Use of an immunomodulator in the preparation of a composition for treating animal cancer

Abstract

The present invention discloses the application of an immunomodulator in the preparation of a composition for treating animal cancers, and relates to the technical field of tumor immunotherapy. The immunomodulator includes Poly IC and QS-21 with a mass ratio of (1.3 to 60):1. Compared with existing immunotherapies, the immunomodulator provided by the present invention has the advantages of strong broad-spectrum property, low cost, easy operation, and good effect, can effectively inhibit the growth of animal cancers, especially has more advantages in the treatment of canine breast cancer and is more easily promoted and used. In addition, the immunomodulator provided by the present invention can maintain for a long time after drug withdrawal, and there are no serious adverse events during injection in terms of safety, and the animals have good tolerance.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor immunotherapy. Specifically, it relates to the application of an immunomodulator in the preparation of a composition for treating animal cancers. Background Art

[0002] Dogs are the most common companion animals. With the rapid development of pet nutrition and welfare, the lifespan of pet dogs is gradually increasing. More and more tumor diseases occur in middle-aged and elderly dogs, bringing great pain to both pets and their owners. Common types of canine tumors include mammary tumors, skin and soft tissue tumors, etc.

[0003] Canine mammary tumor (CMT) is one of the most common tumor diseases in pet clinics, accounting for about 25% - 42% of tumor diseases in female dogs. Affected by factors such as age, genetics, environmental pollution, and feeding habits, canine mammary tumors show characteristics such as being younger, more complex, and having a higher malignancy rate. Currently, the clinical treatment of canine mammary tumors mainly focuses on surgery, and combined with chemotherapy, the recurrence and metastasis rates can be effectively reduced.

[0004] Veterinary clinics use surgical operation as the main treatment method for CMT. Since simple surgical therapy is difficult to eradicate the lesion, tumors often metastasize and spread, resulting in a short survival period after CMT surgery and a recurrence rate as high as 58%. At the same time, CMT mainly occurs in elderly dogs, and a large number of cases are limited by the organ function and are difficult to operate. Promoting chemotherapy can effectively supplement the limitations of surgical therapy and improve the effectiveness and applicability of clinical treatment. Commonly used chemotherapy drugs mainly include alkylating agents, platinum compounds, anti-tubulin agents, antitumor antibiotics, antimetabolic drugs, targeted therapy drugs, and other drugs. However, current research shows that the most commonly used alkylating agents and platinum drugs for pet tumors have poor efficacy against CMT.

[0005] Abroad, small molecule chemical drugs such as masitinib and toceranib phosphate have been approved as tumor drugs for pet clinics, while in China, there is no approved listing of pet-specific anti-cancer drugs. In pet clinics, there is a situation where there is no legal and compliant anti-mammary tumor drug to use. Therefore, it is necessary to accelerate the development of anti-mammary tumor drugs for dogs to meet the anti-cancer drug needs of pet clinics, improve the quality of life and welfare of diseased dogs, and provide support for the rapid development of the pet economy.

[0006] Canine breast cancer has many biological similarities with human breast cancer. Therefore, it has been proposed that immunotherapy can be used for the treatment of canine breast cancer. Although there is currently a large amount of research on tumor immunotherapy, there are still many problems to be solved when applying immunotherapy to the treatment of canine tumors. (1) The existing targets of immunotherapy are not completely applicable to dogs, and new targets need to be explored. (2) There are a wide variety of dog breeds, with huge differences in body size among different breeds. It is difficult to solve the dosage problem of immunotherapy, and more experimental data are needed for exploration. (3) The high price is a very critical issue in the treatment of canine tumors with immunotherapy. Most of the current immunotherapies require precise one-on-one treatment, which has high technical and conditional requirements for pet hospitals during treatment, and the drug prices are expensive.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The purpose of the present invention is to provide the application of an immunomodulator in the preparation of a composition for the treatment of animal cancers to solve the above technical problems.

[0009] The present invention is implemented as follows:

[0010] In the first aspect, the present invention provides the application of an immunomodulator in the preparation of a composition for the treatment of animal cancers, wherein the immunomodulator includes: Poly IC and QS-21 with a mass ratio of (1.3 - 60):1, the cancer is breast cancer, melanoma, liver cancer, colon cancer, ovarian cancer or lung cancer, and the animal is a dog, mouse or rat.

[0011] In the second aspect, the present invention provides the application of a composition in the preparation of a drug for the treatment of animal cancers, wherein the composition includes: an immunomodulator and a pharmaceutically acceptable carrier, the immunomodulator includes: Poly IC and QS-21 with a mass ratio of (1.3 - 60):1, the cancer is breast cancer, melanoma, liver cancer, colon cancer, ovarian cancer or lung cancer, and the animal is a dog, mouse or rat.

[0012] The present invention has the following beneficial effects:

[0013] The present invention provides the application of an immunomodulator in the treatment of animal cancers. After being administered to the tumor tissue of a subject, it activates antigen-presenting cells in the tumor tissue through the TLR3 receptor, further activates the immune response in the tumor microenvironment, promotes the tumor infiltration of CD4+T and CD8+T cells, thereby exerting an anti-tumor effect. Compared with the existing immunotherapy, the immunomodulator provided by the present invention has the advantages of strong broad-spectrum property, low cost, easy operation and good effect, can effectively inhibit the growth of animal cancers, especially has more advantages in the treatment of canine breast cancer and is more easily promoted and used.

