Related application of MA105 adjuvant in tumor treatment

Abstract

The invention discloses a related application of an MA105 adjuvant in tumor treatment, and relates to the technical field of medical biology.The MA105 adjuvant comprises 50-300 [mu] g / ml of QS-21, 400-3000 [mu] g / ml of Poly I: C and lipid molecules forming a carrier; the MA105 adjuvant can be independently applied, is administrated in an intratumor injection manner, can obviously stimulate immune cells in tumors, especially tumor killing type immune cells, and activates tumor immunity, so that the effect of treating the tumors is achieved.

Description

[0001] This case is a divisional application. The application date of the original application is August 17, 2022, the application number is 202280092157.4, and the invention name is "A vaccine adjuvant, its preparation method and application". This application claims the priority of the Chinese patent application with application number 202111627083.3 and name "A herpes zoster vaccine preparation and its preparation method" filed with the Chinese Patent Office on December 28, 2021, the Chinese patent application with application number 202111627088.6 and name "A method for preparing a QS21 immune adjuvant" filed with the Chinese Patent Office on December 28, 2021, and the Chinese patent application with application number 202210358103X filed with the Chinese Patent Office on April 6, 2022, all of which are incorporated by reference in this application. Technical Field

[0002] The present invention relates to the field of pharmaceutical biotechnology, and in particular to the application of MA105 adjuvant in treating tumors. Background Art

[0003] 1. Immune adjuvant

[0004] In immunology, an adjuvant is a component that enhances the immune response to an antigen and / or modulates the immune response. An immunological adjuvant is defined as any substance that accelerates, prolongs, or enhances the antigen-specific immune response when combined with a specific vaccine antigen. There are many known adjuvants in widespread use, including oils, aluminum salts, and viroids. It is generally believed that activation of cellular immunity (especially CD4 + T cells) can produce more effective therapeutic effects.

[0005] 1.1. QS21 adjuvant

[0006] The structural formula of Quillaja saponin 21 (QS21) is as follows: molecular formula: C92H148O46, molecular weight: 1990.14, and its general properties are colorless transparent liquid.

[0007]

[0008] In the cancer patient population, QS-21 has the best activity and good tolerability in the range of 100-200 μg. The toxicity in this dose range is erythema and induration of 2-10 cm at the injection site in most patients, as well as occasional mild low-grade flu-like symptoms. No dose-limiting toxicity has been reported in this dose range. QS-21 is a new pharmaceutical adjuvant with high safety and good tolerability.

[0009] 1.2 Liposomes

[0010] Liposomes are artificial cell membrane-like spheres composed of lipids. They are monolayer or multilayer bimolecular vesicle structures mainly composed of phospholipids, cholesterol, stearylamine, etc. They can encapsulate a variety of vaccines and effectively introduce them into cells. They tend to be deposited in the reticuloendothelial macrophage system such as the liver, spleen, and lymph nodes in the body, prolonging their residence time in the body, reducing the amount of vaccine used, reducing toxic side effects, and improving immune function. It has obvious adjuvant and carrier effects on a variety of antigens and antibodies, and is one of the more ideal vaccine adjuvant substances.

[0011] 1.3. Immune adjuvants and tumor immunotherapy

[0012] Immunotherapy (including immune checkpoint inhibitor antibodies, immunotherapy, anti-tumor vaccines, etc.) is the current trend and development hotspot in clinical cancer treatment. Current cancer immunotherapy still has many limitations, such as low efficiency of tumor vaccine release, blocked cross-presentation of tumor tissue-specific antigens, and the effects of a series of immunosuppressive cytokines.

[0013] In the development of tumor vaccine products, immune adjuvants play a very important role in improving the immunogenicity of tumor antigens and activating the body's adaptive immune response. As a substance that improves the body's adaptive immune response to antigens, adjuvants can make up for the problem of weak immunogenicity of tumor antigens, enhance the immune response to tumor antigens, and effectively avoid the immune tolerance induced by immature antigen presenting cells (APCs) after contact with antigens, thereby improving the immunotherapy efficiency of tumor vaccines.

[0014] Therefore, the research on adjuvants in tumor immunotherapy has developed rapidly. In addition to traditional aluminum salt adjuvants, new adjuvants such as pattern recognition receptor agonists (such as Toll-like receptor agonists, C-type agglutination receptor agonists), polymer materials, and peptides have been found to have good immune activation effects.

[0015] 2. Shingles vaccine

[0016] In 2017, the Advisory Committee on Immunization Practices (ACIP) of the Centers for Disease Control and Prevention (CDC) of the United States issued an opinion recommending the GlaxoSmithKline recombinant subunit vaccine Replaces Merck's live attenuated vaccine Used for immunization of elderly people aged 50 years and above.

[0017] Varicella-zoster live attenuated vaccine produced by Merck The protective efficacy of the vaccine gradually decreases with the increase of the age of the vaccine recipients. The Phase III clinical results show that the overall protection rate against herpes zoster is about 64% in the 60-69 age group, while in the 70+ age group, the protection rate drops to 38%. At the same time, the vaccine cannot be used for people with impaired immune systems and cannot provide effective protection. It is a recombinant subunit vaccine, the core technology of which comes from its AS01 B Adjuvant system and specific antigens. The results of the Phase III clinical trial showed that the overall effectiveness of the vaccine was 97.2%, and the protection rate in people aged ≥70 years could reach 97.5%. The number of antigen-specific CD4+ T cells induced is approximately Induce 10-fold the number of cells.

[0018] Although aluminum adjuvant is a commonly used vaccine adjuvant, studies have shown that aluminum adjuvant mainly induces Th2 cell-mediated humoral immune response, and cannot induce Th1 cell-mediated cellular immune response (cannot induce the expression of cytokines such as IL-2 and INF-γ related to cellular immunity). Merck's inactivated varicella-zoster vaccine formulated with aluminum adjuvant was ultimately not marketed because its immunogenicity did not meet expectations. This further illustrates that adjuvants that can induce cellular immune responses (especially CD4+T cells) are the key factor in the high protectiveness of varicella-zoster vaccines.

[0019] In view of this, the present invention is proposed. Summary of the invention

[0020] The purpose of the present invention is to provide relevant applications of MA105 adjuvant in treating tumors.

[0021] The present invention is achieved in that:

[0022] The present invention firstly relates to an MA105 immune adjuvant, which comprises:

[0023] (1) QS-21, 50-300 μg / ml;

[0024] (2) Poly I:C, 400-3000 μg / ml; and

[0025] (3) lipid molecules that serve as common carriers for adjuvants and antigens;

[0026] The carrier is a liposome carrier, which can be a cationic liposome or a neutral liposome or a mixture of the two.

[0027] Preferably, the carrier is a mixture of cationic liposomes and neutral liposomes;

[0028] Preferably, the MA105 adjuvant comprises:

[0029] (1) QS-21, 50-200 μg / ml;

[0030] (1)Poly I:C, 400-3000μg / ml;

[0031] The lipid molecules are:

[0032] (3) DOTAP, 70-560 μg / ml;

[0033] (4) DOPC, 500-4000 μg / ml;

[0034] (5) Cholesterol, 175-1400 μg / ml.

[0035] More preferably, the MA105 adjuvant comprises:

[0036] (1) QS-21, 80-120 μg / ml;

[0037] (2)Poly I:C, 780-1600μg / ml;

[0038] The lipid molecules are:

[0039] (3) DOTAP, 140-280 μg / ml;

[0040] (4) DOPC, 1200-2500 μg / ml;

[0041] (5) Cholesterol, 350-600 μg / ml.

[0042] The present invention also relates to a method for preparing the MA105 adjuvant, which comprises the following steps:

[0043] (1) preparing cationic liposomes and neutral liposomes respectively;

[0044] (2) QS-21, Poly I:C, cationic liposomes, and neutral liposomes were mixed and stirred evenly to prepare MA105 adjuvant.

[0045] The preparation method of the cationic liposome is:

[0046] (1) 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP), dioleoylphosphatidylcholine (DOPC) and cholesterol were prepared in a mass ratio of 2:2:1;

[0047] (2) After the three lipid components are evenly dispersed, liposomes are prepared using ethanol injection method, thin film dispersion method, ultrasonic dispersion method, and reverse evaporation method;

[0048] Preferably, the preparation is carried out by ethanol injection method, and the preparation method is as follows: after the three lipid components are evenly dispersed, colostrum is prepared in the aqueous phase, and after ultrafiltration replacement of the buffer solution, extrusion molding is performed using extrusion membranes with pore sizes of 200nm and 100nm in sequence; finally, filtration and sterilization are performed through a sterile filter.

[0049] More preferably, the preparation steps of the cationic liposomes are as follows:

[0050] (1) The three lipid components are dissolved in ethanol and sheared online with the aqueous phase for 1 to 3 times;

[0051] (2) Ultrafiltration replacement using a buffer solution containing sucrose, with a concentration multiple of 2 to 4 times and a diafiltration multiple of 6 to 8 times;

[0052] (3) using a filter membrane with a pore size of 200 nm for extrusion, performing multiple cycles of extrusion to control the uniformity of particle size;

[0053] (4) using a filter membrane with a pore size of 100 nm for extrusion, performing multiple cycles of extrusion to control the uniformity of particle size;

[0054] (5) Sterilize by filtering using a sterile filter.

