Traditional Chinese medicine composition for sensitizing immune checkpoint inhibitor to treat microsatellite stable tumors

Abstract

The invention belongs to the technical field of biological medicines, and discloses a traditional Chinese medicine composition for treating microsatellite stability (MSS) type tumors by a sensitization immune checkpoint inhibitor. The traditional Chinese medicine composition provided by the invention comprises radix scutellariae, rhizoma zingiberis, Chinese herbaceous peony, liquorice root and Chinese date, can increase CD8 + T cell infiltration, promote the CD8 + T cell to secrete cytokines and improve the immunosuppression of MSS type tumors, and further breaks through the conventional range of anti-tumor treatment of immune examination inhibitors; meanwhile, the traditional Chinese medicine composition can also improve adverse reaction of gastrointestinal tracts caused by tumor treatment by an immune examination inhibitor, and the life quality of patients is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a traditional Chinese medicine composition for sensitizing immune checkpoint inhibitors in the treatment of microsatellite stable tumors. Background Technology

[0002] Immune checkpoint inhibitors are an emerging cancer treatment approach in recent years, offering good efficacy and fewer side effects, making them one of the most promising methods for treating and improving the prognosis of various malignant tumors. However, not all cancer patients benefit from them. Microsatellites are DNA sequences, and in gastrointestinal tumors, patients with microsatellite stable (MSS) and microsatellite unstable (MSI) phenotypes have drastically different prognoses. MSI tumors have become a necessary condition for the use of immune checkpoint inhibitors in various guidelines, while MSS tumors are almost ineffective against immune checkpoint inhibitor therapy, and both domestic and international guidelines do not recommend the use of immune checkpoint inhibitors for any line of treatment for MSS tumors.

[0003] However, in gastrointestinal tumors, especially colorectal cancer, MSS-type colorectal cancer accounts for more than 85% of all colorectal cancers. Patients with MSS-type colorectal cancer are still treated with traditional chemotherapy drugs, which produce significant side effects and severely impact their quality of life. Therefore, there is an urgent need to explore drug compositions suitable for MSS-type colorectal cancer patients who can also be treated with immune checkpoint inhibitors. Summary of the Invention

[0004] This invention aims to provide a traditional Chinese medicine composition for sensitizing immune checkpoint inhibitors, enabling their use in the treatment of microsatellite stable tumors (MSS). This invention focuses on MSS-type tumors that are insensitive to immune checkpoint inhibitors, proposing a traditional Chinese medicine composition that can sensitize the efficacy of immune checkpoint inhibitors, thus expanding the conventional clinical application scope of immune checkpoint inhibitors for the treatment of MSS-type tumors. This traditional Chinese medicine composition can alter the efficacy of immune checkpoint inhibitors for this type of tumor by reversing the "immunosuppressive microenvironment" of MSS-type tumors. This traditional Chinese medicine composition can synergistically work with immune checkpoint inhibitors, overturning the conventional view that immune checkpoint inhibitors cannot be used to treat MSS-type tumor subtypes, and breaking through the clinical application scope of immune checkpoint inhibitors not covered in current industry guidelines.

[0005] To achieve the above objectives, the following technical solution is adopted:

[0006] In a first aspect, the present invention provides a traditional Chinese medicine composition for sensitizing immune checkpoint inhibitors in the treatment of microsatellite stable tumors, the traditional Chinese medicine composition comprising Scutellaria baicalensis, dried ginger, peony root, licorice root, and jujube.

[0007] In one optional embodiment, the amounts of each component in the traditional Chinese medicine composition, by weight, are: 6-30 parts of Scutellaria baicalensis, 0-20 parts of dried ginger, 4-20 parts of Paeonia lactiflora, 4-20 parts of Glycyrrhiza uralensis, and 2-30 parts of Ziziphus jujuba. In a preferred embodiment, the traditional Chinese medicine composition comprises Scutellaria baicalensis, dried ginger, Paeonia lactiflora, Glycyrrhiza uralensis, and Ziziphus jujuba, and the amounts of each component in the traditional Chinese medicine composition, by weight, are:

[0008] Scutellaria baicalensis 15 parts, dried ginger 10 parts, peony root 10 parts, licorice root 10 parts, jujube 20 parts.

