solution with a mass concentration of 4%, to a final concentration of 1% of sodium alginate. The mixture was slowly transferred into a calcium chloride solution with a concentration of 10 mmol/L.
  • Example 3 A smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant is used for chemotherapy sensitization of mouse colon cancer subcutaneous tumor model
  • ATP adenosine triphosphate
  • mice were randomly divided into the following 6 groups (6 mice in each group): group 1, control group (untreated, Untreated); group 2, intratumoral injection of sodium alginate, intravenous injection of oxa Liplatin (ALG+OxPt); group 3, intratumoral injection of a mixture of sodium alginate and CpG-ODN (ALG/CpG); group 4, intratumoral injection of a mixture of sodium alginate and CpG-ODN, intravenous injection of Thaliplatin (ALG/CpG+OxPt); group 5, the intelligent sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant obtained by intratumoral injection in step (5) of Example 2 ( ALG-Aapt/CpG); group 6, intratumoral injection of smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP)
  • ATP adenosine triphosphate
  • the final concentration of sodium alginate in each treatment group was 10 mg/mL, the intratumoral injection volume was 25 ⁇ L, and the dose of CpG-ODN per mouse was 15 ⁇ g.
  • Oxaliplatin was administered intravenously at a dose of 3 mg/kg body weight on days 7, 10, 13 and 16. After the mice were treated accordingly, the tumor growth was measured.
  • the schematic diagram of the treatment process is shown in Figure 6(a).
  • Figure 6(b)(c) respectively show the changes of tumor volume with time and the survival rate of mice with time during the experiment.
  • the results showed (Fig. 6) that compared with the control group, the tumor growth of groups 2, 3, 4 and 5 was only partially inhibited, while the tumor growth of group 6 was effectively inhibited, Tumors in some mice completely regressed. It is shown that the intelligent sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant combined with low-dose chemotherapy can achieve efficient and synergistic killing of tumors.
  • ATP adenosine triphosphate
  • Example 4 A smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant is used for the evaluation of immune response in chemotherapy sensitization of mouse colon cancer subcutaneous tumor model
  • ATP adenosine triphosphate
  • mice The tumor-bearing BALB/c mice were randomly divided into the following 4 groups (6 mice in each group): group 1, control group (untreated, Untreated); group 2, intratumoral injection of sodium alginate, intravenous injection of oxa Liplatin (OxPt); Group 3, intratumoral injection of the smart sodium alginate hydrogel preparation (ALG-A-Apt) with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant obtained in step (5) of Example 2 /CpG); group 4, intratumoral injection of the smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant obtained in step (5) of Example 2, intravenous injection of oxaliplatin ( ALG-A-Apt/CpG@OxPt).
  • the final concentration of sodium alginate in each treatment group was 10 mg/mL, the intratumoral injection volume was 25 ⁇ L, and the dose of CpG-ODN per mouse was 15 ⁇ g.
  • Oxaliplatin was administered intravenously at a dose of 3 mg/kg body weight on days 7, 10, 13 and 16.
  • mice in each group were sacrificed and the maturation of dendritic cells in the inguinal lymph nodes, the percentage of CD8-positive T cells in the tumor, and the ratio of CD8-positive T cells and regulatory T cells were determined.
  • Example 5 A smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant is used to evaluate the immune memory function of cured mice in the chemotherapy sensitization of mouse colon cancer subcutaneous tumor model
  • ATP adenosine triphosphate
  • mice with complete tumor regression 70 days after starting treatment, peripheral blood was collected from mice with complete tumor regression (Cured group), and memory CD8-positive T cells were identified.
  • Mouse CT26 colon tumors were then re-inoculated subcutaneously into mice with complete tumor regression, and untreated mice (Naive group) were used as controls, and tumor growth status was monitored after tumor inoculation.
  • Figure 8(a1)(a2)(b) is the result of immune evaluation
  • Figure 8(c)(d) is the change of tumor volume with time and the change of mouse survival rate with time respectively during the experiment.
  • the evaluation showed that the percentage of CD8-positive effector memory T cells in peripheral blood of mice with complete tumor regression (Fig. 8(a2)(b)) was significantly higher than that of untreated mice of the same age (Fig. 8(a1)(b) )). No significant tumor growth was seen in mice with complete tumor regression, while tumors grew rapidly in untreated mice. It was shown that mice cured by chemotherapy combined with a smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant developed effective immune memory.
  • ATP adenosine triphosphate
  • Example 6 A smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant is used for radiosensitization of mouse colon cancer subcutaneous tumor model
  • ATP adenosine triphosphate
  • mice The tumor-bearing BALB/c mice were randomly divided into the following 4 groups (6 mice in each group): group 1, control group (untreated, Unteated); group 2, intratumoral injection of sodium alginate, local X-ray of tumor Irradiation (dose of 8Gray) (RT); group 3, intratumoral injection of the smart sodium alginate hydrogel preparation (ALG) with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant obtained in step (5) of Example 2 -Aapt/CpG); group 4, intratumoral injection of the smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant obtained in step (5) of Example 2, local X-ray irradiation of the tumor (Dose of 8 Gray) (ALG-Aapt/CpG+RT).
  • group 1 control group (untreated, Unteated)
  • group 2 intratumoral injection of sodium alginate, local X-ray of tumor Irradi
  • the final concentration of sodium alginate in each treatment group was 10 mg/mL, the intratumoral injection volume was 25 ⁇ L, and the dose of CpG-ODN per mouse was 15 ⁇ g. Irradiation was performed on days 7, 9, 11 and 13 at a dose of 2Gray per tumor.
  • a schematic diagram of the treatment flow is shown in Figure 9(a).
  • Figure 9(b)(c) respectively show the changes of tumor volume with time and mouse survival rate with time during the experiment.
  • the experimental results ( Figure 9) showed that compared with the control group, the tumor growth of groups 2 and 3 was only partially inhibited, while the tumor growth of group 4 was effectively inhibited, and the tumors of all mice completely disappeared. . It is shown that the intelligent sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant combined with low-dose radiotherapy can achieve efficient and synergistic killing of tumors.
  • ATP adenosine triphosphate
  • Example 7 A smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant is used for radiosensitization of mouse melanoma model
  • mice with B16 melanoma were randomly divided into the following 4 groups (6 mice in each group): group 1, control group (untreated, Untreated); group 2, intratumoral injection of sodium alginate, local tumor administration of 8Gray dose of alginate.
  • X-ray irradiation (RT); group 3, intratumoral injection of intelligent sodium alginate hydrogel preparation (ALG-Aapt/ CpG); group 4, intratumoral injection of the smart sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant obtained in step (5) of Example 2, and local X-rays of 8Gray dose were used for the tumor Irradiation (ALG-Aapt/CpG+RT).
  • the final concentration of sodium alginate in each treatment group was 10 mg/mL
  • the intratumoral injection volume was 25 ⁇ L
  • the dose of CpG-ODN per mouse was 15 ⁇ g.
  • Irradiation was performed on days 7, 9, 11 and 13 at a dose of 2Gray per tumor.
  • a schematic diagram of the treatment process is shown in Figure 10(a).
  • Figure 10(b)(c) shows the changes of tumor volume with time and the survival rate of mice with time during the experiment, respectively.
  • the experimental results (Fig. 10) showed that compared with the control group, the tumor growth in groups 2 and 3 was only partially inhibited, while the tumor growth in group 4 was effectively inhibited, and the survival period of the mice was effectively prolonged. It is indicated that the intelligent sodium alginate hydrogel preparation with slow-release function of adenosine triphosphate (ATP)-responsive immune adjuvant combined with low-dose radiotherapy can effectively inhibit melanoma.
  • ATP adenosine triphosphate
  • Example 8 A smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant is used to enhance the distal tumor suppressive effect of external radiation therapy
  • Tumor-bearing BALB/c mice were randomly divided into the following 6 groups (6 mice in each group): group 1, control group (untreated, Unteated); group 2, intravenous injection of immune checkpoint inhibitor PD1 antibody (aPD1 ); group 3, tumor local X-ray irradiation (dose of 8Gray) (RT); group 4, intratumoral injection of adenosine triphosphate (ATP) responsive immune adjuvant sustained-release function obtained in step (5) of Example 2 Intelligent sodium alginate hydrogel preparation (ALG-Aapt/CpG), local tumor X-ray irradiation (dose of 8Gray) (RT); group 5, intravenous injection of immune checkpoint inhibitor PD1 antibody (aPD1), tumor local X-ray Radiation irradiation (dose of 8Gray) (RT); Group 6, intratumoral injection of the smart sodium alginate hydrogel preparation (ALG-Aapt/CpG), local tumor X-ray irradiation (dose of 8Gray) (RT); group 5, intrave
  • the final concentration of sodium alginate in each treatment group was 10 mg/mL, the intratumoral injection volume was 25 ⁇ L, and the dose of CpG-ODN per mouse was 15 ⁇ g.
  • Irradiation was performed on days 7, 9, 11 and 13 at a dose of 2Gray per tumor.
  • the immune checkpoint inhibitor PD1 antibody was administered intravenously at a dose of 10 ug per mouse on days 8 and 11.
  • a schematic diagram of the treatment flow is shown in Figure 11A.
  • Figure 11(B)(C)(D) respectively shows the changes of the volume of the original tumor (B) and the distal tumor body (C) with time and the survival rate of mice with time (D) during the experiment.
  • the experimental results ( Figure 11), tumor growth curves and corresponding statistics suggest that aPDl alone or RT alone showed limited efficacy in inhibiting tumor growth on both sides.
  • RT+ALG-Aapt/CpG treatment abolished 4 of 6 local tumors on the right side and significantly delayed the progression of distant tumors (left side) due to enhanced RT-induced antitumor immune responses via ALG-Aapt/CpG grow.
  • most distant tumors in this group still showed rapid growth later on.
  • aPD1 further enhanced the efficacy of RT+ALG-Aapt/CpG, eliminating all local tumors on the right side and 5 out of 6 distant tumors on the left side.
  • Example 9 A smart sodium alginate hydrogel formulation with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant is used to enhance the radiotherapy effect of breast cancer in situ tumors
  • ATP adenosine triphosphate
  • Tumor-bearing BALB/c mice were randomly divided into the following 6 groups (6 mice in each group): group 1, control group (untreated, Unteated); group 2, intravenous injection of immune checkpoint inhibitor PD1 antibody (aPD1 ); group 3, local tumor X-ray irradiation (dose of 8Gray) (RT); group 4, intravenous injection of immune checkpoint inhibitor PD1 antibody (aPD1), local tumor X-ray irradiation (dose of 8Gray) (RT) ; Group 5, intratumoral injection of the intelligent sodium alginate hydrogel preparation (ALG-Aapt/CpG) with slow-release function of adenosine triphosphate (ATP) responsive immune adjuvant obtained in step (5) of Example 2, tumor local X Radiation irradiation (dose of 8Gray) (RT); Group 6, intratumoral injection of the smart sodium alginate hydrogel preparation (ALG-Aapt/CpG) with slow-release function of adenosine triphosphate (ATP) responsive immune adju
  • the final concentration of sodium alginate in each treatment group was 10 mg/mL, the intratumoral injection volume was 25 ⁇ L, and the dose of CpG-ODN per mouse was 15 ⁇ g.
  • Irradiation was performed on days 7, 9, 11 and 13 at a dose of 2Gray per tumor.
  • the immune checkpoint inhibitor PD1 antibody was administered intravenously at a dose of 10 ug per mouse on days 8 and 11.
  • a schematic diagram of the treatment flow is shown in Figure 12(A).
  • FIG. 12D Representative lung photographs show that treatment with RT+ALG-Aapt/CpG+aPD1 significantly inhibited tumor metastasis despite dense metastatic nodules (indicated by black arrows) in the lungs of untreated mice ( Figure 12D). Pathological changes in representative lung tissues were further observed in hematoxylin-eosin (H&E) staining, which also confirmed that RT+ALG-Aapt/CpG+aPD1 treatment could significantly inhibit lung metastasis of 4T1 tumors.
  • H&E hematoxylin-eosin

