Anti-tumor composition

Abstract

The invention relates to the technical field of medicines, in particular to an anti-tumor composition which comprises 23-hydroxybetulinic acid and a PD-L1 inhibitor. According to the anti-tumor composition disclosed by the invention, the 23-hydroxybetulinic acid reduces the combination of PD-1 and PD-L1 on the surfaces of tumor-specific CD8 + T cells by down-regulating the expression of an immunosuppressive checkpoint PD-L1 of tumor cells, mobilizes the immune response of a host and changes a tumor microenvironment, so that the curative effect of an anti-PD-L1 immunotherapy is enhanced, and a synergistic anti-tumor effect is obtained. The composition provides a novel potential strategy for tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to an anti-tumor composition. Background Art

[0002] Malignant tumors are difficult to cure, and traditional treatment methods such as surgery, radiotherapy, and chemotherapy have limited efficacy. In addition, due to poor targeting and high incidence of adverse reactions, the quality of life of patients is severely affected. Since the beginning of this century, immunotherapy has made breakthrough progress and become the research frontier and hope in the field of cancer treatment.

[0003] Programmed cell death protein-1 (PD-1) is mainly expressed in immune cells, and its ligand PD-L1 (programmed death ligand-1) can be found in malignant tumors such as renal cancer, melanoma, leukemia, lung cancer, oral cancer, nasopharyngeal cancer, laryngeal cancer, breast cancer, bladder cancer, gastric cancer, and colorectal cancer. When PD-1 binds to its ligand, a co-inhibitory signal is generated, directly inhibiting the activation of T cells and causing T cell dysfunction (T cell exhaustion), thus leading to immune escape of malignant tumor cells. PD-1 / PD-L1 inhibitors can restore T cell function and inhibit tumor growth by specifically blocking PD-1 or PD-L1. Currently, immune checkpoint PD-1 / PD-L1 blockers are used to treat various malignant tumors, including leukemia, lung cancer, gastric cancer, colorectal cancer, breast cancer, and head and neck squamous cell carcinoma. In the past decade, targeting PD-1 (such as pembrolizumab, nivolumab) and PD-L1 (such as durvalumab, atezolizumab) has opened a new era for cancer clinical treatment. However, for many tumor types, single immune checkpoint inhibitor treatment still has problems such as low immune response rate, drug resistance, and immune-related adverse events; even for tumors with good immune response, most patients do not have durable clinical benefits. Therefore, compared with single anti-PD-1 / PD-L1 antibody treatment, developing treatment strategies that combine other drugs with it is currently a research hotspot, and finding new and effective combination strategies has become an urgent problem to be solved.

[0004] 23-Hydroxybetulinic acid (hereinafter referred to as 23-HBA) is a pentacyclic triterpenoid compound. As a natural product, its characteristics of multi-target and low toxicity make it have broad prospects in the fields of tumor treatment and immune regulation. In terms of anti-tumor, 23-hydroxybetulinic acid has inhibitory effects on a variety of tumors. This kind of compound can inhibit the growth of a variety of tumor cells, such as human non-small cell lung cancer cells (NCI-H460), human gastric cancer cells (SGC7901), human liver cancer cells (HepG2), human cervical cancer cells (HeLa), mouse melanoma cells (B16), mouse sarcoma cells (S-180), etc.; it can promote the apoptosis of human leukemia cells (K562) and human promyelocytic leukemia cells (HL-60); 23-hydroxybetulinic acid can regulate M2 macrophage polarization through the STAT6 signal, thereby reducing the resistance of colorectal cancer to 5-fluorouracil, and at the same time can also regulate the polarization of M1 macrophages to enhance the anti-tumor immune ability of the body; 23-hydroxybetulinic acid can enhance the anti-tumor effect of doxorubicin and protect against the cardiotoxicity caused by doxorubicin; in addition, 23-hydroxybetulinic acid can also weaken the drug resistance of chemotherapy drugs in anti-tumor. The chemical structure of 23-hydroxybetulinic acid is as follows:

[0005]

[0006] However, there have been no related reports on combining 23-hydroxybetulinic acid and anti-PD-L1 antibody for anti-tumor treatment so far; in view of this, the present invention is specifically proposed. Summary of the Invention

[0007] (1) Technical problems to be solved

[0008] In view of the above-mentioned disadvantages and deficiencies of the prior art, the present invention provides an anti-tumor composition, which combines two active ingredients, a PD-L1 inhibitor and 23-hydroxybetulinic acid, for anti-tumor treatment. When the two active ingredients are used in combination, the combination index of killing colorectal cancer cells is less than 1, and the two active ingredients can play a good synergistic anti-tumor effect.

[0009] (2) Technical solutions

[0010] The present invention provides an anti-tumor composition, which comprises 23-hydroxybetulinic acid and a PD-L1 inhibitor.

[0011] According to a preferred embodiment of the present invention, the PD-L1 inhibitor is an anti-PD-L1 antibody, a functional fragment of an anti-PD-L1 antibody (polypeptide) or a small molecule compound inhibitor.

[0012] According to a preferred embodiment of the present invention, when the PD-L1 inhibitor is an anti-PD-L1 antibody, the combination of 23-hydroxybetulinic acid and the anti-PD-L1 antibody has a synergistic effect, and the combination ratio ranges from 4:1 to 1:16.

[0013] According to a preferred embodiment of the present invention, the anti-PD-L1 antibody is at least one of Envafolimab, Durvalumab, Atezolizumab, Sugemalimab, Socazolimab, Adebrelimab, Tagitanlimab, Benmelstobart, Cosibelimab, Avelumab, etc.

[0014] According to a preferred embodiment of the present invention, the small molecule compound inhibitor is at least one of INCB086550 (Incyte, an oral PD-L1 inhibitor) and CA-170 (targeting PD-L1 / VISTA).

[0015] According to a preferred embodiment of the present invention, the tumor is a malignant tumor such as colorectal cancer.

[0016] According to a preferred embodiment of the present invention, the anti-tumor composition uses 23-hydroxybetulinic acid and a PD-L1 inhibitor as the sole active ingredients.

[0017] According to a preferred embodiment of the present invention, the anti-tumor composition further comprises pharmaceutically acceptable excipients; the excipients include, but are not limited to, one or more of preservatives, antioxidants, flavoring agents, fragrances, solubilizing agents, emulsifying agents, pH buffers, binders, fillers, and lubricants. For example, when the anti-tumor composition is an injection, the excipients include vegetable oil, emulsifying agent, glycerol, antioxidant, and water for injection.

[0018] The anti-tumor composition can reduce the immunosuppressive effect of immunosuppressive checkpoints, reduce the binding of PD-1 and PD-L1 on the surface of tumor-specific T cells, and enhance the anti-tumor immune effect.

[0019] (III) Beneficial effects

[0020] The present invention discloses an anti-tumor composition, which comprises a PD-L1 inhibitor (such as an anti-PD-L1 antibody, an antibody functional fragment, a small molecule compound inhibitor) and 23-hydroxybetulinic acid. Among them, the PD-L1 inhibitor, as a targeted immune checkpoint inhibitor, reshapes the anti-tumor immune response by specifically blocking the immunosuppressive effect mediated by the PD-1 / PD-L1 signaling pathway in the tumor microenvironment; 23-hydroxybetulinic acid, as a natural product, has the characteristics of anti-tumor, immune regulation, and low toxicity.

[0021] The anti-tumor composition of the present invention, in which 23-hydroxybetulinic acid down-regulates the expression of the immune inhibitory checkpoint PD-L1 on the surface of tumor cells, thereby reducing the binding of PD-1 and PD-L1 on the surface of tumor-specific CD8+ T cells, mobilizing the host's immune response, changing the tumor microenvironment, and thus enhancing the efficacy of anti-PD-L1 immunotherapy to obtain a synergistic anti-tumor effect. This composition provides a new and potential strategy for tumor immunotherapy. Brief Description of the Drawings

[0022] Figure 1 Shows the effect of 23-HBA on the expression of the immune inhibitory checkpoint PD-L1 protein on the surface of human colorectal cancer cell line HCT116.

