A composition for enhancing tumor immunogenicity and use thereof

Abstract

The application belongs to the technical field of tumor immunotherapy, and particularly relates to a composition for enhancing tumor immunogenicity and application thereof. The composition for enhancing tumor immunogenicity comprises, in terms of mass fraction, 10-15 parts of radix stephaniae tetrandrae, 10-20 parts of radix astragali, 3-9 parts of glycyrrhiza uralensis, and 3-12 parts of atractylodes macrocephala. The composition for enhancing tumor immunogenicity provided by the application is a natural traditional Chinese medicine prescription, has good biocompatibility, low toxicity and side effects, and is suitable for long-term treatment. The composition enhances immunogenicity by up-regulating MHC-I expression of tumor cells, activates CD8 + T cell-mediated anti-tumor effect, realizes autologous immunotherapy, and can avoid related side effects of exogenous immunotherapy. The composition can also be used in combination with a PD-1 blocker to synergistically improve anti-tumor efficacy and prolong patient survival.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, specifically relating to a composition that enhances tumor immunogenicity and its application. Background Technology

[0002] In recent years, while immune checkpoint therapies (such as PD-1 / PD-L1 inhibitors) have revolutionized cancer treatment, primary or acquired immune escape is prevalent in clinical practice, leading to poor responses or eventual drug resistance in many patients. Tumors evade immune surveillance through various mechanisms, among which the downregulation of major histocompatibility complex class I molecules (MHC-I) is one of the core mechanisms mediating immune escape. MHC-I molecules are responsible for presenting endogenous antigenic peptides to CD8 on the surface of tumor cells. + The absence or dysfunction of T cells in their expression prevents cytotoxic T lymphocytes (CTLs) from effectively recognizing and eliminating tumor cells. Therefore, finding effective strategies to increase the expression of MHC-I molecules on the surface of tumor cells is an urgent scientific problem that needs to be solved.

[0003] Current mainstream strategies for enhancing MHC-I expression in tumor cells mainly fall into two categories: ① Enhancing synthesis and transport: promoting the expression of MHC-I molecules and related proteins. ② Reducing degradation: blocking the degradation of MHC-I by lysosomes or the ubiquitin-proteasome pathway. However, these strategies currently have significant drawbacks. For example, when MHC-I is upregulated, immunosuppressive molecules are also upregulated simultaneously; existing drugs or treatments such as cytokines, epigenetic drugs, and gene editing / therapy have poor specificity and significant toxicity. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention aims to provide a composition that enhances tumor immunogenicity and its application to solve the above-mentioned technical problems.

[0005] This invention aims to overcome the shortcomings of insufficient response rates in existing immunotherapy technologies by providing a composition that enhances tumor immunogenicity. This composition enhances the immunogenicity of tumor cells by upregulating MHC-I expression, thereby improving the therapeutic effect of the body's own immune system against tumors. Furthermore, this invention demonstrates a synergistic effect between this composition and a PD-1 blocker in anti-tumor therapy, resulting in a significant improvement in the therapeutic efficacy against various tumors.

[0006] According to a first aspect of the present invention, the present invention provides a composition for enhancing tumor immunogenicity, comprising, by weight parts: 10-15 parts of Stephania tetrandra, 10-20 parts of Astragalus membranaceus, 3-9 parts of Glycyrrhiza uralensis, and 3-12 parts of Atractylodes macrocephala.

[0007] In some embodiments, the composition for enhancing tumor immunogenicity comprises, by weight parts: 10-15 parts of Fangji, 10-20 parts of Huangqi, 3-9 parts of Gancao, 3-12 parts of Baizhu, 0-6 parts of Dazao, and 0-12 parts of Shengjiang.

[0008] In some embodiments, the composition for enhancing tumor immunogenicity includes, by weight parts: 10-15 parts of Stephania tetrandra, 10-20 parts of Astragalus membranaceus, 3-9 parts of Glycyrrhiza uralensis, 3-12 parts of Atractylodes macrocephala, 1-6 parts of Ziziphus jujuba, and 6-12 parts of Zingiber officinale.

[0009] In some embodiments, the composition for enhancing tumor immunogenicity comprises, by weight parts: 12 parts of Fangji, 15 parts of Huangqi, 6 parts of Gancao, 9 parts of Baizhu, 3 parts of Dazao, and 9 parts of Shengjiang.

[0010] In some embodiments, the dosage form of the composition for enhancing tumor immunogenicity is an oral liquid preparation, granules, pellets, powders, tablets, capsules, transdermal absorbents, ointments, liposomes, sustained-release preparations, controlled-release preparations, or injections.

[0011] In some embodiments, the tumor is one of melanoma, lung cancer, liver cancer, colon cancer, or stomach cancer.

[0012] According to a second aspect of the present invention, the present invention provides a method for preparing a composition that enhances tumor immunogenicity, comprising the following steps: adding the raw materials required for the composition that enhances tumor immunogenicity to water, heating and decocting to obtain the composition that enhances tumor immunogenicity.

[0013] In some embodiments, the temperature of the decoction process is 90-100 degrees Celsius, and the decoction time is 50-70 minutes.

[0014] In some embodiments, the decoction process is performed at least twice; after the first decoction process, the residue and filtrate are separated by filtration, the residue is mixed with water, and a second decoction process is performed, and so on. After each decoction process is completed, the filtrates are combined to obtain the composition that enhances tumor immunogenicity.

[0015] In some embodiments, the raw materials required for the composition that enhances tumor immunogenicity are added to water, soaked for 30 minutes, and then heated for decoction.

[0016] In some embodiments, the mass-to-volume ratio of the Stephania tetrandra to water is 10-15:1000g / mL.

[0017] In some embodiments, the preparation method of the composition for enhancing tumor immunogenicity includes the following steps: adding the raw materials required for the composition for enhancing tumor immunogenicity to 1000 mL of water, soaking for 30 min, heating for a first decoction treatment (heating over high heat, then simmering over low heat after boiling), filtering to obtain a first residue and a first filtrate, mixing the first residue with 800 mL of water, heating for a second decoction treatment, filtering to obtain a second residue and a second filtrate, and combining the first filtrate and the second filtrate to obtain the composition for enhancing tumor immunogenicity.

[0018] In some embodiments, the preparation method of the composition for enhancing tumor immunogenicity includes the following steps: adding the raw materials required for the composition for enhancing tumor immunogenicity to water, heating and decocting, concentrating, and obtaining the composition for enhancing tumor immunogenicity.

[0019] In some embodiments, the preparation method of the composition for enhancing tumor immunogenicity includes the following steps: adding the raw materials required for the composition for enhancing tumor immunogenicity to water, heating and decocting, concentrating, and freeze-drying to obtain the composition for enhancing tumor immunogenicity.

[0020] According to a third aspect of the invention, the invention provides the use of compositions that enhance tumor immunogenicity in the preparation of antitumor drugs.

[0021] According to a fourth aspect of the present invention, the present invention provides the use of a composition for enhancing tumor immunogenicity in combination with a PD-1 blocker in the preparation of an antitumor drug.

[0022] In some embodiments, the tumor is one of melanoma, lung cancer, liver cancer, colon cancer, or stomach cancer.

[0023] In some embodiments, the present invention provides the use of compositions that enhance tumor immunogenicity in combination with PD-1 blockers in the preparation of antitumor drugs, wherein the tumor is melanoma.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The composition for enhancing tumor immunogenicity provided by the present invention is Fangji Huangqi Decoction, which is a natural Chinese medicine formula with good biocompatibility and low toxic side effects, and is suitable for long-term treatment.

[0025] (2) The composition for enhancing tumor immunogenicity provided by the present invention enhances the immunogenicity of tumor cells by upregulating MHC-I of tumor cells, activating the immune system to clear tumors, and increasing CD8. + T-cell-mediated anti-tumor effects enable autologous immunotherapy, avoiding the immune-related side effects that may be caused by exogenous immunotherapy.

[0026] (3) The composition for enhancing tumor immunogenicity provided by the present invention can be used in combination with PD-1 blockers to improve the therapeutic effect and prolong the survival of patients. Attached Figure Description

[0027] Figure 1 The statistical results of the changes in MHC-I levels in various tumor cells in Example 1, which is a verification example of the effectiveness of this invention, are shown in the figure. P<0.01; P<0.001; ns: not significant).

[0028] Figure 2 The statistical results of the changes in MHC-I levels in A375 cells between the composition and the single herb in Example 2 of the efficacy verification of the present invention are shown in the figure. P<0.001; ns: not significant).

[0029] Figure 3 The statistical results of the changes in the levels of MHC-I on the surface of B16 and LLC cell membranes in Example 3, which is a verification example of the effectiveness of this invention, are shown in the figure. P<0.001; ns: not significant).

[0030] Figure 4 The statistical results of the changes in the presentation levels of B16 and LLC cell antigens in Example 4, which is a verification example of the effectiveness of this invention, are shown in the figure. P<0.001; ns: not significant).

[0031] Figure 5 In Example 5, CD8 is used to verify the effectiveness of this invention. + Statistical results of changes in T cell activation levels (Figure) P<0.05; P<0.001).

[0032] Figure 6 In Example 5, CD8 is used to verify the effectiveness of this invention. + Statistical results of changes in T cell effector factor levels (Figure) P<0.001).