[0055] The physicochemical parameters of the cationic liposome are:

[0056] Average particle size (D50): 30~90nm;

[0057] Solution pH: 4.8-7.0; preferably 4.8-6.0;

[0058] Zeta potential: 40~72mV (pH6.5).

[0059] The preparation method of the neutral liposome is:

[0060] (1) dioleoylphosphatidylcholine (DOPC) and cholesterol are prepared in a mass ratio of 4:1;

[0061] (2) After the lipid components are evenly dispersed, liposomes are prepared using ethanol injection method, thin film dispersion method, ultrasonic dispersion method, and reverse evaporation method;

[0062] Preferably, the preparation is carried out by using an ethanol injection method, wherein the preparation method is as follows: colostrum is prepared in an aqueous phase after being evenly dispersed, and extrusion molding is performed using extrusion membranes with pore sizes of 200 nm and 100 nm in sequence, followed by ultrafiltration to replace the buffer solution, and filtration to sterilize.

[0063] More preferably, the preparation steps of the neutral liposome are as follows:

[0064] (1) The two lipid components are dissolved in ethanol and sheared online with the aqueous phase for 1 to 3 times;

[0065] (2) Filtering with a filter membrane with a pore size of 200 nm and performing multiple cycles of extrusion to control the uniformity of particle size;

[0066] (3) Filtering with a filter membrane with a pore size of 100 nm and performing multiple cycles of extrusion to control the uniformity of particle size;

[0067] (4) ultrafiltration replacement using a buffer solution containing sucrose, with a concentration multiple of 3 to 6 times and a diafiltration multiple of 6 to 8 times;

[0068] (5) Sterilize by filtering using a sterile filter.

[0069] The physicochemical parameters of the neutral liposomes are:

[0070] Average particle size (D50): 80~140nm;

[0071] Solution pH: 5.5-7.0, preferably 6.4-6.6.

[0072] The MA105 adjuvant system contains two immunopotentiators (QS-21 and Poly I:C). In order to reduce the toxicity of QS-21 and Poly I:C and improve immunogenicity, a mixture of cationic liposomes and neutral liposomes is used to adsorb QS-21 and Poly I:C, or a mixed liposome of cationic liposomes and neutral liposomes adsorbed with QS-21 is used to further adsorb Poly I:C. The signal transduction of Poly I:C mainly depends on Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA-5), which can strongly drive cellular immunity and effective type I interferon response, and can induce strong antigen-specific CD4+T and CD8+T cell responses through type I interferon (IFN-α / β) signal transduction and antigen cross-presentation. We used cationic liposomes (composed of DOTAP, DOPC and cholesterol) as carriers to adsorb Poly I:C through electrostatic interaction.

[0073] The present invention also relates to the use of the MA105 adjuvant in the preparation of a vaccine preparation, wherein the vaccine preparation contains a therapeutically effective amount of an antigen and the MA105 adjuvant and necessary pharmaceutical excipients; preferably, the vaccine preparation is a recombinant herpes zoster vaccine preparation.

[0074] The pharmaceutical excipients include, but are not limited to, polysorbate 80, histidine, sodium dihydrogen phosphate, sucrose, and sodium chloride.

[0075] The present invention also relates to a method for preparing a recombinant herpes zoster vaccine preparation using the MA105 adjuvant system, wherein the method comprises the following steps:

[0076] (1) adding sucrose, histidine and Tween-80 to the purified antigen solution according to the formula of the antigen buffer, and adjusting the pH to obtain an antigen stock solution;

[0077] (2) mixing the antigen stock solution and the MA105 adjuvant and stirring them evenly to form a semi-finished product;

[0078] (3) After packaging, the finished product of the recombinant herpes zoster vaccine preparation is obtained.

[0079] The antigen stock solution comprises: gE protein antigen (molecular weight 63530.6, isoelectric point 4.8-5.8) with a sequence as shown in SEQ ID NO.1;

[0080] SEQ ID NO.1:

[0081] SVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDV FKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPG VLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNS GCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRY.

[0082] Preferably, calculated per milliliter of vaccine preparation, the recombinant herpes zoster vaccine preparation prepared with the MA105 adjuvant system comprises:

[0083] (1) a gE protein antigen with a sequence as shown in SEQ ID NO.1, with a content of 95-110 ug / ml;

[0084] (2) A pharmaceutical excipient combination for maintaining the stability of gE protein antigen and liposome, the pharmaceutical excipient combination comprising:

[0085] 1) Polysorbate 80, 450-550 μg / ml;

[0086] 2) Histidine, 200-950 μg / ml;

[0087] 3) sucrose, 52-55 mg / ml;

[0088] (3) MA105 immune adjuvant system, comprising:

[0089] 1) DOTAP, 140-215 μg / ml;

[0090] 2) DOPC, 1200-2200 μg / ml;

[0091] 2) Cholesterol, 320-550 μg / ml;

[0092] 3) QS-21, 80-110 μg / ml;

[0093] 4) Poly I:C, 780-830 μg / ml;

[0094] (4) Other pharmaceutical excipients, including:

[0095] 1) Sodium dihydrogen phosphate, 0-800 μg / ml;

[0096] 2) Sodium chloride, 2.5-6 mg / ml.

[0097] Further;

[0098] The osmotic pressure of the vaccine preparation is 210-350 mOsmol / kg;

[0099] The average particle size of the vaccine preparation is 100 to 300 nm, preferably 130 to 200 nm;

[0100] The pH value of the vaccine preparation is 6.0-7.0.

[0101] The invention also relates to a recombinant herpes zoster vaccine preparation prepared by the method.

[0102] The present invention also relates to the following application of the recombinant herpes zoster vaccine preparation: treating or preventing diseases caused by varicella zoster virus (VZV), wherein the treatment is a single treatment or combined with other drugs or vaccines.

[0103] The present invention also relates to an anti-tumor vaccine product, which comprises:

[0104] (1) an effective amount of a tumor antigen, wherein the tumor antigen is a recombinant polypeptide antigen;

[0105] (2) MA105 adjuvant;

[0106] (3) Necessary pharmaceutical excipients.

[0107] Preferably, the anti-tumor vaccine product is a subcutaneous or intramuscular injection vaccine;

[0108] Preferably, the tumor antigen is an HPV antigen whose amino acid sequence is shown in SEQ ID NO.2, or an HPV antigen whose amino acid sequence is shown in SEQ ID NO.3.

[0109] SEQ ID NO.2:

[0110] HQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKVSEYRYYCYSLY GTTLEQQYNKPLCDLLIRGINCQKPLCPDEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLgsgsgsgsgsgsgMHGDTPTLHEYMLDL QPETTDLYGYGQLHDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP;

[0111] SEQ ID NO.3:

[0112] ARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAGHKCIDFYSRIRELRHYSDSVYGDTLEKL TNTGLYNLLIRGLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQVgsgsgsgsgsgsgMHGPKATLQDIVLHLEPQ NEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVGPWCASQQ.

[0113] Preferably, the pharmaceutical excipients include: stabilizers, lyophilization protectants, and osmotic pressure regulators.

[0114] More preferably, the pharmaceutical excipients include: polysorbate 80, sucrose, and sodium chloride.

[0115] The present invention also relates to an anti-tumor preparation, comprising:

[0116] (1) an effective amount of MA105 adjuvant;

[0117] (2) Necessary pharmaceutical excipients.

[0118] Preferably, the anti-tumor preparation is an intratumor injection preparation or a tumor in situ vaccine;

[0119] Preferably, the tumor is melanoma, colorectal cancer or lung cancer.

[0120] Preferably, the pharmaceutical excipients include: stabilizers, lyophilization protectants, and osmotic pressure regulators.

[0121] More preferably, the pharmaceutical excipients include: polysorbate 80, sucrose, sodium chloride, histidine, and sodium dihydrogen phosphate.

[0122] The present invention also relates to the use of the MA105 immune adjuvant or a preparation containing the MA105 immune adjuvant in the preparation of an anti-tumor combination preparation, wherein the combination preparation comprises a therapeutically effective amount of the MA105 immune adjuvant and other drugs selected from any of the following:

[0123] (1) Cytotoxic chemotherapy drugs;

[0124] (2) Antibody anti-tumor drugs;

[0125] (3) Oncolytic peptides and oncolytic virus drugs;

[0126] (4) Cell therapy preparations, preferably Car-T cell therapy preparations.

[0127] When used as a therapeutic preparation, the MA105 adjuvant of the present invention or the vaccine composition containing the MA105 adjuvant can be administered orally or parenterally. In the case of parenteral administration, the adjuvant or vaccine composition can be administered by in situ injection, in situ injection of the lesion, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc.

[0128] When the MA105 adjuvant of the present invention without antigen or the tumor in situ vaccine prepared by using the MA105 adjuvant is administered, intratumoral injection is adopted to activate the immune protection of the subject.

