WO2020001596A1 - 用于增强免疫响应的复合物的制备方法 - Google Patents

用于增强免疫响应的复合物的制备方法 Download PDF

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WO2020001596A1
WO2020001596A1 PCT/CN2019/093607 CN2019093607W WO2020001596A1 WO 2020001596 A1 WO2020001596 A1 WO 2020001596A1 CN 2019093607 W CN2019093607 W CN 2019093607W WO 2020001596 A1 WO2020001596 A1 WO 2020001596A1
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tumor
antigen
complex
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PCT/CN2019/093607
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French (fr)
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林海祥
刘芳
查力
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信福(北京)医药科技有限公司
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Priority claimed from CN201810700708.6A external-priority patent/CN109078180B/zh
Priority claimed from CN201810698033.6A external-priority patent/CN108743938B/zh
Application filed by 信福(北京)医药科技有限公司 filed Critical 信福(北京)医药科技有限公司
Priority to EP19825645.5A priority Critical patent/EP3818990A4/en
Priority to KR1020217002989A priority patent/KR20210025096A/ko
Priority to JP2020573468A priority patent/JP7296644B2/ja
Priority to CA3105283A priority patent/CA3105283A1/en
Priority to SG11202013185VA priority patent/SG11202013185VA/en
Priority to US17/256,980 priority patent/US20230210893A1/en
Publication of WO2020001596A1 publication Critical patent/WO2020001596A1/zh

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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
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    • 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
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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    • A61K9/513—Organic macromolecular compounds; Dendrimers
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    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the field of biomedicine, and in particular, to a method for preparing a complex for enhancing immune response.
  • Double-stranded RNA (dsRNA) adjuvants are currently considered to include PIC (polyriboinosinic-polyribocytoidylic acid), PICLC (PIC with poly-L-lysine and carboxymethylcellulose), PIC 12 U (PIC with uridylic acid in specific interval, trade name Ampligen) and PICKCa ((PIC-kanamycin-CaCl 2 ) is a ligand of multiple membrane recognition receptors (PRRs), which on the one hand enhances the immune response, and on the other hand, by changing the type of immunity, it is possible to make the preventive vaccine into a therapeutic vaccine .
  • PIC polyriboinosinic-polyribocytoidylic acid
  • PICLC PIC with poly-L-lysine and carboxymethylcellulose
  • PIC 12 U PIC with uridylic acid in specific interval, trade name Ampligen
  • PICKCa (PIC-kanamycin-CaCl 2 ) is
  • PIC polymyocyte
  • PICLC polymyocyte + lysine + carboxymethyl cellulose
  • PIC 12 U was developed at Johns Hopkins University in the mid-1970s. It inserts uracil nucleotides at certain positions in the PIC chain. Similar in efficacy to PIC, but less toxic.
  • Hemispherx Biopharmaceutical Company submitted further original clinical research data, but it was not approved by the US Food and Drug Administration (FDA) due to insufficient safety and efficacy data.
  • PICKCa contains the antibiotic kanamycin. Kanamycin has middle ototoxicity, and its content in the vaccine exceeds the national pharmacopoeia standards.
  • PIC 12 U has not been approved by the US FDA because of poor results, and PICLC actually has strong side effects.
  • the present disclosure relates to a novel complex, and the preparation, application, and other aspects of the complex are studied.
  • vaccine adjuvants (trade names: Pica adjuvant or PIKA adjuvant) were formulated using PIC, kanamycin, and calcium chloride. Kanamycin is used because it contains 4 amino groups to stabilize its structure by binding to the phosphate group in PIC, but this product has limited its use in vaccines because it contains antibiotics.
  • chitosan hydrochloride
  • hydrochloride has a large molecular weight and is not easily absorbed by the human body. It is difficult to obtain the desired effect.
  • the present disclosure provides a complex for enhancing an immune response.
  • the complex is prepared from at least the following components under suitable conditions: polymyocytes, at least one cationic stabilizer, and a soluble calcium salt.
  • the complex is prepared from at least the following components under suitable conditions: polymyocytes, at least one cationic stabilizer, and soluble calcium or / and manganese salts.
  • the cationic stabilizer is a water-soluble non-antibiotic amino compound having a molecular weight of ⁇ 5 kDa, or the water-soluble non-antibiotic amino compound and polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, semi- Graft formed by one or more of lactose.
  • the complex has moderate viscosity and molecular weight, convenient pharmaceutical production, stable chemical properties, long-term storage and is not easily degraded, and is safe to use.
  • the compound alone can significantly enhance the body's non-specific immune response and prevent and cure diseases
  • the purpose is to use it in combination with other drugs to have better anti-tumor, anti-viral and anti- (super) bacterial effects, which can be easily absorbed by patients.
  • Figure 1 is a schematic diagram of the structure of the Pammika complex
  • A a schematic diagram of the complex structure of PolyI: C-COS-Ca 2+
  • B a schematic diagram of the structure of the antigen (Ag) + complex particles
  • C PolyI: C-COS- Schematic diagram of the complex structure of Ca 2+ + Ag
  • FIG. 2 is a molecular weight electrophoresis chart after PIC is heated for different times
  • FIG. 3 is an enzyme degradation curve of a palmika complex in an embodiment of the present disclosure
  • FIG. 4 is a melting curve of a palmika compound in an embodiment of the present disclosure
  • FIG. 12 is an example of the present disclosure in which mice were immunized with aluminum adjuvant / rHBsAg (CHO), ADV20 / rHBsAg (CHO), and Pamica / rHBsAg (CHO) for 21 days. Detection map
  • FIG. 13 shows the humoral immunity of mice immunized with aluminum adjuvant / rHBsAg (CHO), ADV20 / rHBsAg (CHO), and pamica / rHBsAg (CHO) after 21 days in an example of the present disclosure; Swelling: increased number of millimeters (footpad sweating: mm)
  • FIG. 14 is a comparison of the effect of the Pammika complex and the complete Freund's adjuvant in the preparation of MYO antigen corresponding antibodies in an embodiment of the present disclosure
  • FIG. 15 is a comparison of the effect of the Pammika complex and the complete Freund's adjuvant in the preparation of MYO antigen corresponding antibodies in an embodiment of the present disclosure
  • FIG. 16 is a picture of an example of an experiment of stimulating phagocytic function of macrophages by the pamica group in an embodiment of the present disclosure
  • FIG. 17 is a picture of an example of an experiment in which the PBS control group does not stimulate phagocytosis of macrophages in an embodiment of the present disclosure
  • 18 to 21 are experimental results of the anticancer effect of the pamica mucosal immune preparation on a tumor-bearing mouse LL2 lung cancer model in an embodiment of the present disclosure
  • FIG. 18 Tumor volume change curves under different drug treatments
  • Figure 19 Tumor weight under different drug treatments (the tumor volume of the vehicle control group reached 2201.09 ⁇ 68.01 mm3 on the 14th day after the administration, and the experiment was terminated on the 14th day after the administration);
  • Figure 21 Tumor inhibition rate of mouse-derived PD-1 antibody
  • 22 to 35 are in vivo antitumor effects of pamika on a 4T1-luc mouse orthotopic breast cancer model in an embodiment of the present disclosure
  • Figure 26 Effects of different drug treatments on spleen weight
  • Figure 27 Frontal photo of lungs under the influence of different drugs
  • Figure 28 Photographs of the back of the lungs under the influence of different drugs
  • Figures 29 to 35 Photographs of bioluminescence intensity of tumor sites and metastases displayed by small animal imagers
  • FIG. 29 Palmika bioluminescence in mice 7 days in advance
  • Figure 30 Bioluminescence of mice in the Pamika day 0 group
  • FIG. 31 Bioluminescence of mice in the vehicle group
  • Figure 32 Bioluminescence of 200 ⁇ g / mice of Pamika nose drops
  • Figure 33 Bioluminescence of mice in the nose group of 300 ⁇ g / Pamica;
  • Figure 34 Bioluminescence of mice in the 200 ⁇ g / mikamika injection group
  • Figure 35 Bioluminescence of mice in the 300 ⁇ g / mikamika injection group.
  • Pamika refers generally to a complex prepared from polymyocytes, cationic stabilizers, and soluble calcium salts (calcium ions), regardless of the specific physical and immunogenicity of the complex.
  • Polyinositol is also known as polyinosinic acid, polyinosinic acid, polyinosinic acid, polyinosinic acid cytosine nucleotides, polyinosinic acid-polycytidylic acid, PIC or PolyI: C.
  • enhancing immune response refers to inducing or enhancing the host's immune response to antigenic substances, or enhancing the function of immune cells, or promoting the release of inflammatory factors or cytokines by immune cells, or increasing the host's ability to cause disease Physical resistance.
  • inducing an immune response means stimulating, initiating or inducing an immune response.
  • Patentiating immune response means that an existing immune response is improved, fueled, supplemented, amplified, promoted, increased, or prolonged.
  • Enhancing immune response This expression or similar expression means that the immune response is improved, improved, or increased compared to the previous immune response state, which is beneficial to the host, such as given to the present disclosure The state of the previous immune response of the immunogen composition.
  • the term "individual” is used interchangeably herein with “host”, “subject” and “animal” and includes humans and all livestock (such as domestic animals and pets) and wild animals and birds, including, without limitation, cattle, Horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, marten, chicken, duck, goose, turkey, cockfighting, etc.
  • antibody includes polyclonal and monoclonal antibodies, as well as antigen-compound-binding fragments of these antibodies, including Fab, F (ab ') 2 , Fd, Fv, scFv, bispecific antibodies, and minimum recognition units for antibodies, and these antibodies And fragments of single-chain derivatives.
  • the type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
  • antibody includes naturally occurring and non-naturally occurring antibodies, including, for example, chimeric, bifunctional, and humanized antibodies, and related synthetic isoforms (isoforms).
  • antibody is used interchangeably with "immunoglobulin”.
  • the term “antigen compound” refers to any substance that can be recognized by the immune system where appropriate (eg, bound to an antibody or processed to induce a cellular immune response).
  • antigen includes, but is not limited to, cells, cell extracts, proteins, lipoproteins, glycoproteins, nucleoproteins, polypeptides, peptides, polysaccharides, polysaccharide conjugates, polysaccharide peptide phantoms, fats, Glycolipids, sugars, viruses, virus extracts, bacteria, bacterial extracts, fungi, fungal extracts, multicellular organisms such as parasites, and allergens.
  • Antigens can be exogenous (e.g., from a source other than the individual to whom the antigen is administered, e.g., from a different species) or endogenous (e.g., from a host, such as a body's disease factors, cancer antigens) , Antigens produced by virus-infected cells, etc.).
  • Antigens can be natural (e.g., naturally occurring), synthetic, or recombinant.
  • Antigens include cell extracts, whole cells, and purified antigens, where the term "purified" means that the antigen presents more abundantly than the environment in which the antigen normally exists and / or compared to crude extracts (such as cultured forms of the antigen) form.
  • vaccine composition refers to a combination of two or more substances (such as an antigen and an adjuvant), which when administered to a host together trigger an immune response.
  • polypeptide peptide
  • oligopeptide protein
  • proteins are used interchangeably in this specification, and they mean any form of amino acid polymer that may include encoded and non-encoded amino acids, Chemically or biochemically modified or derived amino acids and polypeptides with modified peptide backbones.
  • immune response refers to any response of the immune system of a vertebrate individual to an antigenic or immunogenic compound.
  • Typical immune responses include, but are not limited to, local and systemic cellular and humoral immune responses, such as cytotoxic T lymphocyte (CTL) responses including antigen-specific induction of CD8 + CTLs, T-cell proliferation responses, and cells Helper T-cell responses including factor release, and B-cell immune responses including antibody responses.
  • CTL cytotoxic T lymphocyte
  • adjuvant refers to any substance or mixture of substances that increases or alters the host's immune response to an antigenic compound.
  • treatment refers generally to obtaining the desired pharmacological and / or physiological effect.
  • the effect may be preventive from the point of view of completely and / or partially preventing the disease or its symptoms, and / or the effect is caused by completely and / or partially stabilizing or curing the disease and / or disease
  • the negative effects can be medical in nature.
  • treatment covers any treatment of a disease in an individual (especially a mammalian individual, and more particularly a human) and includes: (a) prevention of a disease that may be predisposed but not yet diagnosed The individual develops a disease or symptom; (b) suppresses the disease symptom, such as preventing the development of the disease symptom; or relieves the disease symptom, such as causing the disease or symptom to subside; (c) reduces the level of products produced by the disease infectious substance (such as toxin , Antigen, etc.); (d) reduce the adverse physiological response to disease infectious substances (such as fever, tissue edema, etc.).
  • the disease infectious substance such as toxin , Antigen, etc.
  • reduce the adverse physiological response to disease infectious substances such as fever, tissue edema, etc.
  • a “pharmaceutically acceptable salt” chemical means that the salt is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound.
  • These salts include: (1) acids that synthesize salts, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and other inorganic acids to form salts together; or with acetic acid, propionic acid, hexanoic acid, cyclopentanoic acid, glycolic acid , Pyruvate, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, Methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzen
  • One aspect of the present disclosure relates to a combined product for enhancing an immune response, which comprises a polymyocyte, at least one cationic stabilizer, and a soluble calcium salt;
  • the cationic stabilizer is a water-soluble non-antibiotic amino compound having a molecular weight of 5 kDa or less, or the water-soluble non-antibiotic amino compound and polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, galactose One or more of the grafts formed.
  • pamika alone can enhance the body's non-specific immune response, and can more effectively cause specific humoral and cellular immune responses, thereby improving protective immunity; combined use with antigen substances can achieve better results .
  • pamika can promote tumor cell apoptosis through signal transduction pathways, and can also stimulate immune cells to express a variety of cytokines and change the tumor cell microenvironment, allowing immune cells to attack tumor cells, viruses, bacteria and other pathogenic substances.
  • palmika is easier to be absorbed by the host, or swallowed by the host cells. Furthermore, more antigens can be brought into cells, thereby enhancing the immune response caused by proteins and peptides.
  • pamika has a significant analgesic effect on patients with cancer pain.
  • An important advantage is that pamika can make the virus titer of HPV infected people from strong positive to negative.
  • the palmika is not a simple composition, as described in the specification of the present disclosure, it is a complex with a completely new structure.
  • the molecular weight of the cationic stabilizer can also be selected from 4kDa, 4.5kDa, 3kDa, 3.5kDa, 2.5kDa, 2kDa, 1.5kDa, 1kDa, 500Da, 400Da, 300Da, 200Da, 100Da.
  • the water-soluble non-antibiotic amino compound is selected from the group consisting of chitooligosaccharides, chitosan oligosaccharides, glucosamine, cationic liposomes, DEAE-dextran, polyacrylamide, polyamines, tetraminefulvene One or more of polyethyleneimine.
  • the cationic stabilizer is selected from the group consisting of a graft of oligosaccharides and polyethylene glycol monomethyl ether (COS-g-MPEG), grafting of chitosan hydrochloride and polyethylene glycol (PEG-g-CS), grafts of folic acid and chitosan hydrochloride (FA-g-CS), galactose and grafts of polyethylene glycol and polyethyleneimine (GAL-g-PEG) -g-PEI), chitosan and polyethylene glycol monomethyl ether and grafts of polyethyleneimine (COS-g-MPEG-g-PEI), chitosan and polyethylene glycol monomethyl ether and Graft of polyethyleneimine (CS-g-PEG-g-PEI), graft of polyethylene glycol and polyethyleneimine (PEI-g-PEG), graft of chitooligosaccharide and polyethyleneimine Branches (
  • the cationic stabilizer is selected from the group consisting of chitooligosaccharide (COS), a graft of chitooligosaccharide and polyethylene glycol monomethyl ether (COS-g-MPEG), chitooligosaccharide and polyethylene glycol Graft of alcohol monomethyl ether and polyethyleneimine (COS-g-MPEG-g-PEI).
  • the molecular weight of the graft is ⁇ 50 kDa.
  • the molecular weight of the graft can also be selected from 45kDa, 40kDa, 35kDa, 30kDa, 25kDa, 20kDa, 15kDa, 10kDa, 9kDa, 8kDa, 7kDa, 8kDa, 5kDa, 4kDa, 3kDa, 2kDa, 1kDa, 500Da, 400Da, 300Da, 200Da, 100Da.
  • the degree of deacetylation of the chitooligosaccharide is ⁇ 70%; 80%, 85%, 90%, or 95% can also be selected; preferably 90% to 100%.
  • the chitosan monomer has a molecular weight of 161, a degree of polymerization of 2-20, and a molecular weight range of 322-3220 is selected.
  • the molecular weight of chitooligosaccharide, chito-oligosaccharide, and glucosamine is ⁇ 3200.
  • the molecular weight of polyethylene glycol monomethyl ether, polyethylene glycol, and polyethyleneimine is ⁇ 40,000, and 30,000, 20000, 15000, 10,000, 8000, 6000, 4000, 2000, 1500 can also be selected. 1000 or 500.
  • the soluble calcium salt is selected from CaCl 2 and / or CaNO 3 .
  • the molecular weight of the polymyocyte is 100bp-3000bp.
  • the molecular weight of the polymyocyte is 100bp-1500bp.
  • the combination product further comprises one or more of a pH adjuster, sodium tripolyphosphate, sodium alginate, phenylboronic acid, catechol, buffer salts / agents, and water.
  • each ingredient in the combined product is packaged separately;
  • At least two ingredients in the combined product are mixed and packaged together, for example, a normal valent ion is packaged with water and / or a buffer salt;
  • polymyocytes are packaged in the form of their raw materials, such as inosine (PI) and cytidylic acid (PC).
  • raw materials such as inosine (PI) and cytidylic acid (PC).
  • the present disclosure also relates to a complex for enhancing an immune response, which is prepared from an agent in a combination product as described above.
  • the preparation is performed in a solution system, and in the reagent, the concentration of the polymyocyte is 0.1 to 10 mg / ml;
  • the concentration of polymyocytes can increase the solubility by grafting, and theoretically can reach higher concentrations
  • the preparation is performed in a solution system, and in the reagent, the concentration of the polymyocyte is 0.5 to 5 mg / ml, and 1 mg / ml, 2 mg / ml, and 3 mg / ml can also be selected. , 4 mg / ml, 5 mg / ml, 6 mg / ml, 6.4 mg / ml, 7 mg / ml, 8 mg / ml or 9 mg / ml.
  • the preparation is performed in a solution system, and in the reagent, the concentration of the cationic stabilizer is 0.5 to 51.2 mg / ml;
  • the concentration of the cationic stabilizer is 0.8 to 25.6 mg / ml, and 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml can be selected ml or 20mg / ml.
  • the preparation is performed in a solution system, and in the reagent, the mass ratio of the polymyocyte to the cationic stabilizer is 1: 0.8 to 25.6; also 1: 6.4 or 1: 12.8.
  • the preparation is performed in a solution system, and in the reagent, the concentration of calcium ions in the soluble calcium salt is 0.1 to 1 mM, and 0.2 mM, 0.3 mM, 0.4 mM, 0.5 can also be selected. mM, 0.6mM, 0.7mM, 0.8mM or 0.9mM.
  • the complex is stored in a solution.
  • the solution is preferably a buffer solution.
  • the pH of the solution is 5.0-7.2.
  • the pH of the solution is 5.9 to 6.9, and 6.0, 6.2, 6.4, 6.8, 7.0, 7.2, 7.4, 7.6, or 7.8 can also be selected.
  • the present disclosure also relates to the non-therapeutic use of the complex as described above as an immune adjuvant.
  • the present disclosure also relates to the use of a complex as described above for the preparation of an antibody, a vaccine formulation or a vaccine composition, or for the use of a vaccine adjuvant or a vaccine adjuvant.
  • the present disclosure also relates to a vaccine composition
  • a vaccine composition comprising a complex as described above and at least one antigen.
  • the antigen is a virus, bacteria, protein, polypeptide, polysaccharide, nucleic acid, or small molecule-protein conjugate.
  • the vaccine composition is, for example, an attenuated vaccine (for example, an attenuated virus or bacteria vaccine), an inactivated vaccine (for example, an inactivated virus or bacteria vaccine), a protein vaccine, a polysaccharide vaccine, Protein subunit vaccine, chimeric vector vaccine, DNA vaccine, RNA vaccine, peptide vaccine or small molecule-protein conjugate vaccine.
  • an attenuated vaccine for example, an attenuated virus or bacteria vaccine
  • an inactivated vaccine for example, an inactivated virus or bacteria vaccine
  • a protein vaccine for example, a polysaccharide vaccine, Protein subunit vaccine, chimeric vector vaccine, DNA vaccine, RNA vaccine, peptide vaccine or small molecule-protein conjugate vaccine.
  • the present disclosure also relates to the use of a complex as described above in regulating immune cell activity, said application being performed in vivo or in vitro.
  • the modulating immune cell activity is specifically enhancing immune cell activity.
  • the immune cells are selected from macrophages, lymphocytes, and dendritic cells.
  • the modulation enhances immune cell activity is to promote the release of inflammatory factors by the immune cells.
  • the inflammatory factors include IL-2, IL-6, IL-12p40, IL-18, IL-22, IFN- ⁇ , IFN- ⁇ , and TNF- ⁇ .
  • the inflammatory factors include IFN- ⁇ and TNF- ⁇ .
  • the present disclosure also relates to a complex as described above for the preparation and treatment of tumors, antivirals, antibacterials, antifungals, antiparasites, reduction of side effects of chemotherapy, antifatigue or boosting immunity 3.
  • the medicament is an injection administration form, a respiratory administration form, a nasal drop, a skin administration form, a mucosal administration form, or a cavity administration form.
