Application of combination of BCL6 inhibitor and PD-L1 antibody in preparation of medicine for treating tumors

Abstract

The invention discloses application of a medicine composition in preparation of a medicine for treating tumors poor in response to PD-L1 antibody immunotherapy, and particularly relates to application of a BCL6 inhibitor FX1 combined with a PD-L1 antibody in preparation of a medicine for treating tumors with complete mismatch repair functions. The tumors comprise malignant tumors such as colorectal cancer, breast cancer, prostate cancer, ovarian cancer, gastric cancer, diffuse large B-cell lymphoma or lung cancer. The combined medication scheme of the BCL6 inhibitor FX1 and the PD-L1 antibody can be used for treating tumors which are complete in mismatch repair function and poor in response to PD-L1 antibody immunotherapy, and compared with single-drug treatment and combined medication, tumor growth is remarkably inhibited, so that tumor patients lack of sensitivity to PD-L1 antibody treatment can be benefited from the combined therapy.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of BCL6 inhibitors combined with PD-L1 antibodies in the preparation of drugs for treating tumors, and more specifically to the application of BCL6 inhibitor FX1 combined with PD-L1 antibodies in the preparation of drugs for treating tumors that do not respond well to PD-L1 antibody immunotherapy. Background Technology

[0002] Immune checkpoint blockade therapy is an emerging cancer treatment strategy in recent years. Significant breakthroughs have been achieved in cancer immunotherapy, represented by immune checkpoint programmed cell death protein 1 (PD-1) / programmed cell death ligand 1 (PD-L1) antibodies, which have become an important tool in clinical cancer treatment. The PD-1 / PD-L1 axis is a crucial mechanism for tumor immune escape. PD-1 is typically highly expressed in tumor-infiltrating T cells. PD-1 interacts with PD-L1 ligands on tumor cells or immune cells, inhibiting initial T cell activation and suppressing the production and function of effector T cells, including cytokine production and cytotoxicity. Blocking the PD-L1 / PD-1 axis can reactivate the anti-tumor immune response, and PD-1 / PD-L1 blocking antibodies have been approved for the treatment of various cancers. However, in clinical practice, most cancer patients do not show a good response.

[0003] Studies show that tumors with intact mismatch repair (pMMR) and deficient mismatch repair (dMMR) exhibit different responses to immunotherapy. dMMR / microsatellite highly unstable (MSI-H) tumors, due to their high mutational burden and generated neoantigens, respond well to PD-1 / PD-L1 inhibitors. Conversely, pMMR / microsatellite stable (MSS) tumors typically have less immune cell infiltration and respond poorly to immune checkpoint inhibitors. In cancer patients, the clinical response to PD-1 / PD-L1 immunotherapy is associated with the level of PD-L1 expression on tumor cell surfaces and the number of tumor-infiltrating lymphocytes. Altering PD-L1 expression on tumor cells and the number of tumor-infiltrating T cells, as well as eliminating tumor-associated myeloid cells (TAMCs)-mediated adaptive immune response deficiencies, provides new insights for immunotherapy in pMMR tumor patients. Therefore, enhancing the response rate of immunotherapy through combined use with other treatments such as chemotherapy, radiotherapy, and targeted therapy is of great significance for cancer patients with poor response to PD-1 / PD-L1 antibodies. Summary of the Invention

[0004] The purpose of this invention is to address the problem of poor response to PD-1 / PD-L1 antibody immunotherapy in cancer patients.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Application of a BCL6 inhibitor combined with a PD-L1 antibody in the preparation of a drug for treating tumors.

[0007] Furthermore, the BCL6 inhibitor is FX1, whose structure is shown in Formula I:

[0008]

[0009] Furthermore, the application of FX1 in upregulating PD-L1 expression in tumor cells.

[0010] Furthermore, BCL6 inhibitors, including peptide or natural product inhibitors, have the same effects as FX1.

[0011] Furthermore, the application of the BCL6 inhibitor combined with the PD-L1 antibody in regulating the tumor immune microenvironment.