[0009] In one alternative implementation, the microsatellite stable tumor includes any one of colorectal cancer, non-small cell lung cancer, endometrial cancer, gastric cancer, small intestinal adenocarcinoma, and pancreatic cancer.

[0010] In one optional embodiment, the immune checkpoint inhibitor includes at least one of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

[0011] Secondly, the present invention provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of sensitizers for immune checkpoint inhibitors in the treatment of microsatellite stable tumors.

[0012] Thirdly, the present invention provides the application of the above-mentioned traditional Chinese medicine composition in the preparation of a medicament for treating microsatellite stable tumors.

[0013] Fourthly, the present invention provides a pharmaceutical preparation for sensitizing immune checkpoint inhibitors in the treatment of microsatellite stable tumors, wherein the raw materials of the pharmaceutical preparation include the above-mentioned traditional Chinese medicine composition.

[0014] In one optional embodiment, the pharmaceutical preparation is an oral dosage form, which includes at least one of decoction, tablet, granule, capsule, powder, pill, ointment, elixir, or novel drug delivery system.

[0015] Fifthly, the present invention provides a pharmaceutical composition for treating microsatellite stable tumors, the pharmaceutical composition comprising the above-mentioned traditional Chinese medicine composition and an immune checkpoint inhibitor.

[0016] Based on the above technical solution, the present invention has at least the following technical effects:

[0017] The traditional Chinese medicine composition provided by this invention includes Scutellaria baicalensis, dried ginger, peony root, licorice root, and jujube, which can increase CD8 levels. +This traditional Chinese medicine composition promotes T-cell infiltration and cytokine secretion, improving immunosuppression in MSS-type tumors and thus enhancing the anti-tumor effects of immunosuppressants, overcoming the limitation of current treatment guidelines that do not recommend its use in the treatment of MSS-type tumors. Simultaneously, this composition can also alleviate gastrointestinal adverse reactions caused by immunosuppressant therapy for tumors, improving patients' quality of life. Specifically, this composition can reverse low CD8 levels. + The immunosuppressive microenvironment caused by T cell infiltration enhances the efficacy of immune checkpoint inhibitors for MSS-type tumor patients, changing the conventional view that immune checkpoint inhibitors cannot be used to treat MSS-type tumor subtypes. Moreover, it can reduce toxicity while enhancing efficacy, with no obvious adverse reactions found and good safety. This is a therapeutic effect that cannot be obviously predicted by those skilled in the art, and it has broad application prospects. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of this application or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This refers to the statistical results of the objective response rate (ORR) of the three groups of patients at the experimental endpoint in Example 1 of this invention. ORR is one of the commonly used endpoint indicators for tumor treatment in clinical practice. It refers to the proportion of patients whose tumor volume shrinks to a predetermined value and can be maintained for a minimum time limit. The calculation formula is the ratio of subjects who have all tumor target lesions disappeared, no new lesions appeared, and tumor markers are normal for at least 4 weeks, or subjects whose sum of the maximum diameter of tumor target lesions decreased by ≥30% for at least 4 weeks to the total number of subjects.

[0020] Figure 2 The CD8+ levels in the peripheral blood of the three groups of patients in Experiment Example 1 of this invention at the end of the experiment. + Statistical results of T cell count;

[0021] Figure 3 The CD8+ levels in the peripheral blood of the three groups of patients in Experiment Example 1 of this invention at the end of the experiment. + Statistical results of the amount of IFN-γ secreted by T cells;

[0022] Figure 4 The CD8+ levels in the peripheral blood of the three groups of patients in Experiment Example 1 of this invention at the end of the experiment. + Statistical results of the amount of TNF-α secreted by T cells;

[0023] Figure 5 This is a statistical result of gastrointestinal adverse reactions occurring in the three groups of patients during the experiment in Experiment Example 1 of this invention;

[0024] Figure 6 These are the tumor volume growth curves of each group of mice in Experiment Example 2 of this invention during the experiment;

[0025] Figure 7 In Experiment 2 of this invention, the CD8+ levels in the tumors of mice in each group were measured at the experimental endpoint. + Statistical results of T cell count;