[0014] In addition, the immunomodulator provided by the present invention can maintain its effect for a long time after drug withdrawal. In terms of safety, there are no serious adverse events during injection, and the animals have good tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0016] Figure 1 Tumor growth curves of different ratios of QS21 and Poly(I:C) in the treatment of breast cancer in mice;

[0017] Figure 2 Statistical results chart of tumor weights of different ratios of QS21 and Poly(I:C) in the treatment of breast cancer in mice;

[0018] Figure 3 Tumor growth curves of QS21, Poly IC used alone, and their combination (AYK103);

[0019] Figure 4 Statistical results chart of tumor weights of QS21, Poly IC used alone, and their combination (AYK103);

[0020] Figure 5 Tumor growth curve comparing the effects of the combination of other immune adjuvants and AYK103 in the treatment of melanoma in mice;

[0021] Figure 6 Tumor growth curve of AYK103 in the treatment of breast cancer in mice;

[0022] Figure 7 Statistical results chart of tumor weights of AYK103 in the treatment of breast cancer in mice;

[0023] Figure 8 Tumor tissue diagram of AYK103 in the treatment of breast cancer in mice;

[0024] Figure 9 Results diagram of flow cytometry detection of the effect of AYK103 on the number of immune cells in tumor tissues;

[0025] Figure 10 Results diagram of immunohistochemical detection of the effect of AYK103 on the number of immune cells in tumor tissues;

[0026] Figure 11 Treatment result diagram of the first diseased dog;

[0027] Figure 12 It is the treatment result diagram of the second diseased dog;

[0028] Figure 13 It is the statistical result diagram of the tumor weight of AYK103 in treating mouse lung cancer;

[0029] Figure 14 It is the statistical result diagram of the tumor weight of AYK103 in treating mouse liver cancer;

[0030] Figure 15 It is the statistical result diagram of the tumor weight of AYK103 in treating mouse colon cancer;

[0031] Figure 16 It is the in vivo fluorescence imaging result diagram of AYK103 in treating peritoneal metastasis of mouse ovarian cancer;

[0032] Figure 17 It is the survival curve of AYK103 in treating peritoneal metastasis of mouse ovarian cancer. Detailed implementation manners

[0033] Reference to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided by way of explanation and not limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the present invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0034] The term "immunomodulator" generally refers to a substance that affects the function of the immune system. Immunomodulators can enhance or reduce the immune response. For example, immunomodulators can be active agents of immunotherapy, including but not limited to, for example, cytokines, granulocyte colony-stimulating factor (G-CSF), interferons, imiquimod, cell membrane fragments from bacteria, chemokines, interleukins, Poly IC, QS-21, cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, and recombinant, synthetic, and / or natural preparations of glucan.

[0035] The terms "treat" or "cure" or "prevent" or "alleviate" or "improve" are used interchangeably herein and refer to a method of obtaining a beneficial or desired result (including but not limited to a therapeutic benefit and / or a prophylactic benefit). As used herein, a therapeutic benefit generally refers to eradicating or alleviating the severity of the underlying condition being treated. In addition, a therapeutic benefit is achieved by eradicating, alleviating the severity, or reducing the incidence of one or more physiological symptoms associated with the underlying condition such that an improvement is observed in the subject (although the subject may still be afflicted with the underlying condition).

[0036] The term "subject" or "test subject" generally refers to non-human animals, including but not limited to dogs, mice or rats.

[0037] Poly IC, namely polyinosinic-polycytidylic acid, is synonymous with "Poly(I:C)". Polyinosinic-polycytidylic acid is a toll-like receptor 3 (TLR3) agonist, which is a synthetic double-stranded RNA (dsRNA) with the CAS number: 42524-50-0.

[0038] QS-21, with the CAS number 141256-04-4, has the molecular formula: C 92 H 148 O 46 , and the molecular weight: 1990.14. It is also synonymous with "QS21" and "Quillaja saponin 21".

[0039] In a first aspect, the present invention provides the use of an immunomodulator in the preparation of a composition for treating animal cancers. The immunomodulator includes: Poly IC and QS-21 with a mass ratio of (1.3 to 60):1. The cancers are breast cancer, melanoma, liver cancer, colon cancer, ovarian cancer or lung cancer, and the animals are dogs, mice or rats.

[0040] Through long-term and extensive screening, the inventors found that the combined use of Poly IC and QS-21 has a better anti-tumor effect compared to the use of Poly IC or QS-21 alone or the use of positive chemical anti-cancer drugs. It can inhibit the growth rate of tumors, reduce the size and weight of tumors, and can reverse the tumor immune microenvironment, increase the number of anti-tumor immune cells in tumor tissues, transform the tumor tissue microenvironment from an immune-suppressive type to an immune-activated type, exert an anti-tumor function, and inhibit the growth of tumors.

[0041] In addition, the immunomodulator provided by the present invention can still maintain its effect for a long time after drug withdrawal, has good safety, no serious adverse events after injection, and good tolerance. The immunomodulator also has the advantages of strong broad-spectrum, low cost and easy operation, and has broad application prospects.

[0042] The mass ratio of Poly IC and QS-21 includes but is not limited to 1.3:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 15:1, 16:1, 20:1, 25:1, 30:1, 35:1, 40:1, 42:1, 45:1, 48:1, 50:1, 52:1, 55:1 or 60:1, and the ranges between any two of them.

[0043] At the above ratios, both Poly IC and QS-21 have anti-tumor effects.

[0044] The lung cancer is selected from small cell lung cancer and non-small cell lung cancer, and the non-small cell lung cancer is selected from squamous cell carcinoma and adenocarcinoma.

[0045] In an alternative embodiment, breast cancer includes, but is not limited to, breast epithelial cancer;

[0046] In a preferred embodiment of the application of the present invention, the breast cancer is ductal carcinoma of the breast or lobular carcinoma of the breast.

[0047] In a preferred embodiment of the application of the present invention, the immunomodulator includes Poly IC and QS-21 with a mass ratio of (4 - 32):1; at this ratio, the anti-tumor effect is better, and it has a better effect of inhibiting tumor growth, reducing tumor weight and volume.