[0129] The present invention also relates to a method for preparing a Quillaja saponin 21 (QS21) immune adjuvant using semi-purified commercial grade or food additive grade semi-purified saponin as a raw material, the method comprising the following steps:

[0130] (1) Saponin dissolution and filtration

[0131] Dissolve the saponin raw material with a diluent, and then filter out the insoluble matter;

[0132] (2) Reverse phase crude purification

[0133] Take the saponin filtered sample solution obtained in step (1) and perform reverse phase crude purification according to the following steps:

[0134] 1) Sample loading: Load the sample onto UniPSN 30-300 chromatography medium;

[0135] 2) Elution: Elute the target peak with a linear gradient from 2.5% B to 21.25±5.00% B, collect high-purity samples in sections, and combine the crude pure saponin solution;

[0136] (3) Reverse Phase Purification

[0137] Take the crude pure saponin solution obtained in step (2) and perform reverse phase purification according to the following steps:

[0138] 1) Loading: dilute the sample solution with water to an acetonitrile concentration of 22% to 28%, and load it onto the UniPS10-300 chromatographic medium;

[0139] 2) Elution: 10% B to 25±5% B linear gradient elution of the target peak, collect high-purity samples in sections and merge;

[0140] 3) Repeating the above steps 1) to 2) for 1 to 3 times with the combined samples, preferably, repeating them twice;

[0141] (4) Precipitation and re-dissolution step

[0142] 1) taking the purified saponin sample solution prepared in step (3), adding 2 times the volume of water and mixing, leaving it at room temperature and then centrifuging to collect the precipitate;

[0143] 2) adding a dissolving solution to the precipitate, stirring and dissolving, and then removing the organic solvent sample. Preferably, the amount of the dissolving solution added is the same as the amount of the purified saponin sample solution prepared in step (3);

[0144] (5) Ultrafiltration replacement step

[0145] 1) taking the saponin sample solution obtained after precipitation and redissolution in step (4), and ultrafiltration and concentration of the ultrafiltration replacement solution to obtain a concentrated QS21 adjuvant solution; preferably, ultrafiltration and concentration to 30% to 50% of the original volume;

[0146] In the above steps (1) to (5):

[0147] The diluent was an aqueous solution containing 30% acetonitrile, 5 mM citric acid, pH 5.0;

[0148] Solution B is an aqueous solution containing 99% acetonitrile and 0.1% TFA;

[0149] The dissolving solution is an aqueous solution containing 5 mM histidine at pH 6.0;

[0150] The ultrafiltration replacement fluid was an aqueous solution containing 5 mM histidine at pH 5.0.

[0151] In the saponin raw material, the QS21 content is not less than 8%; the moisture content is not higher than 10%; and the ash content is not higher than 3%;

[0152] The yield of QS21 purified by the method is not less than 50%, and the purity of the finally obtained QS21 is not less than 94%.

[0153] Further:

[0154] In step (1), the saponin raw material is dissolved at a ratio of 1:25 (W / V), and then filtered through a 1.0+0.45 μm filter;

[0155] In step (2), the sample loading capacity is 30-60 mg / ml, the linear flow rate is 90-360 cm / h, and after the sample loading is completed, the sample is washed with A1 solution and the impurities are washed with 2.5% B solution; during gradient elution, samples with a purity higher than 30% are collected and combined;

[0156] In step (3), the sample loading capacity is 10-50 mg / ml, and after the sample loading is completed, the sample is washed with A2 solution, and the impurities are washed with 10% B. The sample with a purity higher than 60% is collected in the first reverse-phase purification, the sample with a purity higher than 80% is collected in the second reverse-phase purification, the sample with a purity higher than 90% is collected in the third reverse-phase purification, and the sample with a purity higher than 95% is collected in the fourth reverse-phase purification;

[0157] In step (4), the mixture is placed at room temperature for not less than 30 min, and the centrifugation parameters are: 9000-11500 g, 2-8° C., 10 min;

[0158] In step (5), the ultrafiltration parameters are: using a 5 kD membrane package, a transmembrane pressure of 0.2 to 0.4 bar, 10 replacement times, and washing with an ultrafiltration replacement fluid of not less than 8 volumes;

[0159] in:

[0160] Solution A1 is an aqueous solution containing 30% acetonitrile and 0.1% TFA;

[0161] Solution A2 is an aqueous solution containing 20% ​​acetonitrile and 0.1% TFA.

[0162] The present invention has the following beneficial effects:

[0163] 1. The MA105 adjuvant system includes mixed liposomes and two immunopotentiators: Poly I:C and QS-21; it has good safety and can induce high levels of antibodies and high frequency CD4+T cell responses.

[0164] 1. When MA105 adjuvant is administered alone by intratumoral injection, it can significantly stimulate the immune cells in the tumor, especially the tumor-killing immune cells, activate tumor immunity, and thus achieve the effect of treating tumors;

[0165] 2. When MA105 adjuvant is used in combination with tumor antigens, only a low dose of antigen is needed to stimulate the body to produce specific cellular immunity and generate memory-type immune protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0166] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0167] Figure 1 Turbidity analysis of the antigen preparations with different formulations after oscillation for 4 hours (the turbidity is detected using a turbidimeter, and the vertical axis unit in the figure is NTU);

[0168] Figure 2 The peak area changes of the polymers after different freeze-thaw times of antigen preparations with different formulations. The peak area changes in the figure are detected by SEC-HPLC (the ordinate is percentage, for example, a change of 0.25 in the ordinate means a change of 0.25% in the peak area); the data of 0 freeze-thaw times have slight changes because there will be certain detection deviations when the proteins are prepared into different formulations and tested before freeze-thaw;

[0169] Figure 3 Analysis of serum antibody subtype IgG2a titer 14 days after the second immunization of each test group;

[0170] Figure 4 Cellular immunity analysis (CD4 + T cell response);

[0171] Figure 5 Cellular immunity analysis (CD4 + T cell response);

[0172] Figure 6 The preparation process roadmap of MA105 adjuvant;

[0173] Figure 7 This is a transmission electron micrograph of the recombinant herpes zoster vaccine;

[0174] Figure 8 This is the particle size distribution diagram of the recombinant herpes zoster vaccine;

[0175] Fig. 9 This is the tumor growth curve of TC-1 tumor-bearing mice after immunization with MA105 adjuvant + antigen;

[0176] Fig.10 This is the body weight change curve of TC-1 tumor-bearing mice after immunization with MA105 adjuvant + antigen;

[0177] Fig.11 The IFN-γ secretion level in TC-1 tumor-bearing mice after immunization with MA105 adjuvant + antigen;

[0178] Fig.12 The antitumor activity of MA105 adjuvant alone (B16F10 tumor model, melanoma);

[0179] Fig.13 The antitumor activity of MA105 alone as an adjuvant (MC38 tumor model, colorectal cancer);

[0180] Fig.14 The antitumor activity of MA105 alone as an adjuvant (LLC tumor model, lung cancer);

[0181] Fig.15 MA105 adjuvant can significantly improve the therapeutic effect of anti-tumor antigens; A, tumor volume after immunization; B, mouse weight after immunization;

[0182] Fig.16 After immunization with MA105 adjuvant + antigen, mice developed significant immune protection;

[0183] Fig.17 This is the HPLC chromatogram of the QS21 sample after the third step of reverse phase purification (after four steps of purification) during the QS21 purification preparation process;

[0184] Fig.18 Analysis of antibodies binding to recombinant herpes zoster vaccines produced by different processes;

[0185] Fig.19 This is a cellular immunity analysis (CD4+T cell response) 14 days after the second immunization with recombinant herpes zoster vaccines using different processes. DETAILED DESCRIPTION

[0186] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0187] Definitions and explanations of terms

[0188] Unless otherwise specified, the terms used herein have the meanings commonly understood by those of ordinary skill in the art. For terms that are explicitly defined herein, the meaning of the term is subject to the definitions.

[0189] The term "effective amount" refers to the amount of therapeutic agent that can effectively prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or object. "Effective amount" also refers to the amount of compound sufficient to relieve symptoms, such as treating, curing, preventing or alleviating a related medical condition, or treating, curing, preventing or alleviating the speed of these conditions. When an active ingredient is administered to an individual alone, a therapeutically effective dose refers to the ingredient alone. When a certain combination is applied, a therapeutically effective dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether it is administered in combination, continuously or simultaneously. The effective amount can be variably specified based on factors such as formulation method, administration method, and age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient.

[0190] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

[0191] The term "anti-tumor agent" refers to anti-tumor drugs, which are a class of drugs used to treat tumor diseases, including chemotherapy drugs, biological agents, etc.

[0192] The term "pharmaceutical preparation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional components that are unacceptably toxic to the subject to whom the preparation is administered. The vaccine adjuvant according to an exemplary embodiment of the present invention or a vaccine composition containing a vaccine adjuvant can be formulated using methods known in the art so that the active ingredient can be quickly released or continuously or delayed when it is administered to a mammal. These preparations may include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injection solutions and sterile powders.

[0193] The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions (e.g., gastrointestinal disorders, dizziness, etc.) or similar reactions when administered to humans. Examples of carriers, excipients, and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, sodium alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, propyl methylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anticoagulants, lubricants, humectants, flavors, emulsifiers, preservatives, etc. may also be further included.

[0194] The term "adjuvant" or "vaccine adjuvant" refers to a drug or immune preparation administered to improve the immune response of a vaccine. In addition to the vaccine adjuvant described in the present application, the vaccine composition may further include a conventionally used vaccine adjuvant. As conventionally used vaccine adjuvants, aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, MF59, virosomes, AS04 [a mixture of aluminum hydroxide and monophosphoryl lipid A (MPLA)], AS03 (a mixture of DL-α-tocopherol, squalene and polysorbate 80), CpG, flagellin, poly I:C, AS01, AS02, ISCOMs, etc. can be used.