[0026] Figure 8 In Experiment 2 of this invention, the CD8+ levels in the tumors of mice in each group were measured at the experimental endpoint. + Statistical results of the amount of IFN-γ secreted by T cells;

[0027] Figure 9 In Experiment 2 of this invention, the CD8+ levels in the tumors of mice in each group were measured at the experimental endpoint. + Statistical results on the amount of TNF-α secreted by T cells. Detailed Implementation

[0028] To further illustrate the technical means and results adopted by this application to achieve the intended inventive purpose, the following preferred embodiments are used to describe in detail the specific implementation methods, technical solutions, and features according to this application. Specific features, structures, or characteristics in the various embodiments described below can be combined in any suitable form.

[0029] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0030] The present application will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed in the present application.

[0031] Example 1: Preparation of a decoction of traditional Chinese medicine composition

[0032] The dosage for a single serving is as follows: 15 g of Scutellaria baicalensis, 10 g of dried ginger, 10 g of peony root, 10 g of licorice root, and 20 g of jujube. Boil these ingredients in 1000 mL of water, then simmer over low heat for 40 minutes. Take 300 mL of the decoction as one dose.

[0033] Example 2: Preparation of a decoction of traditional Chinese medicine composition

[0034] The dosage for a single serving is as follows: 6 g of Scutellaria baicalensis, 20 g of dried ginger, 4 g of Paeonia lactiflora, 20 g of Glycyrrhiza uralensis, and 2 g of jujube. Boil these ingredients in 1000 mL of water, then simmer over low heat for 40 minutes. Take 300 mL of the decoction as one dose.

[0035] Example 3: Preparation of a decoction of traditional Chinese medicine composition

[0036] The dosage for a single serving is as follows: 30 g of Scutellaria baicalensis, 4 g of dried ginger, 20 g of Paeonia lactiflora, 4 g of Glycyrrhiza uralensis, and 10 g of jujube. Boil these ingredients in 1000 mL of water, then simmer over low heat for 40 minutes. Take 300 mL of the decoction as one dose.

[0037] Example 4: Preparation of a decoction of traditional Chinese medicine composition

[0038] The dosage for a single serving is as follows: 15 g of Scutellaria baicalensis, 10 g of Paeonia lactiflora, 10 g of Glycyrrhiza uralensis, and 5 g of Ziziphus jujuba. Boil these ingredients in 1000 mL of water, then simmer over low heat for 40 minutes. Take 300 mL of the decoction as one dose.

[0039] Experimental Example 1

[0040] Sixty patients clinically diagnosed with MSS-type colorectal cancer were recruited as subjects. Imaging examinations of the tumors of each subject were performed, and tumor size was recorded. They were then randomly assigned to three groups (20 patients in each group). Due to clinical data showing ineffectiveness of PD-1 monoclonal antibody therapy, and to comply with ethical requirements, Group 1 received tislelizumab (PD-1 monoclonal antibody) combined with chemotherapy (capecitabine tablets); Group 2 received a traditional Chinese medicine composition (Example 1); and Group 3 received tislelizumab combined with the traditional Chinese medicine composition (Example 1). The treatment duration for all three groups was 63 days (i.e., 3 cycles of tislelizumab treatment). At the trial endpoint, imaging examinations of the tumors of each subject were performed again, and tumor size was recorded and compared with that at enrollment. Results showed that the objective response rate (ORR) in Group 1 was 30%, in Group 2 it was 20%, and in Group 3 it was 60%, significantly higher than the other two groups.

[0041] In this application, Group 1 represents the tislelizumab + capecitabine tablet group, Group 2 represents the traditional Chinese medicine 1 (Example 1) group, and Group 3 represents the tislelizumab + traditional Chinese medicine 1 (Example 1) group.

[0042] Using the scheme described in this application, the applicant also studied CD8 in the peripheral blood of the above three groups of subjects. + The results showed that the number of T cells and the amount of IFN-γ and TNF-α they secreted were significantly lower in the peripheral blood of subjects in group 3. +The number of T cells and the amount of IFN-γ and TNF-α they secrete are significantly higher than those in the other two groups, indicating that the traditional Chinese medicine composition provided in this application can significantly improve the immunosuppressive microenvironment of MSS-type tumors, thereby enhancing the efficacy of PD-1 monoclonal antibody against MSS-type colorectal cancer.