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Polymers & Plastics (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Inorganic Chemistry (AREA)
  • Mycology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种基于免疫佐剂的水凝胶组合物及其应用。基于免疫佐剂的水凝胶组合物在制备外科放疗增敏制剂中的应用。一种基于免疫佐剂的水凝胶组合物，包括海藻酸钠、肿瘤细胞死亡标志物适配体及带有延长序列的水溶性免疫佐剂，海藻酸钠与肿瘤细胞死亡标志物适配体共价连接，延长序列与肿瘤细胞死亡标志物适配体的至少一部分序列碱基互补。基于免疫佐剂的水凝胶组合物能够在体内钙离子的作用下原位成胶，在肿瘤放疗过程中具有肿瘤细胞死亡标志物响应性且同步实现免疫佐剂的释放，在放疗的间隙能够保持免疫佐剂的长时间瘤内滞留，从而在放疗整个疗程中长时间激活肿瘤特异性免疫反应，抑制肿瘤全身转移。

Description

基于免疫佐剂的水凝胶组合物及其应用 技术领域
本发明涉及肿瘤治疗制剂领域，尤其涉及一种基于免疫佐剂的水凝胶组合物及其应用。
背景技术
肿瘤是威胁人类生存的一种恶性疾病。随着世界人口的老龄化以及生活环境和日常生活习惯的变化，因肿瘤死亡的人数每年都在急剧增加。现在，化疗和放疗是临床上除手术切外肿瘤治疗的主要方法。近年来许多研究发现，某些化疗药(蒽环类药物和奥沙利铂)以及电离辐射可以诱导肿瘤细胞免疫原性死亡，这是一种与细胞凋亡相关的独特细胞死亡途径，死亡细胞释放出内源性危险信号。在免疫原性死亡的过程中，肿瘤细胞将发生自噬，导致钙网蛋白(一种内质网钙结合蛋白)暴露在细胞表面，从而刺激树突状细胞吞噬肿瘤抗原。同时，高迁移率族蛋白B1(HMGB1)从细胞中释放，从而促进树突状细胞和垂死的肿瘤细胞之间形成稳定连接。免疫原性死亡的过程中肿瘤细胞代谢产生的高浓度三磷酸腺苷(ATP)也会大量释放，ATP可将树突状细胞招募到肿瘤灶中。上述事件将引发抗肿瘤免疫反应。由于上述机制，在一些临床病例中，放疗有时可能显示出远端效果，即局部肿瘤放疗后远处转移的肿瘤自发消退。
目前在临床开发或使用的化疗药物有很多问题，这些问题包括疏水性强，生物利用度低，不稳定，毒性更大和副作用效果，缺乏针对性等，无法完全满足肿瘤治疗的临床需求等。基于上述问题，原位水凝胶给药系统在肿瘤治疗中的应用近年来引起了越来越多的研究人员兴趣。原位水凝胶制剂一般呈溶液，悬浮液或半固体的状态，被注射到给药部位后水凝胶系统会立即发生相变，由溶液或悬浮液转变成半固体或固态。这个系统的优势包括局部和定点效应，延长的药物输送，减少药物剂量，提高生物利用度，减少副作用，改善患者的舒适度和依从性。当前，研究人员已经开发出响应于pH，温度，离子，声波或光而成胶的水凝胶系统。
海藻酸钠水凝胶系统是一种比较理想的肿瘤原位给药系统，属于离子介导成胶的水凝胶系统。海藻酸钠是海藻酸的钠盐，海藻酸由a-L-甘露糖醛酸(M单元)与b-D-古罗糖醛酸(G单元)依靠1,4-糖苷键连接并由不同比例的GM、MM和GG片段组成的共聚物。海藻酸钠的稳定性、溶解性、粘性和安全性，使其成为良好的药物制剂辅料。在水相中海藻酸钠的羧基可以与钙离子，铜离子等二价金属离子配位，从而形成凝胶。海藻酸钠水凝胶系统作为肿瘤原 位给药系统的一个重要优势在于，其可以利用肿瘤局部组织液中的钙离子等二价金属离子成胶，相比于由pH，温度或光介导成胶，更加容易操作。目前利用海藻酸钠水凝胶直接包裹化疗药和免疫佐剂等进行肿瘤局部给药在动物肿瘤模型中表现出良好的疗效。但是，将包裹免疫佐剂的水凝胶用于增效局部放疗和放射免疫治疗目前还没有报道，特别是具有放射治疗响应性免疫佐剂释放功能的水凝胶体系还有待开发。
免疫佐剂可以通过增强抗原呈递细胞的抗原加工和呈递效率来增强免疫反应，从而显着提高抗原的免疫原性。现已证明在能够诱导肿瘤细胞免疫原性死亡的治疗中将免疫佐剂引入肿瘤可有效增强抗肿瘤免疫反应并产生协同治疗效果。由于免疫佐剂的全身给药可能导致严重的副作用，例如细胞因子风暴，因此经常采用局部注射(例如通过经皮穿刺)直接将免疫佐剂给药至肿瘤。但是，在多数临床肿瘤治疗过程中，都是重复使用低剂量的化疗药或射线，以减少副作用。但是在一个疗程的化疗或放疗中，多次经皮穿刺将免疫佐剂施用于肿瘤患者会给患者造成身体和心理上的压力，而且难以在最佳时间点给于免疫佐剂。因此，需要一种智能载体，一方面实现免疫佐剂在肿瘤内的长时间滞留，另一方面又能在施加放疗/化疗的同时实现免疫佐剂的同步释放，从而达到最理想的免疫刺激效果。
发明内容
为解决上述技术问题，本发明的目的是提供一种基于免疫佐剂的水凝胶组合物及其应用，本发明的基于免疫佐剂的水凝胶组合物能够在体内钙离子的作用下原位成胶，在肿瘤放疗过程中具有肿瘤细胞死亡标志物响应性且同步实现免疫佐剂的释放，而在放疗的间隙能够保持免疫佐剂的长时间瘤内滞留。
本发明的第一个目的是公开基于免疫佐剂的水凝胶组合物在制备外科放疗增敏制剂中的应用，基于免疫佐剂的水凝胶组合物包括海藻酸钠以及包裹于海藻酸钠中的水溶性免疫佐剂。
进一步地，基于免疫佐剂的水凝胶组合物包括海藻酸钠、肿瘤细胞死亡标志物适配体及带有延长序列的水溶性免疫佐剂，海藻酸钠与肿瘤细胞死亡标志物适配体通过肽键共价连接，延长序列与肿瘤细胞死亡标志物适配体的至少一部分序列碱基互补。
进一步地，肿瘤细胞死亡标志物适配体包括ATP核酸适配体，ATP核酸适配体的核苷酸序列包括如SEQ ID No.1所示的序列。放疗是分多次低剂量处理，该基于免疫佐剂的水凝胶组合物的放疗增敏制剂，其对施加于肿瘤的放射治疗具有响应功能，在肿瘤放疗的同步实现免疫佐剂的释放，而在放疗的间隙能够保持免疫佐剂的长时间瘤内滞留。
进一步地，延长序列的核苷酸序列包括如SEQ ID No.2所示的序列。
进一步地，水溶性免疫佐剂包括多聚核苷酸、CpG寡聚脱氧核苷酸、聚肌胞苷酸、polyICLC、脂多糖、胞壁肽、类脂A以及细胞因子中的一种或几种。优选地，水溶性免疫佐剂包括CpG寡聚脱氧核苷酸。
进一步地，外科放疗增敏制剂用于治疗实体肿瘤。优选地，实体肿瘤为结肠癌、黑色素瘤、乳腺癌、肺癌、头颈部中的一种或几种。
进一步地，放疗增敏制剂在单次照射0.5-10Gray放射线的照射剂量下使用，总放射剂量5-80Gray。
进一步地，放疗增敏制剂的给药方式为静脉注射给药。
进一步地，海藻酸钠的粘度为5-1000Cp。优选地，海藻酸钠的粘度为50-200Cp。
本发明的第二个目的是提供一种基于免疫佐剂的水凝胶组合物，包括海藻酸钠、肿瘤细胞死亡标志物适配体及带有延长序列的水溶性免疫佐剂，海藻酸钠与肿瘤细胞死亡标志物适配体通过肽键共价连接，延长序列与肿瘤细胞死亡标志物适配体的至少一部分序列碱基互补。