  • the injectable dosage form is selected from the group consisting of: injections (including intravenous, intramuscular, subcutaneous, and intradermal injection routes).
  • the dosage form for airway administration is selected from the group consisting of: sprays, aerosols, powders, and the like.
  • the dosage form for dermal administration is selected from the group consisting of: external solution, lotion, tincture, ointment, plaster, paste, patch, etc., which acts locally or percutaneously through the system after administration. effect.
  • the dosage form for mucosal administration is selected from the group consisting of: eye drops, nasal drops, ophthalmic ointments, gargles, sublingual tablets, etc.
  • Mucosal administration can play a local role or absorb the whole body through mucosal absorption effect.
  • Cavity administration dosage forms such as suppositories, aerosols, etc., used in rectum, vagina, urethra, nasal cavity, ear canal, etc., cavity administration can play a local role or play a systemic role after absorption.
  • the antigen comprises a tumor, virus, bacterial, fungal or parasite antigen.
  • the host is a mammal.
  • the host is a primate.
  • the host is human.
  • the drug when the antigen is a virus, bacterial, fungal, or parasite antigen, the drug contains 1 to 8 mg per dose;
  • the medicine when the antigen is a tumor antigen, contains 1-10 mg per dose.
  • the present disclosure also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the complex as described above, and the pharmaceutical composition further includes an immune cell therapy drug, an antibody therapy drug, a chemical drug, a booster One or more of a substance for mucosal immune absorption or mucosal adhesion, an immunomodulator, a pathogen antigen, a ligand for a membrane recognition receptor, and a pharmaceutically acceptable excipient.
  • the immune cell therapy drug is selected from tumor infiltrating lymphocytes (TIL), dendritic cells (DC), cytokine induced killer (CIK), One or more of dendritic cells-cytokine-induced killer cells (DC-CIK), natural killer cells (NK), ⁇ T cells, CD3AK, CAR-T, and TCR-T.
  • TIL tumor infiltrating lymphocytes
  • DC dendritic cells
  • CIK cytokine induced killer
  • DC-CIK cytokine induced killer
  • NK natural killer cells
  • ⁇ T cells CD3AK, CAR-T, and TCR-T.
  • the antibody treatment drug is selected from the group consisting of an anti-PD1 antibody, an anti-PDL1 antibody, an anti-CTLA4 antibody, and an anti-CD antigen antibody.
  • the chemical drug is selected from one or more of alkylating agents, antimetabolites, antitumor antibiotics, plant antitumor drugs, hormone drugs, and miscellaneous drugs;
  • the heterodrug is selected from the group consisting of L-asparaginase, cisplatin, carboplatin, platinum oxalate, azalimidamine, hexamethylamine, or derivatives of the above drugs.
  • the alkylating agent is selected from the group consisting of cyclophosphamide, busulfan, ammonium amine, cisplatin, dichloromethanediethylamine, phenylalanine mustard, nitrosoureas, and Derivatives of the aforementioned drugs;
  • the antimetabolite is selected from the group consisting of 5-fluorouracil, methotrexate, cytarabine, cyclocytidine, hydroxyurea, and derivatives of the aforementioned drugs;
  • the anti-tumor antibiotic is selected from the group consisting of actinomycin, mitomycin, lutein, adriamycin, erythromycin, dactinomycin, bleomycin, and derivatives of the above drugs ;
  • the hormonal drug is selected from sex hormones, corticosteroids, and derivatives of the above drugs.
  • the substance that promotes mucosal immune absorption or mucosal adhesion is selected from anionic surfactants (such as carboxylates, sulfonates, sulfates, phosphates, etc.), cationic surfactants (Such as amine salts, quaternary ammonium salts, heterocycles, onium salts, etc.), zwitterionic surfactants such as carboxylate, sulfonate, phosphate, betaine, imidazoline, amino acids Type), non-ionic surfactants (such as alkyl polyglycoside, polyoxyethylene type, polyol type, alkanolamide type, block polyether type), special surfactants (such as fluorine-containing, silicon-containing Type, boron-containing type, polymer type, etc.), chelating agents (such as polyphosphate, aminocarboxylic acid, 1,3-diketone, hydroxycarboxylic acid, polyamine, etc.), adhesives [water-soluble adhesive
  • the immunomodulatory agent is selected from the group consisting of cytokines, chemokines, stem cell growth factors, lymphotoxin, hematopoietic factors, colony-stimulating factors (CSF), interferons, erythropoietin, thrombopoietin, One or more of tumor necrosis factor (TNF), interleukin (IL), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), and stem cell growth factor.
  • TNF tumor necrosis factor
  • IL interleukin
  • G-CSF granulocyte-colony stimulating factor
  • GM-CSF granulocyte macrophage-colony stimulating factor
  • the pathogen antigen is selected from a tumor, virus, bacterial, fungal or parasite antigen.
  • the tumor includes: bone, bone connection, muscle, lung, trachea, pharynx, nose, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx , Esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gall, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, uterus, vagina, vulva Department, scrotum, testis, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brain stem, medulla oblongata, inferior medulla, cerebral inferior fluid, nerve, thyroid, parathyroid gland, adrenal gland, pituitary, pineal gland, Tumors resulting from lesions in any of the islets, thymus, go
  • the bacteria include: Staphylococcus, Streptococcus, Listeria, Rhizoctonia, Renal, Bacillus, Clostridium, Mycobacterium, Actinomyces, Nocardia, Corynebacterium, Rhodococcus, Anthrax, Bacillus tetanus, Tetanus, Listeria, Monerobacterium aeruginosa, Mycobacterium tuberculosis, Escherichia coli, Proteus, Shigella, Pneumoniae, Brucella, Clostridium perfringens, Haemophilus influenzae, Haemophilus parainfluenzae, Moraxella catarrhalis, Acinetobacter, Yersinia, Legionella pneumophila, Pertussis, One or more of B. parapertussis, Shigella, Pasteurella, Vibrio cholerae, and hemolytic bacteria.
  • the parasites include: parasites in the digestive tract (such as roundworms, hookworms, roundworms, amoeba and Yarrow's flagellates, etc.), parasites (such as Trichomonas vaginalis) , Intrahepatic parasites (e.g. liver fluke, echinococcus), intrapulmonary parasites (e.g. Westerman parasites), brain tissue parasites (e.g. Cysticercus cellulosae, Toxoplasma gondii), intravascular parasites (e.g.
  • parasites in the digestive tract such as roundworms, hookworms, roundworms, amoeba and Yarrow's flagellates, etc.
  • parasites such as Trichomonas vaginalis
  • Intrahepatic parasites e.g. liver fluke, echinococcus
  • intrapulmonary parasites e.g. Westerman parasites
  • brain tissue parasites
  • lymphatic parasites such as filariasis
  • muscle tissue parasites such as Trichinella larvae
  • intracellular parasites such as Plasmodium, Leishmania
  • bone tissue parasites such as hydatids
  • skin parasites such as chigger mites, hair follicle mites
  • intraocular parasites such as sucking nematodes, cysticercus suis
  • the viruses include: adenoviridae, arenaviridae, astroviridae, bunyaviridae, projectiviridae, flaviviridae , Hepatitis D virus (hepatitis, delta virus), hepatitis virus (hepeviridae), single molecule negative strand RNA virus (mononegavirales), nest virus (nidovirales), small RNA virus (piconaviridae), orthomyxovirus (orthomyxoviridae), papilloma virus (papillomaviridae), parvoviridae, polyomaviridae, poxviridae, reoviridae, retroviridae, or togaviridae or Multiple.
  • the virus is Human papillomavirus.
  • the fungi include: Coccus sphaeroides, Pseudosphaera spp., Histoplasma capsular, Histoplasma spp., Lobobacterium, Paracoccus brasiliensis, Dermatitis Bacillus, Schenckia sp., Penicillium marneffei, Candida albicans, Candida glabrata, Candida tropicalis, Candida portugis, Pneumocystis carinii, Aspergillus sp Pycnidium spp., Pycnidium versicolor, Pycnidium verrucous, Pycnidium dermatitis, Geotrichum candidum, B.
  • the ligand of the membrane recognition receptor is selected from a ligand of a TLR receptor, a ligand of an RLR receptor, a ligand of a CLR receptor, and a ligand of an NLR receptor.
  • the ligands associated with the TLR receptor are: peptidoglycan, disaccharide, mannan, lipopeptide, glycolipid, atypical lipopolysaccharide, serum amyloid, CPG DNA, dsRNA, ssRNA , LPS, PGN, saturated fatty acids, lipoteichoic acid, resistin, lactoferrin, surfactant protein, flagellin, hyaluronic acid, RNA-related antigens, and profilin-like molecules.
  • the ligands associated with the RLR receptor are: RNA, PIC, PICLC, PIC12u, and the like.
  • the ligands that bind the CLR receptor are: mannose and ⁇ -glucan on the surface of fungal cells, and the like.
  • the ligands that bind the NLR receptor are: such as MDP, DAP, etc.
  • the present disclosure also relates to a method for preparing a complex for enhancing an immune response, including:
  • the cationic stabilizer is a water-soluble non-antibiotic amino compound having a molecular weight of 5 kDa or less, or the water-soluble non-antibiotic amino compound and polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, galactose One or more of the grafts formed.
  • the polymyocytes are made from polycytidylic acid and polyinosinic acid via a base-pairing reaction.
  • the molecular weight of the polycytidylic acid and polyinosinic acid is greater than 23,000 Daltons.
  • the molecular weight of the polycytidylic acid ranges from 66,000 Daltons to 660,000 Daltons.
  • the molecular weight of the polyinosinic acid is in the range of 66,000 Daltons to 660,000 Daltons.
  • the base-pairing reaction is performed at a temperature of 40 ° C to 50 ° C, and 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, or 48 ° C can also be selected. 49 ° C.
  • the temperature may also be selected from 82 ° C, 84 ° C, 86 ° C, 88 ° C, 90 ° C, 92 ° C, 94 ° C, 96 ° C, or 98 ° C;
  • the heating time may also be selected from 80 min, 90 min, 100 min, or 110 min.
  • the temperature of the liquid reaction system is 40 ° C to 50 ° C, and 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, or 49 ° C can also be selected.
  • the method for preparing the graft includes:
  • polyethylene glycol monomethyl ether, polyethylene glycol, polyethyleneimine, folic acid, and galactose are activated with carbonyldiimidazole, and then the activated product is used with the water-soluble non-antibiotic amino group.
  • the compound undergoes a grafting reaction in the ionic liquid [bmim] Cl.
  • the graft is a graft of chitooligosaccharide and polyethylene glycol monomethyl ether, and polyethylene glycol monomethyl ether (MPEG) is activated with carbonyldiimidazole (CDI) before using
  • MPEG polyethylene glycol monomethyl ether
  • CDI carbonyldiimidazole
  • the activated MPEG was grafted with chitooligosaccharide (COS) in ionic liquid [bmim] Cl.
  • the grafting reaction is performed at 60 ° C to 80 ° C in a non-oxidizing atmosphere.
  • the method further includes:
  • the cross-linking agent is selected from at least one of sodium tripolyphosphate, sodium alginate, phenylboronic acid, and catechol.
  • the cross-linker solution contains a (pathogen) antigen.
  • the method further comprises co-incubating the complex or the nanoparticle with an antigen.
  • the antigen is a protein or polypeptide antigen.
  • the present disclosure also relates to a method for promoting an immune response to an antigen in a host, or for enhancing the activity of a host's immune cells, or helping the host to reduce fatigue, or alleviating host pain, the method comprising: The complex as described above, or the vaccine composition as described above, or the pharmaceutical composition as described above is administered to the host.
  • the host is suffering from an infectious disease, and the administration of the antigenic compound provokes an immune response against the pathogen causing the infectious disease.
  • the administration is performed by parenteral injection, intramuscular injection, intraperitoneal injection, intravenous injection, subcutaneous injection, local administration, transdermal administration, or intradermal administration.
  • the host is a tumor patient, a virus-infected patient, a bacterial-infected patient, a parasitic-infected patient, or a rhinitis patient who has failed surgical chemotherapy, radiotherapy or immunotherapy or the medical institution has abandoned treatment.
  • the method can be used in combination with surgery, radiation therapy, chemotherapy, and various immunotherapies, or in combination with patients with viral infections, patients with bacterial infections, and patients with parasitic infections can also be used with traditional therapies.
  • the pain is pain caused by a microbial or parasite infection, pain caused by cancer, or neuropathic pain.
  • the drug when the antigen is a viral, bacterial, fungal, or parasite antigen, the drug is administered at 1 to 8 mg / kg each time; preferably, or every day or every other day or two or three days Dosing once
  • the drug is administered at 1 to 10 mg / kg each time; preferably, the administration period is at least 360 days or at least 180 days or at least 60 days or at least 30 days.
  • the present disclosure provides a complex for enhancing an immune response prepared according to the methods described in the present disclosure.
  • the present disclosure provides a complex for enhancing immune response prepared according to the method disclosed in the present disclosure, used for preparing antibodies, vaccine preparations or vaccine compositions, enhancing immune cell activity, treating and / or preventing tumors, anti-virus, anti-bacterial, anti- Application of fungi, anti-parasites, reducing side effects of chemotherapy, anti-fatigue or improving immunity, alleviating host pain, and promoting host immune response to antigens.
  • the preparation method of the COS-g-MPEG graft is as follows: preparing a chito-oligosaccharide-grafted polyethylene glycol monomethyl ether (COS-g-MPEG) graft copolymer as an auxiliary material to prepare an anticancer drug.
  • COS-g-MPEG polyethylene glycol monomethyl ether
  • COS-g-MPEG is prepared by carbonyl diimidazole (CDI) coupling method.
  • carbonyl diimidazole is used to activate polyethylene glycol monomethyl ether (MPEG) to prepare activated MPEG, and then activated MPEG is reacted with chitooligosaccharide (COS) in an ionic liquid to synthesize a COS-g-MPEG copolymer, specifically
  • the reaction steps include the following three steps:
  • the activated MPEG and COS undergo graft polymerization in an ionic liquid.
  • the synthesis reaction equation is as follows:
  • Methylimidazole chlorobutane, toluene, polyethylene glycol monomethyl ether, carbonyldiimidazole, anhydrous ether, 4A molecular sieve (2-3mm), dimethylsulfoxide, 1,4-dioxane, shell Oligosaccharide, heat-collecting constant temperature magnetic heating stirrer (DF-101S), electronic balance, electric blast drying oven, circulating water vacuum pump, automatic triple pure water still, vacuum drying oven, freeze dryer, glass instrument airflow Dryer, single-phase capacitor starter motor, rotary vane vacuum pump, six-link magnetic heating stirrer, cellulose dialysis bag, ready-to-use dialysis bag 45-2000RC membrane, three-necked flask (500mL, 1000mL), glass stopper, magnet , Disposable paper cup, 500mL beaker, 2L beaker, disposable dropper, medicine spoon, reagent bottle, dryer, etc.
  • DFS constant temperature magnetic heating stirrer
  • the three-necked flask, glass stopper, petri dish, magnets, etc. were first washed with tap water, then rinsed with distilled water three times, and finally placed in a glass instrument air dryer to dry.
  • Solvent drying Take an appropriate amount of molecular sieve in a disposable beaker, take an appropriate amount of dimethyl sulfoxide, 1,4-dioxane, and anhydrous ether into the beaker and remove water.
  • the solution is added to a 1000 mL three-necked flask and placed in a water bath.
  • a vacuum distillation device is installed, and the distillation temperature is gradually increased from room temperature to 25 ° C, 30 ° C, 35 ° C, 40 ° C, and 45 ° C. , 50 ° C, 55 ° C, 60 ° C, distill to the remaining 50mL, remove the distillation device, pour the sample into a disposable beaker while it is hot, cover the mouth of the cup with disposable gloves, and put it in a refrigerator at -18 ° C to freeze a lot In 8 hours.
  • MPEG, PEG, PEI, etc. all have good water solubility, and have good compatibility with many organic components.
  • This embodiment uses MPEG as an example.
  • a graft of MPEG and a cationic stabilizer (such as chitosan) is formulated with PIC, and the compatibility is significantly increased.
  • a cationic stabilizer such as chitosan and PIC are grafted with PEG.
  • the compatibility will also increase; after grafting PEG on the cationic stabilizer, the graft itself will also have the characteristics of PEG.
  • TPP Buy sodium tripolyphosphate
  • the PIC-COS-g-MPEG-CaCl 2 complex is stirred at a constant speed on a constant temperature magnetic stirrer, and different concentrations of TPP aqueous solution are added dropwise. It was observed that the obvious opalescence stopped immediately, and the reaction was maintained for 30 minutes. Nanoparticles were formed by ion-crosslinking self-assembly and obtained by high-speed centrifugation, and the particle size was less than 1000 nm. And passed various tests.
  • each component enters the PEG-COS graft or the COS matrix, and the polypeptide or protein antigen enters the TPP-containing water phase to bind.
  • the components must be in a proper ratio and under a certain pH environment, the magnetic beads are stirred and combined to form composites and nanoparticles.
  • Scheme 2 Incubate the peptide or protein antigen with the pre-formed complexes and nanoparticles, so that the peptide or protein antigen is bound to the nano-complex and rice particle surface polypeptide, or the peptide or protein antigen and the complexes and nanoparticles are in a certain ratio Mix, magnetically stir for 5 minutes, set it at room temperature for 1 hour, and ultracentrifuge under a glycerol matrix at 20,000 rcf at 4 ° C for 2 hours.
  • the above-mentioned compound / graft-containing compound / complex nanoparticle / polypeptide or protein antigen nanoparticle is aseptically packed in a suitable / qualified packaging material, and prepared into various dosage forms such as injection, spray or aerosol. A variety of products are qualified to prepare products.
  • the compound / graft-containing compound / complex nanoparticle / polypeptide or protein antigen nanoparticle is aseptically packed in a suitable / qualified packaging material to prepare a paste.
  • Example of making a spray Pamica is prepared according to the method described above, and the solution is packed in a spray bottle. Twenty bottles are taken, and the spray mode detection and the droplet distribution data detection of the medicinal solution are respectively performed.
  • the spray mode is shown in the following table:
  • the form of the spray is evaluated by the ratio of the longest diameter to the shortest diameter (the closer to 1.0, the better the spray form).
  • the droplet distribution is shown in the following table:
  • Precipitation occurs after increasing the amount of COS in Pammica, which affects the uniformity of its administration. Precipitation can be avoided after adding PEI, and COS can be increased. Dosage, further enhance its immune effect.
  • CN105396130A patent application literature discloses a "dermal calcium adjuvant and a vaccine containing the same", and discloses that the non-antibiotic amino compound may be selected as chitosan.
  • water-soluble chitosan (chitosan hydrochloride, CS for short) was used to replace the chitosan oligosaccharide in Example 1 to compare the effects of the two on the uniformity of administration.
  • the 70-120 minutes (preferably 120 minutes) of PIC compound (Pamika) and its vaccine can pass the abnormal toxicity of mice and guinea pigs according to the "1141 Abnormal Toxicity Test Method" of the four sections of the Chinese Pharmacopoeia 2015 edition.
  • the PIC used for the preparation of Pamica should be heated at 90 ° C for at least 70 minutes before it can be used for product preparation.
  • the heating time at 90 ° C is 120 minutes.
  • the compounds and their vaccines prepared with unheated PIC (strip 2) and heated for 60 minutes (strip 9) failed to pass the guinea pig abnormal toxicity test.
  • the results are shown in Figure 2.
  • Control band 1 100bp, 300bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 3000bp, 5000bp from bottom to top.
  • Control band 8 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp from bottom to top, respectively.
  • Example 1 of the present disclosure The method of preparation of Example 1 of the present disclosure was used to prepare the pamika complex. After the preparation was completed, the pamika complex and polymyocyte injection (polymyocyte-kanamycin-calcium chloride) were sampled separately. Dilute to 0.04mg / ml, take 13 10ml tubes, add 5ml sample diluent to each tube, add 25 ⁇ g of sigma RNase (Cat. No. R4642) to each tube, place in 37 ° C water bath, and take out 1 tube every 5min Measure the OD value at 248nm and draw a curve.
  • polymyocyte injection polymyocyte-kanamycin-calcium chloride
  • Example 5 The pamika complex of the present disclosure is a new structure complex
  • the test measurement results show that the peak of the melting curve of the pamica complex (PIC-cationic stabilizer-calcium chloride) of the present disclosure is 85 ° C, and the melting of polymyocyte injection (PIC-kanamycin-calcium chloride) The peak value of the curve is 80 ° C, which indicates that the pamica complex of the present disclosure is a brand new complex.
  • PI solution, PC solution, PIC solution, PIC-COS solution, and PIC-COS-CaCl 2 solution were prepared, and the above samples were diluted to 0.04 mg / ml with PBS buffer solution, respectively, with ultraviolet light.
  • the scanning absorption spectrum was measured.
  • the results in Figure 5 show that the peaks appearing at 240-260nm are PI, PC, PIC, PIC-COS, PIC-COS-CaCl 2 in order from high to low, of which PIC-COS and PIC-COS-
  • Pamica was prepared according to the method of Example 1.
  • the cationic stabilizer was COS, and the metal cation was calcium chloride. It can be seen from the transmission electron microscope pictures ( Figures 6 and 7) that nanoparticles are formed in the Pamika solution, most of which are spherical, have a particle size of about 50 nm, are relatively uniform, and are individually square, with sides longer than 100 nm.
  • the pamica was prepared according to the method of Example 1.
  • the cation stabilizer was COS-g-MPEG, and the metal cation was calcium chloride. It can be seen from the transmission electron microscope pictures ( Figures 8 and 9) that nanoparticles are formed in the Pamika solution, most of which are square, the side length exceeds 100 nm, and a few are spherical.