[0033] Figure 7 In Example 5, CD8 is used to verify the effectiveness of this invention. + Statistical results of changes in the killing level of T cells against lung cancer cells (survival rate, P<0.001; ns: not significant).

[0034] Figure 8 In Example 5, CD8 is used to verify the effectiveness of this invention.+ Statistical results of changes in the killing level of T cells against lung cancer cells (killing rate, P<0.01; P<0.001).

[0035] Figure 9 The tumor images, growth curves, and tumor weight statistics of each group of tumor-bearing mice in Example 6, which is a verification example of the effectiveness of this invention, are shown in the figure. P<0.05; P<0.001).

[0036] Figure 10 The tumor images, growth curves, and tumor weight statistics of each group of tumor-bearing mice in Example 7, which is a verification example of the effectiveness of this invention, are shown in the figure. P<0.01; P<0.001).

[0037] Figure 11 The tumor images, growth curves, and tumor weight statistics of each group of tumor-bearing mice in Example 8, which is a verification example of the effectiveness of this invention, are shown in the figure. P<0.01; P<0.001). Detailed Implementation

[0038] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] The statistical analysis in the following validation examples was performed using GraphPad Prism software for statistical analysis and graph creation. All data are expressed as mean ± standard error. Differences were analyzed using t-tests or one-way ANOVA. A two-tailed p-value < 0.05 was considered statistically significant.

[0040] The cell information used in the following efficacy verification examples is as follows: B16 cells (mouse melanoma cells) were purchased from the JCRB cell bank in Japan; A375 cells (human melanoma cells) were purchased from Wuhan Pronosai Biotechnology Co., Ltd.; LLC cells (mouse lung cancer cells) were purchased from the ATCC cell bank in the United States; Hepa1-6 cells (mouse liver cancer cells) were purchased from the ATCC cell bank in the United States; MC38 cells (mouse colon cancer cells) were purchased from Wuhan Pronosai Biotechnology Co., Ltd.; and SGC-7901 cells (human gastric cancer cell line) were purchased from the ATCC cell bank in the United States.

[0041] The C57BL / 6 mice used in the following efficacy verification examples were provided by the Guangdong Provincial Experimental Animal Center, aged 7-8 weeks and weighing 20-22g. All mice in this invention were housed in the pathogen-free animal facility of the Animal Experiment Center, School of Basic Medical Sciences, Guangzhou University of Chinese Medicine, in individually ventilated cages with a maximum density of 5 mice per cage. The length and width of all subcutaneous tumors were measured daily using calipers, and the tumor volume was calculated using the formula (tumor volume = 0.5 × major axis × (minor axis × minor axis)). At the end of the experiment, all mice were euthanized. All animal experiments were approved by the Ethics Review Committee of Guangzhou University of Chinese Medicine.

[0042] In the following efficacy verification examples, B16 cells and SGC-7901 cells used 1640 complete medium, while A375 cells, LLC cells, Hepa1-6, and MC38 cells used DMEM complete medium. All culture conditions were performed in a cell culture incubator at 37°C and 5% CO2. Preparation of 1640 or DMEM complete medium: 10 wt% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution were added to 1640 or DMEM basal medium and mixed thoroughly.

[0043] The main reagents and consumables used in the following examples, comparative examples, and efficacy verification examples are shown in Table 1 below. All licorice used in the following examples, comparative examples, and efficacy verification examples is processed licorice.

[0044] Table 1 The main experimental instruments used in the following examples, comparative examples, and effect verification examples are shown in Table 2 below.

[0045] Table 2 The following efficacy verification examples evaluate the effect of the composition (Fangji Huangqi Decoction) that enhances tumor immunogenicity through mouse experiments. The equivalent dose of the drug in mice can be calculated by the following method.

[0046] Calculate the equivalent dose for mice: Human / day dose: 54g / human / day, adult weight: 70kg, mouse weight: 20g; Human dose / day: 54g / 70kg = 0.77g / kg; Given that the human-to-mouse equivalent dose ratio is 0.0026, the mouse equivalent dose is: 0.77 / kg × 70kg × 0.0026 ÷ 0.02kg = 7.02g / kg = 7020mg / kg / day; One mouse is given 7020mg / kg × 0.02kg = 140.4mg by gavage per day; One mouse is given 200μl by gavage per day, so the drug concentration is: 140.4mg / 0.2ml = 702mg / ml. Assuming the equivalent dose is a medium concentration (702mg / ml), twice the equivalent dose is a high concentration (1404mg / ml), and half the equivalent dose is a low concentration (351mg / ml).

[0047] Example 1 The tumor immunogenicity-enhancing composition (Fangji Huangqi Decoction) in this embodiment is prepared from Fangji (Stephania tetrandra family), Huangqi (Fabaceae family), Baizhu (Atractylodes macrocephala family), Gancao (Fabaceae family), Shengjiang (Zingiberaceae family), and Dazao (Rhamnaceae family). All medicinal materials were purchased from Guangzhou Kangmei Smart Pharmacy. By weight, 12 parts of Fangji, 15 parts of Huangqi, 6 parts of Gancao, 9 parts of Baizhu, 3 parts of Dazao, and 9 parts of Shengjiang were weighed. As an example, the mass parts in Example 1 are in grams. The preparation of a composition that enhances tumor immunogenicity (a decoction of Fangji Huangqi Tang) specifically includes the following steps: Remove the decoction pot, mix all the medicinal materials according to the above-mentioned mass proportions, add 1000mL of water and soak for 30 minutes, then heat and decoct (temperature 100 degrees Celsius, time 60 minutes), filter with gauze to obtain the first filtrate and the first residue. Mix the first residue with 1000mL of water, heat and decoct a second time (temperature 100 degrees Celsius, time 60 minutes), filter with gauze to obtain the second filtrate and the second residue. Combine the first and second filtrates, and concentrate the decoction by boiling at 100 degrees Celsius to obtain a composition with a volume of 77mL and a concentration of 702mg / ml that enhances tumor immunogenicity (the decoction of Fangji Huangqi Tang).

[0048] The tumor immunogenicity-enhancing composition (the decoction of Fangji Huangqi Decoction) prepared in Example 1 was concentrated and diluted to obtain a high-dose decoction of Fangji Huangqi Decoction (1404 mg / ml) and a low-dose decoction of Fangji Huangqi Decoction (351 mg / ml), respectively, for later use.

[0049] Example 2 Example 2 provides a composition for enhancing tumor immunogenicity and its preparation method, which is basically the same as Example 1, except that the composition for enhancing tumor immunogenicity includes, by mass parts: 10 parts of Stephania tetrandra, 10 parts of Astragalus membranaceus, 3 parts of Glycyrrhiza uralensis, 3 parts of Atractylodes macrocephala, 0 parts of Ziziphus jujuba, and 0 parts of Zingiber officinale.

[0050] Example 3 Example 3 provides a composition for enhancing tumor immunogenicity and its preparation method, which is basically the same as Example 1, except that the composition for enhancing tumor immunogenicity includes, by mass parts: 15 parts of Stephania tetrandra, 20 parts of Astragalus membranaceus, 9 parts of Glycyrrhiza uralensis, 12 parts of Atractylodes macrocephala, 6 parts of Ziziphus jujuba, and 12 parts of Zingiber officinale.

[0051] Example 4 The tumor immunogenicity-enhancing composition (Fangji Huangqi Decoction) in this embodiment is prepared from Fangji (Stephania tetrandra family), Huangqi (Fabaceae family), Baizhu (Atractylodes macrocephala family), Gancao (Fabaceae family), Shengjiang (Zingiberaceae family), and Dazao (Rhamnaceae family). All medicinal materials were purchased from Guangzhou Kangmei Smart Pharmacy. By weight, 12 parts of Fangji, 15 parts of Huangqi, 6 parts of Gancao, 9 parts of Baizhu, 3 parts of Dazao, and 9 parts of Shengjiang were weighed. For example, the mass parts in Example 2 are in grams. The preparation of a composition that enhances tumor immunogenicity (lyophilized powder of Fangji Huangqi Decoction) specifically includes the following steps: Remove the decoction pot, mix all the medicinal materials according to the above-mentioned mass proportions, add 1000mL of water and soak for 30 minutes, then heat and decoct (temperature 100 degrees Celsius, time 60 minutes), filter with gauze to obtain the first filtrate and the first residue. Mix the first residue with 1000mL of water, heat and decoct a second time (temperature 100 degrees Celsius, time 60 minutes), filter with gauze to obtain the second filtrate and the second residue. Combine the first and second filtrates, boil at 100 degrees Celsius and concentrate the decoction to obtain a composition with a volume of 77mL and a concentration of 702mg / ml that enhances tumor immunogenicity (the decoction of Fangji Huangqi Tang).

[0052] (2) The composition for enhancing tumor immunogenicity at a concentration of 702 mg / ml was filtered in a clean bench using a 50 ml syringe and a 70 μm filter to obtain the filtered drug solution; (3) Transfer the filtered drug solution to a 50ml centrifuge tube and centrifuge at room temperature using an ultracentrifuge. Centrifugation parameters: first centrifuge at 10,000 rpm for 15 minutes, then centrifuge at 10,000 rpm for 30 minutes; take the supernatant.

[0053] (4) Take the supernatant and dispense it into a 15cm cell culture dish. Wrap the top layer with plastic wrap, poke a small hole with the syringe needle, and incubate overnight (12 hours) at -80 degrees Celsius.