进一步地，肿瘤细胞死亡标志物适配体包括ATP核酸适配体，ATP核酸适配体的核苷酸序列包括如SEQ ID No.1所示的序列。
进一步地，延长序列的核苷酸序列包括如SEQ ID No.2所示的序列。
进一步地，水溶性免疫佐剂包括多聚核苷酸、CpG寡聚脱氧核苷酸、聚肌胞苷酸、polyICLC、脂多糖、胞壁肽、类脂A以及细胞因子中的一种或几种。
优选地，水溶性免疫佐剂包括CpG寡聚脱氧核苷酸(CpG-ODN)。CpG-ODN是一种能够提高抗原呈递细胞功能的寡聚脱氧核糖核酸(DNA)序列。
进一步地，海藻酸钠的粘度为5-1000Cp。其结构式如下：
Figure PCTCN2020124668-appb-000001
本发明的基于免疫佐剂的水凝胶组合物中包括海藻酸钠，海藻酸钠的羧基可在肿瘤原位由钙离子介导形成水凝胶，免疫佐剂被包裹在凝胶中。利用水凝胶可以在肿瘤局部缓释免疫佐剂，减少给药次数。然而，只有在肿瘤细胞死亡(肿瘤抗原释放)的同时存在较高浓度的免疫佐剂，才能引发最佳的抗肿瘤免疫反应。常规的水凝胶体系虽然能够使免疫佐剂缓慢释放，但是这种释放不受控制，难以保证在最佳的时间点使得肿瘤局部存在合适浓度得免疫佐 剂。由于本发明的组合物中利用肿瘤细胞死亡标志物适配体通过碱基配对连接水溶性免疫佐剂，在对肿瘤施加放射治疗时，放射治疗使得肿瘤细胞死亡并释放处肿瘤细胞死亡标志物(如ATP)，肿瘤细胞死亡标志物与肿瘤细胞死亡标志物适配体的结合力更强，使得水溶性免疫佐剂从水凝胶中释放，从而在肿瘤抗原产生的同时，使得肿瘤局部存在较高浓度的免疫佐剂，原位产生内源性肿瘤疫苗。在没有接受放疗的时候，免疫佐剂不释放而在肿瘤内长时间滞留。因此，该基于免疫佐剂的水凝胶组合物可应用于制备放疗增敏制剂以辅助增强多次低剂量放疗引发的抗肿瘤免疫反应。
本发明的第三个目的是提供一种上述基于免疫佐剂的水凝胶组合物的制备方法，包括以下步骤：
(1)将海藻酸钠和氨基修饰的肿瘤细胞死亡标志物适配体在溶液中进行反应，得到海藻酸钠-三磷酸腺苷核酸适配体耦联物；优选肿瘤细胞死亡标志物适配体为ATP核酸适配体，其核苷酸序列包括如SEQ ID No.1所示的序列；
(2)将海藻酸钠-三磷酸腺苷核酸适配体耦联物与带有延长序列的水溶性免疫佐剂在缓冲液中进行DNA杂交反应，得到基于免疫佐剂的水凝胶组合物，其中，延长序列与肿瘤细胞死亡标志物适配体的至少一部分序列碱基互补。
进一步地，在步骤(1)中，海藻酸钠中的羧基和氨基修饰的肿瘤细胞死亡标志物适配体中的氨基的摩尔比为100-5000：1。优选地，海藻酸钠中的羧基和氨基修饰的肿瘤细胞死亡标志物适配体中的氨基的摩尔比为1000-1100：1。
进一步地，在步骤(1)中，将海藻酸钠活化后再与氨基修饰的肿瘤细胞死亡标志物适配体反应。
进一步地，采用EDC在酸性条件下活化海藻酸钠，再将活化的海藻酸钠在碱性条件下与氨基修饰的肿瘤细胞死亡标志物适配体反应。具体地，步骤(1)包括如下步骤：
(S1)将海藻酸钠的水溶液和氨基修饰的肿瘤细胞死亡标志物适配体的水溶液混匀，并调节混合溶液的pH值为4-6，然后向混合溶液中加入EDC，在37℃下反应；
(S2)向步骤(S1)的产物中加入乙酸钠溶液，混匀后加入乙醇，随后在-80℃下反应，得到海藻酸钠-三磷酸腺苷核酸适配体耦联物。
进一步地，在步骤(S1)中，海藻酸钠的水溶液浓度为0.1-0.2mg/mL。
进一步地，在步骤(S1)中，氨基修饰的肿瘤细胞死亡标志物适配体的水溶液浓度为0.1mmol/L。
本发明的基于免疫佐剂的水凝胶组合物的制备过程中，通过氨基修饰的肿瘤细胞死亡标志物适配体上的氨基和海藻酸钠上的羧基形成共价键而连接到海藻酸钠上，该反应由活化剂 介导。水溶性免疫佐剂通过延长序列与肿瘤细胞死亡标志物适配体发生DNA杂交而组装到海藻酸钠上。水溶性免疫佐剂从海藻酸钠上释放是由于肿瘤细胞死亡标志物与肿瘤细胞死亡标志物适配体特异性结合，进而打开水溶性免疫佐剂与肿瘤细胞死亡标志物适配体形成的DNA双链。
进一步地，在步骤(1)中，氨基连接于肿瘤细胞死亡标志物适配体的5’端。优选地，在步骤(1)中，氨基修饰的肿瘤细胞死亡标志物适配体包括肿瘤细胞死亡标志物适配体及其一端的单个氨基，肿瘤细胞死亡标志物适配体是一段经由指数富集的配体系统进化(SELEX)技术筛选得到的寡聚脱氧核糖核酸(DNA)序列，具体序列为5’-acctgggggagtattgcggaggaaggt-3’(SEQ ID No.1)，该序列能够与ATP发生特异性的结合形成特定三维结构。
在步骤(2)中，带有延长序列的水溶性免疫佐剂优选为带有延长序列的CpG-ODN，其中CpG-ODN是一种能够提高抗原呈递细胞功能的寡聚脱氧核糖核酸(DNA)序列，其序列为：5’-tccatgacgttcctgacgtt-3’(SEQ ID No.3)，其3’端来延长一段延长序列，该延长序列由与ATP核酸适配体部分互补的序列：5’-accttcctccgcaa-3’(SEQ ID No.2)组成。
以肿瘤细胞死亡标志物适配体为ATP核酸适配体为例，如图1所示，带有延长序列的CpG-ODN通过DNA杂交与海藻酸钠-ATP核酸适配体形成具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂，该制剂注射到肿瘤中，由肿瘤内的钙离子介导形成水凝胶，将免疫佐剂包裹其中，肿瘤接受化疗或放疗后，一方面释放大量ATP，一方面形成肿瘤疫苗。肿瘤释放的ATP与ATP核酸适配体结合，从而将免疫佐剂释放。肿瘤内的树突状细胞摄取肿瘤疫苗和免疫佐剂后成熟，并随后迁移到淋巴结去刺激抗原特异性T细胞活化，活化的抗原特异性T细胞进而杀伤肿瘤。
借由上述方案，本发明至少具有以下优点：
本发明提供了一种具有免疫佐剂缓释功能的水凝胶组合物，其可以在水相中由钙离子介导迅速形成凝胶，并响应于肿瘤细胞死亡标志物以缓慢释放出水溶性免疫佐剂。
本发明的基于免疫佐剂的水凝胶组合物可用于制备放疗增敏制剂，相比较系统给药，利用水凝胶进行肿瘤局部免疫佐剂缓释可以延长佐剂在肿瘤部位的存在时间，减少佐剂用量，提高佐剂生物利用度率，减少副作用，从而更好的增强局部放疗引发的抗肿瘤免疫反应。免疫佐剂的释放是由肿瘤细胞死亡释放的信号分子控制的，从而在肿瘤抗原产生的同时使得局部具有较高浓度的免疫佐剂，抗原加免疫佐剂可以更好的刺激抗原呈递细胞，从而激发更强的抗肿瘤免疫反应，达到1+1大于2的效果。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依 照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1是具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂的工作原理图。