[0048] In a preferred embodiment of the application of the present invention, the immunomodulator includes Poly IC and QS-21 with a mass ratio of (4 - 16):1;

[0049] In a preferred embodiment of the application of the present invention, the immunomodulator includes Poly IC and QS-21 with a mass ratio of (6 - 8):1. At the above ratio, the anti-tumor effect is better, and it has a better effect of inhibiting tumor growth, reducing tumor weight and volume.

[0050] In a preferred embodiment of the application of the present invention, the composition further includes: a pharmaceutically acceptable carrier.

[0051] The pharmaceutically acceptable carrier is selected from at least one of protective agents, excipients, binders, disintegrants, lubricants, fragrances, preservatives, stabilizers, suspending agents, dispersing agents, and diluents.

[0052] Examples include: protective agents such as liposomes; excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; flavors such as citric acid, menthol, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series such as diethylenetriaminepentaacetic acid (DTPA); suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents such as water, organic solvents; waxes, fats and oils such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0053] In a preferred embodiment of the application of the present invention, the carrier is a liposome. A liposome is a nano-vesicle formed by self-assembly of phospholipid or phospholipid-like molecules, having a bilayer structure and a long-chain alkyl hydrophobic end.

[0054] In a preferred embodiment of the application of the present invention, the liposome is a cationic liposome;

[0055] In a preferred embodiment of the application of the present invention, the lipid component of the liposome is a phospholipid or a phospholipid-like molecule.

[0056] Phospholipid-like molecules refer to block polymers of bonded phospholipids with amphiphilic properties, such as substances like PCL-b-(PBrCL-g-PMPC) disclosed in patent CN202010717453.1.

[0057] In a preferred embodiment of the application of the present invention, the lipid component in the liposome refers to: an empty liposome containing phosphatidylcholine and sphingomyelin or composed of them, wherein phosphatidylcholine is present in an amount of 15-98% (w / w).

[0058] In a preferred embodiment of the application of the present invention, the phospholipid is selected from glycerophospholipids and sphingomyelins.

[0059] In a preferred embodiment of the application of the present invention, the phospholipid is selected from any one or a combination of at least two of dioleoyl phosphatidylcholine (DOPC), soy lecithin, sunflower lecithin, DSPG (distearoyl phosphatidylglycerol), DPPC, HSPC (hydrogenated soy phospholipid), DSPC (distearoyl phosphatidylcholine), and EPC (egg yolk lecithin); such as the combination of HSPC and DSPC, the combination of HSPC and DSPC, the combination of HSPC and EPC, etc. Any other combination methods are not elaborated here one by one.

[0060] In a preferred embodiment of the application of the present invention, when the phospholipid is lecithin, the phosphatidylcholine (PC) content in the lecithin includes at least one of 15%, 30%, 35%, 50%, 60%, 70%, 80%, 90%, and 98%.

[0061] In a preferred embodiment of the application of the present invention, in the composition, the mass ratio of Poly IC, QS-21 to liposome is (1.3 - 60):1:(15 - 25). Different mass ratios affect the interaction between QS21 and the liposome. QS21 has hemolytic effects, and QS21 that can be effectively adsorbed on the liposome can significantly reduce this side effect. For example, the mass ratio of Poly IC, QS-21 to liposome is (1.3 - 60):1:15, (1.3 - 60):1:18, (1.3 - 60):1:20, (1.3 - 60):1:22, (1.3 - 60):1:25.

[0062] In a preferred embodiment of the application of the present invention, in the composition, the mass ratio of Poly IC, QS-21 to liposome is (6 - 10):1:(15 - 25).

[0063] In a preferred embodiment of the application of the present invention, the liposome is prepared from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dioleoyl phosphatidylcholine (DOPC), and cholesterol (DC-Chol).

[0064] For example, 1,2-dioleoyl-3-trimethylammonium propane, dioleoyl phosphatidylcholine, and cholesterol are mixed in a mass ratio of 1:8 - 12:3 - 4 to obtain a first mixture; using the first mixture as a raw material, the liposome is prepared by any one of the ethanol injection method, thin film dispersion method, ultrasonic dispersion method, and reverse evaporation method.

[0065] In some embodiments, the specific mixing mass ratio of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dioleoyl phosphatidylcholine (DOPC) and cholesterol can be any one of 1:8:3, 1:8:4, 1:9:3, 1:9:4, 1:10:3, 1:10:4, 1:11:3, 1:11:4, 1:12:3, 1:12:4 or the range between any two of them.

[0066] In a preferred embodiment of the application of the present invention, the composition is prepared by the following method: blending a carrier with Poly IC and QS-21. In one embodiment, Poly IC and QS-21 are first blended to obtain a mixture, and then the mixture is blended with the carrier, and through self-assembly, Poly IC and QS-21 are assembled in the carrier.

[0067] In a preferred embodiment of the application of the present invention, when the carrier is a liposome, the particle size of the liposome is 100 - 300 nm. The particle size can specifically be any one of 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 nm or the range between any two of them. The optimal uptake particle size in vivo is 60 - 300 nm. If the particle size is too small, it is easily metabolized rapidly by the kidneys, reducing the residence time in the tumor, thereby reducing the effect.

[0068] In a preferred embodiment of the application of the present invention, the composition is a drug;

[0069] In a preferred embodiment of the application of the present invention, the administration method of the drug is injection;

[0070] In a preferred embodiment of the application of the present invention, the administration method of the drug is intratumoral injection.