[0012] Furthermore, the application of the BCL6 inhibitor in combination with the PD-L1 antibody in enhancing the response rate of immunotherapy.

[0013] A method for regulating the immune microenvironment of colorectal cancer in vivo by combining BCL6 inhibitors with PD-L1 antibodies: Combining with PD-L1 antibodies can increase T cell recruitment and reduce MDSC infiltration.

[0014] Furthermore, the application of the BCL6 inhibitor in combination with the PD-L1 antibody in increasing the recruitment of CD4-positive T cells.

[0015] Furthermore, the application of the BCL6 inhibitor in combination with the PD-L1 antibody in increasing the recruitment of CD8-positive T cells.

[0016] Furthermore, the BCL6 inhibitor combined with the PD-L1 antibody is used to reduce the infiltration of granulocyte-like MDSCs.

[0017] Furthermore, the BCL6 inhibitor combined with the PD-L1 antibody is used to reduce the infiltration of monocyte-like MDSCs.

[0018] Furthermore, the tumor is a malignant tumor.

[0019] Furthermore, the tumor is one that has not responded well to PD-L1 antibody immunotherapy.

[0020] Furthermore, the tumor is colorectal cancer.

[0021] Furthermore, the malignant tumors are tumors with intact mismatch repair function: colorectal cancer, breast cancer, prostate cancer, ovarian cancer, gastric cancer, diffuse large B-cell lymphoma, and lung cancer.

[0022] This invention also provides a method for upregulating PD-L1 expression in tumor cells in vitro using the FX1 compound. FX1 can upregulate the expression of the PD-L1 gene in colon cancer cells in a concentration-dependent manner using qPCR (real-time quantitative nucleic acid amplification detection system).

[0023] Compared with the prior art, the present invention, employing the above technical solution, has the following technical effects:

[0024] BCL6 inhibitors enhance the efficacy of PD-L1 antibody therapy for cancer, exhibiting the following effects both in vitro and in vivo:

[0025] First: Inhibition of BCL6 in colon cancer cell lines can activate PD-L1 expression;

[0026] Second: In a mouse subcutaneous tumor-bearing model of colon cancer, the combination with PD-L1 antibody significantly inhibited tumor growth and sensitized the immunotherapy of colon cancer;

[0027] Third: Combining with PD-L1 antibodies increases T cell recruitment in the tumor microenvironment and reduces MDSC infiltration.

[0028] This demonstrates the novel function of BCL6 inhibitors and the novel mechanism of the transcriptional repressor BCL6. Inhibition of BCL6 can activate PD-L1 expression, and the combination of BCL6 inhibitors and PD-L1 antibodies can synergistically inhibit tumor growth and enhance the efficacy of PD-L1, which is of great significance for the clinical treatment of tumor patients with poor PD-L1 antibody response. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The diagram shows that FX1 inhibition of BCL6 can promote PD-L1 expression. Figure 1 A, Figure 1 B and Figure 1 C represents the effect of formula (I)FX1 on upregulating PD-L1 expression in colon cancer cell lines HCT116, HT29, and CT26 after 24 h of treatment. In the figure, * indicates p < 0.05, and ** indicates p < 0.01.

[0031] Figure 2 The image shows the combination of FX1 and PD-L1 antibodies in vivo to inhibit colon cancer growth and enhance the sensitization of colon cancer immunotherapy. Among them, Figure 2 A indicates that the combination of FX1 and PD-L1 antibodies inhibits tumor growth in the CT26 subcutaneous tumor-bearing model of colon cancer, demonstrating a significant synergistic effect. Figure 2 B shows the tumor volume changes in mice treated with a combination of FX1 and PD-L1 antibodies. Figure 2 C shows the weight of the tumors at the end of the experiment, and the statistical graph of the tumor weight for each group. In the graph, ** indicates p < 0.01, and *** indicates p < 0.001.