[0054] (5) Take the cell culture dish out to a cold vacuum freeze dryer, freeze dry it, grind it into powder, and obtain 12g of freeze-dried powder of the composition that enhances tumor immunogenicity (freeze-dried powder of Fangji Huangqi Decoction), and store it in a refrigerator at -20 degrees Celsius; wherein, the freeze drying parameters are set as follows: temperature is -40 degrees Celsius, and freeze drying time is 24h.

[0055] Comparative Example 1 Comparative Example 1 provides a method for preparing Astragalus freeze-dried powder, which can be carried out with reference to the method described in Example 4, except that: the medicinal materials added in Example 4, by weight, include 12 parts of Stephania tetrandra, 15 parts of Astragalus membranaceus, 6 parts of Glycyrrhiza uralensis, 9 parts of Atractylodes macrocephala, 3 parts of Ziziphus jujuba, and 9 parts of Zingiber officinale; while the medicinal materials added in this comparative example, by weight, are only 15 parts of Astragalus membranaceus. The weight of the Astragalus freeze-dried powder prepared in this comparative example is 4.6g.

[0056] Comparative Example 2 Comparative Example 2 provides a method for preparing freeze-dried Atractylodes macrocephala powder, which can be carried out with reference to the method described in Example 4, except that: the medicinal materials added in Example 4, by weight, included 12 parts of Stephania tetrandra, 15 parts of Astragalus membranaceus, 6 parts of Glycyrrhiza uralensis, 9 parts of Atractylodes macrocephala, 3 parts of Ziziphus jujuba, and 9 parts of Zingiber officinale; while the medicinal materials added in this comparative example, by weight, are only 9 parts of Atractylodes macrocephala. The weight of the freeze-dried Atractylodes macrocephala powder prepared in this comparative example is 7.3g.

[0057] Comparative Example 3 Comparative Example 3 provides a method for preparing frost-dried Stephania tetrandra powder, which can be carried out with reference to the method described in Example 4, except that: the medicinal materials added in Example 4, by weight, included 12 parts Stephania tetrandra, 15 parts Astragalus membranaceus, 6 parts Glycyrrhiza uralensis, 9 parts Atractylodes macrocephala, 3 parts Ziziphus jujuba, and 9 parts Zingiber officinale; while the medicinal materials added in this comparative example, by weight, are only 9 parts Stephania tetrandra. The weight of the frost-dried Stephania tetrandra powder prepared in this comparative example is 1.95g.

[0058] Comparative Example 4 Comparative Example 4 provides a method for preparing freeze-dried jujube powder, which can be carried out with reference to the method described in Example 4, except that: the medicinal materials added in Example 4, by weight, included 12 parts of Stephania tetrandra, 15 parts of Astragalus membranaceus, 6 parts of Glycyrrhiza uralensis, 9 parts of Atractylodes macrocephala, 3 parts of jujube, and 9 parts of ginger; while the medicinal materials added in this comparative example, by weight, are only 3 parts of jujube. The freeze-dried jujube powder prepared in this comparative example weighs 2.43g.

[0059] Comparative Example 5 Comparative Example 5 provides a method for preparing freeze-dried ginger powder, which can be carried out with reference to the method described in Example 4, except that: the medicinal materials added in Example 4, by weight, included 12 parts of Stephania tetrandra, 15 parts of Astragalus membranaceus, 6 parts of Glycyrrhiza uralensis, 9 parts of Atractylodes macrocephala, 3 parts of Ziziphus jujuba, and 9 parts of ginger; while the medicinal materials added in this comparative example, by weight, are only 3 parts of ginger. The freeze-dried ginger powder prepared in this comparative example weighs 0.26g.

[0060] Comparative Example 6 Comparative Example 6 provides a method for preparing licorice freeze-dried powder, which can be carried out with reference to the method described in Example 4, except that: the medicinal materials added in Example 4, by weight, included 12 parts of Stephania tetrandra, 15 parts of Astragalus membranaceus, 6 parts of licorice, 9 parts of Atractylodes macrocephala, 3 parts of jujube, and 9 parts of ginger; while the medicinal materials added in this comparative example, by weight, are only 6 parts of licorice. The weight of the licorice freeze-dried powder prepared in this comparative example is 2.48g.

[0061] Example 1 of effect verification In vitro pharmacological experiments: To evaluate whether Fangji Huangqi Decoction could increase the expression level of MHC-I in tumor cells, the following cell experiments were performed, and then the protein was extracted and detected by Western blot.

[0062] 1. The preparation of cell-based drugs includes the following steps: (1) Prepare a 15ml centrifuge tube, weigh 2g of the lyophilized powder of the tumor immunogenicity-enhancing composition (lyophilized powder of Fangji Huangqi Decoction) prepared in Example 4 and put it into the 15ml centrifuge tube; add 10mL of basal culture medium to the centrifuge tube and mix it with the lyophilized powder of the tumor immunogenicity-enhancing composition (lyophilized powder of Fangji Huangqi Decoction) (use a vortex shaker to shake 3 times, each shaking time is 5min, and then sonicate for 20min) so that the concentration of the lyophilized powder of the tumor immunogenicity-enhancing composition (lyophilized powder of Fangji Huangqi Decoction) in the basal culture medium is 200mg / ml, and obtain the drug solution for cell administration; wherein, the basal culture medium is 1640 basal culture medium or DMEM basal culture medium; (2) Centrifuge the cell drug solution at 2500 rpm for 5 min, collect the supernatant, filter the supernatant through a 0.22 μm filter to obtain the sterilized drug solution, and store it in a refrigerator at -20 degrees Celsius. When subsequent groups need to use the cell drug solution, add 10 wt% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution to the cell drug solution to form a complete culture medium containing the drug; 2. Cell culture conditions settings: The complete culture medium used was 1640 complete medium or DMEM complete medium. B16 cells (mouse melanoma cells) and SGC-7901 cells (human gastric cancer cell line) were cultured in 1640 complete medium, while LLC cells (mouse lung cancer cells), Hepa1-6 cells (mouse liver cancer cells), and MC38 cells (mouse colon cancer cells) were cultured in DMEM complete medium. Each basal medium was supplemented with 10 wt% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution to form the corresponding complete medium. All culture conditions were in a cell culture incubator at 37 degrees Celsius and 5% CO2.

[0063] 3. Grouping and concentration settings: (1) Experimental group: Take the above sterilized drug solution (the composition that enhances tumor immunogenicity is labeled as FJHQ, with a concentration of 200mg / ml), take the corresponding basic culture medium, dilute it, add 10wt% fetal bovine serum (FBS) and 1% penicillin-streptomycin double antibiotic solution, mix well, and obtain complete culture medium containing FJHQ. Three concentration gradients are set in the complete culture medium containing FJHQ: 200μg / ml, 400μg / ml, and 800μg / ml; (2) Solvent control group: No drug was added, only an equal volume of complete culture medium was added; (3) Positive control group: Baf (bafloxacin) was added. Baf (bafloxacin) was dissolved in the basal medium and filtered through a 0.22 μm filter to remove bacteria. 10 wt% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution were added and mixed well to obtain a complete medium containing 15 nM Baf.

[0064] 4. Cell preparation and drug experiments B16 cells (mouse melanoma cells), SGC-7901 cells (human gastric cancer cell line), LLC cells (mouse lung cancer cells), Hepa1-6 cells (mouse liver cancer cells), and MC38 cells (mouse colon cancer cells) were cultured to the logarithmic growth phase in their respective complete culture media, and then seeded into 6-well plates at a cell density of 2 × 10⁻⁶ cells / well. 5Cells / mL, with 3 replicates for each cell type; then, according to the grouping and concentration settings described above, the culture medium for the experimental groups was replaced with complete medium containing FJHQ, the culture medium for the positive control group was replaced with complete medium containing 15 nM Baf, and the solvent control group was added with complete medium without the drug. After culturing each group for 24 hours, the culture medium was discarded, and the cells were rinsed twice with 1×PBS buffer. 200 µL of RIPA lysis buffer containing protease inhibitors was added to each well, and the cells were lysed on ice for 30 minutes, with continuous shaking during the lysis. The protein samples were then placed on crushed ice for later use.

[0065] 5. Protein extraction (1) Protein quantification (BCA) Prepare standards of different concentrations according to the BCA kit instructions. Mix reagent A and reagent B at a ratio of 50:1 to prepare the BCA working solution; prepare and use immediately. Add 20 µL of standards of different concentrations, tissue protein samples (diluted 40-fold with RIPA lysis buffer), and 200 µL of BCA working solution to a 96-well plate, with three replicates for each sample. After incubation at 37°C for 30 minutes, measure the absorbance at 562 nm using a multi-plate reader.

[0066] (2) Protein dilution and denaturation A standard curve was plotted based on absorbance to calculate the protein concentration of the samples. The samples were then adjusted to the same concentration using RIPA lysis buffer. 1 / 4 volume of 5× Loading Buffer was added to each sample, and the mixture was boiled in a 100°C metal bath for 10 minutes to denature the protein. Protein extraction was then complete, yielding the corresponding protein samples for each group.