[0043] During the above study, the applicant also monitored all subjects for gastrointestinal adverse reactions, such as nausea, vomiting, constipation, and diarrhea. The results showed that the incidence of adverse reactions in groups 2 and 3 was significantly lower than that in group 1, indicating that the traditional Chinese medicine composition can play a role in reducing toxicity while exerting a synergistic effect.

[0044] To verify the accuracy of the above results, the results and data from the specific experimental process are described and analyzed below. To clearly and explicitly introduce the drugs and reagents used in the experimental tests, Table 1 provides the names, catalog numbers, and manufacturer information for different reagents.

[0045] Table 1. Main materials and their sources used in Experimental Example 1 of this application.

[0046]

[0047] 1. Preparation of decoctions based on traditional Chinese medicine compositions

[0048] The dosage for a single serving is as follows: 15 g of Scutellaria baicalensis, 10 g of dried ginger, 10 g of peony root, 10 g of licorice root, and 20 g of jujube. Boil these ingredients in 1000 mL of water, then simmer over low heat for 40 minutes. Take 300 mL of the decoction as one dose.

[0049] 2. Drug therapy

[0050] Group 1: Tislelizumab was administered once every 3 weeks, with a single intravenous infusion of 200 mg, followed by oral capecitabine tablets for 2 weeks, then discontinued for 1 week. The dosage was based on body surface area, specifically 1250 mg / m². 2 Take it once in the morning and once in the evening every day.

[0051] Group 2: Traditional Chinese medicine decoction (Example 1), 1 dose per day.

[0052] Group 3: Tislelizumab was administered once every 3 weeks, with a single intravenous infusion of 200 mg, and the traditional Chinese medicine decoction (Example 1) was administered once daily.

[0053] 3. Drug efficacy evaluation

[0054] All subjects must undergo an abdominal CT scan before starting medication and at the end of the trial to assess tumor size (long diameter) and calculate the ORR ratio. Specifically, the ORR is defined as a reduction of 30% or more in tumor size compared to before medication. The ORR ratio is the percentage of subjects who achieve the ORR.

[0055] The results are as follows Figure 1 As shown, the ORR rate in group 1 was 30%, meaning that 6 out of 20 subjects achieved the ORR; the ORR rate in group 2 was 20%, meaning that 4 out of 20 subjects achieved the ORR; while in group 3, 12 subjects achieved the ORR, meaning that the ORR rate in this group was 60%. It is evident that the ORR rate in group 3 was significantly higher than the other two groups, indicating that the traditional Chinese medicine composition of this application has a significant synergistic effect on the efficacy of PD-1 monoclonal antibodies.

[0056] 4. Flow cytometry detection of CD8 in peripheral blood of subjects + T cells and the IFN-γ and TNF-α they secrete

[0057] Peripheral blood (500 μL) was collected from each subject and mixed with 500 μL of basal cell culture medium (RPMI 1640 medium) to obtain a blood-culture mixture. 1 mL of human lymphocyte separation medium was added to a clean flow cytometry tube, and the blood-culture mixture was added to the supernatant of the separation medium (ensuring a clear interface). The tube was centrifuged at 500 g for 30 min with a rapid ascent and slow descent. After centrifugation, the intermediate lymphocyte layer was transferred to a clean centrifuge tube, 2 mL of phosphate buffer was added, and the mixture was vortexed. The tube was then centrifuged at 500 g for 5 min, the supernatant was discarded, and the cell pellet was obtained. The cells were resuspended in 0.5 mL of basal cell culture medium and counted. The pellet was stored at 4°C for later use.