[0023] Figure 2 Shows the effect of 23-HBA on the expression of the immune inhibitory checkpoint PD-L1 protein on the surface of human colorectal cancer cell line HCT116 induced by IFN-γ.

[0024] Figure 3 Shows the dose-effect curve of the combined inhibition of human colorectal cancer cell line HCT116 by 23-HBA and anti-PD-L1 antibody (1:4) measured by the lactate dehydrogenase method.

[0025] Figure 4 Shows the CI curve of the combined inhibition of human colorectal cancer cell line HCT116 by 23-HBA and anti-PD-L1 antibody (1:4) calculated by the combination index method.

[0026] Figure 5 Shows the dose-effect curve of the combined inhibition of human colorectal cancer cell line HCT116 by 23-HBA and anti-PD-L1 antibody (1:16) measured by the lactate dehydrogenase method.

[0027] Figure 6 Shows the CI curve of the combined inhibition of human colorectal cancer cell line HCT116 by 23-HBA and anti-PD-L1 antibody (1:16) calculated by the combination index method.

[0028] Figure 7 Shows the dose-effect curve of the combined inhibition of human colorectal cancer cell line HCT116 by 23-HBA and anti-PD-L1 antibody (4:1) measured by the lactate dehydrogenase method.

[0029] Figure 8 Shows the CI curve of the combined inhibition of human colorectal cancer cell line HCT116 by 23-HBA and anti-PD-L1 antibody (4:1) calculated by the combination index method.

[0030] Figure 9 Shows the effect of the combined inhibition of human colorectal cancer cell line HCT116 by 23-HBA and anti-PD-L1 antibody detected by fluorescence staining method. Detailed implementation manners

[0031] For better explaining the present invention for easy understanding, the present invention will be described in detail below in conjunction with the accompanying drawings through specific implementation manners. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained by purchasing in the market. Unless otherwise specified, the practice of the present invention will adopt conventional techniques in cell biology, molecular biology (including recombinant technology), microbiology, biochemistry and immunology, and the said conventional techniques are within the capabilities of those skilled in the art.

[0032] Some implementation manners of the present invention provide an anti-tumor composition, which includes a PD-L1 inhibitor and 23-hydroxybetulinic acid. It should be noted that 23-hydroxybetulinic acid is an existing raw material and can be obtained through existing commercial channels. The PD-L1 inhibitor can be an anti-PD-L1 antibody, a functional fragment (polypeptide) of an anti-PD-L1 antibody or a small molecule compound.

[0033] The inventors found through a large number of studies and practices that when 23-hydroxybetulinic acid is combined with a drug that inhibits PD-L1, it can effectively down-regulate the expression of the immunosuppressive checkpoint PD-L1 on the surface of human colorectal cancer cell line HCT116, thereby reducing the binding of PD-1 on the surface of tumor-specific CD8 + T cells, mobilize the host's immune response, change the tumor microenvironment, and thus enhance the efficacy of anti-PD-L1 immunotherapy, obtaining a synergistic anti-tumor effect.