[0067] 6. Western blot experiment (1) Making adhesive Prepare one long glass plate and one short glass plate with a clean, spotless surface and undamaged lower edges. Align the lower edges of the two plates, place them in clips, cut sealing adhesive, and attach it to the lower edge of the glass plates. Ensure the sealing adhesive between the two plates is smooth and without creases to prevent leakage. Using a one-step gel preparation kit (12%), prepare a 12% separating gel and a 4.5% stacking gel according to the kit's instructions. Add them to the glass plates, inserting a comb from one side at an angle to avoid air bubbles. Allow the gel to solidify to obtain the prepared glass plate.

[0068] (2) Electrophoresis Weigh 30g Tris base, 188g glycine, and 10g SDS powder sequentially using an electronic balance, then place them in a 1000mL glass beaker. Add 900mL of pure water, mix well using a magnetic stirrer, and then add pure water to bring the volume to 1000mL for later use, obtaining a 10× electrophoresis buffer. Measure 100mL of the 10× electrophoresis buffer using a measuring cup, add 900mL of pure water, mix well, and set aside for later use, obtaining a 1× electrophoresis buffer.

[0069] After clamping and leak-testing the prepared gel glass plates, place them into a clean electrophoresis tank and remove the comb. Add freshly prepared 1× electrophoresis buffer, ensuring the buffer level is above the short glass plate to prevent leakage. Boil the corresponding protein samples for each group in a 100°C metal bath heater for 5 minutes, then load the samples. Use 20µg of protein sample per well, and add 3µL of pre-stained protein marker (10-180 kDa) to the pre-drilled wells on both sides of the glass plate. Electrophoresis at a constant voltage of 100V for 80 minutes until the bromophenol blue is 1-2 cm from the bottom edge of the glass plate, then disconnect the power.

[0070] (3) Transfer membrane Weigh 33g of Tris base and 144g of glycine powder sequentially using an electronic balance, then place them in a 1000mL glass beaker. Add 900mL of pure water, mix well using a magnetic stirrer, and then add pure water to bring the volume to 1000mL for later use, obtaining 10× transfer buffer. Measure 100mL of the 10× transfer buffer using a measuring cup, add 200mL of methanol, and then add pure water to bring the volume to 1000mL. Mix well and pre-cool at -20°C for later use, obtaining 1× transfer buffer.

[0071] Remove the pre-cooled 1× transfer buffer. Immerse the electrophoresis plate, transfer clamp, filter paper, and sponge in the transfer buffer. Take the PVDF membrane and activate it by soaking it in methanol. Open the transfer clamp and place it flat with the black side down. Place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the following order. Align the edges of the transfer clamp and clamp it tightly, avoiding air bubbles. Place the transfer clamp into the transfer tank, aligning black with black and white with red, and pour in all the transfer buffer. Place the transfer tank in a basin containing an ice-water mixture and electrophoretically transfer at a constant current of 300mA for 90 minutes.

[0072] (4) Closed Measure 50 mL of 20×TBS using a measuring cup, add 1 mL of Tween20, and dilute to 1000 mL with pure water. Mix well and set aside to obtain 1×TBST. Use 1×TBST to add skim milk powder to prepare a 5wt% skim milk solution. Immerse the PVDF membrane in 5wt% skim milk and incubate on a shaker for 3 hours.

[0073] (5) Incubation of primary antibody Dilute H-2K with 5wt% skim milk solution at a ratio of 1:1500. b Primary antibodies, HLA-A / B primary antibodies, and β-actin primary antibodies were added. The blocked PVDF membrane was then immersed in a solution containing the primary antibodies and incubated overnight (12 hours) at 4°C with shaking. H-2K... b The MCH-I molecules are from B16 cells, LLC cells, Hepa1-6 cells, and MC38 cells; HLA-A / B is the MCH-I molecule from SGC-7901 cells. Therefore, different primary antibodies need to be added to the protein samples corresponding to different cells, while β-actin primary antibody needs to be added to all of them.

[0074] (6) Incubation of secondary antibodies After incubation with the primary antibody, the membrane was immersed in 1×TBST and shaken on a shaker for 10 minutes to elute any unbound primary antibody. The 1×TBST was discarded, and new 1×TBST was added, followed by three elutions. The corresponding species secondary antibody (Goat anti-rabbit IgG-HRP / Goat anti-mouse IgG-HRP) was diluted with 5wt% skim milk solution at a ratio of 1:5000. The membrane was then immersed in the secondary antibody and incubated on a shaker for 1 hour.

[0075] (7) Development After incubation with the secondary antibody, the PVDF membrane was immersed in 1×TBST and shaken on a shaker for 10 minutes to elute the unbound secondary antibody. After discarding the 1×TBST, fresh 1×TBST was added, and the elution was repeated 3 times. The chemiluminescence solution was prepared according to the ratio of solution A to solution B = 1:1 (volume ratio), and images were formed and photographed using a Tannon fully automated chemiluminescence imaging system.

[0076] Results analysis: The results are as follows Figure 1 As shown, Figure 1 FJHQ in the formula represents Fangji Huangqi Decoction (a composition that enhances tumor immunogenicity); Baf is a positive control drug; B16 represents melanoma cells; LLC represents lung cancer cells; Hepa1-6 represents mouse liver cancer cells; MC38 represents mouse colon cancer cells; and SGC-7901 represents a human gastric cancer cell line. Figure 1 Part A shows the WB electrophoresis results for B16 and LLC. Figure 1 Part B shows the WB electrophoresis results for Hepa1-6, MC38, and SGC-7901. Figure 1 Part C is a bar chart of the WB quantitative results corresponding to Hepa1-6, MC38, and SGC-7901; Depend on Figure 1As shown in Part A, with increasing concentrations of the composition enhancing tumor immunogenicity (Fangji Huangqi Decoction) (200 μg / ml, 400 μg / ml, 800 μg / ml), the MHC-I protein levels in B16 cells and LLC cells gradually increased; Figure 1 As shown in Parts B and C, as the concentration of the composition (Fangji Huangqi Decoction) that enhances tumor immunogenicity increases (200 μg / ml, 400 μg / ml, 800 μg / ml), the MHC-I protein level in Hepa1-6 cells, MC38 cells and SGC-7901 cells gradually increases.

[0077] Example 2 of effect verification In vitro pharmacological experiments: To evaluate the difference in the effect of the composition (Fangji Huangqi Decoction) which enhances tumor immunogenicity and the single herbal medicine in increasing the expression level of MHC-I in tumor cells, the following cell experiments were performed, and then the protein was extracted and detected by Western blot.

[0078] 1. Cell culture conditions settings: A375 (human melanoma cells) were cultured in DMEM complete medium containing 10 wt% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution in a cell culture incubator at 37 degrees Celsius and 5% CO2.

[0079] 2. Group settings: Based on the quality of the lyophilized powder obtained in Example 4 and the lyophilized powder obtained in Comparative Examples 1-6, and as shown in Effect Verification Example 1, when the lyophilized powder of the composition enhancing tumor immunogenicity (lyophilized powder of Fangji Huangqi Decoction) was at 800 μg / mL in the culture medium, it could significantly enhance the expression of MHC-1 in tumor cells. Therefore, in subsequent experiments, the concentration of the lyophilized powder of the composition enhancing tumor immunogenicity (lyophilized powder of Fangji Huangqi Decoction) was set to 800 μg / mL. Based on the amount of lyophilized powder obtained in each comparative example, the proportion of the amount of lyophilized powder of a single herb to the total lyophilized powder of the compound (lyophilized powder of Fangji Huangqi Decoction) was first calculated, and this proportion was multiplied by 800 μg / mL to obtain the concentration setting of each single herb lyophilized powder as the equivalent dose, as shown in Table 3 below.

[0080] Table 3 The following solutions were prepared according to the process described in Part 1 (Preparation of Cell Drugs) of Example 1, which describes the composition for enhancing tumor immunogenicity prepared in Example 4 (lyophilized powder of Fangji Huangqi Decoction), as well as the solutions of Astragalus membranaceus lyophilized powder (Comparative Example 1), Atractylodes macrocephala lyophilized powder (Comparative Example 2), Stephania tetrandra lyophilized powder (Comparative Example 3), Jujube lyophilized powder (Comparative Example 4), Ginger lyophilized powder (Comparative Example 5), and Licorice lyophilized powder (Comparative Example 6). All these solutions were filtered through a 0.22 μm filter for sterilization.

[0081] The grouping is as follows.

[0082] Experimental group 1: Complete culture medium containing a composition (Fangji Huangqi Decoction) with a concentration of 800 μg / ml that enhances tumor immunogenicity was used; Experimental group 2: Complete culture medium containing lyophilized Astragalus membranaceus powder at a concentration of 307 μg / ml was used; Experimental group 3: Complete culture medium containing lyophilized Atractylodes macrocephala powder at a concentration of 487 μg / ml was used; Experimental group 4: Complete culture medium containing 130 μg / ml of lyophilized Stephania tetrandra powder was used; Experimental group 5: Complete culture medium containing 162 μg / ml jujube freeze-dried powder was used; Experimental group 6: Complete culture medium containing lyophilized ginger powder at a concentration of 17 μg / ml was used; Experimental group 7: Complete culture medium containing lyophilized licorice powder at a concentration of 165 μg / ml was used; Control group: The same volume of complete culture medium was used, without the addition of any drugs.