  • the Pamica complex solution contains substances in two states at the same time, one is a nanoparticle (the result of an electron microscope), and the other is a non-nanoparticle (the electrophoresis of Example 3) Result) solution.
  • nanoparticles are that they can directly penetrate the cell membrane and enter the cell without going through endocytosis, and the effect is fast; non-nanoparticle solutions need to pass through the endocytosis to enter the cell, which is slower than the nanoparticles.
  • Pamika works by both endocytosis and direct access to cells.
  • the structure of the nanoparticles can protect palmika from the degradation of PIC by ribonuclease in primates and above, including human serum, in order to replace the breakthrough of antiviral and antitumor to obtain a larger effect.
  • the pamica refers to the pamica solution of the PIC, COS and calcium chloride solution prepared according to Example 1.
  • rHBsAg (CHO): 20ug / ml, pamika adjuvant: 1mg / ml, ADV20 adjuvant 400ug / ml, aluminum hydroxide adjuvant: 10mg / ml, physiological saline
  • Aluminum adjuvant / rHBsAg (CHO): aluminum adjuvant 0.07ml + rHBsAg (CHO) 0.5ml + normal saline 0.43ml
  • ADV20 cytokine adjuvant
  • rHBsAg CHO
  • Palmika adjuvant / rHBsAg (CHO): Palmika adjuvant 0.5ml + rHBsAg (CHO) 0.5ml
  • mice were immunized with 0.1ml of aluminum adjuvant / rHBsAg (CHO), ADV20 (cytokine adjuvant) / rHBsAg (CHO) and pamika / rHBsAg (CHO) on day 0 and day 14. Immunity and humoral immunity.
  • mice were immunized with PBS, polymyocyte injection + inactivated brucella antigen, pamika complex of the disclosure + inactivated brucella antigen, immunized once on day 0, and treated with brucella after 45 days.
  • the virulent strain was challenged to mice, and the spleen of the mice was isolated by killing the mice 15 days after the challenge, and the spleen was cultured for 3 days with Brucella, and counted to evaluate the Pamika complex + inactivated Brucella antigen. Protection effectiveness.
  • MYO antigen human myoglobin
  • Kits were produced by immunizing rabbits with complete Freund's adjuvant + antigen, pamika adjuvant + complete freund adjuvant + antigen, pamika adjuvant + antigen.
  • Clinically validated antibodies produced by complete Freund's adjuvant or complete Freund's adjuvant + pamika adjuvant antigen groups do not match clinical samples and cannot pass clinical tests. Only antibodies produced by pamika adjuvant antigen can pass Clinical tests will completely break the monopoly of the domestic human myoglobin latex turbidimetric kit by Dako in the Netherlands, which will solve the common immunogenicity of domestic products using Freund ’s adjuvant with low immunopotency and diverse epitopes. Insufficient sex to effectively detect the serious situation of clinical samples.
  • the following table is a comparison of the kits prepared by using pamicar adjuvant + antigen set to produce antibodies and the kits currently being used in clinical preparation by antibodies provided by Dako of the Netherlands.
  • mice were immunized with the pamika complex (1 mg / ml) + antigen, polymyocyte injection (1 mg / ml) + antigen, PBS + antigen and antigen respectively on day 0, challenged on day 14, 28 Efficacy was determined after several days.
  • a The pamika complex + CTN strain rabies vaccine antigen protection effect of the present disclosure is 3.6 times that of the pure CTN strain rabies vaccine antigen and saves 1/5 of the antigen;
  • the protective effect of the pamika complex + CTN strain rabies vaccine antigen of the present disclosure is 1.8 times that of Juji injection + CTN strain rabies vaccine antigen, and the effect is outstanding.
  • mice were immunized with the pamika complex and polymyocyte injection of the disclosure respectively, and the eyes of the mice were removed 1 hour, 2 hours, and 5 hours after the immunization, and the blood was removed into sterile 2ml centrifuge tubes at room temperature. Let stand for 30 minutes, centrifuge at 3500 rpm for 5 minutes, draw the supernatant into a new centrifuge tube, and freeze the serum at -20 ° C.
  • TNF- ⁇ can kill and inhibit tumor cells, promote neutrophil phagocytosis, and resist infection. It is a type of cytokine that can directly cause tumor cell death. IFN- ⁇ can induce cells to resist virus infection. Preventing or limiting viral infection by interfering with viral gene transcription or translation of viral protein components is currently the most important anti-virus infection and anti-tumor cytokine.
  • TNF- ⁇ and IFN- ⁇ cytokines produced by pamika-induced mice are higher than those of polymyocyte injection, indicating that the TNF- ⁇ and IFN- ⁇ produced by the pamika complex disclosed in the present disclosure synergistically kill Tumor cells and resistance to infection are even more powerful.
  • Sample injection 18 to 22 grams of healthy SPF Kunming mice, 5 per sample, 0.5 ml per intraperitoneal injection, and 5 healthy mice with 18 to 22 g were weighed as a blank control.
  • mice were injected intraperitoneally with 0.5 ml of the test article and observed for 7 days. During the observation period, all the mice should survive, and there should be no abnormal reactions. At the expiration, the weight of each mouse should be increased, and the test product was judged as qualified. If the above requirements are not met, 10 mice can be used for retesting, and the determination criteria are the same as before.
  • Sample injection 250 to 350 g of healthy SPF Hartely guinea pigs, 2 per sample, 5 ml per head intraperitoneally, and 2 healthy guinea pigs of 250 to 350 g were weighed as a blank control.
  • Each guinea pig was injected intraperitoneally with 5 ml of the test article and observed for 7 days. During the observation period, all guinea pigs should survive and there should be no abnormal reactions. At the expiration, the weight of each guinea pig should be increased, and the test product is qualified. If the above requirements are not met, the test can be repeated with 4 guinea pigs.
  • Sample injection 250 to 350 g of healthy SPF Hartely guinea pigs, 2 per sample, 5 ml per head intraperitoneally, and 2 healthy guinea pigs of 250 to 350 g were weighed as a blank control.
  • Each guinea pig was injected intraperitoneally with 5 ml of the test article and observed for 7 days. During the observation period, all guinea pigs should survive and there should be no abnormal reactions. At the expiration, the weight of each guinea pig should be increased, and the test product is qualified. If the above requirements are not met, the test can be repeated with 4 guinea pigs.
  • Sample injection 250 to 350 g of healthy SPF Hartely guinea pigs, 2 per sample, 5 ml per head intraperitoneally, and 2 healthy guinea pigs of 250 to 350 g were weighed as a blank control.
  • Each guinea pig was injected intraperitoneally with 5 ml of the test article and observed for 7 days. During the observation period, all guinea pigs should survive and there should be no abnormal reactions. At the expiration, the weight of each guinea pig should be increased, and the test product is qualified. If the above requirements are not met, the test can be repeated with 4 guinea pigs.
  • Hepatitis B vaccine antigen yeast expression (not limited to yeast expression, recombinant CHO engineered cells can also be used to express hepatitis B antigen).
  • PIC double-stranded nucleic acid
  • this product can also be administered by nasal spray, which has a significant effect on metastatic cancer.
  • the pamica complex of the present disclosure After the production of the pamica complex of the present disclosure is completed, it shall be stored in a room protected from light, and the samples shall be tested every 6 months.
  • endotoxin In tap water, the amount of endotoxin in tap water is 1 to 100 EU / ml.
  • endotoxin When endotoxin enters the human body through the digestive tract, it does not cause harm, but endotoxin can cause different diseases when it enters the blood by injection. After a small amount of endotoxin enters the blood, it is inactivated by liver Kupffer cells without causing body damage. Endotoxin enters the blood in large quantities and causes a fever reaction "pyrogenic reaction".
  • Dropper Aseptically aspirate the abdominal cavity wash solution from a test tube, drop it on a glass slide, place the dropper horizontally on a wet gauze, and incubate in a 37 ° C incubator for half an hour. At this time, a large number of macrophages adhere. Attach to the slide, rinse the red blood cells and other tissue cells on the slide without phagocytosis with 0.85% physiological saline, and blow dry with cold air.
  • Percentage of phagocytosis total number of macrophages phagocytosing red blood cells ⁇ total number of macrophages ⁇ 100%.
  • Phagocytic index total number of macrophages phagocytosing red blood cells ⁇ total number of macrophages phagocytosing red blood cells ⁇ 100% The value is the phagocytic index).
  • results In the PBS control group, the percentage of phagocytosis was 12%, and the phagocytic index was 0.11. The percentage of phagocytosis in the Pamika group was 66%, and the phagocytic index was 1.2, indicating that Pamika has a strong stimulating effect on phagocytosis of macrophages. Red blood cells are phagocytosed, Figure 17 Blue macrophages (indicated by arrows) do not phagocytize red blood cells.
  • Influenza virus Mice lung adaptive strain FM1, purchased from Institute of Viral Disease Control, Chinese Academy of Preventive Medicine.
  • Ribavirin a positive control drug, purchased from Shenyang Yanfeng Pharmaceutical Factory.
  • mice Kunming species, 8-10g for FM1 virus passage, 14-20g for the following formal experiments.
  • Influenza virus Mice lung adaptive strain FM1, purchased from Institute of Viral Disease Control, Chinese Academy of Preventive Medicine.
  • Influenza vaccine Influenza virus split vaccine Hualan Biological Products Co., Ltd.
  • Mucosal immune adjuvant of the present disclosure Xinfu (Beijing) Medical Technology Co., Ltd.
  • mice Kunming species, 8-10g for FM1 virus passage, 14-20g for the following formal experiments.
  • Complete Freund's adjuvant influenza vaccine In a centrifuge tube, add an equal volume of vaccine and complete Freund's adjuvant as a water-in-oil emulsion in a vortex.
  • the nasal drops of the combined influenza vaccine of the present disclosure are mixed in equal amounts to form a water solvent.
  • Influenza vaccine The same amount of influenza vaccine is mixed with PBS to form a water solvent.
  • Immunization by subcutaneous injection Immunized mice were injected subcutaneously on days 0 and 28, 0.1ml / head, and on day 42, some mice were drawn to isolate serum and test antibody titers, and some mice were suspended from the lungs of influenza virus FM1 strain. Liquid 5LD 50 / nasal infection, on the 5th day after infection, the lung tissue virus titer was measured.
  • Nasal immunization Immune mice on the 0th and 28th days of the nasal drip method, 0.1ml / mouse, on day 42. Some mice draw blood to isolate serum and test antibody titers. Other mice are suspended by the influenza virus FM1 virus Liquid 5LD 50 / nasal infection, on the 5th day after infection, the lung tissue virus titer was measured.
  • Complete Freund's adjuvant is the gold standard for testing to promote cellular immunity in the body.
  • the test results show that subcutaneous immunization of the disclosed mucosal immune preparations combined with influenza vaccine produces antibodies that are 10 times lower than full Freund's adjuvant influenza vaccine, but more complete Freund's adjuvant influenza
  • the vaccine reduced the influenza virus titer by 31.6 times; especially through nasal mucosal immunization of mice, it was shown that the nasal drops of the disclosed mucosal immune preparation combined with the antigen were 31.6 times higher than the simple influenza vaccine antibody, and the influenza virus titer was reduced by more than 3100 times. Extremely significant effect.
  • Target cells A549 effector cells: culture JSCIK2016042614
  • the comprehensive analysis of the killing ability of JSCIK2016042614 cells is medium (when the target effect ratio is 1:10, the killing rate of target cell A549 (51.4%).
  • the LL2 mouse transplanted tumor model was used to test the antitumor effect of Pamica through nasal spray.
  • the model tumor cells grew rapidly.
  • the tumor volume had reached 2201.9 ⁇ 68.01mm 14 days after inoculation.
  • the experiment was over.
  • the reduction was the best, followed by the Pammika intramuscular injection group, but the Pamika nasal spray except 0.1mg / mouse.
  • the remaining 0.2 mg / only nasal spray group and the vehicle negative control group had a ratio P ⁇ 0.0001, which had significant differences.
  • mouse PD1 has little effect. What needs to be explained is that the cisplatin group is more likely to show its efficacy in this model of very rapid cell division.
  • the effect of such a tumor-bearing mouse model under the new anti-cancer mechanism of Pammica is compelling. In addition, no adverse effects on mice were found in this model.
  • the tumor formation rate of the cells was high, and the tumor in the vehicle control group grew rapidly.
  • the tumor volume reached 2201.09 ⁇ 68.01mm 3.
  • the positive control group cisplatin showed a significant tumor suppressive effect, indicating that the experiment was successful. The results are credible.
  • test results show that in this study of mouse lung cancer LL2 cell C57BL / 6 mouse transplanted tumor model, except for pamika, 150 ⁇ g / mouse group, the other groups showed obvious nasal or intramuscular administration.
  • the statistical effect of the inhibitory effect on tumor growth was very significant, P ⁇ 0.0001; meanwhile, it had no obvious toxic and side effects on tumor-bearing mice.
  • This pharmacodynamic experiment consists of a total of 9 groups: the vehicle group, the PD1 group, the 6 pamika treatment groups, and the combined administration of pamika and PD1.
  • the vehicle group was intranasally administered with PBS solution 66.7 ⁇ L / head 16 days after inoculation and was administered once every two days;
  • the PD1 group was intraperitoneally injected with 100 ⁇ g PD1 solution per day 16 days after inoculation and administered once a week;
  • the mika treatment group was: nasal administration of 200 ⁇ g / m of pamika once a day before inoculation, once every two days; administration of 200 ⁇ g / m of pamika once a day on inoculation, once every two days; 16 after inoculation Intranasal administration of 200 ⁇ g / m of pamika every day, once every two days; Intranasal administration of 300 ⁇ g / m of pamika once every two days after in
  • the experiment used female Balb / c mice, tumor volume was measured every three days, and body weight was measured every two days. The experiment was terminated when the average tumor volume of the vehicle control group exceeded 2000 mm 3 , and the bioluminescence of the tumor was measured by a small animal live imager. Finally, the organs of each group of mice were stained with HE.
  • the results of pharmacodynamic tests showed that the ability to inhibit tumor growth and metastasis was weaker in the 7-day and 0-day groups, and the inhibitory effect of the two pamika muscle injection groups on tumor growth and metastasis was stronger than that of the two pamika nose drops
  • the 300 ⁇ g / pamika nose drop group, the 200 ⁇ g / pamika muscle injection group, and the 300 ⁇ g / pamika muscle injection group can significantly inhibit tumor growth and Transfer.
  • the tumor inhibition rates of the 200 ⁇ g / pamika nose drop group and the 300 ⁇ g / pamika nose drop group were 25% and 35%, respectively.
  • the tumor inhibition rates of the 200 ⁇ g / pammikam injection group and the 300 ⁇ g / pamika injection group were 56% and 61%, respectively.
  • mice 7 days before the vaccination group (7 days in advance), 10 mice were randomly selected 7 days before tumor implantation, and administration was started according to Table 1.
  • mice On the 0th day of vaccination, the pamika nasal administration group (day 0 group for short), 10 mice were randomly selected on the day of seeding tumor, and administration was started according to Table 1.
  • the tumor volume grew to about 80 mm 3 , that is, on the 16th day after tumor implantation, administration was started according to Table 1.
  • the tumor volume grew to about 80 mm 3 , that is, on the 16th day after tumor implantation, administration was started according to Table 1.
  • 200 ⁇ g / Pamika nasal drip group the tumor volume grew to about 80 mm 3 , that is, on the 16th day after tumor implantation, administration was started according to Table 1;
  • 300 ⁇ g / Pamika nasal drip group the tumor volume grew to about 80 mm 3 , that is, on the 16th day after tumor implantation, administration was started according to Table 1;
  • 300 ⁇ g / Pamika intramuscular injection group the tumor volume grew to about 80 mm 3 , that is, on the 16th day after tumor implantation, administration was started according to Table 1.
  • the entire experiment was terminated on the 30th day after the administration because the tumor volume exceeded 2000 mm 3 .
  • Tumor volume was measured every three days, and body weight was weighed every two days.
  • the heart, liver, spleen, lung, kidney, and tumor were stripped.
  • the heart, liver, spleen, and kidney were fixed with 4% neutral formaldehyde, and paraffin sections were used for HE staining analysis.
  • the tumor was photographed and weighed; the lung was used for Bouin's fixing solution was fixed for 16 hours, then dipped in 50% alcohol for 2 hours, and then photographed. After fixed with 4% neutral formaldehyde, paraffin sections were analyzed by HE staining.
  • Luciferin a luciferin substrate at a concentration of 30 mg / mL
  • the image acquisition parameters are: acquisition time, 0.2 seconds; Bin value is 4; F value is 2.
  • Image processing software Living software (version 4.3.1; Caliper Life Sciences Inc.).
  • the experimental data are expressed as "mean ⁇ standard deviation", and the data are analyzed using SPSS Statistics 19 (version 4.0.100.1124; SPSS Inc., IBM Company, USA) software. Data were compared using ANOVA with one-way analysis of variance. Significant differences between groups were tested using t-tests: * p ⁇ 0.05; ** p ⁇ 0.01; *** p ⁇ 0.001.
  • the tumor volume change curve is shown in FIG. 22.
  • the Pamica 7-day advance group and the 0-day group had a certain tumor suppressive effect in the later stage, and there was almost no tumor suppressive effect in the early stage, indicating that there was no obvious tumor-killing effect in the early administration or immediate administration.
  • An oncology vaccine not a preventative vaccine.
  • the tumor suppression rates of the 7-day advance group and the 0-day group were 36% and 26%, respectively.
  • the tumor volume of the PD1 group on the 12th day was significantly different from that of the vehicle group (** p ⁇ 0.01), indicating that PD1 has a certain inhibitory effect on breast cancer in 4T1 mice.
  • mice in the treatment groups of the PD1 and pamika combination administration group died 20 days after administration, so no data was available for the two groups in the later measurement.
  • the tumor volume of the combined administration group on day 6, 12, 18 was significantly different from that of the vehicle group (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, respectively).
  • the inhibitory effect of the 200 ⁇ g / Pamika nasal drops group was more obvious in the early stage of administration, and the tumor suppressive effect gradually weakened in the later stage of administration.
  • the tumor volumes on the 6th and 12th days were significantly different from the vehicle group ( ** p ⁇ 0.01). Except for the 18th day in the 300 ⁇ g / mica pamika nose drops group, the tumor volume at the other time was significantly different from that in the vehicle group. This shows that nasal administration is effective and the tumor suppressive effect is dose-dependent.
  • the tumor inhibition rates of the 200 ⁇ g / pamika nose drop group and the 300 ⁇ g / pamika nose drop group were 25% and 35%, respectively.
  • the intratumoral inhibitory effect of the two intramuscular injection groups of Pamika was similar, and there was a significant difference compared with the vehicle group, and the antitumor effect of the intramuscular injection group was better than that of the two drops nose group.
  • the tumor suppression rates of the 200 ⁇ g / pammikam injection group and the 300 ⁇ g / pamika injection group were 56% and 61%, respectively.
  • mice The weight change curve of mice is shown in FIG. 23.
  • the body weight of the mice in the PD1 and co-administration groups had no significant difference compared with the vehicle group within the effective measurement time, suggesting that their side effects are small.
  • the weight of the 7-day advance group, the 0-day group, the 200 ⁇ g / pamika nose drop group and the 300 ⁇ g / pamika nose drop group was significantly lower than that of the vehicle group at the rest of the time except for the late administration period. There was no significant difference between the 200 ⁇ g / pamica intramuscular injection group and the vehicle group during the entire administration period.
  • the body weight of the 300 ⁇ g / mikamika injection group was lower than that of the vehicle group in the middle stage of administration, and there was no significant difference between the early and late stage of administration compared with the vehicle group.
  • the tumor weight of the 7-day advance group and the 0-day group was not significantly different from that of the control group.
  • the 300 ⁇ g / pamika intranasal group, the 200 ⁇ g / pamika intramuscular injection group, and the 300 ⁇ g / pamika intramuscular injection group were all significantly effective. Inhibition of tumor growth was statistically different from the vehicle group (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001, respectively).
  • the spleen is the body's largest immune organ, accounting for 25% of total lymphoid tissue in the body. It contains a large number of lymphocytes and macrophages, and is the center of cellular and humoral immunity. It can be seen from FIG. 26 that the spleen weight of the 200 ⁇ g / Pamika intramuscular injection group and the 300 ⁇ g / Pamika intramuscular injection group were significantly higher than those of the vehicle group, with statistical differences (respectively ** p ⁇ 0.01, * p ⁇ 0.05), indicating that the immune response in the intramuscular injection group may be stronger.
  • the bioluminescence intensity of tumor sites and metastases was strong in the vehicle group, the 7-day advance group, and the 0-day group.
  • the bioluminescence intensity of the site and metastases was weakened, and the bioluminescence intensity of tumor sites and metastases was the weakest in the 200 ⁇ g / pamika intramuscular injection group and the 300 ⁇ g / pamika intramuscular injection group, indicating that the intramuscular injection group inhibited 4T1 breast cancer growth and metastasis The ability is stronger than the nose drops group, which is consistent with the above results.
  • the test results show that in this study of a mouse breast cancer 4T1-luc cell Balb / c mouse orthotopic tumor model, except PD1 and the combined administration group, due to PD1, a large area of mouse death occurred. Except only part of the experimental data, the remaining groups have a certain tumor suppressive effect at a certain time.
  • the antitumor effect was not obvious in the 7-day advance group, the 0-day group, and the 200 ⁇ g / pamika nose drop group, and the 200 ⁇ g / pamika nose drop group, 200 ⁇ g / pamika intramuscular injection group, and 300 ⁇ g / pamika
  • the card muscle injection group showed obvious antitumor effect.