[0195] The term "antigen" refers to a substance that induces an immune response. Therefore, in the present invention, any substance that exhibits such activity of inducing an immune response can be used without limitation. Antigen can be a peptide, protein, nucleic acid, sugar, pathogen, attenuated pathogen, inactivated pathogen, virus, virus-like particle (VLP), cell or cell fragment. Antigen can be selected from the group consisting of: antigens of Japanese encephalitis virus, antigens of Haemophilus influenzae type B (HIB), antigens of Middle East respiratory syndrome (MERS) virus, antigens of Zika virus, antigens of Pseudomonas aeruginosa, antigens of pertussis, antigens of Mycobacterium tuberculosis, antigens of Bacillus anthracis, antigens of Hepatitis A virus (HAV), antigens of Hepatitis B virus (HBV), antigens of Hepatitis C virus (HCV), antigens of human immunodeficiency virus (HIV), antigens of herpes simplex virus (HSV), antigens of Neisseria meningitidis, antigens of Corynebacterium diphtheriae, Bordetella pertussis Antigens of bacteria, antigens of Clostridium tetani, antigens of human papillomavirus (HPV), antigens of varicella virus, antigens of enterococci, antigens of Staphylococcus aureus, antigens of Klebsiella pneumoniae, antigens of Acinetobacter baumannii, antigens of Enterobacter, antigens of Helicobacter pylori, antigens of malaria, antigens of dengue virus, antigens of Orientia tsutsugamushi, antigens of severe febrile thrombocytopenia syndrome bunya virus (SFTS bunya virus), antigens of severe acute respiratory syndrome-coronavirus (SARS-CoV), antigens of influenza virus, antigens of Ebola virus and antigens of pneumococcus.

[0196] 1. Herpes zoster antigen

[0197] The gE protein is the most abundant protein on the surface of the VZV virus. It is a transmembrane glycoprotein composed of 623 amino acid residues. The 1-30 amino acids at the N-terminus are a signal peptide, amino acids 31-538 are an extramembrane segment, amino acids 539-559 are a transmembrane segment, and amino acids 560-623 are an intramembrane region. The gE protein forms a dimer with the glycoprotein gI on the surface of the virus and plays an important role in the process of virus infection of host cells. Natural infection or vaccination with VZV attenuated vaccine can induce the production of gE-specific neutralizing antibodies and CD4 + T cell immune response, so gE is an important target of human immune response. The most important site of antibody response to gE protein is located in the 50-135 amino acid fragment at its N-terminus, which has at least one linear epitope that can be recognized by neutralizing antibodies. The sites where T cells recognize gE protein are distributed throughout the 31-538 extracellular fragment. The transmembrane region of gE protein does not contain known antigenic epitopes and T cell recognition sites.

[0198] The gE protein sequence used in the antigen of the present application is the gE protein extracellular region of the common human varicella-zoster virus (Strain NH29_3 (ABH08489.1), Dumas (NP_040190.1), DE10-2480 (AKG56356.1), VZV-VSD (AAG48520.1), with an amino acid sequence of 31-538, excluding the amino acid sequence of the transmembrane region and the intramembrane region.

[0199] Antigen production: Each time 35-55 L of basal culture medium is added, 1.5-3 L of cell suspension is inoculated, 8-10 L of feed is added, and the volume of cell harvest liquid is about 45-65 L. Finally, 5-10 g of gE antigen is obtained.

[0200] Antigen-expressing host cells: Host cell CHO-K1 cells were purchased from Nanjing GenScript Biotechnology Co., Ltd., and the cell number of this cell line is CCL-61.

[0201] 2. Tumor antigens

[0202] The tumor antigen used in the present invention is a recombinant tumor antigen, which may be a tumor specific antigen / unique tumor antigen, a tumor-associated antigen (TAA), or a tumor neoantigen; specifically, the tumor antigen includes but is not limited to: HPV antigen, PSA antigen, HER2 antigen, CEA antigen, VEGF antigen, EGFR antigen, MUC1 antigen, etc. Preferably, the antigen is an HPV antigen with an amino acid sequence as shown in SEQ ID NO.1, or an HPV antigen with an amino acid sequence as shown in SEQ ID NO.2.

[0203] SEQ ID NO.2:

[0204] HQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKVSEYRYYCYSLY GTTLEQQYNKPLCDLLIRGINCQKPLCPDEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQLgsgsgsgsgsgsgMHGDTPTLHEYMLDL QPETTDLYGYGQLHDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVGPICSQKP;

[0205] SEQ ID NO.3:

[0206] ARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAAGHKCIDFYSRIRELRHYSDSVYGDTLEKL TNTGLYNLLIRGLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQVgsgsgsgsgsgsgMHGPKATLQDIVLHLEPQ NEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCKCEARIELVVESSADDLRAFQQLFLNTLSFVGPWCASQQ.

[0207] 3. Other raw materials

[0208] It only needs to be a commercially available product that meets pharmaceutical standards.

[0209] DOPC: dioleoylphosphatidylcholine;

[0210] DOTAP: 1,2-dioleoyl-3-trimethylammonium propane chloride;

[0211] cholesterol;

[0212] Poly I:C: (polyinosinic acid-cytidylic acid).

[0213] Example 1. Screening of excipients for antigen stability

[0214] In order to ensure the long-term stability of gE protein at -70℃, we screened a variety of auxiliary materials in the early stage, studied the protective effect of the protein under 50℃ heating conditions and room temperature oscillation conditions, and evaluated the changes in OD350 value, turbidity, IEC-HPLC main peak area and SDS-PAGE before and after sample treatment.

[0215] 1. Experiments have found that Tween-80 can effectively reduce the aggregation of antigenic proteins in the preparation under oscillating conditions, and glycerol, histidine, sorbitol and sucrose can effectively reduce the degradation of proteins under high temperature conditions.

[0216] 2. Since glycerol is not usually used for stock solution preservation, sorbitol and histidine were compared and histidine was selected as the buffer system. Considering that sucrose is the first choice of formulation excipients in most recombinant biological products, 10% sucrose was selected as the heat-stable protective agent. Finally, 10% sucrose + 10mM histidine + 0.025% polysorbate 80 (Tween-80), pH 6.5 was selected as the stock solution formula.

[0217] We screened multiple formulations and investigated the turbidity of the solutions under different formulations. The results are shown in Table 1. Figure 1 As shown; the antigen aggregation after repeated freezing and thawing of solvent systems with different formulations was further investigated, and the results are shown in Table 1, Figure 2 shown.

[0218] Table 1. Antigen solution formulation screening

[0219]

[0220]

[0221] According to the above formula, the sample batch Y202009-15 was prepared and stability tested. The specific stability test parameters and steps are as follows:

[0222] Long-term conditions (-70°C), sampling points: 0 days, 3 months, and 6 months;

[0223] Accelerated conditions (2-8°C), sampling points: 0 days, 3 days, 7 days, 14 days.

[0224] The test results showed that the sample was stable, the appearance was qualified, the protein content was qualified, the antigen activity was qualified, the electrophoresis purity test and HPLC purity test were qualified, the sterility test was qualified, and the isoelectric point, peptide map and N-terminal sequencing results showed that the properties of the antigen protein had not changed. This shows that the recombinant herpes zoster vaccine stock solution can withstand at least 4 freeze-thaw cycles.

[0225] Example 2: Screening of prescriptions for herpes zoster vaccine preparations

[0226] The herpes zoster vaccine preparation of the present invention is a suspension composed of recombinant varicella zoster virus glycoprotein E (gE antigen) and MA105 adjuvant system. MA105 adjuvant system is a liposome preparation containing two immune enhancing components, namely, Quillaja saponin (QS-21) and poly I:C. We determined the formula of MA105 adjuvant system and the content of effective antigen through formula screening, and determined the preparation process of semi-finished product through production process development.

[0227] The adjuvant system contains two immunopotentiators (QS-21 and Poly I:C). In order to reduce the toxicity of QS-21 and Poly I:C and improve the immunogenicity, we used a mixture of cationic liposomes and neutral liposomes to adsorb QS-21 and Poly I:C.

[0228] The signal transduction of Poly I:C mainly depends on Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA-5), which can strongly drive cellular immunity and effective type I interferon response, and can induce strong antigen-specific CD4+T and CD8+T cell responses through type I interferon (IFN-α / β) signal transduction and antigen cross-presentation. Since the receptor of Poly I:C is located in the cell membrane, and in order to target Poly I:C to the lymph nodes after intramuscular injection and reduce the adverse reactions caused by its diffusion to the blood, we use cationic liposomes (composed of DOTAP, DOPC and cholesterol) as carriers, and adsorb Poly I:C through electrostatic interaction. The transmission electron microscopy image of the recombinant herpes zoster vaccine is shown in Figure 7 , particle size distribution diagram see Figure 8 .

[0229] It was found in the study that large floccules or precipitates appeared after the cationic liposomes alone adsorbed Poly I: C. In order to maintain the stability of the physical properties of the product, the present invention selected mixed liposomes containing cationic liposomes and neutral liposomes (composed of DOTAP and cholesterol) to adsorb Poly I: C. Poly I: C after intramuscular injection of mixed liposome adsorption can be passively or actively transferred to antigen presenting cells (APCs), limiting their interaction with nonspecific phagocytes and regulating the resulting cytokine secretion characteristics. Toxicological studies of this product have confirmed that adsorbing Poly I: C on mixed liposomes can effectively improve the safety of the product.