[0083] 3. Cell preparation and Western blot Cells in good logarithmic growth phase were seeded into 6-well plates at a density of 2 × 10⁶ cells / well. 5 The sample was cultured at a concentration of 800 μg / mL, with three parallel groups. Based on the experimental groupings described above, complete culture medium containing 800 μg / mL of Astragalus membranaceus and complete culture medium containing an equivalent concentration of the single herb were added for culturing. After culturing for 24 hours, protein samples were extracted for Western blot detection of HLA-A / B (human MHC-I) levels. The experimental method was the same as described in Example 1 of the effect verification.

[0084] The results are as follows Figure 2 As shown, Figure 2 The left side shows the results of WB protein electrophoresis. Figure 2The right side of the chart is a bar chart of the Western blotting quantitative results. CT represents the control group; FJHQ represents Fangji Huangqi Decoction (experimental group 1); HQ represents Astragalus membranaceus (experimental group 2); BZ represents Atractylodes macrocephala (experimental group 3); FJ represents Fangji (experimental group 4); DZ represents jujube (experimental group 5); SJ represents ginger (experimental group 6); and ZGC represents prepared licorice root (experimental group 6). Figure 2 As shown, cell experiments confirmed that FJHQ upregulates MHC-I expression better than single herbs.

[0085] Example 3 of effect verification In vitro pharmacological experiments: To evaluate whether the composition that enhances tumor immunogenicity can increase the expression level of MHC-I in tumor cells, flow cytometry was used to observe the level of MHC-I on the surface of tumor cells, and the following cell experiments were performed.

[0086] 1. Cell culture conditions settings: The culture methods for B16 cells (mouse melanoma cells) and LLC cells (mouse lung cancer cells) were as described in Part 2 (Cell Culture Conditions) of Example 1, which was used to verify the effectiveness.

[0087] 2. Grouping and concentration settings: (1) Experimental group: Take the sterilized drug solution (containing the lyophilized powder of the composition that enhances tumor immunogenicity, with a concentration of 200 mg / ml) prepared in Part 1 (preparation of cell drug) of the effect verification example 1, and dilute it with the corresponding complete culture medium to set three concentration gradients of 200 μg / ml, 400 μg / ml and 800 μg / ml; (2) Solvent control group: No drug was added, only an equal volume of complete culture medium was added; (3) Positive control group: Add 15 nM Baf (bafloxacin), Baf (bafloxacin) dissolved in complete culture medium, and filter sterilize with a 0.22 μm filter for later use; (4) Isotype group: The treatment is the same as that of the solvent control group, that is, no drug is added, only the same volume of complete culture medium is added. The Isotype group is the isotype control group, which is a negative control set up to exclude false positive signals.

[0088] 3. Cell preparation and drug experiments B16 cells (mouse melanoma cells) and LLC cells (mouse lung cancer cells) were cultured in complete culture medium until they reached the logarithmic growth phase, and then seeded into 6-well plates at a cell density of 2 × 10⁶ cells / well. 5Cells / mL, with 3 parallel groups for each cell type; then, according to the above grouping and concentration settings, the culture medium of the experimental group was replaced with complete culture medium containing FJHQ, the culture medium of the positive control group was replaced with complete culture medium containing 15 nM Baf, and the solvent control group was added with complete culture medium without the drug. After each group was cultured for 24 hours, the culture medium was discarded.

[0089] (1) Cell collection Wash the cells twice with 1×PBS, add trypsin along the cell wall to digest the cells for 1-3 minutes, observe under a microscope that the cells have been completely digested, add 3 times the volume of complete culture medium to stop the digestion, and centrifuge at 1500 rpm for 5 minutes.

[0090] (2) Cell washing After centrifugation, discard the waste liquid in the tube, take the precipitate, add 1 ml of pre-cooled BD Buffer to resuspend the precipitate, and centrifuge again at 1500 rpm for 5 minutes at room temperature. Discard the waste liquid in the tube.

[0091] (3) Cell staining Dilute FITC-conjugated anti-mouse H-2K with BD Buffer b Antibody (mouse MHC-I, diluted 200-fold). After discarding the waste liquid in the tubes, 200 µL of BD Buffer was added to the solvent control group, and 200 µL of the prepared antibody-containing BD Buffer was added to both the experimental group and the positive control group. The mixtures were incubated at room temperature in the dark for 20 minutes. An Isotype group was also included, consisting of 200 µL of BD Buffer and 1 µL of FITC Isotype antibody.

[0092] (4) Flow cytometry testing After staining, cells in each group were washed with 800 µL of BD Buffer and centrifuged at 1500 rpm for 5 minutes. The waste liquid was discarded, and cells were resuspended in 800 µL of BD Buffer in each group. H-2K cells were then detected using a BD LSR Fortessa flow cytometer. b The level of expression.

[0093] The results are as follows Figure 3 As shown. Figure 3 In this context, FJHQ represents Fangji Huangqi Decoction; Baf represents the positive control drug; B16 represents melanoma cells; and LLC represents lung cancer cells. Figure 3 Part A represents the results corresponding to LLC cells. Figure 3 Part B represents the results corresponding to B16 cells. For example... Figure 3As shown, with the increase of the concentration of Fangji Huangqi Decoction (200 μg / ml, 400 μg / ml, 800 μg / ml), the level of MHC-I protein on the surface of lung cancer cells and B16 cells gradually increased.

[0094] Example 4 of effect verification In vitro pharmacological experiments: To evaluate whether the composition enhancing tumor immunogenicity could increase antigen presentation by tumor cells, flow cytometry was used to observe H-2K on the surface of tumor cells. b The expression level of the -SIINFEKL complex was determined through the following cell experiments.

[0095] 1. Cell culture conditions settings: The culture methods for B16 cells (mouse melanoma cells) and LLC cells (mouse lung cancer cells) were as described in Part 2 (Cell Culture Conditions) of Example 1, which was used to verify the effectiveness.

[0096] 2. Grouping and concentration settings: (1) Experimental group: Take the sterilized drug solution (containing the lyophilized powder of the composition that enhances tumor immunogenicity, with a concentration of 200 mg / ml) prepared in Part 1 (preparation of cell drug) of the effect verification example 1, and dilute it with the corresponding complete culture medium to set three concentration gradients of 200 μg / ml, 400 μg / ml and 800 μg / ml; (2) Solvent control group: No drug was added, only an equal volume of complete culture medium was added; (3) Positive control group: Add 15 nM Baf (bafloxacin), Baf (bafloxacin) dissolved in complete culture medium, and filter sterilize with a 0.22 μm filter for later use; (4) Isotype group: The treatment is the same as that of the solvent control group, that is, no drug is added, only the same volume of complete culture medium is added. The Isotype group is the isotype control group, which is a negative control set up to exclude false positive signals.

[0097] 3. Cell preparation and drug experiments B16 cells (mouse melanoma cells) and LLC cells (mouse lung cancer cells) were cultured in complete culture medium until they reached the logarithmic growth phase, and then seeded into 6-well plates at a cell density of 2 × 10⁶ cells / well. 5Cells / mL, with 3 replicates for each cell type; then, according to the grouping and concentration settings described above, the culture medium for the experimental groups was replaced with complete medium containing FJHQ, the culture medium for the positive control group was replaced with complete medium containing 15 nM Baf, and the solvent control group was added with complete medium without the drug. At the same time, OVA-SIINFEKL peptide was added to each group for incubation (OVA-SIINFEKL powder was dissolved in PBS buffer, filtered for sterilization, and the concentration of OVA-SIINFEKL peptide in the complete medium was 2 μg / ml). After culturing each group for 24 hours, the culture medium was discarded.

[0098] 4. Flow cytometry detection of H-2K on cell surface b -SIINFEKL complex level The flow cytometry method was the same as in Example 3 for efficacy verification, but the PE-conjugated anti-mouse H-2K was diluted with BD Buffer instead. b -SIINFEKL antibody (diluted 200-fold before use). After discarding the waste liquid in the tube, add 200µL of BD Buffer to the solvent control group, and add 200µL of the prepared antibody-containing BD Buffer to both the experimental group and the positive control group. Incubate at room temperature in the dark for 20 minutes. An Isotype group was also prepared by adding 200µL of BD Buffer + 1µL of FITC Isotype antibody.

[0099] The results are as follows Figure 4 As shown. Figure 4 H-2K in b SIINFEKL is an MHC-I-antigen peptide complex presented to the cell surface, and its concentration represents the cell's antigen presentation capacity. Figure 4 Part A represents the results corresponding to LLC cells. Figure 3 Part B represents the results corresponding to B16 cells. For example... Figure 4 As shown, with the increase of Fangji Huangqi Decoction concentration (200μg / ml, 400μg / ml, 800μg / ml), the MHC-I mediated antigen also increased accordingly, that is, Fangji Huangqi Decoction can increase MHC-I mediated antigen presentation in melanoma cells and lung cancer cells.

[0100] Example 5 of effect verification In vitro pharmacological experiments: To evaluate whether the composition enhancing tumor immunogenicity could increase CD8 levels by increasing the immunogenicity of tumor cells. + The killing function of T cells was investigated through the following cell experiments.