  • PBS In the PBS group, a few days after the tumor was implanted, PBS was administered nasally, 100 ⁇ L / head, once every other day;
  • PD1 group A few days after tumor implantation, PD1 was administered intraperitoneally, 100 ⁇ g / head, once a week;
  • 3Paclitaxel group a few days after tumor implantation, paclitaxel was administered by tail vein injection, 10 mg / kg, once a week;
  • paclitaxel was injected intravenously (10 mg / kg, administered once a week) + pamika nose drops (100 ⁇ L / head, administered once every other day).
  • An in situ 4T1-luc breast cancer model was established, and early detection grouping was performed with a small animal live imager, and the drug was administered according to the group setting and dosing schedule under 6.1.
  • the tumor volume (measured once every 3 days), weight change (measured once every 3 days), tumor weight (end point detection), spleen weight (end point detection), TUNEL staining (end point detection), lungs were fixed with Bouin's fixative and photographed H & E staining (endpoint detection) was performed after neutral formaldehyde was fixed in each tissue and organ, and the bioluminescence intensity of tumors and metastatic sites in situ at different time points and final time points were observed with a small animal live imager. The effects of inhibiting the growth and metastasis of orthotopic tumors were compared between the groups.
  • pamika has significant effects in reducing breast cancer tumor volume, controlling tumor weight, spleen weight, and promoting tumor cell apoptosis.
  • the compound of the present disclosure has moderate viscosity and molecular mass, convenient pharmacy, stable chemical properties, not easily degraded during long-term storage, and safe to use.
  • the compound alone can significantly enhance the body's non-specific immune response and achieve the purpose of preventing and treating diseases.
  • the combination of drugs has better anti-tumor, anti-viral and anti- (super) bacterial effects, and is easily absorbed by patients.

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Abstract

一种新颖的复合物，并对该复合物的制备、应用等方面进行研究。所述复合物的制备方法包括：将聚肌胞、至少一种阳离子稳定剂以及可溶性钙盐在液体反应体系中接触；所述阳离子稳定剂为分子量≤5kDa的水溶性非抗生素氨基化合物，或所述水溶性非抗生素氨基化合物与聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺、叶酸、半乳糖中的一种或多种所形成的接枝物。该复合物黏度和分子质量适中，制药方便，化学性质稳定，长期储存不易降解，使用安全，该复合物单独使用即可显著地增强机体非特异性的免疫反应，达到防治疾病的目的，与其他药物联合使用具有更好的抗肿瘤、抗病毒和抗(超级)细菌功效，易被患者所吸收。

Description

用于增强免疫响应的复合物的制备方法
相关申请的交叉引用
本申请要求于2018年06月29日提交中国专利局的申请号为201810698033.6、名称为“用于增强免疫响应的复合物的制备方法”的中国专利申请,以及于2018年06月29日提交中国专利局的申请号为201810700708.6、名称为“用于增强免疫响应的复合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本公开涉及生物医药领域，具体而言，涉及一种用于增强免疫响应的复合物的制备方法。
背景技术
双链RNA(dsRNA)佐剂目前一般认为包括PIC(polyriboinosinic-polyribocytoidylic acid)、PICLC(PIC with poly-L-lysine and carboxymethylcellulose)、PIC 12U(PIC with uridylic acid in specific interval，商品名Ampligen)和PICKCa((PIC-kanamycin-CaCl 2)，是多种膜式识别受体(PRRs)的配体，一方面增强免疫反应，另一方面通过改变免疫类型，有可能使预防性疫苗成为治疗性疫苗。
PIC(聚肌胞)是上世纪60年代由美国默克公司研制的。在小白鼠中，PIC是IFN-α诱生剂，有抗病毒活性。PIC可以保护小白鼠鼻腔和肺部致死性的感染。然而由于灵长类动物和人血清核酸酶对PIC的降解，减低PIC结构的稳定性，只产生很少IFN-α，也不具有抗肿瘤活性。
PICLC(聚肌胞+赖氨酸+羧甲基纤维素)是Levy HB等上世纪70年代研制的，即PIC与聚赖氨酸(PolyL-lysine，相对分子质量27 000)和羧甲基纤维素(CMC，相对分子质量700 000)的结合物，使相对分子质量增大了许多，较PIC能5-10倍抵抗灵长目动物核酸酶水解，并在猴体内产生显著的干扰素(15)。PICLC初步的临床研究表明治疗剂量就引起中到重度反应，发烧(100％)，肌痛(50％)，低血压(50％)，白细胞明显下降等。这造成分子量大毒性大的认识误区。
PIC 12U是上世纪70年代中期在美国约翰霍普金斯大学研制的，是在PIC链一定位置插入尿嘧啶核苷酸。效力类似PIC，但毒性较少。2012年8月Hemispherx生物药品公司呈送了进一步原始的临床研究资料，但还是以安全性和有效性资料不足没有被美国食品药品管理局(FDA)批准。
PICKCa含有抗生素卡那霉素，卡那霉素具有中有耳毒性，并且在疫苗中含量超过国家药典规定的标准。
可见，单纯PIC不能用于灵长类以上动物包括人，PIC 12U则因为效果差没有被美国FDA批准，而PICLC实际上也具有很强的副作用。
有鉴于此，特提出本公开。
发明内容
本公开涉及一种新颖的复合物，并对该复合物的制备、应用等方面进行研究。
在发明人之前的工作中，采用PIC、卡那霉素、氯化钙配制疫苗佐剂(商品名叫皮卡佐剂或PIKA佐剂)。使用卡那霉素是缘于其含有4个氨基与PIC中磷酸基结合稳定其结构，但该商品却因含有抗生素而限制了在疫苗中的应用。
进一步的，发明人发现使用壳聚糖(盐酸盐)替换卡那霉素也同样可以起到阳离子稳定剂的作用，然而壳聚糖(盐酸盐)分子量大，不易被人体所吸收，因而难以获得理想的药效。
因此，本公开提供一种用于增强免疫响应的复合物。该复合物至少由以下成分在适宜条件下制备得到：聚肌胞、至少一种阳离子稳定剂以及可溶性钙盐。
该复合物至少由以下成分在适宜条件下制备得到：聚肌胞、至少一种阳离子稳定剂以及可溶性钙盐或/及锰盐。
其中，所述阳离子稳定剂为分子量≤5kDa的水溶性非抗生素氨基化合物，或所述水溶性非抗生素氨基化合物与聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺、叶酸、半乳糖中的一种或多种所形成的接枝物。
与现有技术相比，该复合物黏度和分子质量适中，制药方便，化学性质稳定，长期储存不易降解，使用安全，该化合物单独使用即可显著地增强机体非特异性的免疫反应，达到防治疾病的目的，与其他药物联合使用具有更好的抗肿瘤、抗病毒和抗(超级)细菌功效，易被患者所吸收。
附图说明
为了更清楚地说明本公开具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本公开的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为帕米卡复合物结构示意图；A：PolyI:C-COS-Ca 2+的复合物结构示意图；B：抗原(Ag)+复合物粒子的结构示意图；C：PolyI:C-COS-Ca 2++Ag的复合物结构示意图；
图2为PIC加热不同时间后的分子量电泳图；
图3为本公开一个实施例中帕米卡复合物的酶降解曲线；
图4为本公开一个实施例中帕米卡复合物的融化曲线；
图5为本公开一个实施例中在240-260nm处各物质的扫描吸收光谱；
图6为本公开一个实施例中PIC-COS-CaCl 2复合物的透射电镜图；scale bar＝500nm；
图7为本公开一个实施例中PIC-COS-CaCl 2复合物的透射电镜图；scale bar＝200nm；
图8为本公开一个实施例中PIC-COS-g-MPEG-CaCl 2复合物的透射电镜图；scale bar＝200nm；
图9为本公开一个实施例中PIC-COS-g-MPEG-CaCl 2复合物的透射电镜图；scale bar＝100nm；
图10为本公开一个实施例中PIC-COS-CaCl 2复合物与TPP形成的纳米粒子的透射电镜图；scale bar＝1000nm；
图11为本公开一个实施例中PIC-COS-CaCl 2复合物与TPP形成的纳米粒子的透射电镜图；scale bar＝200nm；
图12为本公开一个实施例中分别用铝佐剂/rHBsAg(CHO)、ADV20/rHBsAg(CHO)、帕米卡/rHBsAg(CHO)免疫小白鼠21天后，Elisa法检测小鼠血清中IgG抗体检测图；
图13为本公开一个实施例中分别用铝佐剂/rHBsAg(CHO)、ADV20/rHBsAg(CHO)、帕米卡/rHBsAg(CHO)免疫小白鼠21天后的体液免疫情况；纵坐标为脚垫肿胀：增加的毫米数(footpad swealling：mm increase)；
图14为本公开一个实施例中帕米卡复合物与完全弗氏佐剂在MYO抗原对应抗体制备中的效果比较；
图15为本公开一个实施例中帕米卡复合物与完全弗氏佐剂在MYO抗原对应抗体制备中的效果比较；
图16为本公开一个实施例中帕米卡组刺激巨噬细胞吞噬功能的实验示例图片；
图17为本公开一个实施例中PBS对照组无刺激巨噬细胞吞噬功能的实验示例图片；
图18～21为本公开一个实施例中帕米卡粘膜免疫制剂对荷瘤小白鼠LL2肺癌模型的抗癌作用实验结果；
图18：不同药物处理下肿瘤体积变化曲线；
图19：不同药物处理下肿瘤重量(给药后14天溶媒对照组的肿瘤体积达到2201.09±68.01mm3，在给药后的第14天结束实验)；
图20：A：溶媒对照，滴鼻，两天一次；B：顺铂(5mg/kg)，尾静脉注射，一周一次；C：Pamica-1(即帕米卡)，200μg/鼠，滴鼻，两天一次；D：Pamica-1，200μg/鼠，滴鼻(接种后随即给药)，两天一次；E：Pamica-1，150μg/鼠，滴鼻，两天一次；F：Pamica-1，200μg/鼠，肌肉注射，两天一次；G：Pamica-1+顺铂，200μg/鼠+5mg/kg，滴鼻+尾静脉注射，两天一次+一周一次；scale bar＝1cm；
图21：鼠源性的PD-1抗体的抑瘤率；
图22～35为本公开一个实施例中帕米卡对4T1-luc小鼠原位乳腺癌模型的体内抗肿瘤效果；
图22：肿瘤体积变化曲线；
图23：小鼠体重变化曲线；
图24：不同药物处理对瘤重的影响；
图25：不同药物影响下的肿瘤照片；
图26：不同药物处理对脾重的影响；
图27：不同药物影响下的肺脏正面照片；
图28：不同药物影响下的肺脏反面照片；
图29～35：小动物成像仪显示的瘤部位和转移灶生物发光强度的照片；
图29：帕米卡提前7天组小鼠生物发光；
图30：帕米卡第0天组小鼠生物发光；
图31：溶媒组小鼠生物发光；
图32：200μg/只帕米卡滴鼻组小鼠生物发光；
图33：300μg/只帕米卡滴鼻组小鼠生物发光；
图34：200μg/只帕米卡肌注组小鼠生物发光；
图35：300μg/只帕米卡肌注组小鼠生物发光。
具体实施方式
本公开可通过后续对于本公开一些实施方案描述以及其中所包括的实施例的详细内容而更容易被了解。
在进一步叙述本公开之前，应明了本公开不会被局限于所述特定实施方案中，因为这些实施方案必然是多样的。亦应明了本说明书中所使用的用语仅是为了阐述特定实施方案，而非作为限制，因为本公开的范围将会被仅仅界定在所附的权利要求中。
名词定义
在陈述本公开的详细内容之前，应当了解被使用于本说明书中的数个用语。
“帕米卡”此用语是一般性地指称由聚肌胞、阳离子稳定剂以及可溶性钙盐(钙离子)制备得到的复合物，而和该复合物的特定物理和免疫原性无关。
“聚肌胞”亦称聚肌胞苷酸、聚肌苷酸聚胞苷酸、聚肌苷酸胞嘧啶核苷酸、聚肌苷酸-聚胞苷酸、PIC或PolyI:C。
本说明书中所使用的“增强免疫响应”此用语是指诱导或增强宿主对抗原物质的免疫反应，或者增强免疫细胞的功能，或促进免疫细胞释放炎症因子或细胞因子，或提高宿主对致病物质的抵抗力。
“诱导免疫反应”此用语是指刺激、起始或诱导一个免疫反应。
“增强(potentiating)免疫反应”是指一个既存的免疫反应被改善、助长、补充、扩增、促进、增加或延长。
“增强免疫反应”此表达方式或类似表达方式的意思是，相较于先前的免疫反应状态，免疫反应被提高、改善或上升，而对宿主有利，所述先前的免疫反应状态例如给予本公开的免疫原组合物之前的免疫反应状态。
“个体”此用语在此和“宿主”、“主体”和“动物”互换使用，包括人类及所有畜养(如家畜和宠物)和野生的动物及禽鸟，其非限制性地包括牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、骆驼、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡等。
“抗体”此用语包括多克隆抗体及单克隆抗体以及这些抗体的抗原化合物结合片段，包括Fab、F(ab’) 2、Fd、Fv、scFv、双特异抗体和抗体最小识别单位，以及这些抗体和片段的单链衍生物。抗体的类型可以选择IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD。此外，“抗体”此用语包括天然发生的抗体以及非天然发生的抗体，包括例如嵌合型(chimeric)、双功能型(bifunctional)和人源化(humanized)抗体，以及相关的合成异构形式(isoforms)。“抗体”此用语可和“免疫球蛋白”互换使用。
如本说明书中所使用，“抗原化合物”此用语是指可在适当情形下被免疫系统所辨识(例如结合至抗体或被加工，以诱导细胞免疫反应)的任何物质。
如本说明书中所使用，“抗原”包括但不限于细胞、细胞提取物、蛋白质、脂蛋白、糖蛋白、核蛋 白、多肽、肽、多糖、多糖缀合物、多糖的肽仿真体、脂肪、糖脂质、糖类、病毒、病毒提取物、细菌、细菌提取物、真菌、真菌提取物、多细胞生物例如寄生虫、以及过敏原。抗原可为外源性(例如来自于被给予该抗原的个体以外的其它来源，例如来自于一个不同的物种)或是内源性(例如源自于宿主体内，例如身体的疾病因子、癌抗原、病毒感染细胞所产生的抗原等等)。抗原可以是天然型(例如天然产生的)、合成型或重组型。抗原包含细胞提取物、完整细胞和纯化抗原，其中“纯化”此用语是指该抗原呈现相较于抗原通常存在的环境和/或相较于粗提物(例如抗原的培养形式)更为丰富的形式。
本说明书中所使用的“疫苗组合物”此用语是指由二或更多种物质(例如抗原和佐剂)所构成的组合，当将这些物质给予宿主时会共同激发免疫反应。
“多肽”、“肽”“寡肽”和“蛋白”等用语在本说明书中可互换使用，它们的意思是任何长度氨基酸聚合物形式，该聚合物形式可包括编码和非编码性氨基酸、经化学或生化修饰或衍生的氨基酸以及具有修饰肽主链的多肽。
“免疫反应”此用语是指脊椎动物个体的免疫系统对于抗原性或免疫原性化合物的任何反应。典型的免疫反应包括但不限于局部及系统性细胞及体液免疫反应，例如包括CD8+CTLs的抗原特异性诱导作用在内的细胞毒性T淋巴细胞(CTL)反应、包括T-细胞增殖反应和细胞因子释出作用在内的辅助T-细胞反应，以及包括抗体反应在内的B-细胞免疫反应。
使用于此处的“佐剂”此用语是指增加或改变宿主对于抗原化合物的免疫反应的任何物质或物质混合物。
本说明书中所使用的“治疗”此用语是一般性地指称获得所需药理和/或生理效应。该效应从完全地和/或部分地防止疾病或其症状的角度来看可以是属于预防性质的，和/或该效应从完全地和/或部分地稳定或治愈疾病和/或疾病所导致的负面效果来看可以是属于医疗性质的。本说明书中所使用的“治疗”此用语涵盖对于个体(特别是哺乳动物个体，更特别是人类)体内的疾病的任何处理，且包括：(a)预防可能有罹病倾向但尚未诊断出罹病的个体发生疾病或症状；(b)遏制疾病症状，例如阻止该疾病症状的发展；或是舒缓疾病症状，例如致使该疾病或症状消退；(c)降低疾病传染物质所产生的产物水平(例如毒素、抗原等)；(d)降低对于疾病传染物质的不利生理反应(例如发烧、组织水肿等)。
“药学上可接受的盐”的化学物意指该盐是药用上可接受的，且拥有母化合物的所需药理活性。这些盐包括：(1)合成盐的酸，例如盐酸、氢溴酸、硫酸、硝酸、磷酸等无机酸共同形成盐；或是和例如乙酸、丙酸、己酸、环戊丙酸、羟乙酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、3-(4-羟基苯酰基)苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1，2-乙二磺酸、2-羟基乙磺酸、苯磺酸、4-氯苯磺酸、2-萘磺酸、4-甲苯磺酸、樟脑磺酸、葡庚糖酸、4，4′-亚甲基双-(3-羟基-2-烯-1-羧酸)、3-苯基丙酸、三甲基乙酸、三级丁基乳酸、月桂基硫酸、葡萄糖酸、谷氨酸、羟基萘甲酸、水杨酸、硬脂酸、粘康酸等有机酸等共同形成盐；或是(2)当母化合物中所存在的酸性质子被如碱金族金属离子、碱土族金属离子或铝离子等金属离子所置换，或是配位有如乙醇胺、二乙醇胺、三乙醇胺、氨基丁三醇(tromethamine)、甲基葡萄糖胺(N-methylglucamine)等有机碱时，所形成的盐。
本公开的示例性实施方案
本公开一方面涉及一种用于增强免疫响应的组合产品，其包括聚肌胞、至少一种阳离子稳定剂以及可溶性钙盐；