[0071] In a preferred embodiment of the application of the present invention, the drug has the following uses:

[0072] (1) Activating the immune response of the tumor microenvironment and reversing the tumor immunosuppressive microenvironment;

[0073] (2) Inhibiting the tumor growth rate;

[0074] (3) Reducing the tumor weight;

[0075] (4) Reducing the tumor volume;

[0076] In a preferred embodiment of the application of the present invention, the use of the drug to activate the immune response of the tumor microenvironment includes: promoting the tumor infiltration of CD4 T and CD8 T cells;

[0077] In a preferred embodiment of the application of the present invention, the use of the drug to activate the immune response of the tumor microenvironment is selected from at least one of the following:

[0078] (1) Increasing the number of cytotoxic T cells CD8+T in tumor tissues;

[0079] (2) Increasing the number of effector memory T cells CD8+T in tumor tissues;

[0080] (3) Increasing the number of helper T cells CD4+T in tumor tissues;

[0081] (4) Increasing the number of macrophages among antigen-presenting cells in tumor tissues;

[0082] (5) Increasing the number of dendritic cells among antigen-presenting cells in tumor tissues.

[0083] In a preferred embodiment of the application of the present invention, the final concentration of QS-21 in the composition is 50-300 μg / mL, and this final concentration can specifically be any one or the range between any two of 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300 μg / mL.

[0084] The final concentration of Poly I:C is 400-3000 μg / mL. This final concentration can specifically be any one or the range between any two of 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000 μg / mL.

[0085] In a second aspect, the present invention provides the use of a composition in the preparation of a drug for treating animal cancer. The composition includes: an immunomodulator and a pharmaceutically acceptable carrier. The immunomodulator includes: Poly IC and QS-21 with a mass ratio of (1.3-60):1. The cancer is breast cancer, melanoma, liver cancer, colon cancer, ovarian cancer or lung cancer, and the animal is a dog, a mouse or a rat.

[0086] In a preferred embodiment of the application of the present invention, the carrier is a liposome;

[0087] In a preferred embodiment of the application of the present invention, the liposome is a cationic liposome;

[0088] In a preferred embodiment of the application of the present invention, in the drug, the mass ratio of Poly IC, QS-21 to liposome is (1.3 - 60):1:(15 - 25); for example, the mass ratio of Poly IC, QS-21 to liposome is (1.3 - 60):1:15, (1.3 - 60):1:18, (1.3 - 60):1:20, (1.3 - 60):1:22, (1.3 - 60):1:25, (5 - 50):1:25, (10 - 25):1:25, or (10 - 32):1:25, and it can also be any one or the range between any two of 1:6:16, 1:6:18, 1:6:20, 1:6:22, 1:6:24, 1:8:16, 1:8:18, 1:8:20, 1:8:22, 1:8:24, 1:10:16, 1:10:18, 1:10:20, 1:10:22, 1:10:24.

[0089] In a preferred embodiment of the application of the present invention, in the composition, the mass ratio of Poly IC, QS-21 to liposome is (6 - 10):1:(15 - 25).

[0090] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0091] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0092] Example 1

[0093] This example provides an immunomodulator and a drug (AYK103) comprising the immunomodulator and liposome. Specifically, the immunomodulator is composed of Poly IC (CAS No.: 42524 - 50 - 0) and QS-21 (CAS No. 141256 - 04 - 4) with a mass ratio of 4:1.

[0094] The liposome was prepared by the ethanol injection method as follows: 1,2-dioleoyl-3-trimethylammonium propane, dioleoylphosphatidylcholine and cholesterol were dissolved in ethanol according to a mass ratio of 1:8:3, and the first immunoadjuvant Poly IC was added and loaded with one syringe; the aqueous phase PBS was loaded with another syringe; and they were injected into a T-shaped microchannel mixer by controlling the flow rate to obtain a liposome solution containing Poly IC. The method can refer to the literature;

[0095] After mixing the Poly IC liposome solution and the QS21 aqueous solution in proportion, mix them with a vortex mixer for 30 min to obtain the AYK103 preparation.

[0096] Example 2

[0097] This example provides an immunomodulator and a drug comprising the immunomodulator and liposomes.

[0098] Specifically, the immunomodulator consists of Poly IC and QS-21 with a mass ratio of 8:1.

[0099] The liposomes are prepared by the following method: Dissolve 1,2-dioleoyl-3-trimethylammonium propane, dioleoyl phosphatidylcholine, and cholesterol in ethanol at a mass ratio of 1:8:3, add the first immunoadjuvant Poly IC, and load it with a syringe; load the aqueous phase PBS with another syringe; inject it into a T-shaped microchannel mixer by controlling the flow rate to obtain a Poly IC liposome solution. The method can refer to the literature (Maritim S, Boulas P, Lin Y. International journal of pharmaceutics, 2021, 592:120051.);

[0100] After mixing the Poly IC liposome solution and the QS21 aqueous solution in proportion, mix them with a vortex mixer for 30 min to obtain the AYK103 preparation.

[0101] Example 3

[0102] Compared with Example 2, the only difference is that the ratio of the immunomodulator is different. This example consists of Poly IC and QS-21 with a mass ratio of 16:1.

[0103] Example 4

[0104] Compared with Example 2, the only difference is that the ratio of the immunomodulator is different. This example consists of Poly IC and QS-21 with a mass ratio of 32:1.

[0105] Comparative Example 1

[0106] Compared with Example 2, the only difference is that the ratio of the immunomodulator is different. This example consists of Poly IC and QS-21 with a mass ratio of 1:1.

[0107] Comparative Example 2

[0108] Compared with Example 2, the only difference is that the ratio of the immunomodulator is different. This example consists of Poly IC and QS-21 with a mass ratio of 2:1.

[0109] Comparative Example 3

[0110] Compared with Example 2, the only difference is that the components of the immunomodulator are different. This example consists of Poly IC and MPLA (CAS No. 1246298-63-4, prepared by Anyikang) with a mass ratio of 4:1.

[0111] Comparative Example 4

[0112] Compared with Example 2, the only difference is that the components of the immunomodulator are different. This example consists of QS-21 and MPLA with a mass ratio of 1:2.

[0113] Experimental Example 1

[0114] In this experimental example, the anti-tumor effects of different ratios of two active ingredients, QS-21 and Poly I:C, were screened.