图2是实施例1包裹免疫佐剂CpG-ODN的海藻酸钠水凝胶放疗/化疗增敏制剂用于小鼠结肠癌皮下瘤模型化疗增敏结果图；
图3是实施例2海藻酸钠-三磷酸腺苷核酸适配体耦联物电泳分析结果图：泳道1为三磷酸腺苷核酸适配体，泳道2-7为海藻酸钠-三磷酸腺苷核酸适配体耦联物；
图4是实施例2具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂电泳分析结果图：泳道1为三磷酸腺苷核酸适配体(A-Apt)，泳道2为CpG-ODN，泳道3为三磷酸腺苷核酸适配体和CpG-ODN形成的双链(A-Apt/CpG)，泳道4、5、6依次为海藻酸钠-三磷酸腺苷核酸适配体耦联物和CpG-ODN以不同比例杂交后产物(ALG-A-Apt/CpG(Apt:CpG＝2:1)、ALG-A-Apt/CpG(Apt:CpG＝1:1)、ALG-A-Apt/CpG(Apt:CpG＝1:2))，泳道7为海藻酸钠-三磷酸腺苷核酸适配体耦联物(ALG-A-Apt)；
图5是实施例2中ATP响应性免疫佐剂CpG-ODN释放测试结果图；
图6是实施例3中具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型的化疗增敏结果图；
图7是实施例4中具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型的化疗增敏中免疫反应评价结果图；
图8为实施例5中具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型化疗增敏中治愈小鼠的免疫记忆功能评价结果图；
图9为实施例6中具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型的放疗增敏结果图；
图10为实施例7中具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠黑色素瘤模型的放疗增敏结果图；
图11为实施例8中具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂联合免疫检查点抑制剂用于增强外放射治疗的远端瘤抑制效应结果图；
图12为实施例9中具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂联合免疫检查点抑制剂用于增强乳腺癌原位瘤放射治疗效果的结果图。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1、一种包裹免疫佐剂CpG-ODN的海藻酸钠水凝胶放疗/化疗增敏制剂的制备及应用
(1)称取400mg海藻酸钠，溶解于9.6mL无菌去离子水中，反复震荡溶解，制成4％海藻酸钠水溶液。
(2)CpG寡聚脱氧核苷酸(CpG-ODN，核苷酸序列如SEQ ID No.3所示)干粉用无菌去离子水溶解，终浓度1mmol/L。
(3)将步骤(1)和步骤(2)中的溶液按照一定比例混合，并加去离子水调整海藻酸酸钠的终浓度为10-20mg/mL，得到包裹免疫佐剂CpG-ODN的海藻酸钠水凝胶放疗/化疗增敏制剂。
包裹免疫佐剂CpG-ODN的海藻酸钠水凝胶放疗/化疗增敏制剂用于小鼠结肠癌皮下瘤模型化疗增敏：小鼠CT26结肠癌肿瘤接种后第7天开始进行治疗。将携带肿瘤的BALB/c小鼠随机分为以下4组(每组6只)：第1组，对照组(不治疗，Untreated)；第2组，瘤内注射海藻酸钠，静脉注射奥沙利铂(ALG+OxPt)；第3组，瘤内注射包裹免疫佐剂CpG-ODN的海藻酸钠水凝胶放疗/化疗增敏制剂(ALG/CpG)；第4组，瘤内注射包裹免疫佐剂CpG-ODN的海藻酸钠水凝胶放疗/化疗增敏制剂，静脉注射奥沙利铂(ALG/CpG+OxPt)；每组治疗中海藻酸钠终浓度为10mg/mL，瘤内注射体积为25μL，每只小鼠的CpG-ODN剂量为15μg。在第7、10、13和16天以3mg/kg体重的剂量静脉注射奥沙利铂。对小鼠进行相应的治疗后，测量其肿瘤的生长。
图2(a)(b)分别为实验过程中肿瘤体积随时间变化情况及小鼠存活率随时间变化情况。结果表明(图2)，相比较对照组，第3组肿瘤生长仅得到了部分抑制，第2组肿瘤生长得到了较明显的抑制，而第4组的肿瘤生长则得到更为明显的抑制，有1/6肿瘤完全清除。表明包裹免疫佐剂CpG-ODN的海藻酸钠水凝胶放疗/化疗增敏制剂能够在一定程度上增强化疗地效果。
实施例2、具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂的制备
(1)海藻酸钠与ATP核酸适配体耦联物的制备：将0.2mg/mL低粘度海藻酸钠(粘度为20-100Cp)和0.1mmol/L5’端修饰单个氨基的ATP核酸适配体(SEQ ID No.1： 5’-acctgggggagtattgcggaggaaggt-3’)混合，其中海藻酸钠中的羧基与ATP核酸适配体耦联物中的氨基的摩尔比等于1000：1，用5倍浓缩MES缓冲液将pH调至5。
(2)称所需质量的EDC·HCl粉末迅速加入上述混合物溶液，其中EDC·HCl与海藻酸钠羧基的摩尔比等于100：1，反应在37℃下进行，期间不断震荡反应。
(3)加入3M乙酸钠溶液，其体积为上述步骤(2)得到的反应混合物体积的1/8，然后加入乙醇，其体积为前述混合物体积的3.75倍，随后在-80℃下放置10分钟，然后在4℃下以10,000g离心10分钟。
(4)离心所得沉淀用去离子水溶解，重复步骤(3)1次，所得沉淀即海藻酸钠-三磷酸腺苷核酸适配体耦联物，用磷酸盐缓冲液溶解。反应原理如图3a所示。
对海藻酸钠-三磷酸腺苷核酸适配体耦联物进行非变性聚丙烯酰胺凝胶电泳分析，结果显示(图3b)，与三磷酸腺苷核酸适配体相比，实施例1制备的海藻酸钠-三磷酸腺苷核酸适配体耦联物表现出明显的电泳迁移滞后现象，表明海藻酸钠与三磷酸腺苷核酸适配体发生耦联，分子量变大，导致电泳迁移率变慢。
(5)带有延长序列的CpG-ODN与海藻酸钠-三磷酸腺苷核酸适配体耦联物组装：其中，带有延长序列的CpG-ODN的序列如SEQ ID No.4所示：5’-tccatgacgttcctgacgttaccttcctccgcaa-3’。将延长序列的CpG-ODN溶解于磷酸盐缓冲液，浓度为0.1mM。将得到的溶液与海藻酸钠-三磷酸腺苷核酸适配体耦联物混合，放4℃冰箱中反应2小时以上，其中带有延长序列的CpG-ODN与三磷酸腺苷核酸适配体的摩尔比为1：2。所得样品即为具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂。反应原理如图4a所示。