所述阳离子稳定剂为分子量≤5kDa的水溶性非抗生素氨基化合物，或所述水溶性非抗生素氨基化合物与聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺、叶酸、半乳糖中的一种或多种所形成的接枝物。
一个重要优点在于帕米卡单独使用即可增强机体非特异免疫反应，更可有效地引起特异体液免疫反应和细胞免疫反应，从而增进保护性免疫力；和抗原物质联合使用可达到更佳的效果。
一个重要优点在于帕米卡能通过《中国药典》2015版四部“1141异常毒性的检查法”，能够安全应用于人体。未加热处理分子量的PIC配制的复合物(如皮氨钙佐剂或皮氨钙佐剂疫苗)不能通过异常毒性的检测。
一个重要优点在于帕米卡具有更佳的化学和/或物理稳定性，从而更便于保存。
一个重要优点在于帕米卡可以通过信号传导通路促进肿瘤细胞凋亡，也可以刺激免疫细胞表达多种 细胞因子并改变肿瘤细胞微环境，让免疫细胞攻击肿瘤细胞、病毒、细菌等病原物质。
一个重要优点在于帕米卡更易于被宿主所吸收，或被宿主细胞所吞噬。进而还可以把抗原更多地带入到细胞中，从而增强蛋白和多肽引起的免疫响应。
一个重要优点在于帕米卡对癌痛患者止疼效果明显。
一个重要优点在于帕米卡可以使HPV感染者的病毒滴度从强阳性转为阴性。
一个重要优点在于帕米卡+布氏杆菌灭活疫苗保护效果很好。
需要注意的是，帕米卡并非简单的组合物，通过本公开说明书所述，其是一种全新结构的复合物。
在一些实施方式中，所述阳离子稳定剂的分子量还可以选择4kDa、4.5kDa、3kDa、3.5kDa、2.5kDa、2kDa、1.5kDa、1kDa、500Da、400Da、300Da、200Da、100Da。
在一些实施方式中，所述水溶性非抗生素氨基化合物选自壳寡糖、几丁寡糖、氨基葡萄糖、阳离子脂质体、DEAE-葡聚糖、聚丙烯酰胺、聚胺、四氨富烯、聚乙烯亚胺中的一种或多种。
在一些实施方式中，所述阳离子稳定剂选自壳寡糖和聚乙二醇单甲醚的接枝物(COS-g-MPEG)、壳聚糖盐酸盐与聚乙二醇的接枝物(PEG-g-CS)、叶酸和壳聚糖盐酸盐的接枝物(FA-g-CS)、半乳糖和聚乙二醇和聚乙烯亚胺的接枝物(GAL-g-PEG-g-PEI)、壳寡糖和聚乙二醇单甲醚和聚乙烯亚胺的接枝物(COS-g-MPEG-g-PEI)、壳聚糖和聚乙二醇单甲醚和聚乙烯亚胺的接枝物(CS-g-PEG-g-PEI)、聚乙二醇和聚乙烯亚胺的接枝物(PEI-g-PEG)、壳寡糖和聚乙烯亚胺的接枝物(PEI-g-COS)、壳聚糖盐酸盐和聚乙烯亚胺的接枝物(PEI-g-CS)、壳寡糖和聚乙二醇的接枝物(COS-g-PEG)、壳寡糖和聚乙二醇和聚乙烯亚胺的接枝物(COS-g-PEG-g-PEI)。
在一些实施方式中，所述阳离子稳定剂选自壳寡糖(COS)、壳寡糖与聚乙二醇单甲醚的接枝物(COS-g-MPEG)、壳寡糖与聚乙二醇单甲醚和聚乙烯亚胺的接枝物(COS-g-MPEG-g-PEI)。
在一些实施方式中，所述接枝物的分子量≤50kDa。
在一些实施方式中，所述接枝物的分子量还可以选择45kDa、40kDa、35kDa、30kDa、25kDa、20kDa、15kDa、10kDa、9kDa、8kDa、7kDa、8kDa、5kDa、4kDa、3kDa、2kDa、1kDa、500Da、400Da、300Da、200Da、100Da。
在一些实施方式中，所述壳寡糖脱乙酰度≥70％；还可以选择80％，85％，90％或95％；优选90％～100％。
在一些实施方式中，壳寡糖单体分子量为161，聚合度为2-20，选用分子量范围为322-3220。
在一些实施方式中，壳寡糖、几丁寡糖、氨基葡萄糖的分子量≤3200。
在一些实施方式中，聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺的分子量为≤40000，也可以选择30000，20000，15000，10000，8000，6000，4000，2000，1500，1000或500。
在一些实施方式中，所述可溶性钙盐选自CaCl 2和/或CaNO 3。
在一些实施方式中，所述聚肌胞的分子量为100bp-3000bp。
在一些实施方式中，所述聚肌胞的分子量为100bp-1500bp。
在一些实施方式中，所述的组合产品还包括pH调节剂、三聚磷酸钠、海藻酸钠、苯基硼酸、苯邻二酚、缓冲盐/试剂以及水中的一种或多种。
在一些实施方式中，所述组合产品中的各成分单独包装；
在一些实施方式中，所述组合产品中的至少两种成分混合包装在一起，例如将正价离子与水和/或缓冲盐包装在一起；
在一些实施方式中，聚肌胞以其原料的形式进行包装，例如肌苷酸(PI)与胞苷酸(PC)。
根据本公开的一方面，本公开还涉及一种用于增强免疫响应的复合物，其由如上所述的组合产品中的试剂制备得到。
在一些实施方式中，所述制备在溶液体系中进行，且在所述试剂中，所述聚肌胞的浓度为0.1～10mg/ml；
聚肌胞的浓度可通过接枝的方式提升溶解度，理论上可达到更高浓度；
在一些实施方式中，所述制备在溶液体系中进行，且在所述试剂中，所述聚肌胞的浓度为0.5～5mg/ml，还可以选择1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml、6mg/ml、6.4mg/ml、7mg/ml、8mg/ml或9mg/ml。
在一些实施方式中，所述制备在溶液体系中进行，且在所述试剂中，所述阳离子稳定剂的浓度为0.5～51.2mg/ml；
在一些实施方式中，所述阳离子稳定剂的浓度为0.8～25.6mg/ml，还可以选择1mg/ml、2mg/ml、3mg/ml、4mg/ml、5mg/ml、10mg/ml、15mg/ml或20mg/ml。
在一些实施方式中，所述制备在溶液体系中进行，且在所述试剂中，所述聚肌胞与所述阳离子稳定剂的质量比为1：0.8～25.6；还可以选择1：6.4或1：12.8。
在一些实施方式中，所述制备在溶液体系中进行，且在所述试剂中，所述可溶性钙盐中钙离子的浓度为0.1～1mM，还可以选择0.2mM、0.3mM、0.4mM、0.5mM、0.6mM、0.7mM、0.8mM或0.9mM。
在一些实施方式中，所述的复合物保存于溶液中。
所述溶液优选为缓冲溶液。
在一些实施方式中，所述溶液的pH＝5.0～7.2。
在一些实施方式中，所述溶液的pH＝5.9～6.9，还可以选择6.0、6.2、6.4、6.8、7.0、7.2、7.4、7.6或7.8。
根据本公开的一方面，本公开还涉及如上所述的复合物作为免疫佐剂的非治疗用途。
根据本公开的一方面，本公开还涉及如上所述的复合物用于制备抗体、疫苗制剂或疫苗组合物的用途，或用于制备疫苗辅料或疫苗佐剂的用途。
根据本公开的一方面，本公开还涉及一种疫苗组合物，其含有如上所述的复合物以及至少一种抗原。
在一些实施方式中，所述抗原为病毒、细菌、蛋白、多肽、多糖、核酸或小分子-蛋白偶合物。
在一些实施方式中，所述的疫苗组合物例如为减毒疫苗(例如为病毒或细菌的减毒疫苗)、灭活疫苗(例如为病毒或细菌的灭活疫苗)、蛋白疫苗、多糖疫苗、蛋白质亚单位疫苗、嵌合载体疫苗、DNA疫苗、RNA疫苗、多肽疫苗或小分子-蛋白偶合物疫苗。
根据本公开的一方面，本公开还涉及如上所述的复合物在调节免疫细胞活性中的应用，所述应用于体内或体外进行。
在一些实施方式中，所述调节免疫细胞活性具体为增强免疫细胞活性。
在一些实施方式中，所述免疫细胞选自巨噬细胞、淋巴细胞和树突细胞。
在一些实施方式中，所述调节增强免疫细胞活性为促进所述免疫细胞释放炎症因子。
在一些实施方式中，所述炎症因子包括IL-2、IL-6、IL-12p40、IL-18、IL-22、IFN-α、IFN-γ以及TNF-α。
在一些实施方式中，所述炎症因子包括IFN-γ以及TNF-α。
根据本公开的一方面，本公开还涉及如上所述的复合物在制备用于治疗和/预防肿瘤、抗病毒、抗细菌、抗真菌、抗寄生虫、降低化疗副作用、抗疲劳或提升免疫力、缓解宿主疼痛、促进宿主对于抗原的免疫反应的药物中的应用。
在一些实施方式中，所述药物为注射给药剂型、呼吸道给药剂型、滴鼻剂、皮肤给药剂型、黏膜给药剂型或腔道给药剂型。
在一些实施方式中，注射给药剂型选自：如注射剂(包括静脉注射、肌内注射、皮下注射、皮内注射多种注射途径)。
在一些实施方式中，呼吸道给药剂型选自：如喷雾剂、气雾剂、粉雾剂等。
在一些实施方式中，皮肤给药剂型选自：如外用溶液剂、洗剂、搽剂、软膏剂、硬膏剂、糊剂、贴剂等，给药后在局部起作用或经皮吸收发挥全身作用。
在一些实施方式中，黏膜给药剂型选自：如滴眼剂、滴鼻剂、眼用软膏剂、含漱剂、舌下片剂等，黏膜给药可起局部作用或经黏膜吸收发挥全身作用。⑤腔道给药剂型：如栓剂、气雾剂等，用于直肠、阴道、尿道、鼻腔、耳道等，腔道给药可起局部作用或吸收后发挥全身作用。
在一些实施方式中，所述抗原包括肿瘤、病毒、细菌、真菌或寄生虫抗原。
在一些实施方式中，所述宿主为哺乳动物。
在一些实施方式中，所述宿主为灵长类动物。
在一些实施方式中，所述宿主为人类。
在一些实施方式中，当所述抗原为病毒、细菌、真菌或寄生虫抗原时，所述药物每剂含药量为1～8mg；
在一些实施方式中，当所述抗原为肿瘤抗原时，所述药物每剂含药量为1～10mg。
根据本公开的一方面，本公开还涉及一种药物组合物，所述药物组合物包含如上所述的复合物，所述药物组合物还包括免疫细胞治疗药物、抗体治疗药物、化学药物、促进粘膜免疫吸收或粘膜粘附的物质、免疫调节剂、病原体抗原、膜式识别受体的配体、药物可接受的赋形剂中的一种或多种。
在一些实施方式中，所述免疫细胞治疗药物选自肿瘤浸润淋巴细胞(tumor infiltrating lymphocyte,TIL)、树突状细胞(dendritic cells,DC)、细胞因子诱导杀伤细胞(cytokine induced killer,CIK)、树突细胞-细胞因子诱导的杀伤细胞(DC-CIK)、自然杀伤细胞(natural killer cell，NK)、γδT细胞、CD3AK、CAR-T和TCR-T中的一种或多种。
在一些实施方式中，所述抗体治疗药物选自抗PD1抗体、抗PDL1抗体、抗CTLA4抗体和抗CD抗原抗体。
在一些实施方式中，所述化学药物选自烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗肿瘤药、激素类药物和杂类药物中的一种或多种；
其中所述杂类药物选自左旋门冬酰胺酶、顺铂、卡铂、草酸铂、氮烯咪胺、六甲嘧胺类药物，或上述药物的衍生物。
在一些实施方式中，所述烷化剂选自环磷酰胺、白消安、弹烯咪胺、顺氯氨铂、二氯甲二乙胺、苯丙氨酸氮芥、亚硝脲类和上述药物的衍生物；
在一些实施方式中，所述抗代谢药选自5-氟脲嘧啶、家氨蝶呤、阿糖胞苷、环胞苷、羟基脲和上述药物的衍生物；
在一些实施方式中，所述抗肿瘤抗生素选自放线菌素、丝裂霉素、黄胆素、阿霉素、红必霉素、更生霉素、博来霉素和上述药物的衍生物；
在一些实施方式中，所述激素类药物选自性激素、皮质类固醇激素和上述药物的衍生物。
在一些实施方式中，所述促进粘膜免疫吸收或粘膜粘附的物质选自阴离子表面活性剂(如羧酸盐类、磺酸盐类、硫酸酯类、磷酸酯类等)、阳离子表面活性剂(如胺盐类、季铵盐类、杂环类、鎓盐类等)、两性离子表面活性剂如羧酸盐型、磺酸盐型、磷酸酯型、甜菜碱型、咪唑啉型、氨基酸型等)、非离子表面活性剂(如烷基多苷型、聚氧乙烯型、多元醇型、烷醇酰胺型、嵌段聚醚型)、特种表面活性剂(如含氟型、含硅型、含硼型、高分子型等)、螯合剂(如多磷酸盐、氨基羧酸、1,3-二酮，羟基羧酸、多胺等)、粘合剂【水溶型粘合剂(如淀粉、糊精、聚乙烯醇、羧甲基纤维素等)、热熔型粘合剂(如聚氨酯、聚苯乙烯、聚丙烯酸酯、乙烯-醋酸乙烯共聚物等)、溶剂型粘合剂(如虫胶、丁基橡胶等)、乳液型粘合剂(如醋酸乙烯树脂、丙烯酸树脂、氯化橡胶等)、无溶剂液体粘合剂(如环氧树脂等)】、聚乳酸-羟基乙酸共聚物、右旋糖酐、多聚糖中的一种或多种。
在一些实施方式中，所述免疫调节剂选自细胞因子、趋化因子、干细胞生长因子、淋巴毒素、造血因子、集落刺激因子(CSF)、干扰素、促红细胞生成素、促血小板生成素、肿瘤坏死因子(TNF)、白介素(IL)、粒细胞-集落刺激因子(G-CSF)、粒细胞巨噬细胞-集落刺激因子(GM-CSF)和干细胞生长因子中的一种或多种。
在一些实施方式中，所述病原体抗原选自肿瘤、病毒、细菌、真菌或寄生虫抗原。
在一些实施方式中，所述肿瘤包括：骨、骨连接、肌肉、肺、气管、咽、鼻、心脏、脾脏、动脉、静脉、血液、毛细血管、淋巴结、淋巴管、淋巴液、口腔、咽、食管、胃、十二指肠、小肠、结肠、直肠、肛门、阑尾、肝、胆、胰腺、腮腺、舌下腺、泌尿肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、外阴部、阴囊、睾丸、输精管、阴茎、眼、耳、鼻、舌、皮肤、脑、脑干、延髓、瘠髓、脑瘠液、神经、甲状腺、甲状旁腺、肾上腺、垂体、松果体、胰岛、胸腺、性腺、舌下腺、腮腺中任一处病变生成的肿瘤。
在一些实施方式中，所述细菌包括：葡萄球菌属、链球菌属、李式杆菌属、丹毒丝菌属、肾杆菌属、芽孢杆菌属、梭菌属、分支杆菌属、放线菌属、奴卡菌属、棒状杆菌属、红球菌属、炭疽杆菌、丹毒杆菌、破伤风杆菌、李氏杆菌、产气荚莫杆菌、气肿疽杆菌结核杆菌、大肠杆菌外、变形杆菌、痢疾杆菌、肺炎杆菌、布氏杆菌、产气夹膜杆菌、流感嗜血杆菌、副流感嗜血杆菌、卡他摩拉克氏菌、不动杆菌属、 耶尔森菌属、嗜肺军团菌、百日咳杆菌、副百日咳杆菌、志贺菌属、巴斯德菌属、霍乱弧菌、副溶血性杆菌中的一种或多种。
在一些实施方式中，所述寄生虫包括：消化道内寄生虫(如蛔虫、钩虫、绦虫、溶组织内阿米巴和雅尔氏鞭毛虫等)、腔道内寄生虫(如阴道毛滴虫)、肝内寄生虫(如肝吸虫、棘球蚴)、肺内寄生虫(如卫斯特曼氏并殖吸虫)、脑组织寄生虫(如猪囊尾蚴、弓形虫)、血管内寄生虫(如血吸虫)、淋巴管内寄生虫(如丝虫)、肌肉组织寄生虫(如旋毛虫幼虫)、细胞内寄生虫(如疟原虫、利什曼氏原虫)、骨组织寄生虫(如包虫；皮肤寄生虫，如疥螨、毛囊螨)、眼内寄生虫(如吸吮线虫、猪囊虫)中的一种或多种。
在一些实施方式中，所述病毒包括：腺病毒(adeniviridae)、沙粒病毒(arenaviridae)、星状病毒(astroviridae)、本扬病毒(bunyaviridae)、杯状病毒(cliciviridae)、黄病毒(flaviviridae)、D型肝炎病毒(hepatitis delta virus)、肝炎病毒(hepeviridae)、单分子负链RNA病毒(mononegavirales)、巢病毒(nidovirales)、小RNA病毒(piconaviridae)、正黏液病毒(orthomyxoviridae)、乳头瘤病毒(papillomaviridae)、细小病毒(parvoviridae)、多瘤病毒(polyomaviridae)、痘病毒(poxviridae)、呼肠孤病毒(reoviridae)、反转录病毒(retroviridae)或披膜病毒(togaviridae)中的一种或多种。
在一些实施方式中，所述病毒为人乳头瘤病毒(Human papillomavirus)。
在一些实施方式中，所述真菌包括：粗球孢子菌、普赛德斯球抱子菌、荚膜组织胞浆菌、杜氏组织胞浆菌、洛博芽生菌、巴西副球孢子菌、皮炎芽生菌、申克氏孢子丝菌、马尔尼菲青霉菌、白色念珠菌、光滑念珠菌、热带念珠菌、葡萄牙假丝酵母、卡氏肺孢子虫病、曲霉菌、甄氏外瓶霉、裴氏着色霉、紧密着色霉、疣状着色霉、皮炎着色霉、白地霉、波氏足肿菌、新型隐球菌、丝孢酵母菌、米根霉、印度毛霉、伞枝犁头霉、总状共头霉、蛙粪霉、冠状耳霉、异孢耳霉、比氏肠胞微孢子虫、肠脑炎微孢子虫、西伯鼻孢子菌、透明丝孢霉、暗色丝孢霉中的一种或多种。
在一些实施方式中，所述膜式识别受体的配体选自TLR受体的配体、RLR受体的配体、CLR受体的配体、NLR受体的配体。
在一些实施方式中，联合TLR受体的配体有：如肽聚糖、二聚糖、甘露聚糖、脂肽、糖脂、非典型脂多糖、血清淀粉样蛋白、CPG DNA、dsRNA、ssRNA、LPS、PGN、饱和脂肪酸、脂磷壁酸、抵抗素、乳铁蛋白、表面活性蛋白、透鞭毛蛋白、明质酸、RNA相关抗原、Profilin样分子等。
在一些实施方式中，联合RLR受体的配体有：如RNA、PIC、PICLC、PIC12u等。
在一些实施方式中，联合CLR受体的配体有：如真菌细胞壁表面的甘露糖和β-葡聚糖等。
在一些实施方式中，联合NLR受体的配体有：如MDP、
Figure PCTCN2019093607-appb-000001
DAP等。
根据本公开的一方面，本公开还涉及一种用于增强免疫响应的复合物的制备方法，包括：
将聚肌胞、至少一种阳离子稳定剂以及可溶性钙盐在液体反应体系中接触；
所述阳离子稳定剂为分子量≤5kDa的水溶性非抗生素氨基化合物，或所述水溶性非抗生素氨基化合物与聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺、叶酸、半乳糖中的一种或多种所形成的接枝物。
在一些实施方式中，所述聚肌胞由聚胞苷酸和聚肌苷酸经碱基配对反应制得。
在一些实施方式中，所述聚胞苷酸、聚肌苷酸分子量大于2.3万道尔顿。
在一些实施方式中，所述聚胞苷酸的分子量范围是在6.6万道尔顿～66万道尔顿。
在一些实施方式中，所述聚肌苷酸的分子量范围是在6.6万道尔顿～66万道尔顿。
在一些实施方式中，所述碱基配对反应在40℃～50℃的温度下进行，还可以选择41℃、42℃、43℃、44℃、45℃、46℃、47℃、48℃或49℃。
在一些实施方式中，所述碱基配对反应在pH＝6.8～7.6下进行，还可以选择7.0、7.2、7.4。
在一些实施方式中，在进行接触反应之前，将所述聚肌胞80℃～99℃下加热70～120min；
在一些实施方式中，温度还可以选择82℃、84℃、86℃、88℃、90℃、92℃、94℃、96℃或98℃；
在一些实施方式中，加热时间还可以选择80min、90min、100min或110min。
在一些实施方式中，所述液体反应体系的温度为40℃～50℃，还可以选择41℃、42℃、43℃、44℃、45℃、46℃、47℃、48℃或49℃。
在一些实施方式中，所述接枝物的制备方法包括：
先用羰基二咪唑活化聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺、叶酸、半乳糖中的一种或多种，再用活化好的产物与所述水溶性非抗生素氨基化合物在离子液体[bmim]Cl中进行接枝反应。
在一些实施方式中，所述接枝物为壳寡糖与聚乙二醇单甲醚的接枝物，先用羰基二咪唑(CDI)活化聚乙二醇单甲醚(MPEG)，再用活化好的MPEG与壳寡糖(COS)在离子液体[bmim]Cl中进行接枝反应。
在一些实施方式中，所述接枝反应在60℃～80℃、非氧化气氛下反应。
在一些实施方式中，所述方法还包括：
将交联剂溶液在搅拌条件下逐滴加入制得的复合物中直至观察到反应体系中出现丁达尔现象后停止滴加，搅拌得到纳米粒子；
所述交联剂选自三聚磷酸钠、海藻酸钠、苯基硼酸、苯邻二酚中的至少一种。
在一些实施方式中，所述交联剂溶液中含有(病原体)抗原。
在一些实施方式中，所述方法还包括，将所述复合物或所述纳米粒子与抗原共孵育。
在一些实施方式中，所述抗原为蛋白质或多肽抗原。
根据本公开的一方面，本公开还涉及一种用以促进宿主体内对于抗原的免疫反应，或调节增强宿主免疫细胞活性，或帮助宿主降低疲劳度，或减轻宿主疼痛的方法，该方法包含将如上所述的复合物，或如上所述的疫苗组合物，或如上所述的药物组合物给予该宿主。
在一些实施方式中，该宿主罹患传染病，且给予该抗原化合物激发免疫反应以对抗造成该传染病的病原体。
在一些实施方式中，所述给药通过胃肠道外注射、肌肉内注射、腹腔内注射、静脉注射、皮下注射、局部给药、透皮给药或皮内给药来进行。
在一些实施方式中，该宿主为手术化疗放疗或免疫疗法失败或医疗机构放弃治疗的肿瘤患者、病毒感染患者、细菌感染患者、寄生虫感染患者或鼻炎患者。
在一些实施方式中，所述方法可与手术、放疗、化疗以及各种免疫疗法联合使用，或与病毒感染患者、细菌感染患者、寄生虫感染患者也可以与传统疗法联合使用。
在一些实施方式中，所述疼痛为微生物或寄生虫感染引起的疼痛、癌症引起的疼痛患者或神经性引起的疼痛。
在一些实施方式中，当所述抗原为病毒、细菌、真菌或寄生虫抗原时，所述药物每次给药1～8mg/kg；优选的，或每天或隔1天或2天或3天给药一次；
当所述抗原为肿瘤抗原时，所述药物每次给药1～10mg/kg；优选的，给药周期或至少360天或至少180天或至少60天或至少30天。
本公开提供根据本公开所述方法制备的用于增强免疫响应的复合物。