[0101] After pretreating LLC-OVA cells with FJHQ for 24 hours, they were then fused with OVA-specific CD8 cells derived from OT-I transgenic mice. +T cells were co-cultured for 24 hours. CD8+ was detected by flow cytometry. + T cell activation level, IFN-γ secretion level in co-culture supernatant detected by ELISA, and CD8 quantitative assessment by crystal violet staining. + T-cell cytotoxicity against tumor cells and a flow cytometry dual-labeling strategy (Annexin V / PI) for quantitative detection of dead tumor cells.

[0102] 1. Cell culture conditions settings: LLC-OVA (mouse lung cancer cells overexpressing OVA) were cultured in DMEM complete medium containing 10 wt% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution in a cell culture incubator at 37°C and 5% CO2.

[0103] 2. Grouping and concentration settings: Experimental group: The sterilized drug solution obtained in the efficacy verification example 1 (the composition that enhances tumor immunogenicity is labeled FJHQ, with a concentration of 200 mg / ml) was diluted with DMEM complete culture medium to set three concentration gradients: 200 μg / ml, 400 μg / ml, and 800 μg / ml. Solvent control group: No drugs were added, only an equal volume of complete culture medium was added.

[0104] 3. Cell preparation and treatment (1) Preparation of spleen single-cell suspension Irradiate the clean bench with ultraviolet light for 30 minutes, then disinfect all reagents and consumables with 75% alcohol before placing them in the clean bench. Under aseptic conditions, harvest the spleen from OT-1 mice (avoid touching the intestines during harvesting, remove connective tissue and fat from the spleen tissue, pay attention to necrotic areas, and check for swelling, discoloration, or lesions). After rinsing in 1×PBS, place the spleen in a 6cm dish containing mouse lymphocyte separation medium (ensuring the spleen and obtained cells are in a liquid environment). Tilt a 70µm cell filter sieve onto the 6cm dish. Cut the spleen into small pieces with sterile scissors and place them on the sieve. Grind the spleen using a 2mL syringe plunger until no visible lumps remain. Clean the sieve of any remaining fragments and filter the separation solution through the sieve into a new 15mL centrifuge tube (using the same sieve).

[0105] (2) T lymphocyte isolation Add 1 ml of 1640 basal medium to a centrifuge tube to allow for cell separation. Centrifuge at 800 g for 30 minutes at room temperature using a low-speed multi-tube automatic balancing centrifuge. During centrifugation, prepare MACS buffer (50:1 v:v) of the stock solution and FBS. After centrifugation, four cell layers can be observed from top to bottom: the first layer is the culture medium layer; the second layer is a ring-shaped milky white lymphocyte layer; the third layer is the clear separation medium layer; and the fourth layer is the erythrocyte layer. Transfer the second layer of ring-shaped milky white lymphocytes to another clean 15 mL centrifuge tube, add 10 mL of 1640 basal medium, and centrifuge at 3000 rpm for 10 minutes. Discard the supernatant, add 4 mL of MACS buffer containing FBS, and centrifuge again at 3000 rpm for 8 minutes. Discard the supernatant, add 90 µL of MACS buffer containing FBS and 10 µL of Mouse CD8a (Ly-2) magnetic beads. Mix thoroughly and incubate at 4°C for 10 minutes. Place the immunomagnetic bead sorting column on a magnetic rack and rinse with 500µL of pure water. Remove the cells from the 4°C refrigerator and add 900µL of MACS buffer containing FBS. Mix the cells thoroughly, then add 500µL of cell suspension to the immunomagnetic bead sorting column each time, allowing the liquid to drip through the column automatically. After the liquid has completely drained, resuspend the cells in 1ml of MACS buffer containing FBS, centrifuge, and resuspend in 1640 complete medium supplemented with interleukin-2 (IL-2: 15ng / mL). Incubate at 37°C in a 5% CO2 incubator for subsequent experiments.

[0106] (3) Constructing a co-culture system of LLC-OVA cells and T lymphocytes from OT-1 mice LLC-OVA cells in good logarithmic growth phase were seeded at a density of 200,000 per well in six-well plates. After the cells reached 40% confluence, different concentrations of Astragalus membranaceus solution were added according to the experimental design. After 24 hours of drug treatment, the drug-containing culture medium was discarded, and the cells were washed twice with 1× PBS. Then, T lymphocytes from OT-1 mice were added to the wells and co-cultured for 24 hours.

[0107] 4. Flow cytometry detection of CD8 + CD69 expression in T cells (1) Cell collection Collect suspended cells from 6-well plates of co-cultured T lymphocytes from LLC-OVA and OT-1 mice into corresponding 5 mL EP tubes.

[0108] (2) Cell washing After centrifuging at 1500 rpm for 5 minutes, discard the waste liquid in the tube, add 1 ml of pre-cooled BD Buffer to resuspend the cells, and then centrifuge again at 1500 rpm for 5 minutes at room temperature.

[0109] (3) Cell staining Dilute the APC-conjugated anti-mouse CD8 antibody and the T-cell activation marker CD69 antibody with BD Buffer (200-fold dilution). After discarding the waste liquid in the tubes, add 200 µL of BD Buffer to the negative control group, and add 200 µL of BD Buffer + 1 µL of APC Isotype antibody + 1 µL of PE Isotype antibody to the experimental group. Add 200 µL of the prepared antibody-containing BD Buffer to the experimental group. Incubate at room temperature in the dark for 20 minutes.

[0110] (4) Flow cytometry testing After staining, the cells were washed with 800 µL of BD Buffer and centrifuged at 1500 rpm for 5 minutes. The waste liquid was discarded, and the cells were resuspended in 800 µL of BD Buffer and analyzed using a BD LSR Fortessa flow cytometer.

[0111] 5. ELISA detection of IFN-γ (1) Collect the supernatant Collect the co-culture supernatant of T lymphocytes from LLC-OVA and OT-1 mice. Collect 1 mL of culture supernatant from each group into a 1.5 mL EP tube and store it in a -80°C freezer for later use.

[0112] (2) Preparation of standard products Different concentrations of IFN-γ standards (1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.2 pg / mL, 15.6 pg / mL, 0 pg / mL) were prepared according to the kit instructions.

[0113] (3) ELISA kit detection The collected supernatant and IFN-γ standards of different concentrations were added to the wells of a 96-well plate pre-coated with anti-IFN-γ antibody. The plate was incubated at room temperature for 2 hours to allow the IFN-γ in the supernatant to fully bind with the anti-IFN-γ antibody coated in the 96-well plate. After washing to remove unbound components, biotinylated anti-IFN-γ detection antibody was added and incubated at room temperature for 1 hour. After washing again to remove unbound components, streptavidin-HRP conjugate was added to bind the detection antibody, and the plate was incubated at room temperature for 30 minutes. After washing to remove unbound components, substrate solution was added and incubated for 15-20 minutes to produce a colorimetric reaction proportional to the amount of IFN-γ. The reaction was terminated with stop solution, and the optical density was measured using a multi-mode microplate reader.

[0114] 6. Crystal violet detection of tumor cell apoptosis (1) Cell collection After 24 hours of co-culturing T lymphocytes from LLC-OVA and OT-1 mice, remove the 6-well plate, discard the residual culture medium, and wash three times with 1×PBS to remove non-adherent cells and debris.

[0115] (2) Fixing cells Fix adherent cells with 4% paraformaldehyde at room temperature for 15 minutes. Discard the 4% paraformaldehyde, add 500µL of pure water, let stand for at least 30 seconds, discard the pure water, replace with fresh pure water and let stand again, repeat 3 times.

[0116] (3) Crystal violet staining Add just enough 0.5% crystal violet solution to cover the cells and stain at room temperature for 10 minutes. Crystal violet binds to DNA and proteins, causing surviving cells to appear purple. Carefully wash away excess dye with pure water and air dry the plate completely to remove any residual moisture. Add dimethyl sulfoxide and gently shake the plate to dissolve the bound dye. Observe the intensity of the color to determine cell viability.

[0117] (4) Detection using a multi-functional microplate reader The absorbance of the obtained solution was measured at 595 nm using a multi-functional microplate reader.

[0118] 7. Flow cytometry detection of tumor cell apoptosis (1) Construction of a co-culture system of LLC-OVA cells and OT-1 mouse T lymphocytes LLC-OVA cells were pretreated with drugs according to the experimental groups to construct a co-culture system.

[0119] (2) Cell collection Wash the cells twice with preheated 1×PBS, add trypsin along the cell wall to digest the cells, add 3 times the volume of complete culture medium to stop digestion, and centrifuge at 1500 rpm for 5 minutes.

[0120] (3) Cell washing After centrifuging at 1500 rpm for 5 minutes, discard the waste liquid in the tube, add 1 ml of pre-cooled BD Buffer to resuspend the cells, and then centrifuge again at 1500 rpm for 5 minutes at room temperature.

[0121] (4) Cell staining Dilute APC-conjugated anti-mouse CD8 antibody (200-fold dilution) and FVS620 viability dye (1000-fold dilution) with BD Buffer to distinguish between live and dead cells. After discarding the waste liquid in the tubes, add 200 µL of BD Buffer to the negative control group and 200 µL of the prepared antibody-containing BD Buffer to the experimental group, and incubate at room temperature in the dark for 20 minutes.

[0122] (5) Flow cytometry testing After staining, the cells were washed with 800 µL of BD Buffer and centrifuged at 1500 rpm for 5 minutes. The waste liquid was discarded, and the cells were resuspended in 800 µL of BD Buffer and analyzed using a BD LSR Fortessa flow cytometer.