[0230] Free QS-21 will bind to cholesterol on the cell membrane to form an irreversible complex, which will cause hemolysis when it binds to red blood cells. When QS-21 is adsorbed with cholesterol in the mixed liposomes, it prevents it from binding to the cell membrane, thereby significantly reducing the hemolytic effect. Experiments have shown that the amount of cholesterol added to the adjuvant system of the present invention can adsorb at least 3 times the dose of QS-21 in the finished product without causing hemolysis, indicating that the cholesterol content is excessive, which can reduce the potential risks caused by the hemolytic activity of QS-21 and improve the safety margin of the process.

[0231] The vaccines formulated with a single immunopotentiator (Poly I:C or QS-21) in combination with the mixed liposomes can induce a higher level of binding antibodies and a higher frequency of CD4+T cell responses. When the two immunopotentiators are combined with the mixed liposomes, the vaccine induces a higher level of binding antibodies and a higher frequency of CD4+T responses, indicating that Poly I:C and QS-21 have a synergistic effect. Therefore, the combination of the two immunopotentiators is more helpful to improve the immunogenicity of this product and the number of CD4+T cells, as shown in Example 4.

[0232] Polysorbate 80 (Tween-80) was selected to avoid the appearance of flocculent aggregates in the finished product, and sodium chloride was used to adjust the osmotic pressure of the vaccine and maintain the osmotic pressure of the system (210-350 mOsmol / kg). Sucrose can maintain the stability of liposomes.

[0233] Specifically, the specific information of active ingredients and excipients in each 1 ml of preparation is shown in Table 2 below.

[0234] Table 2. List of main ingredients in vaccine preparations

[0235]

[0236]

[0237] DOPC: dioleoylphosphatidylcholine;

[0238] DOTAP: 1,2-dioleoyl-3-trimethylammonium propane chloride;

[0239] cholesterol;

[0240] QS-21: Quillaja saponin 21;

[0241] Poly I:C: poly-inosin.

[0242] Example 3: Preparation and property testing of herpes zoster vaccine preparation

[0243] The preparation steps of the vaccine preparation are as follows:

[0244] 3.1. Preparation of cationic liposomes;

[0245] 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP), dioleoylphosphatidylcholine (DOPC) and cholesterol are prepared in a mass ratio of 2:2:1 by an ethanol injection method. The specific preparation steps are as follows:

[0246] (1) The three lipid components are dissolved in ethanol and sheared online with the aqueous phase for 1 to 3 times;

[0247] (2) Ultrafiltration replacement using sucrose buffer, concentration times 2 to 4 times, diafiltration times 6 to 8 times;

[0248] (3) 200 nm liposome extrusion, using a filter membrane with a pore size of 200 nm for extrusion, and performing multiple cycles of extrusion to control the uniformity of particle size;

[0249] (4) 100 nm liposome extrusion, using a filter membrane with a pore size of 100 nm for extrusion, and performing multiple cycles of extrusion to control the uniformity of particle size;

[0250] (5) Sterilize by filtering using a sterile filter.

[0251] The physicochemical parameters of the prepared cationic liposomes are:

[0252] Average particle size (D50): 60~120nm;

[0253] Solution pH: 4.8-7.0;

[0254] Zeta potential: 40~72mV (pH6.5).

[0255] After 12 months of long-term stability study at 2-8°C, the morphology and composition of the cationic liposomes were stable.

[0256] 3.2. Preparation of neutral liposomes;

[0257] Neutral liposomes are spherical lipid bilayers composed of dioleoylphosphatidylcholine (DOPC) and cholesterol, wherein the mass ratio of DOPC to cholesterol is 4:1. The specific preparation steps are as follows:

[0258] (1) The two lipid components are dissolved in ethanol and added to the aqueous phase online by shearing for 1 to 3 times;

[0259] (2) 200 nm liposome extrusion, using a filter membrane with a pore size of 200 nm for filtration, and multiple cycles of extrusion to control the uniformity of particle size;

[0260] (3) 100 nm liposome extrusion, using a filter membrane with a pore size of 100 nm for filtration, and multiple cycles of extrusion to control the uniformity of particle size;

[0261] (4) Ultrafiltration replacement using sucrose buffer, concentration times 3 to 6 times, and diafiltration times 6 to 8 times;

[0262] (5) Sterilize by filtering using a sterile filter.

[0263] The physicochemical parameters of the prepared neutral liposomes are:

[0264] Average particle size (D50): 80~140nm;

[0265] Solution pH: 5.5-7.0, preferably 6.4-6.6;

[0266] After 12 months of long-term stability study at 2-8°C, the morphology and composition of the cationic liposomes were stable.

[0267] 3.3 Preparation of vaccine preparations

[0268] The final contents of the components of the vaccine preparation were prepared in the proportions described in Table 2 of Example 2:

[0269] Cationic liposomes, neutral liposomes and each component excipient were added in the amount calculated according to the concentration in Table 2, and then QS-21, Poly I:C and gE antigen solution were added respectively to adjust the pH to 6.0-7.0.

[0270] The complete vaccine preparation process is as follows Figure 6 shown.

[0271] 3.4. Quality testing of vaccine preparations

[0272] Multiple batches of vaccine preparations were prepared according to the above method, as shown in Table 3 below.

[0273] Table 3. Vaccine preparation production batch records

[0274]

[0275]

[0276] The product was quality tested to determine the content of each major component. The test results are shown in Table 4 below.

[0277] Table 4. Vaccine preparation quality test results

[0278]

[0279] Example 4: Testing of the properties of herpes zoster vaccine preparations and comparison of immunogenicity of different herpes zoster vaccine formulations

[0280] The vaccine of the present invention and the aluminum hydroxide adjuvant formula (gE / nano aluminum hydroxide adjuvant / QS-21 / Poly I:C) were selected as control group 1. The Shingrix formula was used as control group 2, and the neutral liposome formula (gE / neutral liposome / QS-21 / Poly I:C) was used as control group 3. The effects after immunization of mice were investigated, and the results showed that:

[0281] (1) The four different gE antigen vaccines were able to stimulate the production of high titers of binding antibodies in mice, and the mixed liposome formula of the present invention was superior to the other formulas; the test results are detailed in Figure 3 , in the figure:

[0282] The gE antigen concentration in all reference preparation groups was consistent (100ug / ml);

[0283] MPL, 3M-52 (TLR7 / 8 agonist), aluminum hydroxide, and Poly I:C were immune adjuvants;

[0284] The gE group was a pure antigen solution without liposome preparation;

[0285] The nanoemulsion group was an oil-in-water formulation;

[0286] The formulation and preparation method of neutral liposomes are as described in Section 3.2 of Example 3, and the amount of neutral liposomes in the reference preparation of neutral liposomes is consistent with that in the nanoemulsion group;

[0287] The buffer, solvent, QS-21, and Poly I:C used in all groups (except the high- and low-dose Poly I:C groups) were consistent, and the dosage of the components is shown in Table 2.

[0288] (2) the mixed liposome formulation of the present invention, the neutral liposome formulation and All formulas can induce CD4+T cell response. There is no significant difference between the groups, but they are better than the aluminum hydroxide formula group. For details of the test results, see Figure 4 , in the figure:

[0289] The gE antigen concentration in all reference preparation groups was consistent (100ug / ml);

[0290] The blank control contained no antigen, but the vaccine preparation was the complete formulation;

[0291] The virus control contained only antigen but not mixed liposomes, Poly I:C, and QS-21;

[0292] Synanlis is a group of commercially available vaccine products;

[0293] The mixed liposome group was a mixed formulation of cationic liposomes and neutral liposomes described in Section 3.3 of Example 3;

[0294] The formulation and preparation method of neutral liposomes are as described in Section 3.2 of Example 3, and the amount of neutral liposomes in the reference preparation of neutral liposomes is consistent with that in the nanoemulsion group;

[0295] The buffer, solvent, QS-21, and Poly I:C used in all groups remained consistent, and the amounts of the components are shown in Table 2.

[0296] In order to study the immune effects of different preparation methods and formulations, we conducted the experiments shown in the following table, two of which are shown in Table 2, and two preparation methods of MA105 adjuvant are shown in Figure 6 The sample information is shown in the following table.

[0297]

[0298]

[0299] The results showed that different formulations and preparation methods could induce the production of high levels of binding antibodies, and there was no significant difference between the groups ( Fig.18 ); however, sample 4 induced a higher frequency of (IL-2 / IFN-γ) CD4+T cell responses ( Fig.19 ).

[0300] Example 5: Comparison of properties of vaccines with different formulations

[0301] 5.1. Synergistic Effects of Immunoenhancers

[0302] In order to study the effect of a single immunopotentiator (Poly I:C or QS-21) on the immunogenicity of the vaccine, and whether Poly I:C and QS-21 in the formulation have a synergistic effect on the immunogenicity of the vaccine, based on the vaccine formulation determined in Example 2 and the preparation route determined in Example 3, the immunogenicity of the vaccine formulation containing a single immunopotentiator (Poly I:C or QS-21) and mixed liposomes was compared with the vaccine formulation containing Poly I:C, QS-21 and mixed liposomes.