本公开提供根据本公开所述方法制备的用于增强免疫响应的复合物，用于制备抗体、疫苗制剂或疫苗组合物，增强免疫细胞活性，治疗和/预防肿瘤、抗病毒、抗细菌、抗真菌、抗寄生虫、降低化疗副作用、抗疲劳或提升免疫力、缓解宿主疼痛、促进宿主对于抗原的免疫反应中的应用。
下面将结合实施例对本公开的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本公开，而不应视为限制本公开的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1帕米卡的制备
一、帕米卡复合物的制备
1、配制PBS溶液(pH7.2)：
1.1配制氯化钠溶液(0.85％，1500ml)：称量氯化钠12.75g，放入2000ml量筒中，用注水定容至1500ml；
1.2配制磷酸氢二钠溶液(0.006mol/L，500ml)：称量磷酸氢二钠0.006×0.5×141.96＝0.4259g，放入500ml容量瓶中，用0.85％生理盐水定容至500ml；
1.3配制磷酸二氢钠溶液(0.006mol/L，500ml)：称量磷酸二氢钠0.006×0.5×137.99＝0.4140g，放入500ml容量瓶中，用0.85％生理盐水定容至500ml；
1.4配制pH值为7.2的PBS溶液：取“1.2液”273.6ml+“1.3液”126.4ml。
2、配制PIC液(2.0mg/ml，100ml)：
2.1称取PI 2.0mg/ml*100ml*【1.04/(1.04+1)】/91.5％/(1-2.7％)＝114.5mg，放入250ml三角瓶中，加50ml PBS溶液溶解，并用40-60℃水浴平衡；
2.2称取PC 2.0mg/ml*100ml*【1/(1.04+1)】/90.4％/(1-4.2％)＝113.2mg，放入250ml三角瓶中，加50ml PBS溶液溶解，并用40-60℃水浴平衡；
2.3配制PIC液：将50ml PI溶液倒入50ml PC溶液中，并在45℃水浴反应30分钟。
2.4 80-99℃对PIC液进行分子量加热15～300分钟
3、配制氯化钙溶液(0.16mol/L，25ml)：
称取CaCl 2.2H 2O(MW:147.02)0.5881g，放入100ml三角瓶中，加约25ml注射用水溶解并定容。
4、COS-g-MPEG的制备：
其中COS-g-MPEG接枝物制备方法如下：制备壳寡糖接枝聚乙二醇单甲醚(COS-g-MPEG)接枝共聚物，以此为辅料制备抗癌药物。
原理：采用羰基二咪唑(CDI)偶联法制备COS-g-MPEG。首先用羰基二咪唑活化聚乙二醇单甲醚(MPEG)，制备活化的MPEG，再用活化的MPEG与壳寡糖(COS)在离子液体中反应，合成COS-g-MPEG共聚物，具体反应步骤包括以下三步：
4.1离子液体1-丁基-3-甲基咪唑氯盐([BMIM]Cl)的制备
1-甲基咪唑和氯丁烷反应制备离子液体[BMIM]Cl，合成反应方程式如下：
Figure PCTCN2019093607-appb-000002
4.2聚乙二醇单甲醚(MPEG，分子量1000)的活化
用CDI活化MPEG，合成反应方程式如下：
Figure PCTCN2019093607-appb-000003
4.3 COS-g-MPEG共聚物的合成；
活化的MPEG与COS在离子液体中进行接枝聚合，合成反应方程式如下：
Figure PCTCN2019093607-appb-000004
4.4器材试剂：
甲基咪唑、氯丁烷、甲苯、聚乙二醇单甲醚、羰基二咪唑、无水乙醚、4A分子筛(2-3mm)、二甲基亚砜、1,4-二氧六环、壳寡糖、集热式恒温磁力加热搅拌器(DF-101S型)、电子天平、电热鼓风干燥箱、循环水式真空泵、自动三重纯水蒸馏器、真空干燥箱、冷冻干燥机、玻璃仪器气流烘干器、单相电容起动电动机、旋片式真空泵、六联磁力加热搅拌器、纤维素透析袋、即用型透析袋45-2000RC膜、三口烧瓶(500mL、1000mL)、玻璃塞、磁子、一次性纸杯、500mL烧杯、2L烧杯、一次性滴管、药匙、试剂瓶、干燥器等。
4.5准备：
4.5.1蒸馏水的制备
用SZ-97A自动三重纯水蒸馏器制备三次蒸馏水。
4.5.2玻璃器皿的清洗
将三口烧瓶、玻璃塞、培养皿、磁子等先用自来水洗刷，再用三次蒸馏水冲洗，最后放在玻璃仪器 气流烘干器烘干。
4.5.3溶剂的干燥：取适量分子筛于一次性烧杯中，取适量二甲基亚砜、1,4-二氧六环、无水乙醚放入烧杯中，除水。
4.5.4提前冷冻无水乙醚。
4.6.操作(每步注意事项)：
4.6.1离子液体的制备
(1)分别取1-甲基咪唑100g、氯丁烷148.5mL依次加入500mL的三口烧瓶中，装上冷凝管，通氩气30min，磁力搅拌，油浴加热至80℃，反应24h；
(2)反应完成后，取出，冷却至室温，放入-18℃冰箱中冷冻2h，可以看到溶液分层，倒去上层液体(主要是除氯丁烷)；
(3)放入80℃鼓风干燥箱中，待固体完全融化，趁热加入适量甲苯，摇晃，使甲苯与溶液充分混合，冷却至室温，放入冰箱中冷冻，取出，倒去上层液体(1-甲基咪唑、氯丁烷溶于甲苯，除去甲苯、1-甲基咪唑、氯丁烷)；
(4)再重复步骤(3)两次，除尽未反应的氯丁烷；
(5)将样品放入真空干燥箱中，加热至90℃，待完全融化后，90℃真空干燥8h(除甲苯)，取出放冷置室温，再放入干燥器中备用。
4.6.2 MPEG的活化
(1)取二甲基亚砜10mL、1,4-二氧六环20mL放入500mL三口烧瓶中，磁力搅拌，加入MPEG 20g，待MPEG完全融化，加入CDI 3.24g，水浴加热至37℃反应18h；
(2)反应完成后，在冰水浴、磁力搅拌的条件下，将样品加入预冷的无水乙醚中，用保鲜膜盖住烧杯口，将样品放入冰箱中30min；
(3)30min后取出，倒去上层溶液，在沉淀物中加入预冷的无水乙醚，磁力搅拌30min，放入冰箱中30min。
(4)再重复步骤(3)两次，充分洗去未反应的CDI；
(5)倒去上层溶液，放入40℃鼓风干燥箱中6h，初步除去乙醚；
(6)放入40℃真空干燥箱中真空干燥2.5h，充分除去乙醚，取出放冷置室温后，再放入干燥器中备用。
4.6.3 COS-g-MPEG的制备
(1)将离子液体在80℃鼓风干燥箱中融化；
(2)称取离子液体105g放入三口烧瓶中，油浴加热至70℃，通氩气，缓慢加入COS 9g，待COS完全溶解，加入活化MPEG 6g，磁力搅拌，所有原料加完后在氩气保护下反应6h，反应完成后，将反应瓶冷却至室温；
(3)先用夹子夹住透析袋一侧，加入适量蒸馏水清洗三次，并检查透析袋是否漏水，然后将反应瓶中样品放入透析袋(截留分子量为2000)中透析72小时，蒸馏水的用量以没过透析袋为宜。第一天2～3h换一次水，以后每隔12h换一次水。
(4)透析完成后，将溶液加入1000mL三口烧瓶中并置入水浴锅中，安装好减压蒸馏装置，蒸馏温度从室温分梯度升到25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃，蒸馏至剩余50mL左右，撤去蒸馏装置，趁热将样品倒入一次性烧杯中，用一次性手套盖住杯口，放入-18℃冰箱中冷冻不少于8小时。
(5)将冷冻干燥机预冷30min，冷冻温度达到-52℃。将样品砸碎，平铺在培养皿中，放入预冷的冷冻干燥机中，-52℃冷冻干燥30h。冷冻完成后，关机，取出样品，称重，放入试剂袋中，放入干燥器保存。
(6)红外分析：取适量样品，用溴化钾压片，扫描范围为400～4000cm-1，测定样品的红外光谱。
5、配制COS-g-MPEG的PBS液：
5.1 5.12％：称取0.128g的COS-g-MPEG放入5ml离心管中，加PBS液溶解并定容至2.5ml；
5.2 2.56％：5.12％液1.2ml+PBS液1.2ml；
5.3 1.28％：2.56％液1.2ml+PBS液1.2ml；
5.4 0.64％：1.28％液1.2ml+PBS液1.2ml；
5.5 0.32％：0.64％液1.2ml+PBS液1.2ml；
5.6 0.16％：0.32％液1.2ml+PBS液1.2ml；
6、配制PIC、COS-g-MPEG和氯化钙溶液的帕米卡溶液：
6.1取PIC液1.0ml放入45℃水浴锅中，滴加5.1 1.0ml，再滴加氯化钙溶液0.005ml使其终浓度为0.0004mol/L。
6.2取PIC液1.0ml放入45℃水浴锅中，滴加5.2 1.0ml，再滴加氯化钙溶液0.005ml使其终浓度为0.0004mol/L。
6.3取PIC液1.0ml放入45℃水浴锅中，滴加5.3 1.0ml，再滴加氯化钙溶液0.005ml使其终浓度为0.0004mol/L。
6.4取PIC液1.0ml放入45℃水浴锅中，滴加5.4 1.0ml，再滴加氯化钙溶液0.005ml使其终浓度为0.0004mol/L。
6.5取PIC液1.0ml放入45℃水浴锅中，滴加5.5 1.0ml，再滴加氯化钙溶液0.005ml使其终浓度为0.0004mol/L。
6.6取PIC液1.0ml放入45℃水浴锅中，滴加5.6 1.0ml，再滴加氯化钙溶液0.005ml使其终浓度为0.0004mol/L。
7、结果：
MPEG，PEG，PEI等均具有良好的水溶性，并与许多有机物组份有良好的相溶性。本实施例以MPEG为例，用MPEG和阳离子稳定剂(如壳寡糖)的接枝物与PIC配制，相容性明显增加，理论上用PEG等接枝壳聚糖等阳离子稳定剂和PIC配制，相容性也会增加；阳离子稳定剂上接枝上PEG后，接枝物本身也会兼有PEG的特性。
Figure PCTCN2019093607-appb-000005
试验结果表明，PIC：COS-g-MPEG在1：25.6mg时候仍为澄清溶液，但是增色效应结果表明并不是接枝物越多越好；另外，我们将2018-05-07配制完的样品6.1、6.、6.3、6.4、6.5、6.6放置室温，2018-05-14观察发现6.6的样品出现片状析出物，重新溶解不了。综合考虑，我们限定帕米卡处方中PIC与COS-g-MPEG的处方在1：6.4mg范围内。
二、帕米卡纳米粒子的制备
购买具有药用标准的三聚磷酸钠(TPP)在适宜比例下PIC—COS-g-MPEG－CaCl 2复合物在恒温磁力搅拌器上以一定转速匀速搅拌，逐滴滴加不同浓度TPP水溶液，观察到明显的乳光立即停止，维持反应30分钟，通过离子交联自组装形成纳米粒子，经高速离心获得，粒径小于1000nm。并经各种检定合格。
三、多肽或蛋白抗原纳米粒子
方案1：多肽或蛋白抗原在上述纳米粒子形成过程中加入：各成分进入到PEG-COS接枝物或COS基质中，多肽或蛋白抗原进入含有TPP水相中结合。其中各成分必须合适比例并在一定pH环境下，磁珠搅拌结合，形成复合物及纳米粒子。
方案2：多肽或蛋白抗原与上述预先形成的复合物及纳米粒子孵育，使多肽或蛋白抗原结合在纳复合物及米粒子表面多肽或将多肽或蛋白抗原与上述复合物及纳米粒子以一定比例混合，磁力搅拌5分钟， 置室温1小时，在甘油基质下超速离心20000rcf 4℃2小时获得。
方案1/方案2复合物及纳米粒子需经各项检定合格。
四、帕米卡的制剂形式
将上述复合物/含有接枝物复合物/复合物纳米粒子/多肽或蛋白抗原纳米粒子无菌装入在适宜/合格包材，制备成注射、喷雾剂或气雾剂等各种剂型，经各种产品检定合格制备成产品。
将上述复合物/含有接枝物复合物/复合物纳米粒子/多肽或蛋白抗原纳米粒子无菌装入在适宜/合格包材，制备成膏剂。
制成喷雾剂的实施例：根据上述方法配制帕米卡，将其溶液装于喷雾剂瓶中，取20瓶，分别进行药液的喷雾模式检测和雾滴分布的数据检测。
喷雾模式如下表所示：
Figure PCTCN2019093607-appb-000006
说明：通过最长径与最短径的比值评价喷雾的形态(越接近1.0，喷雾形态越佳)。
雾滴分布如下表所示：
Figure PCTCN2019093607-appb-000007
实施例2帕米卡联合PEI增加溶解度试验
参考“帕米卡联合PEG”的配制方法(即实施例1)进行配制，帕米卡中增加COS用量后出现沉淀，影响其给药的均一性，添加PEI后可避免出现沉淀，并增加COS的用量，进一步增强其免疫效果。
Figure PCTCN2019093607-appb-000008
Figure PCTCN2019093607-appb-000009
试验清楚表明帕米卡联合PEI后，在PIC中COS溶解度从1.6mg/ml提高到6.4mg/ml，至少提高4倍。
CN105396130A专利申请文献中公开了一种“皮氨钙佐剂及含有皮氨钙佐剂的疫苗”，并公开了非抗生素氨基化合物可选为壳聚糖。
本对比例采用水溶性壳聚糖(壳聚糖盐酸盐，简称CS)替换实施例1中的壳寡糖，以比较二者对给药均一性方面的影响，结果见下表。
Figure PCTCN2019093607-appb-000010
研究发现，水溶性壳聚糖的加入导致在配制过程中出现沉淀并且摇不散，影响其给药的均一性，而选用壳寡糖可以解决上述问题。此外，壳聚糖接枝PEG或水溶性壳聚糖均需要降解成小分子量壳寡糖才易被人体吸收，而壳寡糖可以直接吸收。
实施例3 PIC加热及分子量检测
根据实施例1配制的方法，配制PIC液，取PIC液260ml，分装成20ml/管，共13管，待恒温水浴锅温度升至80-99℃时(优选90℃)，放入样品12管，放入后分别开始计时10分钟(条带3)、20分钟(条带4)、30分钟(条带5)、40分钟(条带6)、50分钟(条带7)、60分钟(条带9)、70分钟(条带10)、80分钟(条带11)、90分钟(条带12)、100分钟(条带13)、110分钟(条带14)和120分钟(条带15)，每次取出1管。未加热的样品为条带2。
取加热70-120分钟(优选加热时间120分钟)PIC配制的复合物(帕米卡)及其疫苗按《中国药典》2015版四部“1141异常毒性检查法”均能通过小鼠和豚鼠异常毒性的检测，配制帕米卡所用的PIC应在90℃加热至少70分钟才能用于产品的配制，优选90℃加热时间为120分钟。选择未加热PIC(条带2)及加热60分钟(条带9)配制的复合物及其疫苗不能通过豚鼠异常毒性的检测。结果如图2所示，对照条带1：从下向上分别为100bp、300bp、500bp、750bp、1000bp、1500bp、2000bp、3000bp、5000bp。对照条带8：从下向上分别为100bp、200bp、300bp、400bp、500bp、600bp、700bp、800bp、900bp、1000bp。
实施例4帕米卡复合物的酶降解试验
方法：将本公开实施例1的制备方法制备帕米卡复合物，制备完成后将帕米卡复合物和聚肌胞注射液(聚肌胞-卡那霉素-氯化钙)各样品分别稀释至0.04mg/ml，取13个10ml管，每管加5ml样品稀释液，每管再分别加sigma的RNA酶(货号R4642)25μg，放置37℃水浴锅中，每隔5min中取出1管测248nm处的OD值，绘制曲线。
结果如图3所示。
实施例5本公开的帕米卡复合物是全新结构的复合物
(一)融化曲线峰值测定
方法：将本公开的帕米卡复合物和聚肌胞注射液各样品稀释至0.04mg/ml，转移至250ml试剂瓶中，放置水浴锅，水浴锅持续升温。每隔5℃，取3ml放置石英比色杯中，测248nm处的OD值，绘制曲线。
结果如图4所示。
试验测定结果表明本公开的帕米卡复合物(PIC-阳离子稳定剂-氯化钙)的融化曲线峰值是85℃，聚肌胞注射液(PIC-卡那霉素-氯化钙)的融化曲线峰值是80℃，这说明本公开的帕米卡复合物是一种全新的复合物。
(二)吸收峰扫描
按实施例1的制备方法，分别制备PI溶液、PC溶液、PIC溶液、PIC-COS溶液、PIC-COS-CaCl 2溶液，用PBS缓冲液将上述样品分别稀释至0.04mg/ml，用紫外分别测定其扫描吸收光谱，图5结果显示在240-260nm处出现的峰从高到低依次是PI、PC、PIC、PIC-COS、PIC-COS-CaCl 2，其中PIC-COS和PIC-COS-CaCl 2峰重叠，这说明本公开的帕米卡复合物(PIC-COS-CaCl 2)是一种全新结构的复合物。
实施例6 PIC、COS和氯化钙形成的部分纳米粒子
按实施例1的方法制备帕米卡，其中阳离子稳定剂选用COS，金属阳离子选用氯化钙。从透射电镜图片(图6、图7)中可以看出，帕米卡溶液中形成了纳米粒子，大部分为球形，粒径为50nm左右，较均匀，个别为方形，边长超过100nm。
实施例7 PIC、COS-g-MPEG和氯化钙形成的部分纳米粒子
按实施例1的方法制备帕米卡，其中阳离子稳定剂选用COS-g-MPEG，金属阳离子选用氯化钙。从透射电镜图片(图8、图9)中可以看出，帕米卡溶液中形成了纳米粒子，大部分为方形，边长超过100nm，少数为球形。
实施例8 PIC、COS、TPP和氯化钙形成的部分纳米粒子
COS溶解在磷酸盐缓冲溶液中，按实施例1的方法制备PIC的TPP溶液，将PIC的TPP溶液在搅拌下缓慢滴加到COS溶液中，再滴加氯化钙溶液。从透射电镜图片(图10、图11)中可以看出，形成纳米粒子为梭形。
通过实施例5～8可以发现，令人惊奇的是，帕米卡复合物溶液中同时含有两种状态的物质，一种是纳米粒子(电镜结果)，一种是非纳米粒子(实施例3电泳结果)溶液。
纳米粒子的优点是不经过胞吞作用可以直接穿透细胞膜，进入胞内，见效快；非纳米粒子溶液要通过胞吞，才能进入胞内，相对于纳米粒子见效慢。帕米卡可以通过胞吞和直接进入细胞两种模式发挥效果。此外，更为重要的是，纳米粒子的结构可以保护帕米卡，使得其免受灵长类以上包括人体血清中的核糖核酸酶对PIC的降解以求取代抗病毒抗肿瘤的突破获得更大效果。这些效果使得帕米卡在人体和小鼠的抗癌效果上均得以获得突出的效果。
实验例对帕米卡复合物的免疫效果评价
在下述实验例中，若无特别提及，则帕米卡均指按实施例1制备的PIC、COS和氯化钙溶液的帕米卡溶液。
实验例1帕米卡复合物在重组乙肝疫苗【rHBsAg(CHO)】上的免疫效果评价
材料：rHBsAg(CHO)：20ug/ml，帕米卡佐剂：1mg/ml，ADV20佐剂400ug/ml，氢氧化铝佐剂：10mg/ml，生理盐水
铝佐剂/rHBsAg(CHO)：铝佐剂0.07ml+rHBsAg(CHO)0.5ml+生理盐水0.43ml
ADV20(细胞因子佐剂)/rHBsAg(CHO)：ADV20 0.25ml+rHBsAg(CHO)0.5ml+生理盐水0.25ml
帕米卡佐剂/rHBsAg(CHO)：帕米卡佐剂0.5ml+rHBsAg(CHO)0.5ml
方法：分别用铝佐剂/rHBsAg(CHO)、ADV20(细胞因子佐剂)/rHBsAg(CHO)、帕米卡/rHBsAg(CHO)于0、14天肌肉免疫小白鼠0.1ml，21天检测细胞免疫和体液免疫情况。
结果：本公开的帕米卡复合物免疫效果突出，尤其是在ELISA抗体和细胞免疫均有大幅度提高，显著优于铝佐剂和ADV20(细胞因子佐剂)，帕米卡是一有发展前景的免疫佐剂，详见图12和图13。
实验例2帕米卡复合物与灭活的布氏杆菌抗原上的免疫效果评价
方法：分别用PBS、聚肌胞注射液+灭活布氏杆菌抗原、本公开的帕米卡复合物+灭活布氏杆菌抗原免疫小鼠，0天免疫1次，免疫45天后用布鲁氏菌强毒株攻击小白鼠，攻毒15天后杀鼠分离小鼠脾脏，并将脾脏的布氏杆菌培养3天，计数，评价本公开的帕米卡复合物+灭活布氏杆菌抗原的保护效力。
Figure PCTCN2019093607-appb-000011
Figure PCTCN2019093607-appb-000012
结果：本公开的帕米卡复合物免疫效果突出，在灭活布氏杆菌抗原开发上有一定前景，本公开的帕米卡复合物+灭活布氏杆菌抗原与空白对照组的细菌分离数相差3.03个log，与聚肌胞注射液+灭活布氏杆菌抗原比较，分离数相差1.35，帕米卡复合物在灭活布氏杆菌保护效果突出。
实验例3帕米卡复合物与MYO抗原(人肌红蛋白)在制备抗体上应用
材料：MYO抗原(人肌红蛋白)，浓度1mg/ml
本公开的帕米卡复合物，浓度1mg/ml
完全弗氏佐剂(CFA，Sigma)
方法：准备2.3-2.5kg兔子，免疫前采阴性血分离血清做为对照，腹背部皮下分别多点注射不同样品后分离血清进行抗体检测。其中抗原+佐剂按0.5ml+0.5ml进行配制，每次免疫剂量为1ml。
结果：
(1)分别用本公开的帕米卡复合物+MYO抗原、完全弗氏佐剂+MYO抗原免疫家兔，间隔7天免疫注射1次。加完全弗氏佐剂免疫2次(10天)ELTSA抗体效价13000，加完全弗氏佐剂免疫3次(18天)ELTSA抗体效价39000，加完全弗氏佐剂免疫6次(3个月)ELTSA抗体效价25000；加本公开的帕米卡复合物免疫2次(10天)ELTSA抗体效价45000，免疫3次(18天)ELTSA抗体效价51000，免疫6次(3个月)ELTSA抗体效价36000，帕米卡佐剂在MYO抗原早期抗体滴度较高，的持续期也很好，表明本公开的帕米卡复合物在MYO抗原上明显优于具有免疫佐剂金标准的完全弗氏佐剂，详见图14。