[0123] The results are as follows Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown. Figure 5 The results showed that with increasing concentrations of Fangji Huangqi Decoction (200, 400, 800 μg / ml), activated CD8... + T cells (i.e., CD69) + CD8 + The number of T cells gradually increased; Figure 6 The results showed that as the concentration of Fangji Huangqi Decoction increased (200, 400, 800 μg / ml), CD8... + The release of IFN-γ by T cells gradually increases. Figure 7 This is the result of crystal violet staining. Figure 8 To quantify the results of flow cytometry using a dual-labeling strategy, Figure 7 and Figure 8 The results showed that as the concentration of Fangji Huangqi Decoction increased (200, 400, 800 μg / ml), CD8... + The tumor-killing effect of T cells gradually increases.

[0124] Example 6 of effect verification In vivo pharmacological experiments: To evaluate whether the composition (Fangji Huangqi Decoction) which enhances tumor immunogenicity can inhibit the growth of melanoma and lung cancer in mice, the following animal experiments were conducted.

[0125] 1. Animal experiment grouping: After a week of acclimatization in the SPF-grade animal experimental center of the Basic Research Center for Integrated Traditional and Western Medicine at Guangzhou University of Chinese Medicine, C57BL / 6 mice were transferred to the mouse housing. After ear tagging, the mice were randomly divided into three groups based on their weight: a blank control group (n=8), a low-dose group of Astragalus membranaceus and Stephania tetrandra (n=8), and a high-dose group of Astragalus membranaceus and Stephania tetrandra (n=8).

[0126] 2. Animal experimental modeling: (1) Mouse melanoma model Cell preparation: Mouse melanoma B16 cells were resuscitated in advance and cultured in 1640 complete medium at 37°C in a 5% CO2 incubator. Cell status was observed daily, and medium was changed and passaged according to cell condition. Animal modeling was performed when the cells were in the logarithmic growth phase. The laminar flow hood was irradiated with UV light 30 minutes beforehand. Consumables and reagents were sprayed with 75% alcohol before being placed in the laminar flow hood. Cells were rinsed twice with 1×PBS, then trypsin was added to digest the cells. After digestion, 3 times the volume of complete medium was added to stop the digestion. The cells were centrifuged at 1500 rpm for 5 minutes. After discarding the waste liquid, the cell density was adjusted to 1×10⁶ cells / mL with pre-cooled 1×PBS. 6 After gently mixing the cells by blowing the solution through a pipette, aliquot the cell suspension into 1.5 mL EP tubes (250 μL / tube), one 1.5 mL EP tube per mouse. Place the 1.5 mL EP tubes in an ice box to maintain cell viability.

[0127] Cell inoculation: After anesthetizing C57BL / 6 mice with isoflurane gas, the cell suspension was homogenized using a 200 μL pipette. 200 μL of the cell suspension was drawn up using a 1 mL syringe, and the syringe was purged of air. The mouse's right hand was gently opened, and the needle was inserted under the mouse's right armpit. After injecting all the fluid, the needle was rotated out to prevent leakage. 200 μL of B16 cell suspension was subcutaneously inoculated, with each mouse receiving 2 × 10-1 cells. 5 Each cell.

[0128] (2) Mouse lung cancer model Cell preparation: Mouse lung cancer LLC cells were resuscitated beforehand and cultured in DMEM complete medium at 37°C in a 5% CO2 incubator. The procedure was the same as for melanoma cell preparation, with the cell density adjusted to 1×10⁶ cells / year using 1×PBS. 7 / mL.

[0129] Cell inoculation: The procedure is the same as for melanoma cell inoculation. 200 μL of LLC cell suspension is subcutaneously inoculated, with 2 × 10⁶ cells per mouse. 6 Each cell.

[0130] 3. Dosing in animal experiments After subcutaneous inoculation of B16 / LLC cells into C57BL / 6 mice, tumor formation in the axillae of the mice was observed daily starting the following day.

[0131] The dosage for mice was calculated using the following formula for equivalent drug dosage.

[0132] The formula for equivalent drug dosage is: Mouse dosage = Adult clinical dosage (0.77g / kg) × Adult average weight (70kg) × 0.0026 / Mouse average weight (20g) = 7020mg / kg.

[0133] Blank control group: Starting from the second day after mouse modeling, each mouse was given 200 μL of pure water by gavage every day.

[0134] Low-dose group of Fangji Huangqi: Starting from the second day after mouse modeling, each mouse was administered 200 μL of low-dose Fangji Huangqi decoction (351 mg / ml) by gavage daily. The preparation method is the same as in Example 1.

[0135] High-dose group of Fangji Huangqi: Starting from the second day after mouse modeling, each mouse was administered 200 μL of high-dose Fangji Huangqi decoction (702 mg / ml) by gavage daily. The preparation method is the same as in Example 1.

[0136] 4. Animal experimental material collection Observation indicators: During the animal experiment, the activity status of the mice was observed at regular intervals every day. The weight of the mice was recorded using an electronic scale. The longest and shortest diameters of the tumors were measured using vernier calipers. The tumor volume V was calculated using the formula V = 1 / 2ab. 2 (a: longest diameter, b: shortest diameter). Observe the mice for any special conditions when the tumor volume approaches 800 mm. 3 To gather materials.

[0137] Tumor removal: After anesthetizing the mice, they were euthanized by dislocation of the neck. The subcutaneous tissue was slowly dissected and the tumor was removed using curved forceps and surgical scissors. The tumor was washed with pre-cooled physiological saline, dried to remove excess water, and weighed. The tumors were arranged neatly from largest to smallest and photographed.

[0138] The results are as follows Figure 9 As shown. Figure 9 Part A in the figure shows the size results of B16 melanomas in each group; Figure 9 Part B in the figure shows the curves showing the changes in the volume of B16 melanomas in each group. Figure 9 Part C in the table represents the weight of B16 melanomas in each group. Figure 9 Part D in the figure represents the lung cancer size results for each group; Figure 9 Part E in the figure represents the volume change curves of lung cancer in each group; Figure 9 The F portion represents the weight results for each group of lung cancers. Figure 9 It is known that Fangji Huangqi Decoction has a significant inhibitory effect on B16 melanoma and lung cancer (gross observation), effectively reducing the volume and weight of B16 melanoma and lung cancer. The high concentration (702mg / ml) is more effective than the low concentration (351mg / ml).

[0139] Example 7 of effect verification In vivo pharmacological experiments: To evaluate whether the composition (Fangji Huangqi Decoction) enhancing tumor immunogenicity crosses CD8 pathways. + T-cell-mediated immune killing inhibits the growth of melanoma and lung cancer in mice. The following animal experiments were conducted.

[0140] 1. Animal experiment grouping: After a week of acclimatization in the SPF-grade animal experimental center of the Basic Research Center for Integrated Traditional and Western Medicine at Guangzhou University of Chinese Medicine, C57BL / 6 mice were transferred to the mouse housing. After ear tagging, the mice were randomly divided into four groups based on their body weight: a blank control group (n=8), a Fangji Huangqi Decoction group (n=8), an αCD8 antibody group (n=8), and a Fangji Huangqi Decoction group combined with an αCD8 antibody group (n=8).

[0141] 2. Animal experimental modeling The method for establishing the mouse melanoma model is the same as described in Example 6, which validates the effect.

[0142] 3. Dosing in animal experiments After subcutaneous inoculation of B16 cells into C57BL / 6 mice, tumor formation in the axillae of the mice was observed regularly every day starting the following day.

[0143] Blank control group: Starting from the second day after the mice were modeled, each mouse was given 200 μL of pure water by gavage every day, and 200 μL of 1×PBS was injected intraperitoneally every two days.

[0144] Fangji Huangqi Decoction Group: Starting from the second day after mouse modeling, each mouse was given 200 μL of high-dose Fangji Huangqi Decoction by gavage daily. The preparation method was the same as in Implementation Case 1. Two days later, 200 μL of 1×PBS was injected intraperitoneally.

[0145] αCD8 antibody group: Starting from day 2 after mouse modeling, each mouse was administered 200 μL of pure water by gavage daily. Two days later, 200 μL of in vivo CD8 antibody was injected intraperitoneally. The preparation method was as follows: After 30 minutes of UV irradiation in a clean bench, the materials and reagents were sprayed with 75% alcohol and then placed in the clean bench. The stock solution concentration of CD8α-InVivo antibody was 3.91 mg / mL, which was diluted to 1 mg / mL using pre-cooled 1×PBS. The solution was kept in the dark during dilution and use, stored at 4°C, and prepared fresh for immediate use.

[0146] Fangji Huangqi Decoction combined with αCD8 antibody group: Starting from the second day after mouse modeling, each mouse was given 200μL of high-dose Fangji Huangqi Decoction by gavage daily, and 200μL of in vivo CD8 antibody was injected intraperitoneally every two days. The preparation method is as described above.

[0147] 4. Animal experimental material collection Observation indicators: During the animal experiment, the activity status of the mice was observed at regular intervals every day. The weight of the mice was recorded using an electronic scale. The longest and shortest diameters of the tumors were measured using vernier calipers. The tumor volume V was calculated using the formula V = 1 / 2ab. 2 (a: longest diameter, b: shortest diameter). Observe the mice for any special conditions when the tumor volume approaches 800 mm. 3 To gather materials.

