Combination of chebulagic acid and pd-1 antibodies and uses thereof

Abstract

The application discloses a combination of chebulagic acid and a PD-1 antibody and application thereof, improves a tumor-related macrophage immune suppression phenotype, reduces a tumor growth rate, and further enhances a PD-1 antibody-mediated anti-tumor immunotherapy effect by using chebulagic acid to intervene in a tumor immune microenvironment. Compared with a current simple immunotherapy method, the application has a clear administration scheme, a simple intervention mode, good repeatability, and a good clinical transformation application prospect.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, and relates to a composition of chebulic acid and PD-1 antibody and its application. Background Technology

[0002] In recent years, tumor immunotherapy, especially programmed death receptor 1 (PD-1) antibody therapy, has shown promising application prospects in a variety of solid tumors. However, the overall clinical efficacy is still limited, and some patients have primary or secondary drug resistance, which seriously affects the treatment effect.

[0003] Existing research indicates that the immunosuppressive state in the tumor microenvironment is an important factor limiting the efficacy of PD-1 antibodies. In particular, the massive infiltration of tumor-associated macrophages (TAMs) with an immunosuppressive phenotype can inhibit the killing activity of CD8+ T cells, weaken the body's anti-tumor immune response, and thus reduce the sensitivity to immunotherapy.

[0004] Currently, intervention methods for improving TAM immunosuppression and enhancing PD-1 therapy sensitivity are still relatively limited, especially lacking natural small molecule compounds with clear sources and good safety profiles.

[0005] Therefore, it is of great significance to develop a new method that can improve the tumor immune microenvironment and enhance the anti-tumor effect of PD-1. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a composition of chebulic acid and PD-1 antibody and its application, so as to solve the technical problems of limited response rate, drug resistance in some patients, and irreversible tumor-associated macrophage immunosuppression in existing PD-1 antibody therapy in tumors.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] 1. Application of chebulic acid in the preparation of antitumor drugs.

[0009] Preferably, the antitumor drug is an anti-breast cancer drug.

[0010] 2. A composition of chebulic acid and PD-antibody, wherein the mass ratio of the two is 1:4 to 2:1, and more preferably 1:2 to 2:1.

[0011] Preferably, the PD-1 antibody is an anti-PD-1 monoclonal antibody capable of blocking the PD-1 signaling pathway. The PD-1 antibody may be selected from nivolumab, pembrolizumab, sintilimab, camrelizumab, tislelizumab, toripalimab, cimiprimab, cepalimumab, or other antibodies with PD-1 blocking function.

[0012] More preferably, in animal experiments, the PD-1 antibody is an anti-mouse PD-1 monoclonal antibody; in specific embodiments, the PD-1 antibody is Anti-mouse PD-1 (CD279)-InVivo, catalog number A2122, with a specification of 5 mg × 20, or other antibodies with the same PD-1 blocking function are selected.

[0013] 3. Application of the aforementioned composition in the preparation of antitumor drugs.

[0014] Preferably, the antitumor drug is an anti-breast cancer drug.

[0015] 4. An anti-breast cancer drug, the active ingredient of which is chebulic acid, or which contains the aforementioned composition.

[0016] The beneficial effects of this invention are as follows:

[0017] This invention discloses a composition of chebulic acid and a PD-1 antibody and its application. Chebulic acid intervenes in the tumor immune microenvironment, improving the immunosuppressive phenotype of tumor-associated macrophages, reducing tumor growth rate, and further enhancing the anti-tumor immunotherapy effect mediated by the PD-1 antibody. Compared with existing immunotherapy methods alone, this invention has a clear dosing regimen, a simple intervention method, and good reproducibility, showing promising prospects for clinical translational applications. Attached Figure Description

[0018] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0019] Figure 1 This is a graph showing the results of different doses of chebulic acid inhibiting the growth of EO771 tumors;

[0020] Figure 2 This is a diagram illustrating the therapeutic effect of chebulic acid combined with anti-PD-1 antibody to enhance EO771 tumor immunotherapy.

[0021] Figure 3 This is a flow cytometry result of chebulic acid combined with anti-PD-1 antibody improving the phenotype of EO771 tumor-associated macrophages. The detection indicators include CD206, MHC-II and MHC-II / CD206 ratio.

[0022] Figure 4 This is a flow cytometry result of chebulic acid combined with anti-PD-1 antibody enhancing CD8+ T cell function in EO771 tumors, where the detection indicators include the proportion of TNF-α+CD8+ T cells. Detailed Implementation

[0023] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0024] Reference Implementation Examples

[0025] Source of experimental materials

[0026] EO771 mouse breast cancer cells were obtained from Sun Yat-sen University; DMEM medium was obtained from Rongda and used for EO771 cell culture or serum-free resuspension.