(2)本公开的帕米卡复合物联合完全弗氏佐剂在MYO抗原免疫家兔2次下(0天免疫1次，14天免疫1次)，ELISA抗体效价(免疫后35天)：完全弗氏佐剂10000，本公开的帕米卡复合物60000，本公开的帕米卡复合物+完全弗氏佐剂为160000(图15)。
分别用完全弗氏佐剂+抗原、帕米卡佐剂+完全弗氏佐剂+抗原、帕米卡佐剂+抗原免疫家兔产生的抗体制备试剂盒。经临床验证用完全弗氏佐剂或完全弗氏佐剂+帕米卡佐剂抗原组产生的抗体和临床样品不匹配，不能通过临床检验，只有用帕米卡佐剂抗原产生的抗体能通过临床检验，这将完全打破荷兰Dako公司垄断国内的人肌红蛋白胶乳比浊试剂盒所用抗体原料的供应，解决了国内产品使用完全弗氏佐剂而普遍存在的免疫效价低，表位多样性不足，不能有效检测临床样品的严重局面。
下表是用帕米卡佐剂+抗原组产生抗体制备的试剂盒与荷兰Dako公司提供的抗体制备的临床正在使用的试剂盒进行比较。
Figure PCTCN2019093607-appb-000013
实验例4帕米卡复合物在狂犬病疫苗抗原上的NIH效力评价
名称：本公开的帕米卡复合物在狂犬病疫苗抗原上的NIH效力评价
方法：分别用本公开的帕米卡复合物(1mg/ml)+抗原、聚肌胞注射液(1mg/ml)+抗原、PBS+抗原 和抗原于0天免疫小鼠，14天攻毒，28天后测定效力。
结果：见下表。
Figure PCTCN2019093607-appb-000014
结果表明：
a本公开的帕米卡复合物+CTN株狂犬病疫苗抗原保护效果是单纯CTN株狂犬病疫苗抗原的3.6倍，并节省抗原1/5；
b本公开的帕米卡复合物+CTN株狂犬病疫苗抗原保护效果是聚肌胞注射液+CTN株狂犬病疫苗抗原的1.8倍，效果突出。
实验例5帕米卡复合物诱导小鼠多种细胞因子的产生
方法：分别用本公开的帕米卡复合物、聚肌胞注射液免疫小鼠，免疫后1小时、2小时、5小时，分别摘除小鼠眼球取血到灭菌的2ml离心管中，室温放置30分钟，3500转离心5分种，吸取上清液至一新离心管中，血清-20℃冻存。
结果：本公开的帕米卡复合物诱导的TNF-α、IFN-γ细胞因子产量明显优于聚肌胞注射液，所得到的数据均为几何平均值，具体结果见下表。
Figure PCTCN2019093607-appb-000015
小结：TNF-α，能杀伤和抑制肿瘤细胞，促进中性粒细胞吞噬，抗感染，是一类能直接造成肿瘤细胞死亡的细胞因子；IFN-γ能诱导细胞对病毒感染产生抗性，它通过干扰病毒基因转录或病毒蛋白组分的翻译，从而阻止或限制病毒感染，是目前最主要的抗病毒感染和抗肿瘤的细胞因子。
帕米卡诱导小鼠产生的TNF-α和IFN-γ细胞因子水平均高于聚肌胞注射液，说明本公开的帕米卡复合物诱导产生的TNF-α和IFN-γ，协同杀死肿瘤细胞和抗感染的能力更加强大。
实验例6异常毒性检查
1、本公开帕米卡复合物的小鼠试验法：
1.1试验方法：
样品注射：18～22克健康SPF昆明小鼠，5只/样品，腹腔注射0.5ml/只，同时称取5只18～22g健康小鼠作为空白对照。
1.2判定标准：
每只小鼠腹腔注射供试品0.5ml，观察7天。观察期内，小鼠应全部健存，且无异常反应，到期时每只小鼠体重应增加，供试品判为合格。如不符合上述要求，可用10只小鼠复试一次，判定标准同前。
1.3结果：
Figure PCTCN2019093607-appb-000016
Figure PCTCN2019093607-appb-000017
2本公开帕米卡复合物的豚鼠试验法：
2.1试验方法：
样品注射：250～350g健康SPF级Hartely豚鼠，2只/样品，腹腔注射5ml/只，同时称取2只250～350g健康豚鼠作为空白对照。
2.2.判定标准：
每只豚鼠腹腔注射供试品5ml，观察7天。观察期内，豚鼠应全部健存，且无异常反应，到期时每只豚鼠体重应增加，供试品判为合格。如不符合上述要求，可用4只豚鼠复试一次，判定标准同前。
2.3.试验结果：
Figure PCTCN2019093607-appb-000018
Figure PCTCN2019093607-appb-000019
注：复测仍不合格。
3本公开帕米卡复合物+狂犬病疫苗纯化抗原的豚鼠试验法：
3.1试验方法：
样品注射：250～350g健康SPF级Hartely豚鼠，2只/样品，腹腔注射5ml/只，同时称取2只250～350g健康豚鼠作为空白对照。
3.2.判定标准：
每只豚鼠腹腔注射供试品5ml，观察7天。观察期内，豚鼠应全部健存，且无异常反应，到期时每只豚鼠体重应增加，供试品判为合格。如不符合上述要求，可用4只豚鼠复试一次，判定标准同前。
3.3.试验结果：
Figure PCTCN2019093607-appb-000020
注：1)复测仍不合格；
2)狂犬病疫苗纯化抗原：细胞：Vero细胞，毒种：CTN株，(并不仅限于Vero细胞和
CTN株毒种)。
4本公开帕米卡复合物+乙型肝炎疫苗抗原的豚鼠试验法：
4.1试验方法：
样品注射：250～350g健康SPF级Hartely豚鼠，2只/样品，腹腔注射5ml/只，同时称取2只250～350g健康豚鼠作为空白对照。
4.2.判定标准：
每只豚鼠腹腔注射供试品5ml，观察7天。观察期内，豚鼠应全部健存，且无异常反应，到期时每只豚鼠体重应增加，供试品判为合格。如不符合上述要求，可用4只豚鼠复试一次，判定标准同前。
4.3.试验结果：
Figure PCTCN2019093607-appb-000021
Figure PCTCN2019093607-appb-000022
注：1)复测仍不合格；
2)乙型肝炎疫苗抗原：酵母表达(并不仅限于酵母表达，也可用重组CHO工程细胞表达乙肝抗原)。
总结分析：
PIC(双链核酸)经加热一定温度后解旋，随温度缓慢下降，两条单链又以氢键配对，恢复双链，加热能降低PIC的分子量，使其毒性降低，若PIC未经加热或加热时间不够就进行配制，这种方法配制的复合物本身毒性很大，这种复合物配制的疫苗毒性也很大，故很难应用。
实验例7帕米卡复合物在部分晚期癌症患者及感染治疗上的应用
Figure PCTCN2019093607-appb-000023
Figure PCTCN2019093607-appb-000024
Figure PCTCN2019093607-appb-000025
从多例晚期癌症病例可以看到本品除注射给药途径外，还可通过鼻腔喷雾给药，对转移癌效果明显。
(1)安全性良好，未见明显副作用；帕米卡免疫制剂是非细胞毒性药物。
(2)能降低放化疗副作用：提高白细胞数和血清蛋白含量。
(3)临床效果显著：减轻疼痛、食量增加、体力增加，精神由悲观到乐观、有信心，总生存期已延长数月，有病例延长13个月；
(4)对转移癌体积缩小明显，有病例显示肿瘤缩小1/3-1/2。
实验例8稳定性检测
本公开帕米卡复合物生产完成后避光储存于室内，每隔6个月对样品进行检测一次
Figure PCTCN2019093607-appb-000026
结果：样品室温(北京8-9月室温都超过30度)储存6个月内，产品检测指标未发生明显的变化，说明产品比较稳定；配制1mg/ml的帕米卡至少可达到12个月的稳定性；配制3mg/ml的帕米卡至少可达到9个月的稳定性。该产品的pH比较稳定，放置3年以内，pH变化不大。本公开的帕米卡细菌内毒素小于＜10EU/ml，而聚肌胞注射液的细菌内毒素大于100EU/ml，本领域公知技术细菌内毒素是革兰氏阴性菌的细胞壁成分，当细菌死亡或自溶后便会释放出内毒素。因此，细菌内毒素广泛存在于自然界中。如自来水中含内毒素的量为1至100EU/ml。当内毒素通过消化道进入人体时并不产生危害，但内毒素通过注射等方式进入血液时则会引起不同的疾病。内毒素小量入血后被肝脏枯否细胞灭活，不造成机体损害。内毒素大量进入血液就会引起发热反应"热原反应"。因此，生物制品类、注射用药剂、化学药品类、放射性药物、抗生素类、疫苗类、透析液等制剂以及医疗器材类(如一次性注射器，植入性生物材料)必须经过细菌内毒素检测试验合格后才能使用。
实验例9帕米卡促进巨噬细胞吞噬功能测定
地点：中国医学科学院药物研究所
方法：巨噬细胞的收集：准备20～25克的SPF级昆明小白鼠6只，随机分为2组，每组3只。于0天免疫帕米卡和PBS，每只小鼠滴鼻200μL，2小时后每只小鼠腹腔再注射5.0％鸡红血球的0.85％生理盐水悬液，4小时后每组小鼠各脱臼处死小白鼠3只。消毒后剪开皮肤并经腹膜注射Hanks缓冲液，每只注射2.5mL，轻揉小鼠腹部，使Hanks缓冲液充分涮洗腹腔内的巨噬细胞。然后在腹膜中间部位剪一小孔，用5mL移液管吸取腹腔内液体约2mL置于试管中。
滴片：从试管中无菌吸出腹腔洗液，滴于载玻片上，将滴片水平放入湿纱布上，置37℃恒温培养箱中温育半小时，此时即有大量的巨噬细胞粘附于载玻片上，用0.85％生理盐水冲洗去载玻片上未被吞噬的鸡红细胞及其他组织细胞，冷风吹干。
标本的固定和染色：标本用甲醇固定5分钟，以姬姆萨-瑞氏染液染色。按姬姆萨氏染色法进行，所用PBS缓冲液pH调整为6.5，冷风吹干后用油镜观察并计算。
吞噬百分数：吞噬红血球的巨噬细胞总数÷巨噬细胞总数×100％。
吞噬指数：巨噬细胞吞噬红血球总数÷吞噬红血球的巨噬细胞总数×100％(在油镜下观察100个巨噬细胞，记录每个巨噬细胞吞噬红血球数，求其总和被100除，所得数值即为吞噬指数)。
结果：在PBS对照组吞噬百分数12％，吞噬指数为0.11；帕米卡组吞噬百分数66％，吞噬指数为1.2，表明帕米卡具有强烈刺激巨噬细胞吞噬功能作用，下列图16巨噬细胞吞噬红血球，图17蓝色巨噬细胞(箭头所示)没有吞噬红血球。
实验例10帕米卡滴鼻剂粘膜免疫制剂单独抗流感小白鼠保护试验
流感病毒：亚甲型鼠肺适应株FM1，购于中国预防医学科学院病毒病防治研究所。
病毒唑：阳性对照药，购于沈阳延风药厂。
小白鼠：昆明种，8～10g用于FM1病毒传代，14～20g用于如下正式实验。
将流感病毒FM1株病毒鼠肺悬液用5LD 50/只通过对小白鼠滴鼻可造成致死性肺炎，试验时先感染再给药，按下表进行分组试验。
Figure PCTCN2019093607-appb-000027
该实验结果表明在小白鼠保护试验中本公开单独的滴鼻剂经粘膜免疫途径非特异性抗流感效果就优于公认的抗病毒药病毒唑，统计学分析具有显著的抗流感病毒效果。
实验例11帕米卡粘膜免疫制剂(滴鼻剂)联合流感疫苗经鼻腔粘膜免疫与皮下注射免疫和完全弗氏佐剂比较对体液抗体IgA和对流感病毒繁殖滴度的影响
实验方案如下：
流感病毒：亚甲型鼠肺适应株FM1，购于中国预防医学科学院病毒病防治研究所。
流感疫苗：流感病毒裂解疫苗华兰生物制品有限公司
完全弗氏佐剂：上海伟进生物科技有限公司
本公开的粘膜免疫佐剂：信福(北京)医药科技有限公司
小白鼠：昆明种，8～10g用于FM1病毒传代，14～20g用于如下正式实验。
完全弗氏佐剂流感疫苗：在离心管内，在漩涡混匀加入等体积的疫苗和完全弗氏佐剂呈油包水乳剂。
本公开联合流感疫苗的滴鼻剂：流感疫苗和本公开的粘膜免疫佐剂等量混合呈水溶剂。
流感疫苗：流感疫苗与PBS等量混合成水溶剂。
免疫方法：
皮下注射免疫：皮下0天、28天注射免疫小白鼠，0.1ml/只，第42天，部分小白鼠抽血分离血清，检测抗体滴度，另部分小白鼠通过流感病毒FM1株病毒鼠肺悬液5LD 50/滴鼻感染，感染后第5天，检测肺组织病毒滴度。
鼻腔免疫：滴鼻法0天、28天免疫小白鼠，0.1ml/只，第42天，部分小白鼠抽血分离血清，检测 抗体滴度，另部分小白鼠通过流感病毒FM1株病毒鼠肺悬液5LD 50/滴鼻感染，感染后第5天，检测肺组织病毒滴度。
各组实验结果如下表所示：
粘膜免疫制剂联合流感疫苗滴鼻法抗流感试验
Figure PCTCN2019093607-appb-000028
完全弗氏佐剂是检测促进机体细胞免疫的金标准，试验结果表明皮下免疫本公开粘膜免疫制剂联合流感疫苗产生抗体较完全弗氏佐剂流感疫苗低10倍，但较完全弗氏佐剂流感疫苗降低流感病毒滴度31.6倍；特别是通过小白鼠鼻腔粘膜免疫表明本公开粘膜免疫制剂与抗原结合后的滴鼻剂较单纯流感疫苗抗体高31.6倍，降低流感病毒滴度3100多倍，具有极其显著的效果。
实验例12帕米卡对CIK细胞效靶实验的初步测试具有明确效果
北京京蒙高科干细胞技术有限公司检测本公开对CIK细胞效靶实验的初步测试具有明确效果。
样本编号：JSCIK2016042614；检测日期：2016.04.26；报告日期：2016.04.28。
操作流程：按照常规的CIK细胞效靶实验进行培养和分析，实验方法例如(庄捷等，树突状细胞与CIK细胞共培养诱生的DCCIK细胞群对肿瘤的杀伤作用，细胞生物学杂志，2007,29；237-240)所示。
靶细胞：A549效应细胞：培养JSCIK2016042614
结论：结合荧光显微镜和酶标仪检测方法以及实验本身误差，对JSCIK2016042614细胞杀伤能力进行综合分析，1:10的杀伤效果为中(靶效比为1:10时，对靶细胞A549的杀伤率为51.4％)。
实验例13帕米卡粘膜免疫制剂对荷瘤小白鼠LL2肺癌模型的抗癌作用
用LL2小白鼠移植瘤模型通过鼻腔喷雾检测帕米卡的抗肿瘤效果，该模型瘤细胞生长迅速，接种14天肿瘤体积已达2201.9±68.01mm3，实验即告结束，其中阳性对照顺铂对肿瘤缩小最好，其次是帕米卡肌肉注射组，但帕米卡鼻腔喷雾除0.1mg/只小白鼠外。其余0.2mg/只各鼻腔喷雾组与溶媒阴性对照组比P<0.0001，具有及其显著的差异。特别是与历史资料在相同小白鼠模型中，鼠型PD1几乎没有效果。需要解释的是顺铂组在细胞分裂十分迅速的该模型中更易表现其效力，综合考虑帕米卡在新抗癌机制下的这样的荷瘤小白鼠模型的效果是引人瞩目的。另外在该模型中没有发现对小白鼠的副作用。
实验分组如下表所示：
Figure PCTCN2019093607-appb-000029
Figure PCTCN2019093607-appb-000030
具体实验结果如图18～21所示。
在本次实验中，细胞成瘤率高，溶媒对照组肿瘤生长迅速，实验结束时肿瘤体积达到2201.09±68.01mm 3，阳性对照组顺铂表现出明显的抑瘤效果，表明本次实验成功，结果可信。
试验结果显示，在本次对小鼠肺癌LL2细胞C57BL/6小鼠移植瘤模型的研究中，除帕米卡，150μg/鼠组外，其余组无论是滴鼻还是肌肉给药均表现出明显的抑制肿瘤生长的作用，统计学测定显示具有极其显著差异，P<0.0001；同时对荷瘤小鼠无明显毒副作用。
辉源生物之前的数据显示，鼠源性的PD-1抗体在LL2模型上药效较弱(抑瘤率小于10％)，作为同样作用于免疫系统的帕米卡，展示的效果要优于PD-1抗体。通常认为PD-1抗体的抗肿瘤药效取决于肿瘤细胞PD-L1的表达水平，或者与mutation load,微卫星不稳定性(MSI-H)或者错配修复缺陷(dMMR)有关，进而影响其药物效果。作为免疫佐剂类的帕米卡展示的抑瘤效果，比免疫检查点的范围更为广泛，显示了极大的开发前景。
实验例14帕米卡对4T1-luc小鼠原位乳腺癌模型的体内抗肿瘤效果研究
本次药效学实验共设9个组：溶媒组、PD1组、6个帕米卡治疗组、帕米卡与PD1联合给药组。其中，溶媒组为接种后16天滴鼻给予PBS溶液66.7μL/只，每两天给药一次；PD1组为接种后16天腹腔注射PD1溶液100μg/只，每周给药一次；6个帕米卡治疗组分别为：接种前7天滴鼻给予帕米卡200μg/只，每两天给药一次；接种当天滴鼻给予帕米卡200μg/只，每两天给药一次；接种后16天滴鼻给予帕米卡200μg/只，每两天给药一次；接种后16天滴鼻给予帕米卡300μg/只，每两天给药一次；接种后16天肌注给予帕米卡200μg/只，每两天给药一次；接种后16天肌注给予帕米卡300μg/只，每两天给药一次；帕米卡与PD1联合给药组为接种后16天滴鼻给予帕米卡200μg/只，每两天给药一次+接种后16天腹腔注射PD1溶液100μg/只，每周给药一次。实验采用雌性Balb/c小鼠，每三天测量一次瘤体积，每两天称量一次体重。在溶媒对照组平均瘤体积超过2000mm 3时结束实验，小动物活体成像仪测定肿瘤生物发光情况，最后取各组小鼠脏器进行HE染色。
药效学试验结果显示，提前7天组和第0天组抑制肿瘤生长和转移的能力较弱，两帕米卡肌注组对肿瘤生长和转移的抑制作用强于两帕米卡滴鼻组，除200μg/只帕米卡滴鼻组外，300μg/只帕米卡滴鼻组、200μg/只帕米卡肌注组和300μg/只帕米卡肌注组均能显著的抑制肿瘤生长和转移。实验结束时，200μg/只帕米卡滴鼻组和300μg/只帕米卡滴鼻组的肿瘤抑制率分别为25％和35％。200μg/只帕米卡肌注组和300μg/只帕米卡肌注组的肿瘤抑制率分别为56％和61％。
以上结果说明，帕米卡对4T1乳腺癌具有良好的抑制肿瘤生长和转移的作用。
下面对实验流程和实验结果进行具体论述。
1.实验方法
1.1 4T1-luc小鼠原位乳腺癌瘤模型的构建
取雌性Balb/c小鼠，选择处于对数生长期的4T1-luc乳腺癌细胞，以1×105个/0.2mL/只的数量接种于Balb/c小鼠第四乳腺垫下，构建原位荷瘤小鼠模型。以游标卡尺动态测量瘤块体积。肿瘤体积计算公式：V＝0.5×L×D2(其中，V为肿瘤体积，L为肿瘤长径，D为肿瘤短径)。
1.2给药时间设置
接种前7天帕米卡滴鼻给药组(简称提前7天组)，种瘤前7天随机挑选10只小鼠，按表1开始给药；
接种第0天帕米卡滴鼻给药组(简称第0天组)，种瘤当天随机挑选10只小鼠，按表1开始给药；
溶媒组，肿瘤体积生长至80mm 3左右，即种瘤后第16天，按表1开始给药；
PD1组，肿瘤体积生长至80mm 3左右，即种瘤后第16天，按表1开始给药；
200μg/只帕米卡滴鼻组：肿瘤体积生长至80mm 3左右，即种瘤后第16天，按表1开始给药；
300μg/只帕米卡滴鼻组：肿瘤体积生长至80mm 3左右，即种瘤后第16天，按表1开始给药；
200μg/只帕米卡肌注组：肿瘤体积生长至80mm 3左右，即种瘤后第16天，按表1开始给药；
300μg/只帕米卡肌注组：肿瘤体积生长至80mm 3左右，即种瘤后第16天，按表1开始给药。
表1实验给药剂量及分组
Figure PCTCN2019093607-appb-000031
1.3实验终点
在给药后的第30天因肿瘤体积超过2000mm 3结束整个实验。
1.4观察指标
每三天测量一次瘤体积，每两天称量一次体重。实验终点时剥离心脏、肝脏、脾脏、肺脏、肾脏、肿瘤，其中，心脏、肝脏、脾脏、肾脏用4％中性甲醛固定后，石蜡切片并进行HE染色分析；肿瘤拍照并称重；肺脏用Bouin’s固定液固定16h，然后用50％酒精浸洗2h后拍照，用4％中性甲醛固定后，石蜡切片并进行HE染色分析。
1.5小动物活体成像仪
给药终点时腹腔注射给予100μL浓度为30mg/mL的荧光素底物Luciferin，以异氟烷麻醉小鼠，17min后将小鼠固定于小动物活体成像仪中观察生物发光情况。图像采集参数为：采集时间，0.2秒；Bin值为4；F值为2。图像处理软件：Living
Figure PCTCN2019093607-appb-000032
software(version 4.3.1；Caliper Life Sciences Inc.)。
2.统计学分析
实验数据均以“均值±标准差”表示，数据分析采用SPSS Statistics 19(version 4.0.100.1124；SPSS Inc.,IBM Company,USA)软件。数据比较采用单因素方差分析ANOVA，组间显著性差异采用t检验：*p<0.05；**p<0.01；***p<0.001。
3.实验结果及讨论
3.1肿瘤体积
肿瘤体积变化曲线如图22所示。
表2肿瘤体积t检验结果
Figure PCTCN2019093607-appb-000033
Figure PCTCN2019093607-appb-000034
Note:误差线表示SD；——表示无数据。。*：表示与溶媒组相比p<0.05；**：表示与溶媒组相比p<0.01；***：表示与溶媒组相比p<0.001。
由图22和表2可知，提前7天组和第0天组抑瘤效果较弱，两肌注组的抑瘤效果好于两滴鼻组，具体结果如下。
帕米卡提前7天组和第0天组在后期时具有一定的抑瘤作用，前期基本无抑瘤效果，说明提前给药或者当即给药没有明显的肿瘤杀伤效果，侧面证明帕米卡是一种肿瘤治疗疫苗，而不是预防疫苗。实验结束时，提前7天组和第0天组的肿瘤抑制率分别为36％和26％.