[0148] Tumor removal: After anesthetizing the mice, they were euthanized by dislocation of the neck. The subcutaneous tissue was slowly dissected and the tumor was removed using curved forceps and surgical scissors. The tumor was washed with pre-cooled physiological saline, dried to remove excess water, and weighed. The tumors were arranged neatly from largest to smallest and photographed.

[0149] The results are as follows Figure 10 As shown in the figure. FJHQ represents Fangji Huangqi Decoction; αCD8 represents the anti-CD8 antibody group; Ctrl represents the blank control group; B16 represents melanoma. Figure 10 Part A in the figure shows the size results of B16 melanomas in each group; Figure 10 Part B in the figure shows the curves of change in the volume of B16 melanomas in each group. Figure 10 Part C in the figure represents the weight of B16 melanomas in each group; from Figure 10 It can be seen that Fangji Huangqi Decoction alone can significantly inhibit melanoma growth, while the anti-CD8 antibody group alone can inhibit CD8 growth. + T cells do not have a tumor-killing effect, therefore this group did not have a tumor-suppressing effect, and the size of the tumors in this group was similar to that of the blank control group. The group treated with Fangji Huangqi Decoction combined with anti-CD8 antibody, although using the tumor-suppressing Fangji Huangqi Decoction, did not show any tumor-suppressing effect due to the elimination of CD8 antibodies. + T cells kill tumors, therefore this group also showed no tumor-suppressing effect, and the size of the tumors in this group was similar to that of the blank control group. This demonstrates that the tumor-suppressing effect of Fangji Huangqi Decoction is mediated through CD8. + T cell mediated.

[0150] Example 8 of effect verification In vivo pharmacological experiments: To evaluate whether Fangji Huangqi Decoction combined with PD-1 therapy could further inhibit the growth of melanoma and lung cancer in mice, the following animal experiments were conducted.

[0151] 1. Animal experimental grouping: After a week of acclimatization in the SPF-grade animal experimental center of the Basic Research Center for Integrated Traditional and Western Medicine at Guangzhou University of Chinese Medicine, C57BL / 6 mice were transferred to the mouse housing. After ear tagging, the mice were randomly divided into four groups based on their body weight: a blank control group (n=8), a Fangji Huangqi Decoction group (n=8), an αPD-1 antibody group (n=8), and a Fangji Huangqi Decoction group combined with an αPD-1 antibody group (n=8).

[0152] 2. Animal experimental modeling The methods for establishing mouse melanoma and mouse lung cancer models were the same as those used in Example 6 for efficacy verification.

[0153] 3. Dosing in animal experiments After subcutaneous inoculation of C57BL / 6 mice with B16 cells or mouse lung cancer LLC cells, the tumor formation in the axilla of the mice was observed regularly every day starting the next day.

[0154] Blank control group: Starting from the second day after the mice were modeled, each mouse was given 200 μL of pure water by gavage every day, and 200 μL of 1×PBS was injected intraperitoneally every two days.

[0155] Fangji Huangqi Decoction Group: Starting from the second day after mouse modeling, each mouse was given 200 μL of high-dose Fangji Huangqi Decoction by gavage daily. The preparation method was the same as in Implementation Case 1. Two days later, 200 μL of 1×PBS was injected intraperitoneally.

[0156] αPD-1 antibody group: Starting from day 2 after mouse modeling, each mouse was administered 200 μL of pure water by gavage daily. Two days later, 200 μL of in vivo PD-1 antibody was injected intraperitoneally. The preparation method was as follows: After 30 minutes of UV irradiation in a clean bench, the materials and reagents were sprayed with 75% alcohol and then placed in the clean bench. The stock solution of PD-1-InVivo antibody had a concentration of 3.45 mg / mL, which was diluted to 1 mg / mL using pre-cooled 1×PBS. The solution was kept in the dark during dilution and use, stored at 4°C, and prepared fresh for immediate use.

[0157] Fangji Huangqi Decoction combined with αPD-1 antibody group: Starting from the second day after mouse modeling, each mouse was given 200μL of high-dose Fangji Huangqi Decoction by gavage daily, and 200μL of in vivo PD-1 antibody was injected intraperitoneally every two days. The preparation method is as described above.

[0158] 4. Animal experimental material collection Observation indicators: During the animal experiment, the activity status of the mice was observed at regular intervals every day. The weight of the mice was recorded using an electronic scale. The longest and shortest diameters of the tumors were measured using vernier calipers. The tumor volume V was calculated using the formula V = 1 / 2ab. 2 (a: longest diameter, b: shortest diameter). When the tumor volume is close to 800 mm... 3 To gather materials.

[0159] Tumor removal: Mice were anesthetized and euthanized by dislocation of the neck. The subcutaneous tissue was dissected and the tumor was removed using curved forceps and surgical scissors. The tumors were washed with pre-cooled physiological saline, dried to remove excess water, and weighed. The tumors were arranged neatly from largest to smallest and photographed.

[0160] The results are as follows Figure 11 As shown. Figure 11Part A shows the size results of B16 melanomas in each group; Figure 11 Part B in the figure shows the curves showing the changes in the volume of B16 melanomas in each group. Figure 11 Part C in the table represents the weight of B16 melanomas in each group. Figure 11 Part D in the figure represents the size results of LLC lung cancer in each group; Figure 11 Part E in the figure represents the volume change curves of LLC lung cancer in each group; Figure 11 Part F represents the weight results of LLC lung cancer in each group. Among them, FJHQ represents the Fangji Huangqi Decoction group; αPD-1 represents the αPD-1 antibody group; FJHQ+αPD-1 represents the Fangji Huangqi Decoction group combined with αPD-1 antibody group; Ctrl represents the blank control group; B16 represents melanoma; LLC represents lung cancer.

[0161] Depend on Figure 11 It is known that the combined effect of Fangji Huangqi Decoction and PD-1 antibody can significantly inhibit the growth of melanoma and lung cancer, and the effect of the combination is better than that of Fangji Huangqi Decoction or αPD-1 antibody alone; for melanoma, from Figure 11 Part B shows that tumor volume growth in the combination group (FJHQ + αPD-1) almost stopped, significantly lower than in the Ctrl group, αPD-1 monotherapy group, and FJHQ monotherapy group; from Figure 11 As shown in Part C, the tumor weight in the combination group was significantly lower than that in any single-drug group, and the difference was extremely significant compared to the single-drug group. The inhibition was almost complete, with Q=1.56. In pharmacology, a Q value > 1.15 is considered synergistic, and Q=1.56 indicates significant synergistic enhancement. For LLC lung cancer, from... Figure 11 As shown in Part E, the tumor volume growth in the combination group was significantly slower than that in the single-drug group. Figure 11 As shown in part F, the tumor weight in the combined treatment group was significantly lower than that in the FJHQ monotherapy group and the αPD-1 monotherapy group, with a highly significant difference. ).

[0162] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A composition for enhancing tumor immunogenicity, characterized in that, Based on the weight percentages, it includes: 10-15 parts of Fangji, 10-20 parts of Huangqi, 3-9 parts of Gancao, and 3-12 parts of Baizhu.

2. The composition for enhancing tumor immunogenicity according to claim 1, characterized in that, By weight, it includes: 10-15 parts of Fangji, 10-20 parts of Huangqi, 3-9 parts of Gancao, 3-12 parts of Baizhu, 0-6 parts of Dazao, and 0-12 parts of Shengjiang.

3. The composition for enhancing tumor immunogenicity according to claim 2, characterized in that, By weight, it includes: 12 parts of Fangji, 15 parts of Huangqi, 6 parts of Gancao, 9 parts of Baizhu, 3 parts of Dazao, and 9 parts of Shengjiang.

4. The composition for enhancing tumor immunogenicity according to any one of claims 1-3, characterized in that, The dosage form of the composition is a decoction, lyophilized powder, oral liquid preparation, granules, pills, powder, tablets, capsules, transdermal absorption agent, ointment, liposome, sustained-release preparation, controlled-release preparation, or injection.

5. The composition for enhancing tumor immunogenicity according to any one of claims 1-3, characterized in that, The tumor is one of the following: melanoma, lung cancer, liver cancer, colon cancer, or stomach cancer.

6. A method for preparing the composition for enhancing tumor immunogenicity according to any one of claims 1-3, characterized in that, The process includes the following steps: adding the raw materials required for the composition that enhances tumor immunogenicity to water, heating and decocting to obtain the composition that enhances tumor immunogenicity.

7. The method for preparing the composition for enhancing tumor immunogenicity according to claim 6, characterized in that, The temperature for the decoction process is 90-100 degrees Celsius, and the decoction time is 50-70 minutes.

8. The method for preparing the composition for enhancing tumor immunogenicity according to claim 6, characterized in that, The decoction process is repeated at least twice. After the first decoction, the residue and filtrate are separated by filtration. The residue is mixed with water and then subjected to a second decoction. This process is repeated until all decoctions are completed. The filtrates are then combined to obtain the composition that enhances tumor immunogenicity.

9. The use of the composition for enhancing tumor immunogenicity according to any one of claims 1-3 in the preparation of an antitumor drug.

10. The use of the composition for enhancing tumor immunogenicity according to any one of claims 1-3 in combination with a PD-1 blocker in the preparation of an antitumor drug.

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