将智能海藻酸钠水凝胶制剂进行非变性聚丙烯酰胺凝胶电泳分析，结果显示(图4b)，与海藻酸钠-三磷酸腺苷核酸适配体耦联物相比，智能海藻酸钠水凝胶制剂表现出明显的电泳迁移滞后现象，表明带有延长序列的CpG-ODN与三磷酸腺苷核酸适配体发生杂交而组装到海藻酸钠分子上，从而智能海藻酸钠水凝胶制剂分子量更大，导致电泳迁移率变慢。
(6)ATP响应性免疫佐剂CpG-ODN释放测试：为了能够监测CpG-ODN从水凝胶中的释放，采用一端修饰有荧光团的CpG-ODN，根据实施例1和3，制备具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂。制备好的智能海藻酸钠水凝胶制剂与质量浓度为4％的海藻酸钠溶液混合，至海藻酸钠的最终浓度为1％。将混合物缓慢转入浓度为10mmol/L的氯化钙溶液中，5分钟后，移出氯化钙溶液，加入含有一定浓度ATP溶液的氯化钙溶液，每隔1小时取出溶液进行荧光定量，然后再次补充相同体积的含有一定浓度ATP溶液的氯化钙溶液。释放原理如图5a所示。
测试结果显示(图5b)，ATP可以引发CpG-ODN从海藻酸钠水凝胶中的释放，而且ATP 浓度越高，释放速率越快。
实施例3、一种具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型的化疗增敏
小鼠CT26结肠癌肿瘤接种后第7天开始进行治疗。将携带肿瘤的BALB/c小鼠随机分为以下6组(每组6只)：第1组，对照组(不治疗，Untreated)；第2组，瘤内注射海藻酸钠，静脉注射奥沙利铂(ALG+OxPt)；第3组，瘤内注射海藻酸钠和CpG-ODN的混合物(ALG/CpG)；第4组，瘤内注射海藻酸钠和CpG-ODN的混合物，静脉注射奥沙利铂(ALG/CpG+OxPt)；第5组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂(ALG-Aapt/CpG)；第6组，瘤内注射具有实施例2步骤(5)得到的三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂，静脉注射奥沙利铂(ALG-Aapt/CpG+OxPt)。每组治疗中海藻酸钠终浓度为10mg/mL，瘤内注射体积为25μL，每只小鼠的CpG-ODN剂量为15μg。在第7、10、13和16天以3mg/kg体重的剂量静脉注射奥沙利铂。对小鼠进行相应的治疗后，测量其肿瘤的生长，治疗流程示意图如图6(a)所示。
图6(b)(c)分别为实验过程中肿瘤体积随时间变化情况及小鼠存活率随时间变化情况。结果表明(图6)，相比较对照组，第2组、第3组、第4组和第5组的肿瘤生长仅得到了部分抑制，而第6组的肿瘤生长则得到了有效的抑制，部分小鼠肿瘤完全消退。表明具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂联合低剂量化疗能够实现对肿瘤的高效协同杀灭。
实施例4、一种具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型的化疗增敏中免疫反应评价
小鼠CT26结肠癌肿瘤接种后第7天开始进行治疗。将携带肿瘤的BALB/c小鼠随机分为以下4组(每组6只)：第1组，对照组(不治疗，Untreated)；第2组，瘤内注射海藻酸钠，静脉注射奥沙利铂(OxPt)；第3组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂(ALG-A-Apt/CpG)；第4组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂，静脉注射奥沙利铂(ALG-A-Apt/CpG@OxPt)。每组治疗中海藻酸钠终浓度为10mg/mL，瘤内注射体积为25μL，每只小鼠的CpG-ODN剂量为15μg。在第7、10、13和16天以3mg/kg体重的剂量静脉注射奥沙利铂。治疗开始后第5天，处死每组小鼠，鉴定腹股沟淋巴结中树突状细胞的成熟度，肿瘤中CD8阳性T细胞的百分比，以及CD8阳性T细胞和调节性T细胞的比例。
评价结果显示(图7)，相比较对照组，第2组和第3组治疗没有明显提高淋巴结中树突状细胞(DC)的成熟度，肿瘤中CD8阳性T细胞的百分比，以及CD8阳性T细胞和调节性T细胞的比例，而第4组治疗明显提高了淋巴结中树突状细胞的成熟度，肿瘤中CD8阳性T细胞的百分比，以及CD8阳性T细胞和调节性T细胞的比例。表明具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂能够显著增强多次低剂量化疗引发的抗肿瘤免疫反应。
实施例5、一种具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型化疗增敏中治愈小鼠的免疫记忆功能评价
实施例4中的各组小鼠在开始治疗后70天，从肿瘤完全消退的小鼠(Cured group)中收集了外周血，鉴定记忆性CD8阳性T细胞。然后再次将小鼠CT26结肠肿瘤重新接种到肿瘤完全消退小鼠的皮下，以及未治疗的小鼠(Naive group)作为对照，接种肿瘤后监测肿瘤生长状态。
图8(a1)(a2)(b)为免疫评价结果，图8(c)(d)分别为实验过程中肿瘤体积随时间变化情况及小鼠存活率随时间变化情况。评价显示，肿瘤完全消退的小鼠(图8(a2)(b))外周血中CD8阳性效应记忆T细胞的百分比显着高于相同周龄的未治疗小鼠(图8(a1)(b))。肿瘤完全消退的小鼠的身上也未发现明显的肿瘤生长，而未经过治疗的小鼠身上肿瘤迅速生长。表明化疗联合具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂治愈的小鼠会产生有效的免疫记忆。
实施例6、一种具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠结肠癌皮下瘤模型的放疗增敏
小鼠CT26结肠癌肿瘤接种后第7天开始进行治疗。将携带肿瘤的BALB/c小鼠随机分为以下4组(每组6只)：第1组，对照组(不治疗，Unteated)；第2组，瘤内注射海藻酸钠，肿瘤局部X射线照射(剂量为8Gray)(RT)；第3组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂(ALG-Aapt/CpG)；第4组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂，肿瘤局部X射线照射(剂量为8Gray)(ALG-Aapt/CpG+RT)。每组治疗中海藻酸钠终浓度为10mg/mL，瘤内注射体积为25μL，每只小鼠的CpG-ODN剂量为15μg。在第7、9、11和13天以2Gray每个肿瘤的剂量进行照射。治疗流程示意图如图9(a)所示。