PD1组在第12天时的肿瘤体积与溶媒组相比均有显著性差异(**p<0.01)，说明PD1对4T1小鼠乳腺癌具有一定的抑制作用。
PD1和帕米卡联合给药组各治疗组在给药后20天，大部分小鼠已死亡，因此后期的测定两组均无数据。其中，联合给药组在第6、12、18天时的肿瘤体积与溶媒组相比均有显著性差异(分别为*p<0.05，**p<0.01，***p<0.001)。
200μg/只帕米卡滴鼻组在给药前期抑制效果较明显，给药后期抑瘤效果逐渐减弱，在第6天和第12天的的肿瘤体积与溶媒组相比均有显著性差异(**p<0.01)，300μg/只帕米卡滴鼻组除第18天之外，其余时间时的肿瘤体积与溶媒组相比均有显著性差异。说明滴鼻给药有效且抑瘤作用呈剂量依赖性。实验结束时，200μg/只帕米卡滴鼻组和300μg/只帕米卡滴鼻组的肿瘤抑制率分别为25％和35％。
帕米卡两个肌注剂量组在整个给药过程中的抑瘤效果相似，并且与溶媒组相比均有显著性差异，且两肌注组的抑瘤效果好于两滴鼻组。实验结束时，200μg/只帕米卡肌注组和300μg/只帕米卡肌注组的肿瘤抑制率分别为56％和61％。
3.2体重
小鼠体重变化曲线如图23所示。
表3各组小鼠与溶媒组小鼠体重t检验统计学结果
Figure PCTCN2019093607-appb-000035
Figure PCTCN2019093607-appb-000036
Note:误差线表示SD；——表示无数据。*：表示与溶媒组相比p<0.05；**：表示与溶媒组相比p<0.01；***：表示与溶媒组相比p<0.001。
由图23和表3可知，PD1和联合给药组小鼠体重在有效测定的时间内与溶媒组相比均无显著性差异，提示其副作用较小。提前7天组、第0天组、200μg/只帕米卡滴鼻组和300μg/只帕米卡滴鼻组除在给药后期外，其余时间的体重均明显低于溶媒组。200μg/只帕米卡肌注组在整个给药期间内与溶媒组相比均无显著性差异。300μg/只帕米卡肌注组在给药中期体重低于溶媒组，给药前期和后期与溶媒组相比均无显著性差异。
以上结果说明，肌注给药对小鼠的体重影响较小，副作用较小，滴鼻给药对小鼠具有一定的副作用。
3.3瘤重、脾重及肿瘤照片
由图24和图25可以看出，提前7天组和第0天组的瘤重与对照组相比无显著性差异，两帕米卡肌注组对肿瘤生长的抑制作用强于两帕米卡滴鼻组，除200μg/只帕米卡滴鼻组外，300μg/只帕米卡滴鼻组、200μg/只帕米卡肌注组和300μg/只帕米卡肌注组均能显著的抑制肿瘤生长，与溶媒组相比具有统计学差异(分别为*p<0.05，**p<0.01，***p<0.001)。
脾脏是机体最大的免疫器官，占全身淋巴组织总量的25％，含有大量的淋巴细胞和巨噬细胞，是机体细胞免疫和体液免疫的中心。由图26可以看出，200μg/只帕米卡肌注组和300μg/只帕米卡肌注组脾重均显著高于溶媒组，具有统计学差异(分别为**p<0.01，*p<0.05)，说明肌注组免疫反应可能较强烈。
3.4肺脏照片
由图27和图28可以看出，溶媒组、提前7天组、第0天组肺脏组织表面均有较多白色肿瘤结节，说明提前7天组和第0天组基本无抑制4T1肺转移的作用。200μg/只帕米卡滴鼻组和300μg/只帕米卡滴鼻组肺脏组织表面结节较少，200μg/只帕米卡肌注组和300μg/只帕米卡肌注组肺脏组织表面结节最少，说明此两滴鼻组和两肌注组均能有效抑制4T1肺转移，且肌注组抑制4T1肺转移能力强于滴鼻组。
3.5小动物成像仪
由图29-图35可以看出，溶媒组、提前7天组、第0天组肿瘤部位和转移灶生物发光强度较强，200μg/只帕米卡滴鼻组和300μg/只帕米卡肿瘤部位和转移灶生物发光强度减弱，200μg/只帕米卡肌注组和300μg/只帕米卡肌注组肿瘤部位和转移灶生物发光强度最弱，说明肌注组抑制4T1乳腺癌生长和转移能力强于滴鼻组，与上述结果相符。
4.结论
在本次实验中，我们成功建立了4T1-luc小鼠原位乳腺癌模型，肿瘤生长迅速，实验结束时肿瘤体积超过2000mm 3。
试验结果显示，在本次对小鼠乳腺癌4T1-luc细胞Balb/c小鼠原位肿瘤模型的研究中，除PD1和联合给药组小鼠由于PD1的原因出现了小鼠大面积死亡导致只得到部分实验数据外，其余各组均在一定的时间具有一定的抑瘤作用。提前7天组、第0天组和200μg/只帕米卡滴鼻组抑瘤作用不明显，200μg/只帕米卡滴鼻组、200μg/只帕米卡肌注组和300μg/只帕米卡肌注组均表现出明显的抑瘤作用。
实验例15帕米卡在人乳头瘤病毒(HPV)感染患者治疗上的应用
Figure PCTCN2019093607-appb-000037
实验例16帕米卡在乳腺癌治疗上的应用
1、阳性对照药：
PD1、紫杉醇注射液
2、荷4T1小鼠原位乳腺癌动物模型的构建
取雌性BALB/c小鼠，选择处于对数生长期的4T1-luc乳腺癌细胞，以1×10 5cells/0.2mL/只的数量接种于BALB/c小鼠第四乳腺垫下，构建原位荷瘤小鼠模型。以游标卡尺动态测量瘤块体积。肿瘤体积计算公式：V＝0.5×L×D2(其中，V为肿瘤体积，L为肿瘤长径，D为肿瘤短径)
3、对4T1原位乳腺癌生长和自发转移的影响
3.1组别设置及给药方案
①PBS组，种瘤后数天，PBS滴鼻给药，100μL/只，间隔1天给药1次；
②PD1组：种瘤后数天，PD1腹腔给药，100μg/只，1周给药1次；
③紫杉醇组，种瘤后数天，紫杉醇尾静脉注射给药，10mg/kg，1周1次；
④帕米卡滴鼻组，种瘤后数天，帕米卡滴鼻给药，100μL(0.3mg)/只，间隔1天给药1次，给药当天上午滴鼻50μL，下午滴鼻50μL；
⑤帕米卡肌注组，种瘤后数天，帕米卡肌注给药，100μL(0.3mg)/只，间隔1天给药1次；
⑥联合给药组，种瘤后数天，PD1腹腔(100μg/只，1周给药1次)+帕米卡滴鼻(100μL/只，间隔1天给药1次)；
⑦联合给药组，种瘤后数天，紫杉醇尾静脉注射(10mg/kg，1周给药1次)+帕米卡滴鼻(100μL/只，间隔1天给药1次)。
3.2每组动物数:每组15只(每组多余5只)。
3.3结束实验时间点：根据实际情况(小鼠死亡或者给药组与对照组差异明显)，可能提前结束实验。
3.4实验方案
建立原位4T1-luc乳腺癌模型，以小动物活体成像仪进行早期检测分组，按照6.1项下组别设置和给药方案给药。检测肿瘤体积(3天测1次)、体重变化(3天测1次)、瘤重(终点检测)、脾重(终点检测)、TUNEL染色(终点检测)，肺脏用Bouin’s固定液固定后拍照，各组织器官中性甲醛固定后进行H&E染色(终点检测)，并以小动物活体成像仪观察不同时间点和最终时间点原位肿瘤和转移部位的生物发光强度。综合比较各组之间的抑制原位肿瘤生长和转移的作用强弱。
实验证明，帕米卡在缩小乳腺癌肿瘤体积，控制瘤重、脾重，促进肿瘤细胞的凋亡等方面均具有明显效果。
最后应说明的是：以上各实施例仅用以说明本公开的技术方案，而非对其限制；尽管参照前述各实施例对本公开进行了详细的说明，但本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本公开各实施例技术方案的范围。
工业实用性
本公开的复合物黏度和分子质量适中，制药方便，化学性质稳定，长期储存不易降解，使用安全，该化合物单独使用即可显著地增强机体非特异性的免疫反应，达到防治疾病的目的，与其他药物联合使用具有更好的抗肿瘤、抗病毒和抗(超级)细菌功效，易被患者所吸收。

Claims (20)

  1. 一种用于增强免疫响应的复合物的制备方法，包括：
    将聚肌胞、至少一种阳离子稳定剂以及可溶性钙盐在液体反应体系中接触；
    所述阳离子稳定剂为分子量≤5kDa的水溶性非抗生素氨基化合物，或所述水溶性非抗生素氨基化合物与聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺、叶酸、半乳糖中的一种或多种所形成的接枝物。
  2. 根据权利要求1所述的方法，其中所述水溶性非抗生素氨基化合物选自壳寡糖、几丁寡糖、氨基葡萄糖、阳离子脂质体、DEAE-葡聚糖、聚丙烯酰胺、聚胺、四氨富烯、聚乙烯亚胺中的一种或多种。
  3. 根据权利要求1或2所述的方法，其中所述阳离子稳定剂选自壳寡糖、壳寡糖与聚乙二醇单甲醚的接枝物、壳寡糖与聚乙二醇单甲醚和聚乙烯亚胺的接枝物。
  4. 根据权利要求1-3中任一项所述的方法，其中所述接枝物的分子量≤50kDa。
  5. 根据权利要求1-4中任一项所述的方法，其中，在所述液体反应体系中，所述聚肌胞的浓度为0.1～10mg/ml；
    可选的，在所述液体反应体系中，所述聚肌胞的浓度为0.5～5mg/ml。
  6. 根据权利要求1-5中任一项所述的方法，其中，在所述液体反应体系中，所述阳离子稳定剂的浓度为0.5～51.2mg/ml；
    可选的，在所述液体反应体系中，所述阳离子稳定剂的浓度为0.8～25.6mg/ml。
  7. 根据权利要求1-6中任一项所述的方法，其中，在所述液体反应体系中，所述可溶性钙盐中钙离子的浓度为0.1～1mM。
  8. 根据权利要求1-7中任一项所述的方法，其中，所述壳寡糖脱乙酰度大于等于70％。
  9. 根据权利要求1-8中任一项所述的方法，其中，所述可溶性钙盐选自CaCl 2和/或CaNO 3。
  10. 根据权利要求1-9中任一项所述的方法，其中，所述聚肌胞由聚胞苷酸和聚肌苷酸经碱基配对反应制得。
  11. 根据权利要求10所述的方法，其中，所述聚胞苷酸、聚肌苷酸分子量大于2.3万道尔顿；
    可选的，所述聚胞苷酸的分子量范围是在6.6万道尔顿～66万道尔顿；
    可选的，所述聚肌苷酸的分子量范围是在6.6万道尔顿～66万道尔顿。
  12. 根据权利要求10或11所述的方法，其中，所述碱基配对反应在40℃～50℃的温度下进行。
  13. 根据权利要求10-12中任一项所述的方法，其中，所述碱基配对反应在pH＝6.8～7.6下进行。
  14. 根据权利要求1-13中任一项所述的方法，其中，在进行接触反应之前，将所述聚肌胞80℃～99℃下加热70～120min；
    可选的，在进行接触反应之前，所述聚肌胞88℃～92℃下加热70～120min。
  15. 根据权利要求1-14中任一项所述的方法，其中，所述液体反应体系的温度为40℃～50℃。
  16. 根据权利要求1-15中任一项所述的方法，其中，所述接枝物的制备方法包括：
    先用羰基二咪唑活化聚乙二醇单甲醚、聚乙二醇、聚乙烯亚胺、叶酸、半乳糖中的一种或多种，再用活化好的产物与所述水溶性非抗生素氨基化合物在离子液体[bmim]Cl中进行接枝反应；
    可选的，所述接枝物为壳寡糖与聚乙二醇单甲醚的接枝物，先用羰基二咪唑(CDI)活化聚乙二醇单甲醚(MPEG)，再用活化好的MPEG与壳寡糖(COS)在离子液体[bmim]Cl中进行接枝反应；
    可选的，所述接枝反应在60℃～80℃、非氧化气氛下反应。
  17. 根据权利要求1-16中任一项所述的方法，其中，所述方法还包括：
    将交联剂溶液在搅拌条件下逐滴加入制得的复合物中直至观察到反应体系中出现丁达尔现象后停止滴加，搅拌得到纳米粒子；
    所述交联剂选自三聚磷酸钠、海藻酸钠、苯基硼酸、苯邻二酚中的至少一种；
    可选的，所述交联剂溶液中还含有免疫细胞治疗药物、抗体治疗药物、化学药物、促进粘膜免疫吸收或粘膜粘附的物质、免疫调节剂、抗原、膜式识别受体的配体、药物可接受的盐或赋形剂中的一种或多种；
    可选的，所述方法还包括，将所述复合物或所述纳米粒子与免疫细胞治疗药物、抗体治疗药物、化学药物、促进粘膜免疫吸收或粘膜粘附的物质、免疫调节剂、抗原、膜式识别受体的配体、药物可接受的盐或赋形剂中的一种或多种共孵育；
    可选的，所述抗原均为蛋白质或多肽抗原；
    可选的，所述抗原均包括肿瘤、病毒、细菌、真菌或寄生虫抗原；
    可选的，所述肿瘤包括：骨、骨连接、肌肉、肺、气管、咽、鼻、心脏、脾脏、动脉、静脉、血液、毛细血管、淋巴结、淋巴管、淋巴液、口腔、咽、食管、胃、十二指肠、小肠、结肠、直肠、肛门、阑尾、肝、胆、胰腺、腮腺、舌下腺、泌尿肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、外阴部、阴囊、睾丸、输精管、阴茎、眼、耳、鼻、舌、皮肤、脑、脑干、延髓、瘠髓、脑瘠液、神经、甲状腺、甲状旁腺、肾上腺、垂体、松果体、胰岛、胸腺、性腺、舌下腺、腮腺中任一处病变生成的肿瘤；
    可选的，所述细菌包括：葡萄球菌属、链球菌属、李式杆菌属、丹毒丝菌属、肾杆菌属、芽孢杆菌属、梭菌属、分支杆菌属、放线菌属、奴卡菌属、棒状杆菌属、红球菌属、炭疽杆菌、丹毒杆菌、破伤风杆菌、李氏杆菌、产气荚莫杆菌、气肿疽杆菌结核杆菌、大肠杆菌外、变形杆菌、痢疾杆菌、肺炎杆菌、布氏杆菌、产气夹膜杆菌、流感嗜血杆菌、副流感嗜血杆菌、卡他摩拉克氏菌、不动杆菌属、耶尔森菌属、嗜肺军团菌、百日咳杆菌、副百日咳杆菌、志贺菌属、巴斯德菌属、霍乱弧菌、副溶血性杆菌中的一种或多种；
    可选的，所述寄生虫包括：消化道内寄生虫、腔道内寄生虫、肝内寄生虫、肺内寄生虫、脑组织寄生虫、血管内寄生虫、淋巴管内寄生虫、肌肉组织寄生虫、细胞内寄生虫、骨组织寄生虫、眼内寄生虫中的一种或多种；
    可选的，所述病毒包括：腺病毒(adeniviridae)、沙粒病毒(arenaviridae)、星状病毒(astroviridae)、本扬病毒(bunyaviridae)、杯状病毒(cliciviridae)、黄病毒(flaviviridae)、D型肝炎病毒(hepatitis delta virus)、肝炎病毒(hepeviridae)、单分子负链RNA病毒(mononegavirales)、巢病毒(nidovirales)、小RNA病毒(聚胞苷酸conaviridae)、正黏液病毒(orthomyxoviridae)、乳头瘤病毒(pa聚胞苷酸llomaviridae)、细小病毒(parvoviridae)、多瘤病毒(polyomaviridae)、痘病毒(poxviridae)、呼肠孤病毒(reoviridae)、反转录病毒(retroviridae)或披膜病毒(togaviridae)中的一种或多种；
    可选的，所述真菌包括：粗球孢子菌、普赛德斯球抱子菌、荚膜组织胞浆菌、杜氏组织胞浆菌、洛博芽生菌、巴西副球孢子菌、皮炎芽生菌、申克氏孢子丝菌、马尔尼菲青霉菌、白色念珠菌、光滑念珠菌、热带念珠菌、葡萄牙假丝酵母、卡氏肺孢子虫病、曲霉菌、甄氏外瓶霉、裴氏着色霉、紧密着色霉、疣状着色霉、皮炎着色霉、白地霉、波氏足肿菌、新型隐球菌、丝孢酵母菌、米根霉、印度毛霉、伞枝犁头霉、总状共头霉、蛙粪霉、冠状耳霉、异孢耳霉、比氏肠胞微孢子虫、肠脑炎微孢子虫、西伯鼻孢子菌、透明丝孢霉、暗色丝孢霉中的一种或多种；
    可选的，所述免疫细胞治疗药物选自肿瘤浸润淋巴细胞、树突状细胞、细胞因子诱导杀伤细胞、树突细胞-细胞因子诱导的杀伤细胞、自然杀伤细胞、γδT细胞、CD3AK、CAR-T和TCR-T中的一种或多种；
    可选的，所述抗体治疗药物选自抗PD1抗体、抗PDL1抗体、抗CTLA4抗体和抗CD抗原抗体；
    可选的，所述化学药物选自烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗肿瘤药、激素类药物和杂类药物中的一种或多种；
    其中所述杂类药物选自左旋门冬酰胺酶、顺铂、卡铂、草酸铂、氮烯咪胺、六甲嘧胺类药物，或上述药物的衍生物；
    可选的，所述促进粘膜免疫吸收或粘膜粘附的物质选自阴离子表面活性剂、阳离子表面活性剂、两性离子表面活性剂、非离子表面活性剂、特种表面活性剂、螯合剂、粘合剂、聚乳酸-羟基乙酸共聚物、右旋糖酐、多聚糖中的一种或多种；
    可选的，所述免疫调节剂选自细胞因子、趋化因子、干细胞生长因子、淋巴毒素、造血因子、集落刺激因子(CSF)、干扰素、促红细胞生成素、促血小板生成素、肿瘤坏死因子(TNF)、白介素(IL)、粒细胞-集落刺激因子(G-CSF)、粒细胞巨噬细胞-集落刺激因子(GM-CSF)和干细胞生长因子中的一种或多种；
    可选的，所述膜式识别受体的配体选自TLR受体的配体、RLR受体的配体、CLR受体的配体、NLR受体的配体。
  18. 根据权利要求1-17中任一项所述的方法制备的用于增强免疫响应的复合物。
  19. 根据权利要求1-17中任一项所述的方法制备的用于增强免疫响应的复合物，用于制备抗体、疫苗制剂或疫苗组合物，增强免疫细胞活性，治疗和/预防肿瘤、抗病毒、抗细菌、抗真菌、抗寄生虫、降低化疗副作用、抗疲劳或提升免疫力、缓解宿主疼痛、促进宿主对于抗原的免疫反应中的应用。
  20. 一种用以促进宿主体内对于抗原的免疫反应，或调节增强宿主免疫细胞活性，或帮助宿主降低 疲劳度，或减轻宿主疼痛的方法，所述方法包括将根据权利要求1-17中任一项所述的方法制备的用于增强免疫响应的复合物，或包括根据权利要求1-17中任一项所述的方法制备的用于增强免疫响应的复合物以及至少一种抗原的疫苗组合物，或包括根据权利要求1-17中任一项所述的方法制备的用于增强免疫响应的复合物的药物组合物给予该宿主，
    其中所述药物组合物还包括免疫细胞治疗药物、抗体治疗药物、化学药物、促进粘膜免疫吸收或粘膜粘附的物质、免疫调节剂、病原体抗原、膜式识别受体的配体、药物可接受的赋形剂中的一种或多种。
PCT/CN2019/093607 2018-06-29 2019-06-28 用于增强免疫响应的复合物的制备方法 WO2020001596A1 (zh)

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