[0034] The features and properties of the present invention will be further described in detail below in conjunction with the examples. In the following examples, the anti-PD-L1 antibody is selected as Envafolimab, and 23-hydroxybetulinic acid comes from natural components of plants such as Betula platyphylla and Pulsatilla chinensis. In the following examples of the present invention, the synergy of the killing effect of 23-hydroxybetulinic acid combined with an anti-PD-L1 antibody on human colorectal cancer cell line HCT116 is verified by CompuSyn 2.0 software. In this evaluation method, statistical analysis is carried out using SPSS21.0 statistical software. For the comparison of multiple group means, one-way analysis is used. P < 0.05 indicates that the difference is statistically significant. The evaluation of the synergistic inhibitory effect of two anti-tumor drugs on cells adopts the combination index CI method, and CompuSyn 2.0 software is used to analyze the drug synergistic effect. The combination index CI > 1 indicates that the two drugs act antagonistically; CI = 1 indicates that the two drugs act additively; CI < 1 indicates that the two drugs synergize with each other; CI < 0.3 indicates that the two drugs are highly synergistic.

[0035] Example 1

[0036] In this example, the effect of 23-hydroxybetulinic acid (abbreviated as 23-HBA) on the expression of the immunosuppressive checkpoint PD-L1 protein on the surface of human colorectal cancer cell line HCT116 was detected by Western Blot. HCT116 cells were co-incubated with 23-HBA at concentrations of 0 μg / mL, 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5.0 μg / mL respectively. The detailed experimental method is as follows:

[0037] (1) Seed HCT116 cells into 6-well plates at a density of 5×10 5 / well, and culture them at 37°C and 5% CO2 until 80% confluence. Add drugs (23-HBA / DMSO) according to the grouping. The added concentrations of 23-HBA are set as 6 groups: 0 μg / mL, 0.3125 μg / mL, 0.625 μg / mL, 1.25 μg / mL, 2.5 μg / mL, and 5.0 μg / mL, with 3 replicate wells in each group.

[0038] When studying the effect of 23-HBA on the mRNA and protein expression of the immunosuppressive checkpoint PD-L1 in HCT116 cells induced by IFN-γ, 50 ng / mL of IFN-γ was also pre-added to the wells to construct a cell model.

[0039] (2) After 48 hours, discard the culture medium, wash twice with pre-cooled PBS, add 200 μL of RIPA lysis buffer (containing 1 mM PMSF) to each well, and lyse on ice for 30 min. Centrifuge at 4°C and 12,000×g for 15 min, take the supernatant, and determine the protein concentration by BCA method. Adjust the protein concentration of the samples to 2 μg / μL.

[0040] (3) Prepare 10% separating gel and 5% stacking gel. Load 20 μg of protein (containing Loading Buffer) into each well, using pre-stained Marker as a reference.

[0041] Electrophoresis conditions: Constant voltage of 80 V for 30 min (stacking gel), constant voltage of 120 V for 60 min (separating gel).

[0042] Soak the gel and PVDF membrane in transfer buffer (containing 20% methanol), and set constant current transfer at 300 mA for 90 min (the molecular weight of PD-L1 is about 40 kDa).

[0043] (4) After transfer, block the PVDF membrane with 5% skim milk (prepared with TBST) at room temperature for 1 h.

[0044] Dilute the primary antibodies: PD-L1 (1:1000), GADPH (1:5000), incubate overnight at 4°C, and wash the membrane 3 times with TBST for 5 min each time.

[0045] (5) Incubate the HRP-labeled secondary antibody (1:5000) at room temperature for 1 h. After washing the membrane with TBST, add the ECL reagent and expose it using a chemiluminescence imager (the exposure time was optimized to 30 s - 2 min).

[0046] (6) Use Image Lab 7.0 software to analyze the gray value of the bands, and calculate the relative expression level of PD-L1 using GADPH as the internal reference protein (formula: PD-L1 / GADPH). One-way ANOVA was performed using Graph Pad Prism 10, and *p < 0.05 was considered a significant difference.

[0047] See the experimental results in Figure 1 and Table 1.

[0048] Table 1: Effects of 23-HBA on the expression of the immunosuppressive checkpoint PD-L1 protein on the surface of human colorectal cancer cell line HCT116

[0049]

[0050] Note: *p < 0.05, **p < 0.01 compared with the 0 μg / mL 23-HBA dose group; GAPDH was used as the internal reference protein

[0051] Meanwhile, the effects of IFN-γ (50 ng / mL) on the mRNA and protein expression levels of the immunosuppressive checkpoint PD-L1 in HCT116 cells were detected by Western blotting and quantitative real-time PCR (qRT-PCR); see the experimental results in Table 2 Figure 2 and Table 3.