图9(b)(c)分别为实验过程中肿瘤体积随时间变化情况及小鼠存活率随时间变化情况。实验结果显示(图9)，相比较对照组，第2组和第3组的肿瘤生长仅得到了部分抑制，而第 4组的肿瘤生长则得到了有效的抑制，全部小鼠肿瘤都完全消退。表明具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂联合低剂量放疗能够实现对肿瘤的高效协同杀灭。
实施例7、一种具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于小鼠黑色素瘤模型的放疗增敏
小鼠B16黑色素瘤接种后第7天开始进行治疗。将携带肿瘤的C57小鼠随机分为以下4组(每组6只)：第1组，对照组(不治疗，Untreated)；第2组，瘤内注射海藻酸钠，肿瘤局部采用8Gray剂量的X射线照射(RT)；第3组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂(ALG-Aapt/CpG)；第4组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂，肿瘤局部采用8Gray剂量的X射线照射(ALG-Aapt/CpG+RT)。每组治疗中海藻酸钠终浓度为10mg/mL，瘤内注射体积为25μL，每只小鼠的CpG-ODN剂量为15μg。在第7、9、11和13天以2Gray每个肿瘤的剂量进行照射。治疗流程示意图如图10(a)所示。
图10(b)(c)分别为实验过程中肿瘤体积随时间变化情况及小鼠存活率随时间变化情况。实验结果显示(图10)，相比较对照组，第2组和第3组的肿瘤生长仅得到了部分抑制，而第4组的肿瘤生长则得到了有效的抑制，小鼠生存期有效延长。表明具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂联合低剂量放疗能够实现对黑色素瘤的有效抑制。
实施例8、一种具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于增强外放射治疗的远端瘤抑制效应
如图所示，在小鼠背部两侧皮下分别接种两个结肠癌肿瘤，接种后第7天开始进行治疗。将携带肿瘤的BALB/c小鼠随机分为以下6组(每组6只)：第1组，对照组(不治疗，Unteated)；第2组，静脉注射免疫检查点抑制剂PD1抗体(aPD1)；第3组，肿瘤局部X射线照射(剂量为8Gray)(RT)；第4组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂(ALG-Aapt/CpG)，肿瘤局部X射线照射(剂量为8Gray)(RT)；第5组，静脉注射免疫检查点抑制剂PD1抗体(aPD1)，肿瘤局部X射线照射(剂量为8Gray)(RT)；第6组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂，肿瘤局部X射线照射(剂量为8Gray),静脉注射免疫检查点抑制剂PD1抗体(ALG-Aapt/CpG+RT)。每组治疗中海藻酸钠终浓度为10mg/mL，瘤内注射体积为25μL，每只小鼠的CpG-ODN剂 量为15μg。在第7、9、11和13天以2Gray每个肿瘤的剂量进行照射。在第8天和第11天以10ug每只小鼠的剂量进行静脉注射免疫检查点抑制剂PD1抗体。治疗流程示意图如图11A所示。
图11(B)(C)(D)分别为实验过程中原位瘤(B)和远端肿瘤体(C)积随时间变化情况及小鼠存活率随时间变化情况(D)。实验结果显示(图11)，肿瘤生长曲线和相应的统计数据表明，单独的aPD1或单独的RT在抑制两侧肿瘤生长方面显示出有限的功效。由于通过ALG-Aapt/CpG增强了RT诱导的抗肿瘤免疫反应，RT+ALG-Aapt/CpG处理可消除右侧6个局部肿瘤中的4个，并明显延迟了远处肿瘤(左侧)的生长。然而，该组中大多数远处的肿瘤以后仍显示出快速的生长。值得注意的是，aPD1进一步增强了RT+ALG-Aapt/CpG的功效，消除了右侧的所有局部肿瘤以及消除了左侧的6个远处肿瘤中的5个。
实施例9、一种具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂用于增强乳腺癌原位瘤放射治疗效果
如图所示，在小鼠腹部两侧皮下接种乳腺癌肿瘤，接种后第7天开始进行治疗。将携带肿瘤的BALB/c小鼠随机分为以下6组(每组6只)：第1组，对照组(不治疗，Unteated)；第2组，静脉注射免疫检查点抑制剂PD1抗体(aPD1)；第3组，肿瘤局部X射线照射(剂量为8Gray)(RT)；第4组，静脉注射免疫检查点抑制剂PD1抗体(aPD1)，肿瘤局部X射线照射(剂量为8Gray)(RT)；第5组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂(ALG-Aapt/CpG)，肿瘤局部X射线照射(剂量为8Gray)(RT)；第6组，瘤内注射实施例2步骤(5)得到的具有三磷酸腺苷(ATP)响应性免疫佐剂缓释功能的智能海藻酸钠水凝胶制剂，肿瘤局部X射线照射(剂量为8Gray),静脉注射免疫检查点抑制剂PD1抗体(ALG-Aapt/CpG+RT)。每组治疗中海藻酸钠终浓度为10mg/mL，瘤内注射体积为25μL，每只小鼠的CpG-ODN剂量为15μg。在第7、9、11和13天以2Gray每个肿瘤的剂量进行照射。在第8天和第11天以10ug每只小鼠的剂量进行静脉注射免疫检查点抑制剂PD1抗体。治疗流程示意图如图12(A)所示。
结果如图12所示，单独的aPD1或单独的RT导致抑制肿瘤生长的功效有限(图12B)。如预期的那样，RT+ALG-Aapt/CpG+aPD1治疗导致最显着的肿瘤生长抑制以及大大延长的动物存活，与RT+aPD1或RT+ALG-Aapt/CpG相比，具有更好的治疗反应(图12B-C)。在第27天进一步获得了原发肿瘤体积超过1000mm 3的小鼠肺，用于统计转移性结节。代表性的肺部照片显示，尽管未治疗的小鼠的肺中有致密的转移性结节(黑色箭头所示)，但RT+ALG-Aapt/CpG+aPD1的治疗可显着抑制肿瘤转移(图12D)。在苏木精-曙红(H&E) 染色中进一步观察到代表性肺组织的病理变化，这也证实了RT+ALG-Aapt/CpG+aPD1的治疗可以显着抑制4T1肿瘤的肺转移。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。
Figure PCTCN2020124668-appb-000002
Figure PCTCN2020124668-appb-000003