[0303] Both the immunopotentiator and liposome combination vaccines can induce higher levels of binding antibodies and higher frequencies of (IL-2 / IFN-γ)CD4+T cell responses, but the gE / composite adjuvant candidate vaccine induced higher levels of binding antibodies and higher frequencies of (IL-2 / IFN-γ)CD4+T responses, proving that Poly I:C and QS-21 have synergistic effects. For details of the comparison results, see Figure 5 , in the figure:

[0304] The preparation methods of the reference group preparations are shown in Example 3, and the specific differences in the preparation ingredients are as follows:

[0305] Blank control group: does not contain antigen, but the vaccine preparation is a complete formula;

[0306] gE buffer group: does not contain mixed liposomes, Poly I:C and QS-21;

[0307] gE mixed liposome group: mixed liposome preparation, but without Poly I:C and QS-21;

[0308] gE mixed liposome / QS-21 group: mixed liposome preparation without Poly I:C;

[0309] gE mixed liposome / Poly I:C group: mixed liposome preparation without QS-21;

[0310] gE mixed adjuvant group: the complete preparation prepared in Example 3.

[0311] 5.2. Dose optimization of Poly I:C and QS-21

[0312] 5.2.1. Poly I:C Dose Optimization

[0313] The immunogenicity of gE / composite adjuvant candidate vaccines prepared with different Poly I:C contents (400 μg / ml, 800 μg / ml, 1600 μg / ml) was compared to determine the Poly I:C content in the vaccine formulation.

[0314] The gE / composite adjuvant candidate vaccines containing different doses of Poly I:C were able to produce high levels of binding antibodies, with no significant differences between the groups, and were able to induce a high frequency of (IL-2 / IFN-γ) CD4+T cell responses. Considering that the use of mixed liposomes can improve the stability and delivery efficiency of Poly I:C, the Poly I:C content in the vaccine was temporarily set at 800μg / ml.

[0315] 5.2.2. Dose optimization of QS-21

[0316] The immunogenicity of gE / composite adjuvant candidate vaccines formulated with different contents of QS-21 (100 μg / dose, 50 μg / dose, 25 μg / dose, and 12.5 μg / dose) was compared to determine the QS-21 content in the vaccine formulation (0.5 ml per dose).

[0317] After immunization with the gE / composite adjuvant candidate vaccine containing different doses of QS-21, they were able to stimulate the production of high levels of binding antibodies; all experimental groups were able to induce the production of (IL-2 / IFN-γ) CD4+T cell responses. The concentration of QS-21 in this product is tentatively set at 50 μg / dose (100 μg / ml).

[0318] 5.3 Antigen content study

[0319] This vaccine is a liquid preparation containing a new adjuvant. According to the requirements of the "Technical Guidelines for Preclinical Research of Preventive Vaccines", four groups of gE vaccines prepared with different contents of gE antigen (100μg / dose; 50μg / dose; 25μg / dose; 5μg / dose, 0ug / dose, 0.5ml per dose) and a pure adjuvant control were compared to study the effect of gE antigen content on the immunogenicity of the vaccine.

[0320] Within the scope of the antigen content in this study, gE vaccines prepared with different gE antigen contents were able to induce a higher level of binding antibodies and a higher frequency of (IL-2 / IFN-γ) CD4+T cell responses. The results of humoral immunity and cellular immunity showed that gE antigen content of 50μg / dose and above could induce a good immune response. The final antigen was determined to be 50μg / dose (concentration of 100ug / ml).

[0321] Example 6. Preparation of anti-tumor vaccine preparation

[0322] Cationic liposomes and neutral liposomes in MA105 adjuvant were prepared according to the contents of Sections 3.1-3.2 of Example 3.

[0323] 6.1. Preparation of vaccine preparations

[0324] The final contents of the components of the vaccine preparation refer to the contents of Example 2 and are prepared according to the proportions described in Table 5:

[0325] Table 5. List of the contents of each ingredient in the anti-tumor vaccine preparation (per dose)

[0326]

[0327]

[0328] The antigen is an HPV antigen with an amino acid sequence as shown in SEQ ID NO.2.

[0329] Example 7. Activity detection of anti-tumor vaccine preparation (TC-1 tumor model)

[0330] 7.1 Experimental plan

[0331] TC-1 tumor-bearing mice (C57BL / 6) were randomly divided into groups, with 7 mice in each group, for group immunization and subsequent observation and testing.

[0332] Immunization procedure: The first immunization was performed on the 7th day after tumor implantation (the tumor was excised from the previous generation of TC-1 tumor-bearing mice and inoculated at the lower part of one shoulder blade), when the short diameter grew to about 9 mm. The immunization method was hind limb intramuscular injection, 200 μL per mouse (the formula of the vaccine preparation is shown in Table 6, and 200 μL of each group of preparations was given each time for each immunization). The second and third immunizations were performed on days 14 and 21 after tumor implantation.

[0333] Detection point:

[0334] (1) TC-1 tumor-bearing mice were observed every 3 days after immunization, and the long and short diameters of the tumor, the weight of the mice, and the general condition of the mice were recorded.

[0335] (2) Isolation of spleen lymphocytes from TC-1 tumor-bearing mice and detection of IFN-γ secretion by ELISPOT.

[0336] Table 6. Formula of anti-tumor vaccine preparation

[0337]

[0338] The antigen used is the HPV antigen with the amino acid sequence shown in SEQ ID NO.2.

[0339] 7.2 Experimental Results

[0340] (1) Tumor growth curve of TC-1 tumor-bearing mice

[0341] At the first immunization, the average tumor volume of each group was 0.59 cm 3 ,0.62cm 3 ,0.68cm3 ,0.77cm 3 ,0.68cm 3 , there was no statistical difference. The tumor growth curve of TC-1 tumor-bearing mice during the study period was shown in Fig. 9 The results showed that compared with the NC group, the D group showed significant tumor inhibition, with tumor inhibition rates of 82.16% (24 days) and 73.30% (28 days).

[0342] In the figure, the formula for calculating tumor volume is: V = 4 / 3*π*long diameter*short diameter 2 .

[0343] The formula for calculating the tumor inhibition rate is: inhibition rate = (average volume of transplanted tumors in the NC group - average volume of transplanted tumors in the experimental group) / average volume of transplanted tumors in the NC group * 100%.

[0344] (2) Body weight change curve of TC-1 tumor-bearing mice

[0345] The tumor growth curve of TC-1 tumor-bearing mice during the study period is shown in Fig.10 The results showed that after the administration of each group, the weight of the mice did not decrease significantly (the increase in weight was caused by the growth of the tumor), indicating that the safety of each group of vaccines was good.

[0346] (3) IFN-γ secretion level of TC-1 tumor-bearing mice after three immunizations

[0347] Ten days after the third immunization, three mice were randomly selected from each group, and their spleens were removed for isolation and culture of splenic lymphocytes. 5ug and 50ug of antigen were used for stimulation, respectively, and the IFN-γ secretion level in TC-1 tumor-bearing mice was detected using the Dayou ELISPOT-IFN-γ kit. The results are as follows: Fig.11 As shown, it can be seen that compared with the NC group, the D group showed obvious immune activation efficiency.

[0348] The above results indicate that MA105 adjuvant can significantly enhance the immune effect of tumor antigens and improve the therapeutic activity of vaccines.

[0349] Example 8. Anti-tumor activity detection of MA105 adjuvant without antigen (B16F10 tumor model)

[0350] Experimental protocol

[0351] B16F10 tumor-bearing mice (C57BL / 6) were randomly divided into groups, with 8 mice in each group, for group immunization and subsequent observation and testing.

[0352] Immunization procedure: After tumor implantation (50,000 B16F10 tumor cells were inoculated per mouse), immunization was performed on the 9th, 12th, and 15th days. The administration method was intratumoral injection. Only antigen-free adjuvant was given for immunotherapy, and 100 μL was administered to each mouse each time (the formula is shown in Table 7). On the 18th day, mice without tumors were inoculated with tumor cells for the second time (inoculated with 50,000 B16F10 tumor cells).

[0353] Monitoring points: Observe once every 2 days after the first treatment, and record the long and short diameters of the tumor, the weight of the mice, and the general condition of the mice.

[0354] Table 7. Formulations without antigen adjuvant

[0355]

[0356] The tumor growth curve of B16F10 tumor-bearing mice during the study period is shown in Fig.12 .

[0357] The results showed that the MA105 adjuvant-administered group showed significant tumor inhibition compared with other groups.

[0358] Example 9. Anti-tumor activity detection of antigen-free MA105 adjuvant (colon cancer MC38 cell line homograft tumor model)

[0359] Experimental protocol

[0360] MC38 tumor-bearing mice (C57BL / 6) were randomly divided into groups, with 10 mice in each group, for group immunization and subsequent observation and testing.

[0361] Immunization procedure: Tumor inoculation (1×10 5 MC38 tumor cells), immunization (intratumoral injection) was performed on days 11, 14, and 17. Immunotherapy was given only without antigen adjuvant, and each mouse was given 100 μL each time (the formula is shown in Table 7 above).

[0362] Monitoring points: Observe once every 3 days after the first treatment, record the long and short diameters of the tumor, mouse weight, and general condition of the mouse; at the end of the experiment, dissect the mice, remove the tumor tissue and weigh it.