[0115] Experimental method:

[0116] According to the preparation methods of AYK103 drugs in Examples 1-4 and Comparative Examples 1-2, several drugs with different ratios were prepared. QS21:Poly IC were 1:1, 1:2, 1:4, 1:8, 1:16, 1:32 respectively.

[0117] Construction of 4T1 cell line breast cancer model: Mouse breast cancer cell line 4T1 cells (American Type Culture Collection, i.e., ATCC). Culture conditions: RPMI 1640 medium + 10% FBS + 1% double antibody. Centrifuge the mouse breast cancer cell line 4T1 cells at 1000 r / min for 5 minutes. Detect the cell viability by 0.4% trypan blue exclusion method (viability > 90%), wash the cells 3 times with PBS, and centrifuge at 1000 r / min for 5 minutes. Add PBS to adjust the cell concentration to 5×10 6 cells / ml, aspirate 100 μl of cell suspension and inoculate it under the armpits of 20 female mice. On the 5th day after tumor inoculation in mice, measure the subcutaneous tumor volume of female C57BL / 6J mice respectively.

[0118] Tumor volume measurement: The tumor volume was measured by measuring the tumor diameter, once every three days, to dynamically observe the anti-tumor activity of the test drugs. Measure the long and short diameters of the tumor with a vernier caliper, and calculate the tumor volume. Tumor volume = 1 / 2 × long diameter × short diameter².

[0119] According to the tumor volume, the animals were randomly divided into 8 experimental groups as follows: 0.9% NaCl (100 μL / animal), Vehicle (blank liposome control, 100 μL / animal), 32:1 (Poly I:C:QS-21, Example 4), 16:1 (Poly I:C:QS-21, Example 3), 8:1 (Poly I:C:QS-21, Example 2), 4:1 (Poly I:C:QS-21, Example 1), 2:1 (Poly I:C:QS-21, Comparative Example 2), 1:1 (Poly I:C:QS-21, Comparative Example 1). There were 8 animals in each group, all of which were female. The first administration was carried out on the day after grouping (the 8th day after tumor inoculation), and the dosage was 100 μL / animal. The second administration was carried out on the 11th day after tumor inoculation, and the dosage was 100 μL / animal. The last administration was carried out on the 14th day after tumor inoculation, and the dosage was 100 μL / animal. The general conditions of the animals such as body weight and diet were observed daily, the change of the body weight of the mice was monitored, and the change of the tumor size was detected every three days.

[0120] Experimental results:

[0121] Figure 1 As can be seen from the results, when the ratio of the two active ingredients PolyIC:QS21 in AYK103 is between 1:1 and 32:1, the tumor growth rate is significantly inhibited. Figure 2 The results show that the tumor weight and volume are significantly smaller than those of the 0.9% NaCl and Vehicle groups, and among the six groups of 1:1, 2:1, 4:1, 8:1, 16:1, and 32:1, the optimal ratios for anti-tumor effects can be 4:1, 8:1, 16:1, and 32:1.

[0122] Experimental Example 2

[0123] Experiment on the enhanced anti-tumor effect by the combined use of QS-21 and Poly I:C.

[0124] Experimental method:

[0125] Liposomes containing only QS-21, liposomes containing only Poly I:C, and liposomal AYK103 containing QS-21 / Poly I:C were prepared according to the preparation method of AYK103 in Example 1, wherein the mass ratio of QS-21:Poly I:C in AYK103 was 1:8.

[0126] A mouse breast cancer model was constructed according to the method in Experimental Example 1. The tumors were randomly divided into 4 experimental groups using a random grouping method, with 8 mice in each group, all female. The first administration was carried out on the day after grouping (the 9th day after tumor inoculation), the second administration was carried out on the 12th day after tumor inoculation, and the last administration was carried out on the 15th day after tumor inoculation. The dosage was the same as that in Experimental Example 1. The general conditions of the animals such as body weight and diet were observed daily, the body weight change of the mice was monitored, and the tumor size change was detected once every three days.

[0127] Figure 3 、 Figure 4 From the results in, it can be seen that after injecting QS21 liposome and injecting Poly I:C liposome, tumor growth was inhibited, and the tumor growth rate of injecting Poly I:C liposome was lower than that of injecting QS21 liposome. However, the effects of both were not as good as that of injecting AYK103 (QS21 / Poly I:C), indicating that the combined use of QS21 and Poly I:C has a synergistic effect.

[0128] Experimental Example 3

[0129] Comparison of the effects of combined use of other immune adjuvants with AYK103.

[0130] Three experimental groups were set up, and immune adjuvants MPLA+QS-21 (Comparative Example 4), MPLA+PolyIC (Comparative Example 3), and AYK103 (Example 1) were prepared respectively.

[0131] Experimental method

[0132] 6-week-old C57BL / 6J mice were subcutaneously inoculated with 5×10 5 B16-OVA cells to establish a subcutaneous tumor model of melanoma in mice. On the 5th day after tumor inoculation, the subcutaneous tumor volumes of female C57BL / 6J mice were measured respectively. According to the tumor volume, they were randomly divided into 4 experimental groups as follows: liposome control group (blank liposome negative control group without encapsulated drug, 100 μL / mouse / time), MPLA+QS-21 (100 μL / mouse / time), MPLA+PolyIC (100 μL / mouse / time), AYK103 (100 μL / mouse / time). There were 10 mice in each group, all female. The first administration was carried out on the day after grouping (the 9th day after tumor inoculation), the second intratumoral injection was carried out on the 12th day after tumor inoculation, and the last intratumoral injection was carried out on the 15th day after tumor inoculation. The general conditions of the animals such as body weight and diet were observed daily, the body weight change of the mice was monitored, and the tumor size change was detected once every three days.

[0133] Experimental reference Figure 5 , it can be seen that among these three adjuvant combinations, AYK103 (Poly(I:C)+QS21) has the best effect on inhibiting tumor growth, and the tumor inhibition rate is about 96%.