[0058] After resuspending and counting the cells in 0.5 mL of basal cell culture medium, approximately 1 × 10⁶ cells were collected. 6 Cells were collected and their volume adjusted to 500 μL with complete culture medium. Tetradecanoyl phorbol acetate (50 ng / mL), ionomycin calcium salt (1 μg / mL), and monensin (2 μM) were added, and the mixture was incubated at 37℃ and 5% CO2 for 5 h. After incubation, cells were collected, centrifuged, and the supernatant was discarded. 2 μL each of antibodies CD8 and CD25 were added to the sample tubes, and the mixture was incubated at room temperature in the dark for 15 min. 0.5 mL of cell fixation solution was added, and the cells were fixed at room temperature in the dark for 30 min. 2 mL of 1× cell permeabilization buffer was added, and the mixture was centrifuged at 500 g for 5 min, then the supernatant was discarded. 2 mL of 1× cell permeabilization buffer was added again, and the mixture was centrifuged at 500 g for 5 min, then the supernatant was discarded. 2 μL each of IFN-γ and TNF-α antibodies were added, and the mixture was incubated at room temperature in the dark for 30 min. 2 mL of cell permeabilization buffer was added, and the cells were centrifuged and the supernatant was discarded. Cells were resuspended in 0.4 mL of cell staining buffer and analyzed.

[0059] Test results as follows Figure 2-4 As shown, CD8 levels in the peripheral blood of subjects in group 3 were... +The number of T cells and the amount of IFN-γ and TNF-α they secreted were significantly higher than in the other two groups (**P<0.01, ***P<0.001, ****P<0.0001). This suggests that the traditional Chinese medicine composition of the present invention improves the poor efficacy of immune checkpoint inhibitors against MSS-type colorectal cancer by improving immunity.

[0060] 5. Monitoring of adverse gastrointestinal reactions

[0061] Gastrointestinal adverse reactions in each subject during medication were recorded, and the results are as follows: Figure 5 As shown, up to 90% of the subjects in Group 1 experienced adverse reactions of varying degrees during the medication period, 40% of the subjects in Group 3 experienced adverse reactions, while only 10% of the subjects in Group 2 experienced adverse reactions. This indicates that the traditional Chinese medicine composition has good safety and not only has a synergistic effect on immune checkpoint inhibitor therapy but also a toxicity reduction effect, which is beneficial to improving the quality of life of patients.

[0062] Experimental Example 2

[0063] To recreate clinical MSS-type colorectal cancer, a humanized MSS-type colon cancer mouse model was constructed. Mice were treated with immune checkpoint inhibitors, the traditional Chinese medicine compositions from Examples 1-4, and combinations of immune checkpoint inhibitors and the traditional Chinese medicine compositions from Examples 1-4 (simultaneous administration of the traditional Chinese medicine composition and the immune checkpoint inhibitor, or administration of the traditional Chinese medicine composition followed by the immune checkpoint inhibitor). An equal volume of physiological saline was used as a control group. Changes in tumor volume before and after treatment were studied. The traditional Chinese medicine composition from Example 1 was named Traditional Chinese Medicine 1, the traditional Chinese medicine composition from Example 2 was named Traditional Chinese Medicine 2, the traditional Chinese medicine composition from Example 3 was named Traditional Chinese Medicine 3, and the traditional Chinese medicine composition from Example 4 was named Traditional Chinese Medicine 4.

[0064] The results showed that there was no significant difference in tumor volume between the immune checkpoint inhibitor monotherapy group (PD-1 group) and the traditional Chinese medicine composition monotherapy group (TCM 1-4) and the control group. However, the tumor volume in mice treated with the combined TCM composition and immune checkpoint inhibitor in Examples 1-4 (TCM 1+PD-1 group, TCM 2+PD-1 group, TCM 3+PD-1 group, TCM 4+PD-1 group) was significantly reduced (*P<0.05, **P<0.01, vs. control group), with statistically significant differences. These results indicate that the TCM composition proposed in this application combined with immune checkpoint inhibitors can significantly improve the efficacy of immune checkpoint inhibitors against MSS-type colorectal cancer.