Claims (10)

  1. 基于免疫佐剂的水凝胶组合物在制备外科放疗增敏制剂中的应用，所述基于免疫佐剂的水凝胶组合物包括海藻酸钠以及包裹于所述海藻酸钠中的水溶性免疫佐剂。
  2. 根据权利要求1所述的应用，其特征在于：所述基于免疫佐剂的水凝胶组合物包括海藻酸钠、肿瘤细胞死亡标志物适配体及带有延长序列的水溶性免疫佐剂，所述海藻酸钠与所述肿瘤细胞死亡标志物适配体通过肽键共价连接，所述延长序列与肿瘤细胞死亡标志物适配体的至少一部分序列碱基互补。
  3. 根据权利要求2所述的应用，其特征在于：所述肿瘤细胞死亡标志物适配体包括ATP核酸适配体，所述ATP核酸适配体的核苷酸序列包括如SEQ ID No.1所示的序列。
  4. 根据权利要求2所述的应用，其特征在于：所述延长序列的核苷酸序列包括如SEQ ID No.2所示的序列。
  5. 根据权利要求1-4中任一项所述的应用，其特征在于：所述水溶性免疫佐剂包括多聚核苷酸、CpG寡聚脱氧核苷酸、聚肌胞苷酸、polyICLC、脂多糖、胞壁肽、类脂A以及细胞因子中的一种或几种。
  6. 根据权利要求1-4中任一项所述的应用，其特征在于：所述外科放疗增敏制剂用于治疗实体肿瘤。
  7. 一种基于免疫佐剂的水凝胶组合物，其特征在于：包括海藻酸钠、肿瘤细胞死亡标志物适配体及带有延长序列的水溶性免疫佐剂，所述海藻酸钠与所述肿瘤细胞死亡标志物适配体通过肽键共价连接，所述延长序列与肿瘤细胞死亡标志物适配体的至少一部分序列碱基互补。
  8. 根据权利要求7所述的基于免疫佐剂的水凝胶组合物，其特征在于：所述肿瘤细胞死亡标志物适配体包括ATP核酸适配体。
  9. 根据权利要求7所述的基于免疫佐剂的水凝胶组合物，其特征在于：所述水溶性免疫佐剂包括多聚核苷酸、CpG寡聚脱氧核苷酸、聚肌胞苷酸、polyICLC、脂多糖、胞壁肽、类脂A以及细胞因子中的一种或几种。
  10. 一种权利要求7-9中任一项所述的基于免疫佐剂的水凝胶组合物的制备方法，其特征在于，包括以下步骤：
    (1)将海藻酸钠和氨基修饰的肿瘤细胞死亡标志物适配体在溶液中进行反应，得到海藻酸钠-三磷酸腺苷核酸适配体耦联物；
    (2)将所述海藻酸钠-三磷酸腺苷核酸适配体耦联物与带有延长序列的水溶性免疫佐剂在缓冲液中进行DNA杂交反应，得到所述基于免疫佐剂的水凝胶组合物，其中，所述延长序列与肿瘤细胞死亡标志物适配体的至少一部分序列碱基互补。
PCT/CN2020/124668 2020-09-16 2020-10-29 基于免疫佐剂的水凝胶组合物及其应用 WO2022057018A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/044,565 US20240148874A1 (en) 2020-09-16 2020-10-29 Immunoadjuvant-based hydrogel composition and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010976044.3 2020-09-16
CN202010976044.3A CN114259460B (zh) 2020-09-16 2020-09-16 基于免疫佐剂的水凝胶组合物及其应用