[0134] Experimental Example 4

[0135] Experiment on inhibiting the growth of murine breast cancer by AYK103.

[0136] Experimental method:

[0137] Prepare AYK103 according to the preparation method in Example 2.

[0138] Construct a murine breast cancer model according to the method in Experimental Example 1. On the 8th day after tumor inoculation, measure the subcutaneous tumor volume of female C57BL / 6J mice respectively. According to the tumor volume, randomly divide them into 3 experimental groups by the method of random grouping, specifically as follows: Vehicle (100 μL / mouse), AYK103 group (100 μL / mouse), CTX group (25 mg / kg). Each group has 10 mice, all of which are female. The first administration is carried out on the day after grouping (the 8th day after tumor inoculation), the second administration is carried out on the 11th day after tumor inoculation, and the last administration is carried out on the 14th day after tumor inoculation. Observe the general conditions of the animals such as body weight and diet every day, monitor the change of body weight of the mice, and detect the change of tumor size once every three days.

[0139] Figure 6 It can be seen from the results that after injecting AYK103, the tumor growth was inhibited, and the tumor growth rate of the AYK103 group was significantly lower than that of the negative control Vehicle group and the positive control chemotherapeutic drug cyclophosphamide CTX group. Figure 7 、 Figure 8 The results showed that the tumor weight and the final volume of the mice in the AYK103 group were significantly smaller than those in the cyclophosphamide CTX group. These results indicate that the effect of AYK103 in inhibiting breast cancer growth is better than that of the chemotherapeutic drug cyclophosphamide, and the relative tumor inhibition rate is 70.4%.

[0140] Experimental Example 5

[0141] Experiment on reversing the tumor immunosuppressive microenvironment by AYK103.

[0142] The experimental method is as follows:

[0143] Prepare AYK103 according to the preparation method in Example 2.

[0144] Construct a murine breast cancer model according to the method in Experimental Example 1. On the 4th day after tumor inoculation, measure the subcutaneous tumor volume of female C57BL / 6J mice respectively. According to the tumor volume, randomly divide them into 2 experimental groups by the method of random grouping, specifically as follows: Vehicle (100 μL / mouse), AYK103 group (100 μL / mouse). Each group has 8 mice, all of which are female. After grouping, administer the drug according to the method in Example 3. On the 2nd day after the last administration, take out the tumor tissue for flow cytometry detection and immunohistochemical detection.

[0145] 1) Flow cytometry detection and processing method:

[0146] Tumor tissue sampling: Mice were sacrificed by cervical dislocation. The hair, skin, and tissues of the mice were carefully separated with surgical scissors and curved forceps. The intact subcutaneous breast cancer tumors were gently dissected, weighed, recorded, and photographed. Then, 0.1 - 0.5 g of each tumor tissue was collected and placed in pre-cooled 1×PBS.

[0147] Preparation of single-cell suspension from tumor tissue: Clean 5 mL EP tubes were prepared according to the group numbers. The above tumor tissues were cut into 5 mm tissue blocks in the tubes and resuspended with the prepared tissue digestion solution [RPMI - 1640 medium, 0.5 mg / ml Collagenase D, 0.1 mg / ml Dnase I] at a volume of 4 mL digestion solution per sample, and then transferred to a gentle MACS C tube. The program 37C_Multi_F was run. After the program ended, the C tube was removed from the gentle MACS tissue processor. The filter was rinsed with RPMI - 1640, and the grinding solution was filtered through a 70 μm filter membrane. The cell suspension was collected in a 10 mL EP tube, centrifuged (400 g, 5 min, room temperature), and the supernatant was discarded. 1× red blood cell lysis buffer was prepared with pure water, and the cell pellet was resuspended at a volume of 2 mL 1× red blood cell lysis buffer per sample. After standing at room temperature for 5 min, 5 ml of 1×PBS was added to terminate the lysis, and then centrifuged (400 g, 5 min, room temperature) to discard the supernatant.

[0148] Live / Dead staining: The above cell pellet was resuspended with the prepared Live / Dead dilution solution (Near IR diluted at a ratio of 1:500 with 1×PBS) at a volume of 100 μL Live / Dead dilution solution per sample, incubated at 4°C in the dark for 30 min, and then stained with 1 mL of 1×PBS to terminate the staining. A small amount of cell suspension was taken from each group of samples as a Live / Dead single-positive tube for backup, and then centrifuged (400 g, 5 min, room temperature) to discard the supernatant.

[0149] Flow cytometry antibody staining: The above cell pellet was resuspended with the prepared mixed staining solution (PE / Cy7-CD3, FITC-CD4, BV510-CD8, PE-CD45, BV421-CD62, APC-CD206 diluted at a ratio of 1:800 with 1×PBS) at a volume of 100 μL mixed staining solution per sample, incubated at 4°C in the dark for 30 min, and then stained with 1 mL of 1×PBS to terminate the staining. Each antibody single-positive tube was stained with 0.2 μL of Beads per tube for backup, and the staining method was the same as above.

[0150] Sample loading: For flow cytometry detection, first perform voltage determination on the blank control tube and single-positive tube. After fixing the voltage, record the cell count of each group of samples, and convert it to cells / g tumor using the mass of the sampled tumor tissue.