[0065] Using the scheme of this application, the number of CD8+ T cells and the amount of IFN-γ and TNF-α secreted by them in the tumor tissue of the above-mentioned tumor-bearing mouse model were also studied. The results showed that after treatment with the traditional Chinese medicine composition of Examples 1-4 combined with immune checkpoint inhibitors, the tumor volume of mice that had significantly shrunk significantly decreased. + The number of T cells and the amount of IFN-γ and TNF-α they secreted were significantly increased (**P<0.01, ***P<0.001, vs. control group). These results indicate that the traditional Chinese medicine compositions of Examples 1-4, combined with immune checkpoint inhibitors, can significantly reverse the immunosuppressive microenvironment of MSS-type tumors, thereby improving the efficacy of immune checkpoint inhibitors against MSS-type colorectal cancer.

[0066] In the above study, no significant adverse reactions were observed in the combined treatment group.

[0067] To verify the accuracy of the above results, the results and data from the specific experimental process are described and analyzed below. To clearly and explicitly introduce the drugs and reagents used in the experimental tests, Table 2 provides the names, catalog numbers, and manufacturer information for different reagents.

[0068] Table 2. Main materials and their sources used in Experimental Example 2 of this application.

[0069]

[0070] 1. Preparation of experimental solutions

[0071] (1) Preparation of broad-spectrum antibiotic (ABX) solution: Weigh appropriate amounts of neomycin sulfate, metronidazole, ampicillin and vancomycin, add physiological saline to dissolve and obtain a solution containing 40 mg / mL neomycin sulfate, metronidazole, ampicillin and 20 mg / mL vancomycin, mix well, dispense into 15 mL centrifuge tubes and store at -20℃ for later use.

[0072] (2) Preparation of fecal suspension from clinical patients: Fecal samples from pre-frozen clinical MSS-type and MSI-H-type colon cancer patients were resuspended in sterile sodium chloride solution (0.9%), with a dilution ratio of 1 g feces to 10 mL. The feces were stirred until no large particles were visible. The solution was filtered through a 200-mesh sterile sieve to remove large particles, and the filtrate was collected in sterile centrifuge tubes. The resuspension was then vortexed for 5 minutes to obtain a resuspended solution. The resuspension was centrifuged at 600×g for 5 minutes to remove insoluble matter, and the centrifuged fecal suspension was immediately aliquoted in a clean bench and stored at -20℃ for later use.

[0073] (3) Preparation of αPD-1 solution: Take an appropriate amount of αPD-1 and prepare a suspension with a concentration of 2.5 mg / mL using physiological saline. Prepare and use immediately.

[0074] 2. Establishment of MSS-FMT tumor-bearing mice

[0075] Fifty female SPF-grade BAlb / C mice were purchased from Beijing Huafukang Biotechnology Co., Ltd., and were housed in an SPF environment at the animal experiment center of Beijing Langke Biotechnology Co., Ltd.

[0076] After a week of acclimatization, BAlb / C mice were administered 100 μL of ABX by gavage daily for 3 consecutive days. Two days after ABX administration was discontinued, a frozen fecal suspension (1 g / 10 mL) from a clinical MSS-type colon cancer patient was extracted, heated in a 37°C water bath, and then administered to the ABX-treated mice by gavage at a rate of 100 μL per mouse for 7 consecutive days, thus obtaining BAlb / C mice with fecal microbiota transplantation.

[0077] The cell concentration was approximately 2 × 10⁻⁶. 7 CT26.WT cell (MSS type) suspension at a concentration of 0.05 mL / mice was inoculated into the right axilla of BAlb / C mice following fecal microbiota transplantation. The longest and shortest diameters of the tumor were measured using calipers, and the volume was calculated as 1 / 2 the longest diameter × the shortest diameter. 2 .

[0078] 3. Therapeutic effect of traditional Chinese medicine composition on MSS-type colon cancer-bearing mice

[0079] The mouse tumor grew to 50-100 mm 3 Subsequently, MSS-type tumor-bearing mice modeled using CT26.WT cells were divided into 10 groups (n=5 per group) and administered drugs to each group. The types and dosages of drugs administered to each group are as follows:

[0080] Control group: Normal saline was administered by gavage daily until the end of the experiment (200 μL / day), and normal saline was injected intraperitoneally every 3 days (100 μL / time), for a total of 5 injections.