[0032] Figure 3 The figure shown is a statistical diagram illustrating the regulation of the immune microenvironment in colorectal cancer by the combined use of FX1 and PD-L1 antibodies. Figure 3 A is that the combined use of FX1 and PD-L1 antibodies increases the number of CD4-positive T cells in the tumor; Figure 3 B is that the combined use of FX1 and PD-L1 antibodies increases the number of CD8-positive T cells in the tumor; Figure 3 C is that the combination of FX1 and PD-L1 antibodies reduces the infiltration of granulocyte-like MDSCs in tumors; Figure 3 D represents the reduction in tumor monocyte-like MDSC infiltration caused by the combined use of FX1 and PD-L1 antibodies. In the figure, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only 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.

[0034] Example 1: BCL6 inhibitors can promote the expression of PD-L1 in tumor cells.

[0035] Technical methods:

[0036] Quantitative Real-time PCR (qRT-PCR)

[0037] Quantitative real-time PCR (qRT-PCR) is a method for measuring the total amount of product after each polymerase chain reaction (PCR) cycle using fluorescent chemicals in DNA amplification reactions. In the PCR reaction system, an excess of SYBR fluorescent dye is added. SYBR dye non-specifically incorporates into the DNA double strand, emitting a fluorescent signal, while SYBR dye molecules not incorporated into the strand do not emit any fluorescent signal, thus ensuring that the increase in fluorescence signal is completely synchronized with the increase in PCR product. SYBR binds only to double-stranded DNA; the specificity of the PCR reaction can be determined by analyzing the melting curve. This invention uses qRT-PCR to examine the regulation of PD-L1 gene expression in colon cancer cells by FX1.

[0038] RNA extraction:

[0039] (1) Colon cancer cells HCT116, HT29 and CT26 were inoculated into 6cm dishes. After 24 hours, the cells adhered to the dish. FX1 drug (final concentration 25μM and 50μM) was added. After 24 hours of drug treatment, the cells were collected by centrifugation and washed twice with pre-cooled PBS.

[0040] (2) Add 1 mL of Trizol reagent and lyse the cells by pipetting. The sample can be stored at -80°C for several weeks.

[0041] (3) Centrifuge the lysed cells at 12000 rpm for 5 minutes, take the supernatant into a new 1.5 mL RNase-free EP tube, add 200 μL of chloroform, gently shake several times, let stand for 5 minutes, and then centrifuge at 12000 rpm at 4℃ for 15 minutes.

[0042] (4) Aspirate the supernatant into a clean EP tube free of RNase, add 500 μL of isopropanol, gently shake several times, and let stand for 10 minutes to precipitate RNA.

[0043] (5) Centrifuge the sample at 12000 rpm at 4℃ for 10 min, remove the supernatant as much as possible, add 1 mL of 75% ethanol (prepared with DEPC water) to wash the precipitate twice, air dry at room temperature, and then add an appropriate amount of DEPC water to dissolve the RNA precipitate.

[0044] (6) Determine RNA concentration and purity using a UV-Vis spectrophotometer.

[0045] qRT-PCR:

[0046] (1) Reverse transcription: mRNA is reversed to cDNA. The reaction system is 20 μL, including 15 μL of ddH2O, 4 μL of 5×Mixbuffer, and 1000 ng of RNA. The reaction conditions are: 37℃ for 30 min, 85℃ for 5 s, and stored at 16℃.

[0047] (2) qRT-PCR reaction: performed according to the Takara SYBR kit. The reaction system (25 mL) was as follows: 10 μL ddH2O, 12.5 μL 2×SYBR Mix buffer, 1 μL each of forward and reverse primers (10 μM), and 0.5 μL template cDNA.

[0048] The results are as follows Figure 1 As shown, FX1 treatment of colon cancer cells HCT116, HT29 and CT26 for 24 h upregulated PD-L1 expression in colon cancer cells in a concentration-dependent manner, indicating that inhibiting BCL6 can promote PD-L1 expression.

[0049] Example 2: Formula (I) FX1 combined with PD-L1 antibody synergistically inhibits the growth of colon cancer in mice.