[0151] 2) Immunohistochemical detection processing method:

[0152] Slice the tumor tissue with a thickness of 4 μm and spread the slices in water at 42°C. Immerse the slices in xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, absolute ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and distilled water wash for 5 minutes. Perform EDTA microwave heat repair for 5 - 8 minutes and cool to room temperature. Drop endogenous peroxidase blocking solution and incubate at room temperature for 10 minutes, wash 3 times with PBS buffer for 5 minutes each time. Drop blocking serum and incubate at 37°C for 30 minutes. Drop the primary antibody and incubate in a wet box at 37°C for 2 h, wash 3 times with PBS buffer for 5 minutes each time. Drop HRP-labeled goat anti-rabbit and incubate at 37°C for 30 minutes, wash 3 times with PBS buffer for 5 minutes each time. Prepare DAB chromogenic solution by mixing 1 ml of solution B + 1 drop of solution A, drop the DAB chromogenic solution, and observe under the microscope. Drop Mayer's hematoxylin for 30 s, wash with distilled water, immerse in bluing solution for 1 minute, and wash with water. Dehydrate in a gradient of 75% - 95% - 100% alcohol for 1 minute in each cylinder, clear in three cylinders of xylene for 2 minutes in each cylinder, and mount with neutral balsam.

[0153] Figure 9 It can be seen that after injecting AYK103, the numbers of cytotoxic T cells CD8+T, effector memory cytotoxic T cells Effector memory CD8+T, helper T cells CD4+T, and anti-tumor M1 macrophages in the tumor tissue increase significantly, indicating that the tumor tissue immune response is activated, and the increase in anti-tumor immune cells contributes to the killing of tumors. Figure 10 The results also prove that after injecting AYK103, the numbers of CD8+T, CD4+T cells, and macrophages and DC cells in antigen-presenting cells in the tumor tissue increase significantly, indicating that the microenvironment of the tumor tissue changes from an immunosuppressive type to an immune-activated type.

[0154] Example 6

[0155] Experiment on treating breast cancer in pet dogs with AYK103 prepared in Example 2.

[0156] Adopt a single-arm trial design, collect 6 breast cancer-affected dogs from a pet hospital in Xinxiang, Henan and a pet hospital in Dalian. Administer AYK103 by injecting it into the tumor or subcutaneously around the tumor. Administer 0.5 - 1 mL to each tumor once a week. Measure the length and width of the tumor with a ruler and calculate the tumor volume = (length * width * width) / 2 (mm3 ) Record the tumor volume, body temperature, body weight of the test dogs, and the clinical characteristics of the animals: whether there is fever, whether there is redness, swelling and inflammation at the injection site, and the mental state of the animals.

[0157] The test results show that:

[0158] Among the 6 dogs, 4 dogs dropped out, withdrew or died, and 2 dogs received complete treatment. The preliminary clinical treatment effects are shown in the following table. Figure 11 The results show that the first diseased dog treated is a Teddy dog, with a total of two lesions. After 8 doses of drug administration, the two tumor volumes decreased by 65% and 67% respectively compared with before treatment. When reexamined 3 months after drug withdrawal, the tumors decreased by 90% and 73% respectively compared with before treatment, indicating that AYK103 not only has a good therapeutic effect on canine breast cancer, but also the effect can be maintained for at least 3 months or more.

[0159] Figure 12 The results show that the second diseased dog treated is a Golden Retriever dog, with a total of one lesion. After 12 doses of drug administration, the tumor decreased by 25%, further indicating the effectiveness of AYK103 against canine breast cancer. AYK103 has good safety. The main adverse events that occurred after injection in the 2 treated diseased dogs were local redness, swelling, fever, vomiting, and listlessness at the injection site, all of which could recover on their own within 24 - 48 hours, and no serious adverse events occurred, indicating that AYK103 has good safety.

[0160] Experimental Example 7

[0161] Experiment on treating mouse lung cancer with AYK103 prepared in Example 2.

[0162] Experimental method

[0163] 6-week-old C57BL / 6J mice were subcutaneously inoculated with 6×10 5 LLC cells to establish a subcutaneous tumor model of mouse lung cancer. On the 6th day after tumor inoculation, the subcutaneous tumor volumes of female C57BL / 6J mice were measured respectively, and according to the tumor volume, they were randomly divided into 3 experimental groups by the random grouping method, and grouped and administered according to the method of Experimental Example 4. Observe the general conditions of the animals such as body weight and diet every day, monitor the change of mouse body weight, and detect the change of tumor size once every three days.

[0164] Experimental results

[0165] After injecting AYK103, the growth of LLC tumors was inhibited, and the tumor growth rate in the AYK103 group was significantly lower than that in the negative control Vehicle group and the positive control chemotherapy drug cyclophosphamide CTX group ( Figure 13 ), and the relative tumor inhibition rate was 73.2%.

[0166] Experimental Example 8

[0167] Experiment on treating liver cancer in mice with AYK103 prepared in Example 2.

[0168] Experimental method

[0169] Six-week-old C57BL / 6J mice were subcutaneously inoculated with 2×10 6 H22 cells to establish a subcutaneous tumor model of liver cancer in mice. On the 4th day after tumor inoculation, the subcutaneous tumor volumes of female C57BL / 6J mice were measured respectively, and according to the tumor volume, they were randomly divided into 3 experimental groups as follows: Vehicle (100 μL / mouse), AYK103 group (100 μL / mouse), Sorafenib group. The general conditions of the animals such as body weight and diet were observed daily, the body weight changes of the mice were monitored, and the tumor size changes were detected once every three days.

[0170] Experimental results

[0171] After injecting AYK103, the growth of H22 tumors was inhibited, and the tumor growth rate in the AYK103 group was significantly lower than that in the negative control Vehicle group and the positive control chemotherapeutic drug Sorafenib group ( Figure 14 ), and the relative tumor inhibition rate was 62.6%.

[0172] Experimental Example 9

[0173] Experiment on treating colon cancer in mice with AYK103 prepared in Example 2.

[0174] Six-week-old C57BL / 6J mice were subcutaneously inoculated with 1×10 5 CT26 cells to establish a subcutaneous tumor model of liver cancer in mice. On the 11th day after tumor inoculation, the subcutaneous tumor volumes of female C57BL / 6J mice were measured respectively, and according to the tumor volume, they were randomly divided into 3 experimental groups as follows: Vehicle (100 μL / mouse), AYK103 group (100 μL / mouse), CTX group. The general conditions of the animals such as body weight and diet were observed daily, the body weight changes of the mice were monitored, and the tumor size changes were detected once every three days.