[0081] PD-1 group: 200 μL of normal saline was administered by gavage daily until the end of the experiment. 100 μL of αPD-1 solution was injected intraperitoneally every 3 days for a total of 5 injections.

[0082] Traditional Chinese Medicine Group 1 (Example 1): The decoction of the traditional Chinese medicine composition of Example 1 was administered by gavage daily until the end of the experiment (200 μL / d).

[0083] Traditional Chinese Medicine Group 2 (Example 2): The decoction of the traditional Chinese medicine composition of Example 2 was administered by gavage daily until the end of the experiment (200 μL / d).

[0084] Traditional Chinese Medicine Group 3 (Example 3): The decoction of the traditional Chinese medicine composition of Example 3 was administered by gavage daily until the end of the experiment (200 μL / d).

[0085] Traditional Chinese Medicine Group 4 (Example 4): The decoction of the traditional Chinese medicine composition of Example 4 was administered by gavage daily until the end of the experiment (200 μL / d).

[0086] Traditional Chinese Medicine 1 (Example 1) + PD-1 group: The decoction of the traditional Chinese medicine composition of Example 1 was administered by gavage daily until the end of the experiment (200 μL / d), and αPD-1 solution was injected intraperitoneally once every 3 days (100 μL / time), for a total of 5 injections.

[0087] Traditional Chinese Medicine 2 (Example 2) + PD-1 group: The decoction of the traditional Chinese medicine composition of Example 2 was administered by gavage daily until the end of the experiment (200 μL / d), and αPD-1 solution was injected intraperitoneally once every 3 days (100 μL / time), for a total of 5 injections.

[0088] Traditional Chinese Medicine 3 (Example 3) + PD-1 group: The decoction of the traditional Chinese medicine composition of Example 3 was administered by gavage daily until the end of the experiment (200 μL / d), and αPD-1 solution was injected intraperitoneally once every 3 days (100 μL / time), for a total of 5 injections.

[0089] Traditional Chinese Medicine 4 (Example 4) + PD-1 group: The decoction of the traditional Chinese medicine composition of Example 4 was administered by gavage daily until the end of the experiment (200 μL / d), and αPD-1 solution was injected intraperitoneally once every 3 days (100 μL / time), for a total of 5 injections.

[0090] Mice body weight and tumor volume were monitored every 3 days during the administration period.

[0091] Tumor volume detection results as follows Figure 6 Compared with other treatment groups, the tumor volume of mice in the combined Chinese medicine composition and αPD-1 groups (Chinese medicine 1+PD-1 group, Chinese medicine 2+PD-1 group, Chinese medicine 3+PD-1 group, and Chinese medicine 4+PD-1 group) was significantly reduced, indicating that the Chinese medicine composition of Examples 1-4 of this application can improve the poor efficacy of immune checkpoint inhibitors against MSS-type colorectal cancer.

[0092] 4. Flow cytometry detection of CD8 in tumor tissue + The number of T cells and the amount of IFN-γ and TNF-α they secrete.

[0093] Fresh tumor tissue was collected from mice in each group, minced, and added to 5 mL of tissue digestion solution. Digestion was carried out at 37°C for 30 min. The tissue was then filtered through a 200-mesh filter into a 5 mL centrifuge tube, centrifuged at 500 g for 5 min, and the supernatant was discarded to obtain cell pellet. The cell pellet was thoroughly resuspended in 3 mL of mouse lymphocyte separation medium. 2 mL of phosphate buffer was slowly added to the upper layer of the cell suspension (ensuring a clear interface), and the pellet was centrifuged at 500 g for 30 min with a rapid ascent and slow descent. After centrifugation, the intermediate lymphocyte layer was aspirated into a clean centrifuge tube, 3 mL of phosphate buffer was added, and the mixture was vortexed. The pellet was centrifuged at 500 g for 5 min, and the supernatant was discarded. The resulting cell pellet was resuspended in 0.5 mL of basal cell culture medium and counted. Approximately 1 × 10⁶ cells were collected. 6 Cells were collected and their volume adjusted to 500 μL with complete cell culture medium. Tetradecanoyl phorbol acetate (50 ng / mL), iomycin calcium salt (1 μg / mL), and monensin (2 μM) were added, and the cells were incubated at 37℃ and 5% CO2 for 5 h. After incubation, cells were collected, centrifuged, and the supernatant was discarded. 2 μL each of antibodies CD8 and CD25 were added to the sample tubes, and the cells were incubated at room temperature in the dark for 15 min. 0.5 mL of cell fixation solution was added, and the cells were fixed at room temperature in the dark for 30 min. 2 mL of 1× cell permeabilization buffer was added, and the cells were centrifuged at 500g for 5 min, discarding the supernatant. 2 mL of 1× cell permeabilization buffer was added again, and the cells were centrifuged at 500g for 5 min, discarding the supernatant. 2 μL each of IFN-γ and TNF-α antibodies were added, and the cells were incubated at room temperature in the dark for 30 min. 2 mL of cell permeabilization buffer was added, and the cells were centrifuged and the supernatant was discarded. Cells were resuspended in 0.4 mL of cell staining buffer and analyzed.