[0050] Technical methods:

[0051] This experiment used the CT26 cell line derived from BALB / c mice, specifically 6-8 week old male BALB / c mice. The procedure is as follows:

[0052] 1. Large-scale expansion and culture of CT26 cells were carried out to ensure sufficient tumor-bearing experimental volume, using 1×10⁶ cells per mouse. 6 Cell quantity: To ensure a high success rate in tumor bearing, the cells must be in optimal condition.

[0053] 2. Once a sufficient number of cells have been collected, centrifuge to collect the cells, and wash twice with PBS to remove serum from the culture medium and reduce immune rejection. Transfer the cells to EP tubes and place them on ice to reduce cell metabolism.

[0054] 3. Press 1×10 6 100 μL per mouse was subcutaneously injected into the back of 6-8 week old BALB / c mice.

[0055] 4. Measure the length and width of the tumor approximately one week later, using the formula: Volume = Length × Width. 2 Calculate tumor volume by multiplying by 0.52. Wait until the tumor grows to 100 mm. 3At approximately 10:00 AM, mice were randomly divided into four groups: a blank control group, an FX1 monotherapy group (50 mg / kg / mouse), a PD-L1 antibody group (100 μg / mouse), and a combination therapy group (FX1 - 50 mg / kg / mouse, PD-L1 - 100 μg / mouse), with 7 mice per group. The length and width of the mouse tumors were measured and recorded every three days to calculate the volume. The administration frequency was FX1 once a day and PD-L1 antibody once every three days. After the experiment, the tumors were removed and photographed.

[0056] Experimental results are as follows Figure 2 As shown, after 15 consecutive days of administration, FX1 monotherapy could inhibit tumor growth, but the effect was not significant. The PD-L1 antibody group showed even weaker inhibitory effects on CT26 tumors, consistent with clinical findings. The combination of FX1 and PD-L1 antibody significantly inhibited tumor growth compared to the control group, and demonstrated better efficacy compared to both the PD-L1 monotherapy group and the FX1 monotherapy group. Figure 2 A). Figure 2 (B) is a graph showing the changes in tumor volume measured every 3 days. Figure 2 (C) is a tumor statistics chart, which shows that the combination of FX1 and PD-L1 antibody has a synergistic effect.

[0057] Example 3: Formula (I) FX1 and PD-L1 antibody are used in combination to synergistically regulate the immune microenvironment of colorectal cancer.

[0058] Technical methods:

[0059] In this experiment, single-cell suspensions prepared from tumor samples in Example 2 were used for flow cytometry analysis to detect the proportions of CD4-positive T cells, CD8-positive T cells, granulocyte-like MDSCs, and monocyte-like MDSCs within the tumor cells. The procedure is as follows:

[0060] (1) The mouse tumors excised in Example 2 were placed in a 70 μm cell strainer and ground with a syringe core. The cell suspension was collected by rinsing with MACS buffer (PBS + 2% FBS);

[0061] (2) Centrifuge at 1500 rpm for 5 min and discard the supernatant;

[0062] (3) Add 2 mL of red blood cell lysis buffer (ACKbuffer) to each spleen and process for 2 min;

[0063] (4) Add 4 times the volume of MACS buffer to terminate the lysis, filter with a 70 μm cell strainer to remove impurities, centrifuge at 1500 rpm for 5 min, and remove the supernatant.

[0064] (5) Resuspend cells in MACS buffer and perform cell counting;

[0065] (6) 10 6 The cells were resuspended in 100 μL of MACS buffer for T cell and MDSC staining. For CD4-positive T cell staining, 1 μL of APC-CD3 and FITC-CD4 antibodies were added and incubated at 4°C in the dark for 40 min; for CD8-positive T cell staining, 1 μL of APC-CD3 and PerCP / cy5.5-CD8 antibodies were added and incubated at 4°C in the dark for 40 min; for granulocyte-like MDSC staining, 1 μL of APC-CD11b and FITC-LY6G antibodies were added and incubated at 4°C in the dark for 40 min; for monocyte-like MDSC staining, 1 μL of APC-CD11b and PE-LY6C antibodies were added and incubated at 4°C in the dark for 40 min.