[0175] Experimental results

[0176] After injecting AYK103, the growth of CT26 tumors was inhibited, and the tumor growth rate in the AYK103 group was significantly lower than that in the negative control Vehicle group and the positive control chemotherapeutic drug CTX group ( Figure 15 ), and the relative tumor inhibition rate was 94.4%.

[0177] Experimental Example 10

[0178] Experiment on treating the peritoneal metastasis model of ovarian cancer in mice with AYK103 prepared in Example 2.

[0179] 6-week-old C57BL / 6J mice were intraperitoneally inoculated with 1×10 6 ID-8-Luci mouse epithelial ovarian cancer cells to establish a mouse model of intraperitoneal metastasis of ovarian cancer. On the 3rd day after tumor inoculation, the mice were grouped and administered drugs according to fluorescence imaging as follows: Vehicle (100 μL / mouse), AYK103 group (100 μL / mouse). Fluorescence imaging was performed on the 6th and 9th days to track the mouse ovarian cancer cells. The general conditions of the animals such as body weight and diet were observed daily, the body weight changes of the mice were monitored, and the survival curve was continuously recorded.

[0180] Experimental results

[0181] After injection of AYK103, the growth of ovarian cancer cells in the peritoneal cavity was significantly reduced ( Figure 16 ), and the survival rate of the mice in the AYK103 group remained 100% at 110 days after drug administration, while the survival rate of the control group was 40% ( Figure 17 ), indicating that AYK103 is effective in treating the intraperitoneal metastasis model of mouse ovarian cancer.

[0182] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of an immunomodulator in the preparation of a composition for treating animal cancer, characterized in that: The immunomodulator is: Poly IC and QS-21 in a mass ratio of (1.3-60):1, the cancer is breast cancer, liver cancer or ovarian cancer, and the animal is a dog, a mouse or a rat.

2. The use according to claim 1, characterized in that: The immunomodulator is: Poly IC and QS-21 in a mass ratio of (4-32):

1.

3. The use according to claim 2, characterized in that: The immunomodulator is: Poly IC and QS-21 in a mass ratio of (4-16):

1.

4. The use according to claim 3, characterized in that: The immunomodulator is: Poly IC and QS-21 in a mass ratio of (6-8):

1.

5. The use according to claim 1, characterized in that: The composition further comprises: a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that: The carrier is a liposome.

7. The use according to claim 6, characterized in that: The liposome is a cationic liposome.

8. The use according to claim 6, characterized in that: The lipid component of the liposome is phospholipid or phospholipid-like molecules.

9. The use according to claim 8, characterized in that: The phospholipids are selected from glycerophospholipids and sphingomyelins.

10. The use according to claim 8, characterized in that: The phospholipid is selected from any one or a combination of at least two of DOPC, soybean lecithin, sunflower lecithin, DSPG, DPPC, HSPC, DSPC and EPC.

11. The use according to claim 6, characterized in that In the composition, the mass ratio of Poly IC, QS-21 and liposome is (1.3-60):1:(15-25).

12. The use according to claim 11, characterized in that In the composition, the mass ratio of Poly IC, QS-21 and liposome is (6-10):1:(15-25).

13. The use according to claim 12, characterized in that: The liposome is prepared from 1,2-dioleoyl-3-trimethylammonium chloride propane, dioleoylphosphatidylcholine and cholesterol.

14. The use according to claim 5, characterized in that The composition was prepared by blending the carrier with Poly IC and QS-21.

15. The use according to claim 14, characterized in that: When the carrier is a liposome, the particle size of the liposome is 60-300 nm.

16. The use according to claim 5, characterized in that: The composition is a medicine.

17. The use according to claim 16, characterized in that The drug is administered by injection.

18. The use according to claim 17, characterized in that The drug is administered by intratumoral injection.

19. The use according to any one of claims 16 to 18, characterized in that: The drug has any of the following uses: (1) Activate the immune response of the tumor microenvironment and reverse the tumor immunosuppressive microenvironment; (2) Inhibit tumor growth rate; (3) Reduce tumor weight; (4) Reduce tumor volume.

20. The use according to claim 19, characterized in that The use of the drug to activate the immune response of the tumor microenvironment includes: promoting the tumor infiltration of CD4 T and CD8 T cells.

21. The use according to claim 19, characterized in that The use of the drug to activate the immune response of the tumor microenvironment is selected from at least one of the following: (1) Increase the number of cytotoxic T cells CD8+T in tumor tissue; (2) Increase the number of effector memory T cells CD8+T in tumor tissue; (3) Increase the number of helper T cells CD4+T in tumor tissue; (4) Increase the number of macrophages among antigen-presenting cells in tumor tissue; (5) Increase the number of dendritic cells among antigen-presenting cells in tumor tissue.

22. The use according to claim 1, characterized in that The final concentration of the QS-21 in the composition is 50-300 μg / mL, and the final concentration of the Poly I:C is 400-3000 μg / mL.

23. Use of a composition in the preparation of a drug for treating animal cancer, characterized in that: The composition comprises: an immunomodulator and a pharmaceutically acceptable carrier, wherein the immunomodulator is: Poly IC and QS-21 in a mass ratio of (1.3-60):1, the cancer is breast cancer, liver cancer or ovarian cancer, and the animal is a dog, a mouse or a rat.

24. The use according to claim 23, characterized in that The carrier is a liposome.

25. The use according to claim 24, characterized in that The liposome is a cationic liposome.

26. The use according to claim 24, characterized in that In the drug, the mass ratio of Poly IC, QS-21 and liposome is (1.3~60):1:(15~25).

27. The use according to claim 26, characterized in that In the composition, the mass ratio of Poly IC, QS-21 and liposome is (6-10):1:(15-25).

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