Publications (1)

Publication Number Publication Date
WO2022057018A1 true WO2022057018A1 (zh) 2022-03-24

Family

ID=80777224

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/124668 WO2022057018A1 (zh) 2020-09-16 2020-10-29 基于免疫佐剂的水凝胶组合物及其应用

Country Status (3)

Country Link
US (1) US20240148874A1 (zh)
CN (1) CN114259460B (zh)
WO (1) WO2022057018A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007030619A2 (en) * 2005-09-08 2007-03-15 Nastech Pharmaceutical Company Inc. Pharmaceutical compositions for delivery of ribonucleic acid to a cell
CN104225591A (zh) * 2014-09-15 2014-12-24 兰州大学 一种免疫佐剂及含有该免疫佐剂的疫苗
CN104689325A (zh) * 2013-12-06 2015-06-10 中国科学院深圳先进技术研究院 一种负载寡聚脱氧核苷酸的壳聚糖纳米粒及其制备方法和应用
WO2016138071A1 (en) * 2015-02-24 2016-09-01 Short Jay M Conditionally active biological proteins
CN108697805A (zh) * 2016-02-05 2018-10-23 医药研究产品有限公司 包含核酸及壳聚糖的温敏性水凝胶组合物
CN109939228A (zh) * 2019-03-25 2019-06-28 苏州博特龙免疫技术有限公司 一种新型水溶性免疫佐剂及其制备方法
CN110198985A (zh) * 2017-01-19 2019-09-03 株式会社岛津制作所 水凝胶组合物及其制造方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100558851B1 (ko) * 2004-01-08 2006-03-10 학교법인연세대학교 면역조절능력이 증가된 ＣｐＧ 올리고데옥시뉴클레오티드변형체
CN107153052B (zh) * 2016-03-03 2020-10-09 湖北中医药大学 酶引发的自由基聚合反应及检测应用
EP3506883A1 (en) * 2016-08-31 2019-07-10 Institut National de la Santé et de la Recherche Médicale (INSERM) Immunomodulation after locoregional anti-tumoral treament
CN111035757A (zh) * 2019-12-20 2020-04-21 天津大学 肿瘤免疫-化学联合治疗dna纳米制剂的方法
CN111012909A (zh) * 2019-12-20 2020-04-17 天津大学 肿瘤光热-免疫联合治疗dna纳米制剂的方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007030619A2 (en) * 2005-09-08 2007-03-15 Nastech Pharmaceutical Company Inc. Pharmaceutical compositions for delivery of ribonucleic acid to a cell
CN104689325A (zh) * 2013-12-06 2015-06-10 中国科学院深圳先进技术研究院 一种负载寡聚脱氧核苷酸的壳聚糖纳米粒及其制备方法和应用
CN104225591A (zh) * 2014-09-15 2014-12-24 兰州大学 一种免疫佐剂及含有该免疫佐剂的疫苗
WO2016138071A1 (en) * 2015-02-24 2016-09-01 Short Jay M Conditionally active biological proteins
CN108697805A (zh) * 2016-02-05 2018-10-23 医药研究产品有限公司 包含核酸及壳聚糖的温敏性水凝胶组合物
CN110198985A (zh) * 2017-01-19 2019-09-03 株式会社岛津制作所 水凝胶组合物及其制造方法
CN109939228A (zh) * 2019-03-25 2019-06-28 苏州博特龙免疫技术有限公司 一种新型水溶性免疫佐剂及其制备方法

Also Published As

Publication number Publication date
US20240148874A1 (en) 2024-05-09
CN114259460A (zh) 2022-04-01
CN114259460B (zh) 2024-03-15

Similar Documents

Publication Publication Date Title
Sun et al. ATP‐responsive smart hydrogel releasing immune adjuvant synchronized with repeated chemotherapy or radiotherapy to boost antitumor immunity
JP5971945B2 (ja) カチオン性脂質の鏡像異性体による免疫応答の刺激
JP4535211B2 (ja) コクリエート送達ビヒクル
ES2235839T3 (es) Uso de oligonucleotidos estabilizados para la preparacion de un medicamento de accion antitumoral.
JP2004505009A (ja) 新生物を処置するための組合せ
JP2010507361A (ja) 具体的には免疫刺激剤／アジュバントとしての、一般式（Ｉ）：ＧｌＸｍＧｎ、または一般式（ＩＩ）：ＣｌＸｍＣｎで表される核酸
JPH05503285A (ja) グルカン配達系及び補薬
Cemazar et al. Intratumoral cisplatin administration in electrochemotherapy: antitumor effectiveness, sequence dependence and platinum content
Wu et al. Overview of vaccine adjuvants
ES2565578T3 (es) Vacunas contra el cáncer que contienen epítopos de antígeno oncofetal
CN1810970B (zh) 含CpG的单链脱氧核苷酸与其疫苗组合物及其应用
US6656481B1 (en) Vaccinal preparations
WO2015027915A1 (zh) 体内个体化系统免疫治疗方法和装置
EP3220913B1 (en) Nanoparticle-based vaccine targeting cancer/testis antigens (cta) and its use in solid and hematological malignancies
CN111514306B (zh) 一种增强抗肿瘤免疫治疗的富勒烯纳米颗粒
BR112020017778A2 (pt) Anfifilos de cpg e usos dos mesmos
EP1653992A1 (en) Use of a vegf antagonist in combination with radiation therapy
WO2022057018A1 (zh) 基于免疫佐剂的水凝胶组合物及其应用
WO2018233605A1 (zh) 一种包含过氧化氢酶的组合物、其制备方法及用途和一种杀死肿瘤细胞的方法
Zhou et al. Intranasal administration of CpG DNA lipoplex prevents pulmonary metastasis in mice
JPS6366128A (ja) 動物およびヒト癌の治療に用いられるｃ反応性タンパク質
CN105008399B (zh) 新型的具有5个连接的ctl表位的肽
CN118055775A (zh) 包括hsp90抗原肽的抗癌用疫苗组合物及其用途
Li et al. Emerging nanoparticle platforms for CpG oligonucleotide delivery
JPH03502574A (ja) メチルセルロース製薬学的組成物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20953875

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20953875

Country of ref document: EP

Kind code of ref document: A1