[0066] (7) Add MACS buffer, centrifuge at 1500 rpm for 5 min to wash the cells, resuspend the cells in 500 μL of MACS buffer and transfer them to flow cytometry tubes for detection.

[0067] Experimental results are as follows Figure 3 As shown, flow cytometry was used to detect changes in immune cells in the tumor microenvironment. We found that compared with the control group and the PD-L1 monotherapy group, the proportions of CD4-positive T cells and CD8-positive T cells were significantly increased in the combination therapy group. Figure 3 A and Figure 3 B), while the proportions of granulocyte-like MDSCs and monocyte-like MDSCs were both reduced ( Figure 3 C and Figure 3 D). The above results indicate that the combination of FX1 and PD-L1 regulates the infiltration of T cells and MDSCs in the colorectal cancer immune microenvironment, sensitizes PD-L1 immunotherapy, and BCL6 is a potential target for immunotherapy.

[0068] In summary:

[0069] FX1 can upregulate the expression of PD-L1 in colon cancer cells, specifically HCT116, HT29, and CT26.

[0070] In a mouse subcutaneous CT26 tumor-bearing model, FX1, when used in combination with PD-L1 antibody, showed a synergistic effect and significantly inhibited colon cancer growth.

[0071] The combined use of FX1 and PD-L1 antibodies can regulate the tumor immune microenvironment, increase T cell recruitment, and reduce MDSC infiltration.

[0072] FX1 is a small molecule inhibitor of BCL6 synthesis. Other BCL6 inhibitors, including peptide or natural product inhibitors, have the same effect as FX1.

[0073] FX1 can upregulate the expression of the PD-L1 gene in colon cancer cells in a concentration-dependent manner using qPCR (real-time quantitative nucleic acid amplification detection system).

[0074] FX1 can inhibit the growth of CT26 tumors in a mouse subcutaneous tumor-bearing animal model and enhance the immunotherapy of colon cancer.

[0075] The combined use of FX1 and PD-L1 antibody can increase T cell recruitment and reduce MDSC infiltration.

[0076] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. It should be noted that implementations not illustrated or described in the drawings or the main text of the specification are forms known to those skilled in the art and have not been described in detail.

[0077] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a BCL6 inhibitor combined with a PD-L1 antibody in the preparation of a drug for treating tumors.

2. The use of the BCL6 inhibitor combined with the PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The BCL6 inhibitor is FX1, and its structure is shown in Formula I:

3. The use of the BCL6 inhibitor combined with the PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The application of FX1 in upregulating the expression of PD-L1 in tumor cells.

4. The use of the BCL6 inhibitor combined with the PD-L1 antibody in the preparation of a drug for treating tumors according to claim 1, characterized in that: The BCL6 inhibitor is combined with the PD-L1 antibody to enhance the response rate of tumor immunotherapy.

5. The use of the BCL6 inhibitor combined with PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The use of the BCL6 inhibitor combined with the PD-L1 antibody in increasing the recruitment of CD4-positive T cells.

6. Use of the BCL6 inhibitor combined with PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The use of the BCL6 inhibitor combined with the PD-L1 antibody in increasing the recruitment of CD8-positive T cells.

7. Use of the BCL6 inhibitor combined with PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The BCL6 inhibitor is used in combination with the PD-L1 antibody to reduce the infiltration of granulocyte-like MDSCs.

8. Use of the BCL6 inhibitor combined with PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The BCL6 inhibitor is combined with the PD-L1 antibody to reduce the infiltration of monocytic MDSCs.

9. Use of the BCL6 inhibitor combined with PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The tumor is a tumor that does not respond well to PD-L1 antibody immunotherapy.

10. Use of the BCL6 inhibitor combined with PD-L1 antibody according to claim 1 in the preparation of a drug for treating tumors, characterized in that: The tumor is colorectal cancer.

Images