[0363] The tumor growth curve of MC38 tumor-bearing mice during the study period and the tumor weight analysis of each group at the end of the experiment are shown in Fig.13 .

[0364] The results showed that compared with the saline control group, the MA105 adjuvant administration group showed significant tumor inhibition.

[0365] Example 10. Anti-tumor activity detection of antigen-free MA105 adjuvant (lung cancer LLC cell homograft tumor model)

[0366] Experimental protocol

[0367] LLC tumor-bearing mice (C57BL / 6) were randomly divided into groups, with 10 mice in each group, for group immunization and subsequent observation and testing.

[0368] Immunization procedure: Tumor inoculation (6×10 5 LLC tumor cells), immunization (intratumoral injection) was performed on days 11, 14, and 17. Immunotherapy was given only without antigen adjuvant, and each mouse was given 100 μL each time (the formula is shown in Table 7 above).

[0369] Monitoring points: After the initial treatment, the mice were observed every 3 days, and their weight and general condition were recorded; at the end of the experiment, the mice were dissected, and the tumor tissues were removed and weighed.

[0370] During the study, LLC tumors grew flat and the growth curve could not be calculated. The tumor weight analysis of each group at the end of the experiment is shown in Fig.14 .

[0371] The results showed that compared with the saline control group, the MA105 adjuvant administration group showed significant tumor inhibition.

[0372] Example 11. Detection of the immune protection efficacy of the vaccine preparation (antigen + MA105 adjuvant) (TC-1 tumor model)

[0373] Experimental protocol

[0374] (1) A tumor-bearing mouse model was constructed according to the method of Example 2. TC-1 tumor-bearing mice (C57BL / 6) were randomly divided into groups, with 5 mice in each group. The mice were first immunized (7 days) when the short diameter of the transplanted tumor grew to about 5 mm. The second and third immunizations were performed on days 14 and 21, respectively.

[0375] (2) Each mouse is injected intramuscularly with 200 μL of the following vaccine groups on the left and right hind limbs.

[0376] 1) NC group (PBS 200 μL);

[0377] 2) MKK700+MA105 group: MKK700 antigen is an HPV antigen with an amino acid sequence as shown in SEQ ID NO.1 + MA105 group (PBS buffer 20 μL + MKK700 antigen 80 μL + MA105 adjuvant 100 μL, the concentration and dosage of the antigen and adjuvant are the same as in Example 2);

[0378] 3) MA105 group (PBS buffer 100 μL + MA105 adjuvant 100 μL);

[0379] The formulas of the antigen solution and MA105 adjuvant are shown in Tables 6 and 7 above.

[0380] After administration, the tumor growth curve of mice was as follows Fig.15 As shown, among the mice in the antigen+MA105 adjuvant administration group, 60% (3 / 5) of the mice achieved CR (complete remission) and 40% (2 / 5) of the mice achieved PR (partial remission).

[0381] Subsequently, TC-1 tumors were re-implanted in other locations (under the contralateral scapula) of the three mice that had achieved complete remission after treatment. The results showed that antigen + MA105 adjuvant treatment had a sustained immune protection effect. The mice that had achieved complete remission developed strong immunity to the re-implanted TC-1 tumors, resulting in the inability of the tumors to grow ( Fig.16 ).

[0382] Example 12: Purification and preparation of QS21

[0383] Main raw materials, equipment and reagents

[0384] Saponin (derived from dried extract of Quillaja bark): purchased from Desert King (name: VET-SAP, CAS number: 8047-15-2).

[0385] According to the detection by high performance liquid chromatography, the QS21 content in the raw material is not less than 8%; the moisture content is not higher than 10%, the ash content is not higher than 3%, and the heavy metal content and pesticide residues meet the pharmaceutical standards.

[0386] UniPSN 30-300 chromatography medium: Suzhou Nanovita Technology Co., Ltd.

[0387] UniPS10-300 chromatographic medium: Suzhou Nanovita Technology Co., Ltd.

[0388] Main Equipment

[0389] Device Name Specifications factory High-speed refrigerated centrifuge H5-25KR Hunan Kecheng Instrument Equipment Co., Ltd. Membrane ultrafiltration system <![CDATA[0.5m 2 ]]> Hangzhou Cobetter Filter Equipment Co., Ltd. High performance liquid chromatography DAC150 Jiangsu Hanbang Technology Co., Ltd.

[0390] Main buffer

[0391] Buffer name and composition Cleaning solution 1 (0.5M NaOH) Storage solution 1 (100 mM NaOH) Dilution solution (30% acetonitrile ~ 5mM citric acid pH 5.0) A1 solution (30% acetonitrile 0.1% TFA) A2 solution (20% acetonitrile 0.1% TFA) Solution B (99% acetonitrile 0.1% TFA) Dissolution solution (5 mM histidine pH 6.0) Ultrafiltration replacement solution (5mM histidine pH 5.0)

[0392] Step 1: Dissolution, filtration and crude purification of saponin

[0393] 1.1. Saponin dissolution and filtration

[0394] Feeding amount: saponin 200g, diluent 5L;

[0395] Saponin was dissolved in a diluent (30% acetonitrile-5 mM citric acid pH 5.0) at a ratio of 1:25 (W / V), and filtered through a 1.0+0.45 μm filter to obtain 5.11-5.15 L of saponin filtration sample.

[0396] 1.2. Reverse phase crude purification of saponin solution

[0397] (1) Pretreatment: Connect the chromatography column to the liquid chromatography system, control the A1 liquid to enter the A pump port, and the B liquid to enter the B pump port, respectively, the system linear flow rate is 100-140 cm / h, and after 100% B flushing, 100% A balance;

[0398] (2) Sampling: Take the saponin filtered sample obtained in step 1 (about 5 L) and load it onto UniPSN 30-300 chromatographic medium, with a loading capacity of 30-60 mg / ml and a linear flow rate of 90-360 cm / h. After the loading is completed, rinse with A1 solution (4.5 L), and then rinse the impurities with 2.5% B in sequence;

[0399] (3) Elution: The target peak was eluted with a linear gradient from 2.5% B to 21.25±5.00% B, and the samples were collected in sections for purity testing. The samples with a purity higher than 30% were combined to obtain 4.7 to 5.5 L of crude pure saponin solution.

[0400] The amounts of materials used for loading and elution in the reverse phase crude purification step are shown in Table 8 below.

[0401] Table 8. Reverse Phase Crude Purification Steps

[0402]

[0403] Step 2: Purification of saponin

[0404] 2.1. The first step of reverse phase purification of saponin solution

[0405] (1) Pretreatment: Connect the chromatography column to the liquid chromatography system, control the A2 liquid to enter the A pump port, and the B liquid to enter the B pump port, respectively, the system linear flow rate is 100-140 cm / h, and after 100% B flushing, 100% A balance;

[0406] (2) Loading: dilute the crude pure saponin solution with water, control the acetonitrile concentration to 22% to 28%, and load it on the UniPS10-300 chromatographic medium (load the entire diluted crude pure saponin solution, with a loading of about 10 to 50 mg / ml), with a linear flow rate of 100 to 140 cm / h; after loading, rinse with A2 solution, and then rinse the impurities with 10% B in turn;

[0407] (3) Elution: The target peak was eluted with a linear gradient from 10% B to 25±5% B. The samples were collected in sections and tested for purity. The samples with purity higher than 60% were combined to obtain 4.94-5.30 L of two-step purified saponin sample solution.

[0408] The amounts of materials used for loading and elution in the first step of reverse phase purification are shown in Table 9 below.

[0409] Table 9. Steps of the first reverse phase purification

[0410]

[0411] 2.2. Reverse phase purification of saponin solution, step 2

[0412] (1) Pretreatment: Connect the chromatography column to the liquid chromatography system, control the A2 liquid to enter the A pump port, and the B liquid to enter the B pump port, respectively, the system linear flow rate is 100-140 cm / h, and after 100% B flushing, 100% A balance;

[0413] (2) Loading: dilute the saponin sample solution purified in the second step with water, control the acetonitrile concentration to 22% to 28%, and load it on the UniPS10-300 chromatographic medium (load all the diluted sample, with a loading of about 10 to 50 mg / ml), with a linear flow rate of 100 to 140 cm / h; after loading, rinse with A2 solution, and then rinse the impurities with 10% B solution in turn;

[0414] (3) Elution: 10% B to 25 ± 5% B linear gradient elution of the target peak, and segmented collection for purity testing. Samples with purity higher than 80% were combined to obtain 1.76-2.30 L as the three-step purified saponin sample solution.

[0415] The amounts of materials used for loading and elution in the second step of reverse phase purification are shown in Table 10 below.

[0416] Table 10. Steps of the second reverse phase purification

[0417]

[0418] 2.3. Reverse phase purification of saponin solution, step 3

[0419] (1) Pretreatment: Connect the chromatography column to the liquid chromatography system, control the A2 liquid to enter the A pump port and the B liquid to enter the B pump port respectively, the system linear flow rate is 100-140 cm / h, and after 100% B flushing, 100% A balance.