[0027] The PBS is 1×PBS (Phosphate Buffered Saline), material code G4202-500ML, specification is 500mL / bottle, obtained from Rongda.

[0028] Female C57BL / 6 mice, 6–8 weeks old, were obtained from Enswell and used to establish a subcutaneous xenograft model in EO771 mice.

[0029] The anti-PD-1 antibody is Anti-mouse PD-1 (CD279)-InVivo, catalog number A2122, with a specification of 5 mg × 20, obtained from Shanghai Lanmu Chemical; it is used in the PD-1 immune checkpoint blockade experiment in mice.

[0030] 1) Steps for establishing a tumor model

[0031] EO771 mouse breast cancer cells obtained from Sun Yat-sen University were cultured in DMEM medium until the logarithmic growth phase. After cell collection, the cells were resuspended in 1×PBS or serum-free DMEM medium and the cell density was adjusted to 0.5×10⁻⁶. 7 ~1×10 7 Cells / mL: 100-200 μL of cell suspension was subcutaneously inoculated into 6-8 week old female C57BL / 6 mice obtained from Enswell to establish a subcutaneous xenograft model in EO771 mice.

[0032] 2) Chebulic acid dosage screening steps

[0033] Once the tumor volume reached 50–100 mm³, patients were randomly assigned to groups and administered chebulic acid 2.5 mg / kg, 5 mg / kg, or 10 mg / kg intraperitoneally, once every 3 days for 4 consecutive times. The control group received an equal volume of solvent. The long and short diameters of the tumor were measured every 2–3 days, and the tumor volume change was calculated as tumor volume = long diameter × short diameter² / 2 to screen for an effective dose range.

[0034] 3) Enhance tumor immunotherapy steps

[0035] After determining the effective dosage range, chebulic acid was used in combination with anti-PD-1 antibody for intervention. The experiment was divided into a control group, a chebulic acid group, an anti-PD-1 antibody group, and a chebulic acid combined with anti-PD-1 antibody group. The chebulic acid group was given chebulic acid 5-10 mg / kg intraperitoneally every 3 days for 4 consecutive times; the anti-PD-1 antibody group was given Anti-mouse PD-1 (CD279)-InVivo (product number A2122) 100-200 μg / mouse intraperitoneally every 3 days for 4 consecutive times; the combination group was given chebulic acid and anti-PD-1 antibody respectively during the same treatment cycle, with the same dosage and frequency as the single-drug groups. Based on the weight of mice weighing approximately 20 g per administration, the dosage of chebulic acid was approximately 0.10-0.20 mg / mouse, and the dosage of PD-1 antibody was approximately 0.10-0.20 mg / mouse, with a weight ratio of approximately 1:2-2:1.

[0036] 4) Tumor tissue detection steps

[0037] At the experimental endpoint, tumor tissue was collected, and single-cell suspensions were prepared. Flow cytometry was used to detect changes in the tumor immune microenvironment. Tumor-associated macrophages were defined as CD45+CD11b+F4 / 80+ cell populations, and the expression levels of CD206 and MHC-II, as well as the MHC-II / CD206 ratio, were detected. Simultaneously, CD45+CD8+ T cells and the proportion of TNF-α+CD8+ T cells were detected using CD45, CD8, and TNF-α labeling protocols to evaluate the efficacy of chebulic acid-enhanced PD-1 antibody immunotherapy.

[0038] Example 1: Low-dose example

[0039] Using the EO771 mouse subcutaneous xenograft model, chebulic acid was administered intraperitoneally at a dose of 2.5 mg / kg every 3 days for 4 consecutive times. The results showed that the tumor growth rate was slowed down.

[0040] Example 2: Medium-dose example

[0041] Using the EO771 mouse subcutaneous xenograft model, chebulic acid was administered intraperitoneally at a dose of 5 mg / kg every 3 days for 4 consecutive times. The results showed that the tumor-suppressive effect was further enhanced at the lower dose.

[0042] Example 3: High-dose example

[0043] Using the EO771 mouse subcutaneous xenograft model, chebulic acid was administered intraperitoneally at a dose of 10 mg / kg every 3 days for 4 consecutive times. The results showed that a stable and significant tumor-suppressive effect was obtained.

[0044] like Figure 1As shown, chebulic acid can significantly inhibit the growth of subcutaneous xenografts in EO771 mice within a dose range of 2.5–10 mg / kg, and shows a certain dose-dependent trend. The tumor-suppressing effect is more obvious in the range of 5–10 mg / kg, which provides an effective dose basis for subsequent combined anti-PD-1 antibody therapy.

[0045] Example 4: Chebulic acid combined with PD-1 antibody improves macrophage phenotype.