[0052] Table 2: Effects of 23-HBA on the mRNA expression level of the immunosuppressive checkpoint PD-L1 gene on the surface of human colorectal cancer cell line HCT116 induced by IFN-γ

[0053]

[0054] Note: #p < 0.0001 compared with the control group; *p < 0.05, ****p < 0.0001 compared with the model group; β-actin was used as the internal reference gene

[0055] Table 3: Effects of 23-HBA on the protein expression of the immunosuppressive checkpoint PD-L1 on the surface of human colorectal cancer cell line HCT116 induced by IFN-γ

[0056]

[0057] Note: #p < 0.05 compared with the control group; *p < 0.05, ***p < 0.001 compared with the model group; GAPDH is the internal reference protein

[0058] As can be seen from Figure 1 and Table 1, 23-HBA can significantly down-regulate the expression of the immunosuppressive checkpoint PD-L1 in HCT116 cells; moreover, as can be seen from the results of Table 2, Figure 2 and Table 3, 23-hydroxybetulinic acid can significantly down-regulate the mRNA and protein expression levels of the immunosuppressive checkpoint PD-L1 gene in HCT116 cells induced by IFN-γ (50 ng / mL).

[0059] Example 2

[0060] In this Example 2, a co-culture system of CD8+ T cells and HCT116 cells was constructed to evaluate the anti-tumor effects of 23-HBA, anti-PD-L1 antibody and their combination by the lactate dehydrogenase method. In this example, peripheral blood mononuclear cells (PBMCs) were extracted from healthy human blood, and the cell density of PBMCs was adjusted to 2×10 6 cells / mL with serum-free medium and placed in an incubator for 2 hours to allow monocytes to adhere (inducing the adherent monocytes to differentiate into dendritic cells (DCs), and antigen-loading the DC cells with the HCT116 cell lysate), collecting the suspended cells and purifying and sorting them using a CD8+ T cell sorting kit to obtain CD8+ T cells. After activation and proliferation with CD3 monoclonal antibody (OKT-3 clone), they were co-cultured with DC cells (10:1) to enable the CD8+ T cells to acquire the ability to specifically kill HCT116 cells. Then, the specific CD8+ T cells were co-cultured with HCT116 cells and drug intervention was carried out. The lactate dehydrogenase method was used to calculate the mortality rate of HCT116 cells and detect the anti-tumor effects of 23-HBA, anti-PD-L1 antibody and their combination. The calculation formula for cell mortality rate (lactate dehydrogenase method): Cell mortality rate (%) = (absorbance of treated sample - absorbance of sample control well) / (absorbance of maximum enzyme activity of cells - absorbance of sample control well) × 100.

[0061] The experimental results are shown in Table 4, Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Table 5.

[0062] Table 4: Mortality rate of tumor cells HCT116 under specific killing by CD8+ T cells

[0063]

[0064] Note: The * in () is a significant marker, *p < 0.05, **p < 0.01 compared with the anti-PD-L1 antibody group at the same dose.

[0065] Table 5: CI values after the combination of 23-HBA and anti-PD-L1 antibody

[0066]

[0067] As can be seen from Table 4, the combination of 23-HBA and anti-PD-L1 antibody significantly increased the mortality of HCT116 cells, and was superior to the case of using the two drugs alone; from Figure 3 、 Figure 5 、 Figure 7 it can be seen that the dose-effect curve of the combination of 23-HBA and anti-PD-L1 antibody shifted upward, indicating that the combination of the two drugs was stronger than using the two drugs alone; from Figure 4 、 Figure 6 、 Figure 8 and Table 5, it can be seen that the CI value after the combination of 23-HBA and anti-PD-L1 antibody was less than 1, indicating that it had a synergistic anti-tumor effect.