[0420] (2) Loading: dilute the saponin sample solution purified in three steps with water, control the acetonitrile concentration to 22% to 28%, and load it on the UniPS10-300 chromatographic medium (load the entire diluted sample, with a loading of approximately 10 to 50 mg / ml), with a linear flow rate of 100 to 140 cm / h; after loading, rinse with A2 solution, and then rinse the impurities with 10% B in sequence;

[0421] (3) Elution: 10% B to 25 ± 5% B linear gradient elution of the target peak, and segmented collection for purity testing. Samples with purity higher than 95% were combined to obtain 1.41 to 1.81 L, which was the four-step purified saponin sample solution.

[0422] The amounts of materials used for loading and elution in the third step of reverse phase purification are shown in Table 11 below.

[0423] Table 11. Steps of the third reverse phase purification

[0424]

[0425] Step 3: Precipitation, re-dissolution, ultrafiltration, replacement and subpackaging of saponin

[0426] 3.1. Precipitation and re-dissolution steps

[0427] (1) Take the four-step purified saponin sample solution (1.4-1.8 L) prepared in Example 2, add 2 times the volume of water and mix well, place at room temperature for not less than 30 min, then centrifuge at 9000-11500g, 2-8°C for 10 min, and collect the precipitate;

[0428] (2) 1.5 to 3.0 L of a dissolving solution is added to the precipitate, and after stirring and dissolving, 1.5 to 3.0 L of a sample having the organic solvent removed is obtained.

[0429] 3.2 Ultrafiltration replacement steps

[0430] (1) Rinse the ultrafiltration membrane with ultrafiltration replacement solution (5 mM histidine pH 5.0); take the saponin sample (about 1.5-3.0 L) obtained by precipitation and redissolution in step 1 of this example and remove the organic solvent, and ultrafiltration and concentrate to 0.91-0.93 L; 5 kD membrane package, transmembrane pressure: 0.2-0.4 bar, replacement times 10 times.

[0431] (2) Wash and filter with an ultrafiltration replacement fluid of not less than 8 times the volume to obtain 0.91 to 0.93 L of saponin ultrafiltration sample.

[0432] After using the 5kD membrane package, the yield is no less than 70%, which is significantly improved compared with the conventional 10kD ultrafiltration replacement membrane package.

[0433] 3.3. Packaging

[0434] The saponin ultrafiltration sample after sterilization and ultrafiltration replacement is filtered through a 0.45+0.2 μm filter to obtain the QS21 adjuvant solution described in the present application, which is stored at a temperature below -70°C after being divided into portions.

[0435] Step 4: Key process quality inspection

[0436] 4.1 Detection Method

[0437] The intermediate samples of each step of the three batches of pilot production were taken for RP-HPLC detection to compare and analyze the removal effect of impurity peaks in each chromatography step. The chromatographic conditions are shown in Table 12.

[0438] Table 12. Chromatography steps

[0439]

[0440] 4.2. Removal efficiency of main impurities

[0441] The samples obtained in the first step of reverse phase crude purification and reverse phase fine purification had many HPLC impurity peaks, among which the main peak of QS-21 had a retention time of 22.622 min, and the three peaks with retention times of 20.376 min (impurity No. 1), 20.734 min (impurity No. 2), and 23.109 min (impurity No. 3) were the main impurity peaks.

[0442] The detection chromatogram of the QS21 sample after the third step of reverse phase purification (after four steps of purification) is as follows Fig.17 shown.

[0443] Since there are many sample impurity peaks obtained in the reverse phase crude purification and the first step of reverse phase purification, only the results of the second step of reverse phase purification (after three-step purification) and the third step of reverse phase purification after four steps of purification are used to calculate the chromatographic impurity removal effect. The main impurity removal effect of four-step purification compared with three-step purification is shown in Table 13 below.

[0444] Table 13. Comparison of the removal effects of main impurities between four-step purification and three-step purification

[0445]

[0446]

[0447] Removal rate = total amount of impurity peaks of the chromatographic harvest sample / total amount of impurity peaks of the chromatographic loading sample, where: total amount of impurity peaks = (peak area of ​​UV peak detected by RP-HPLC / injection volume) * chromatography step sample volume.

[0448] 4.3. QS21 purity and yield detection

[0449] The purity of QS21 was tested using the same RP-HPLC, and the results are shown in Table 15 below.

[0450] Table 15. Purity test results of QS21

[0451]

[0452] The same RP-HPLC was used to detect the yield of QS21. The results are shown in Table 16 below.

[0453] Table 16. Yield test results of QS21

[0454] Chromatography steps MA001-20200810 MA001-20200815 MA001-20200819 Reverse phase crude purification 76.77％ 75.37％ 80.60％ Reversed Phase Purification Step 1 82.64％ 83.21％ 71.87％ Reversed Phase Purification Step 2 37.87％ 51.13％ 60.10％ Reversed Phase Purification Step 3 80.46％ 53.78％ 63.17％

[0455] Step 5: QS-21 functional verification

[0456] The QS21 sample prepared in the above steps was used to prepare MA105 adjuvant, herpes zoster vaccine, and anti-tumor vaccine products according to the methods of the above Examples 1-11, and the activities were compared with those of the existing commercial QS21 products.

[0457] 5.1. Herpes zoster vaccine products

[0458] The homemade QS-21 of this example and the purchased QS-21 were respectively formulated into gE / compound adjuvant candidate vaccines, and the difference in immunogenicity between the two was compared.

[0459] Experimental method: At the beginning of the study, pre-immunization was first performed and then divided into 3 groups, with 10 mice in each experimental group and 5 mice in the gE / buffer control group.

[0460] Pre-immunization method: 104-5TCID50 VZV attenuated viruses were subcutaneously administered to 5-6 week old female C57BL / 6 mice.

[0461] After 5 weeks of pre-immunization, mice were injected intramuscularly on days 0 and 28, 0.05 ml / 0.1 dose / mouse each time. Blood was collected from mice 14 days after the second immunization, and the binding antibodies in individual sera were detected by ELISA. At the same time, the spleen was isolated, and the frequency of CD4+T cells producing cytokines (IL-2 / IFN-γ) was evaluated by intracellular cytokine staining (flow cytometry).

[0462] The experimental results showed that: (1) there was no significant difference in the binding antibody titer after immunization with the herpes zoster vaccine prepared with two different sources of QS-21; (2) both vaccine preparations were able to induce stimulation to produce (IL-2 / IFN-γ) CD4+T cell responses; it can be seen that there was no significant difference in inducing activation immunity between the QS-21 prepared by the method of this embodiment and the gE / composite adjuvant candidate vaccine prepared with the purchased QS-21.

[0463] Finally, the same therapeutic activity results were also verified in anti-tumor vaccines based on MA105 adjuvant and antigen-free therapeutic agents (Examples 8-10).

[0464] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The use of MA105 adjuvant in the preparation of anti-tumor preparations, characterized in that: The MA105 adjuvant includes the following components and their final concentrations: 50-300 μg / ml QS-21, 400-3000 μg / ml PolyI:C, and lipid molecules constituting the carrier.

2. The use according to claim 1, characterized in that: The anti-tumor preparations include intratumor injection preparations or tumor in situ vaccines.

3. The use according to claim 1, characterized in that: The tumor is melanoma, colorectal cancer or lung cancer.

4. The use according to any one of claims 1 to 3, characterized in that: The concentration of QS-21 is 50-200 μg / ml; Optionally, the concentration of QS-21 is 80-120 μg / ml; Optionally, the concentration of the Poly I:C is 780-1600 μg / ml; Optionally, the carrier is a mixture of cationic liposomes and neutral liposomes; Optionally, the lipid molecules include: 70-560 μg / ml DOTAP, 500-4000 μg / ml DOPC and 175-1400 μg / ml cholesterol; Optionally, the concentration of DOTAP is 140-280 μg / ml; Optionally, the concentration of DOPC is 1200-2500 μg / ml; Optionally, the concentration of cholesterol is 350-600 μg / ml.

5. The use according to any one of claims 1 to 3, characterized in that: The anti-tumor preparation also includes: pharmaceutical excipients.

6. The use according to claim 5, characterized in that: The pharmaceutical excipients include any one or more of a stabilizer, a lyophilization protectant and an osmotic pressure regulator; Optionally, the pharmaceutical excipients include any one or more of polysorbate 80, sucrose, sodium chloride, histidine and sodium dihydrogen phosphate.

7. An anti-tumor preparation, characterized in that: It includes: The MA105 adjuvant as claimed in any one of claims 1 to 4, and pharmaceutical excipients; Optionally, the pharmaceutical excipients include any one or more of a stabilizer, a lyoprotectant and an osmotic pressure regulator; Optionally, the pharmaceutical excipients include any one or more of polysorbate 80, sucrose, sodium chloride, histidine and sodium dihydrogen phosphate.

8. The use of MA105 adjuvant and other drugs in the preparation of anti-tumor combination preparations, characterized in that: The MA105 adjuvant is the MA105 adjuvant described in any one of claims 1 to 4, and the other drugs include any one of the following drugs: (1) Cytotoxic chemotherapy drugs; (2) Antibody anti-tumor drugs; (3) Oncolytic peptides and oncolytic virus drugs; (4) Cell therapy preparations.

9. The use according to claim 8, characterized in that: The cell therapy preparation includes Car-T cell therapy preparation.

10. An anti-tumor combination preparation, characterized in that: It comprises the MA105 adjuvant as claimed in claim 8 or 9 and other drugs.

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