[0094] Test results as follows Figure 7-9 As shown, compared to other treatment groups, the CD8 values ​​of the combined traditional Chinese medicine (TCM) composition and αPD-1 groups (TCM 1 + PD-1, TCM 2 + PD-1, TCM 3 + PD-1, and TCM 4 + PD-1) were significantly higher. + The number of T cells and the amount of IFN-γ and TNF-α they secrete are significantly increased, indicating that the traditional Chinese medicine composition of Examples 1-4 of this application improves the poor efficacy of immune checkpoint inhibitors against MSS-type colorectal cancer by reversing the immunosuppressive microenvironment.

[0095] The above animal experiment results lead to the same conclusion as the clinical trial results: the combination of PD-1 monoclonal antibody and traditional Chinese medicine composition can effectively treat MSS-type colorectal cancer, reversing the current situation where PD-1 monotherapy is ineffective in treating MSS-type colorectal cancer.

[0096] The above description is merely a preferred embodiment of this application; however, the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and its improved concept, should be covered within the scope of protection of this application.

Claims

1. A traditional Chinese medicine composition for sensitizing immune checkpoint inhibitors in the treatment of microsatellite stable tumors, characterized in that, The traditional Chinese medicine composition includes Scutellaria baicalensis, dried ginger, peony root, licorice root, and jujube.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The dosage of each component in the traditional Chinese medicine composition by weight is as follows: 6-30 parts of Scutellaria baicalensis, 0-20 parts of dried ginger, 4-20 parts of Paeonia lactiflora, 4-20 parts of Glycyrrhiza uralensis, and 2-30 parts of Ziziphus jujuba.

3. The traditional Chinese medicine composition according to any one of claims 1 to 2, characterized in that, The microsatellite stable tumors include any one of colorectal cancer, non-small cell lung cancer, endometrial cancer, gastric cancer, small intestinal adenocarcinoma, and pancreatic cancer.

4. The traditional Chinese medicine composition according to any one of claims 1 to 2, characterized in that, The immune checkpoint inhibitors include at least one of PD-1 inhibitors, PD-L1 inhibitors, and CTLA-4 inhibitors.

5. The use of the traditional Chinese medicine composition according to any one of claims 1 to 2 in the preparation of an immune checkpoint inhibitor sensitizer for the treatment of microsatellite stable tumors.

6. The use of the traditional Chinese medicine composition according to any one of claims 1 to 2 in the preparation of a medicament for treating microsatellite stable tumors.

7. A pharmaceutical formulation for sensitizing immune checkpoint inhibitors in the treatment of microsatellite stable tumors, characterized in that, The raw materials for the pharmaceutical preparation include the traditional Chinese medicine composition as described in any one of claims 1 to 2.

8. The pharmaceutical preparation according to claim 7, characterized in that, The pharmaceutical preparation is an oral dosage form, which includes at least one of the following: decoction, tablet, granule, capsule, powder, pill, ointment, elixir, or novel drug delivery system.

9. A pharmaceutical composition for treating microsatellite stable tumors, characterized in that, The pharmaceutical composition comprises the traditional Chinese medicine composition according to any one of claims 1 to 2 and an immune checkpoint inhibitor.

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