[0068] Example 3

[0069] In this Example 3, a co-culture system of CD8+ T cells and HCT116 cells was constructed to evaluate the anti-tumor effects of 23-HBA, anti-PD-L1 antibody and their combination by cell fluorescence staining method. In this example, peripheral blood mononuclear cells (PBMCs) were extracted from healthy human blood, and the cell density of PBMCs was adjusted to 2×10 6cells / mL, placed in an incubator and cultured for 2 hours to allow monocytes to adhere (inducing the adhered monocytes to differentiate into dendritic cells (DCs), and antigen-loading the DC cells with HCT116 cell lysate), collecting the suspended cells and purifying and sorting them using a CD8+ T cell sorting kit to obtain CD8+ T cells. After activation and proliferation with anti-CD3 monoclonal antibody (OKT-3 clone), the CD8+ T cells were co-cultured with DC cells (10:1) to endow the CD8+ T cells with the ability to specifically kill HCT116 cells. Then, the specific CD8+ T cells were co-cultured with HCT116 cells labeled with CFDA-SE fluorescence, and drug intervention was performed. Using the cell fluorescence staining method, the fluorescence area of HCT116 cells was statistically analyzed and calculated using Image J software. The smaller the fluorescence area, the more cell death indicated. The mortality rate of HCT116 cells was reflected by the relative reduction area of fluorescence, and the anti-tumor effects of 23-HBA, anti-PD-L1 antibody, and their combination were detected.

[0070] The experimental results are shown in Figure 9 , Table 6.

[0071] Table 6: Detection of the combined inhibitory effect of 23-HBA and anti-PD-L1 antibody on human colorectal cancer cell line HCT116 by fluorescence staining

[0072]

[0073] Note: * within () is the significance marker, *p < 0.05, **p < 0.01 compared with the anti-PD-L1 antibody group at the same dose.

[0074] As shown in Figure 9 and Table 6, the combination of 23-HBA and anti-PD-L1 antibody significantly increased the mortality rate of HCT116 cells, and was superior to the case of using the two drugs alone.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements, or in the case where the technical features in the above embodiments do not conflict with each other, can be combined in the manner described in the embodiments, and these modifications, replacements or combinations do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antitumor composition, characterized in that: It contains 23-hydroxybetulinic acid and a PD-L1 inhibitor.

2. The antitumor composition according to claim 1, characterized in that The PD-L1 inhibitor is an anti-PD-L1 antibody, an anti-PD-L1 antibody fragment or a small molecule compound inhibitor.

3. The antitumor composition according to claim 1, characterized in that When the PD-L1 inhibitor is an anti-PD-L1 antibody, the combination of 23-hydroxybetulinic acid and the anti-PD-L1 antibody has a synergistic effect, and the ratio of the combination ranges from 4:1 to 1:

16.

4. The antitumor composition according to claim 2 or 3, characterized in that: The anti-PD-L1 antibody is at least one of Envafolimab, Durvalumab, Atezolizumab, Sugemalimab, Socazolimab, Adebrelimab, Tagitanlimab, Benmelstobart, Cosibelimab, Avelumab, etc.

5. The antitumor composition according to claim 2 or 3, characterized in that: The small molecule compound inhibitor is at least one of INCB086550 and CA-170.

6. The antitumor composition according to claim 1, characterized in that: The tumor is a malignant tumor such as colorectal cancer.

7. The antitumor composition according to claim 1, characterized in that: The anti-tumor composition uses 23-hydroxybetulinic acid and a PD-L1 inhibitor as active ingredients.

8. The antitumor composition according to claim 1, characterized in that: The anti-tumor composition further comprises a pharmaceutically acceptable excipient.

9. The antitumor composition according to claim 8, characterized in that: The auxiliary materials include one or more of preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, pH buffers, adhesives, fillers and lubricants.

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