[0046] Using an EO771 mouse subcutaneous xenograft model, once the tumor volume reached 50–100 mm³, mice were randomly divided into a control group, a chebulic acid group, an anti-PD-1 antibody group, and a chebulic acid combined with an anti-PD-1 antibody group. The chebulic acid group received chebulic acid 5–10 mg / kg intraperitoneally every 3 days for 4 consecutive treatments. The anti-PD-1 antibody group received Anti-mouse PD-1 (CD279)-InVivo (catalog number A2122) 100–200 μg / mouse intraperitoneally every 3 days for 4 consecutive treatments. The combination group received chebulic acid and anti-PD-1 antibody, respectively, within the same treatment cycle, with the same dosage and frequency as the single-drug groups. At the experimental endpoint, tumor tissue was used to prepare single-cell suspensions. Flow cytometry was used to detect the expression levels of CD206 and MHC-II in CD45+CD11b+F4 / 80+ tumor-associated macrophages, and the MHC-II / CD206 ratio was calculated. The results showed that, compared with the control group, the chebulic acid group, and the anti-PD-1 antibody group, the combination therapy group could further reduce CD206 expression and / or increase MHC-II expression, and increase the MHC-II / CD206 ratio, suggesting that chebulic acid combined with PD-1 antibody can further improve the immunosuppressive phenotype of tumor-associated macrophages.

[0047] Example 5: Chebulic acid combined with PD-1 antibody enhances CD8+ T cell function.

[0048] Using an EO771 mouse subcutaneous xenograft model, once the tumor volume reached 50–100 mm³, mice were randomly divided into a control group, a chebulic acid group, an anti-PD-1 antibody group, and a chebulic acid combined with anti-PD-1 antibody group. The chebulic acid group received chebulic acid 5–10 mg / kg intraperitoneally every 3 days for 4 consecutive treatments. The anti-PD-1 antibody group received Anti-mouse PD-1 (CD279)-InVivo (catalog number A2122) 100–200 μg / mouse intraperitoneally every 3 days for 4 consecutive treatments. The combined group received chebulic acid and anti-PD-1 antibody, respectively, within the same treatment cycle, with the same dosage and frequency as the single-drug groups. During the experiment, the long and short diameters of the tumor were measured every 2–3 days, and the tumor volume was calculated. At the experimental endpoint, tumor tissue was used to prepare a single-cell suspension, and the proportions of CD45+CD8+ T cells and TNF-α+CD8+ T cells were detected by flow cytometry. The results showed that, compared with the control group and the monotherapy group, the tumor growth rate was further reduced and the proportion of TNF-α+CD8+T cells was further increased in the chebulic acid combined with anti-PD-1 antibody group, suggesting that chebulic acid can enhance the anti-tumor immune response mediated by PD-1 antibody.

[0049] like Figure 2 As shown, compared with the control group, the chebulic acid group and the anti-PD-1 antibody group, the chebulic acid combined with the anti-PD-1 antibody group can further enhance the effect of tumor immunotherapy, which is manifested by a significant slowdown in tumor growth rate.

[0050] like Figure 3 As shown, flow cytometry analysis revealed that CD206 expression was decreased and / or MHC-II expression was increased in tumor-associated macrophages in the combined treatment group, and the MHC-II / CD206 ratio was increased, suggesting that chebulic acid combined with anti-PD-1 antibody can improve the tumor immunosuppressive microenvironment.

[0051] like Figure 4 As shown, the proportion of TNF-α+CD8+ T cells was increased after combined treatment, suggesting that chebulic acid can further enhance the anti-tumor function of CD8+ T cells, thereby improving the sensitivity of tumor immunotherapy.

[0052] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Application of chebulic acid in the preparation of antitumor drugs.

2. A composition of chebulic acid and PD-1 antibody, characterized in that, The mass ratio of the two is 1:4 to 2:

1.

3. The composition according to claim 2, characterized in that, The PD-1 antibody is an anti-PD-1 monoclonal antibody that can block the PD-1 signaling pathway.

4. The composition according to claim 3, characterized in that, The PD-1 antibody is selected from nivolumab, pembrolizumab, sintilimab, camrelizumab, tislelizumab, toripalimab, cimiprimab, cepalimumab, or other antibodies with PD-1 blocking function; in animal experiments, the PD-1 antibody is an anti-mouse PD-1 monoclonal antibody.

5. Use of the composition according to any one of claims 2 to 4 in the preparation of an antitumor drug.

6. The application according to claim 5, characterized in that, The anti-tumor drug is an anti-breast cancer drug.

7. An anti-breast cancer drug, characterized in that, Its active ingredient includes chebulic acid, or contains the composition described in any one of claims 2 to 4.

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