A pharmaceutical composition for anti-tumor

Abstract

This invention discloses a pharmaceutical composition for antitumor use, comprising pericidatinib and a compound for inhibiting PD-L1 activity. The invention's research found that the combination of pericidatinib and a PD-L1 antibody exhibits good inhibitory effects on various tumor cells and can be used to prepare antitumor drugs. Pericidatinib, as an antitumor drug, has already been applied clinically and has good application prospects. Furthermore, it can be used as an adjuvant drug to improve the therapeutic efficacy of PD-L1 antibody drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for anti-tumor purposes. Background Technology

[0002] After binding to its ligand, the immune checkpoint molecule PD-1 recruits SHP-2 tyrosine phosphatase, weakening the phosphorylation of the ZAP70 / CD3ζ signal transducer, thereby inhibiting the activation of the TCR signaling pathway ([1] Sheppard, KA, et al., PD-1 inhibits T-cell receptor induced phosphorylation of the ZAP70 / CD3zetasignalosome and downstream signaling to PKCtheta. FEBS Lett, 2004. 574(1-3): p. 37-41.). PD-1 has two ligands, PD-L1 and PD-L2. PD-L1 can be expressed on various types of tumor cells. In addition, tumor-infiltrating lymphocytes can also promote the expression of PD-L1 in tumor cells by releasing interferon-γ ([2] Dong, H., et al., Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion. Nat Med, 2002. 8(8): p. 793-800. [3] Taube, JM, et al., Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immuneescape. Sci Transl Med, 2012. 4(127): p. 127ra37.). Currently, clinical immune checkpoint inhibitors include neutralizing antibodies against PD-1 and PD-L1 (αPD-1 and αPD-L1), which can effectively block PD-1-mediated inhibitory signals, promote the normalization of T cell function, and enhance the body's anti-tumor immunity. Although αPD-1 and αPD-L1 have achieved great success in clinical practice, they are only effective in 10-30% of cancer patients ([4] Page, DB, et al., Immunemodulation in cancer with antibodies. Annu Rev Med, 2014. 65: p. 185-202.).Primary and secondary resistance are the main reasons for the failure of αPD-1 and αPD-L1 treatment ([5] Sharma, P., et al., Primary, Adaptive, and Acquired Resistance to Cancer Immunotherapy. Cell, 2017. 168(4): p. 707-723. [6] Zaretsky, JM, et al., Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med, 2016. 375(9): p. 819-29.). Therefore, elucidating the mechanism of αPD-1 and αPD-L1 treatment resistance and formulating new and effective strategies to improve the response rate and efficacy of αPD-1 and αPD-L1 treatment remains a research hotspot in the field of tumor treatment.

[0003] Extracellular vesicles (EVs) are nanoscale vesicles with lipid bilayer membrane structures released by various cells. Since EVs carry proteins, nucleic acids, lipids and metabolites from the maternal cell, they participate in the communication between different cells ([7] Chen, J., et al., Tumor-derived extracellular vesicles: Regulators of tumor microenvironment and the enlightenment in tumor therapy. Pharmacol Res, 2020. 159: p. 105041. [8] Kalluri, R. and VS LeBleu, The biology, function, and biomedical applications of exosomes. Science, 2020. 367(6478).). Numerous studies have shown that tumor-derived extracellular vesicles (TEVs) contain a large amount of membrane PD-L1, which can suppress the body's systemic anti-tumor immunity ([9] Chen, G., et al., Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature, 2018. 560(7718): p. 382-386.

[10] Poggio, M., et al., Suppression of Exosomal PD-L1 Induces Systemic Anti-tumor Immunity and Memory. Cell, 2019. 177(2): p. 414-427. e13.). More importantly, the membrane PD-L1 of TEVs is associated with αPD-L1 therapeutic resistance. Inhibiting the secretion of TEVs in mouse MC38 colon cancer and mouse 4T1 breast cancer significantly enhances the antitumor effect of αPD-L1 (

[11] Yang, Y., et al., Exosomal PD-L1 harbors active defense function to suppress T cell killing of breast cancer cells and promote tumor growth. Cell Res, 2018.28(8):p.862-864.).Therefore, TEVs play an important role in the development and treatment of tumors. How to improve the therapeutic effect by changing the level of TEVs and increasing the duration of drug residence in the body has always been a difficult and hot topic in clinical research.

[0004] Pexidartinib (PLX3397) is a tyrosine kinase inhibitor developed by Daiichi Sankyo Co., Ltd. for the treatment of adult tenosynovial giant cell tumor (TGCT). On August 2, 2019, the FDA approved pexidartinib for the systemic treatment of TGCT patients with severe functional limitations who are not suitable for surgical improvement (

[12] FDA. FDA approves pexidartinib fortenosynovial giant cell tumor [EB / OL]. [2019-08-08].). TGCT is a rare tumor associated with overexpression of colony-stimulating factor-1 receptor (CSF-1R), which promotes the proliferation and accumulation of cells in the synovium. Percidatinib selectively inhibits the internal tandem repeats of CSF-1R, c-KIT proto-oncogene receptor tyrosine kinase, and Fms-like tyrosine kinase-3 genes, thereby inhibiting tumor cell proliferation (

[13] Tap WD, Gelderblom H, Stacchiotti S, et al. Final results of ENLIVEN: aglobal, double-blind, randomized, placebo-controlled, phase 3 study of pexidartinib in advanced tenosynovial giant cell tumor (TGCT)[J]. 2018, 36(15_suppl):11502.). Percidatinib was the first systemic therapy drug approved for TGCT patients, but its application in other tumors and its combination therapy with other drugs have not been reported. Therefore, finding new treatment strategies for percidatinib and the possibility of combining it with other drugs can provide new ideas for the treatment of other tumors. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a pharmaceutical composition for anti-tumor purposes, which, by combining pericidatinib with a PD-L1 antibody, exhibits good inhibitory effects against a variety of tumors.

[0006] This invention first provides a pharmaceutical composition for antitumor purposes, comprising pericidatinib and a compound for inhibiting PD-L1 activity. Specifically, the pharmaceutical composition targets prostate cancer or colon cancer.

[0007] The compound used to inhibit PD-L1 activity can be any pharmaceutically viable compound that has PD-L1 activity inhibitory function. Preferably, the compound used to inhibit PD-L1 activity is a PD-L1 antibody drug. For example, PD-L1 antibody drugs include atezolizumab (Roche), sugemalimab (CStone Pharmaceuticals), envorimab (Simcere Pharmaceuticals / Kangning Biopharmaceuticals / 3D Pharmaceuticals), or durvalumab (AstraZeneca).

[0008] This invention also provides the application of pericidatinib in combination with a compound for inhibiting PD-L1 activity in the preparation of an antitumor drug. Specifically, the target tumor type is prostate cancer or colon cancer. The compound for inhibiting PD-L1 activity can be any pharmaceutically viable compound that has PD-L1 activity inhibitory function. Preferably, the compound for inhibiting PD-L1 activity is a PD-L1 antibody drug. For example, PD-L1 antibody drugs include atezolizumab (Roche), sugemalimab (CStone Pharmaceuticals), envorimab (Simcere Pharmaceuticals / Kangning Biopharmaceuticals / 3D Pharmaceuticals), or durvalumab (AstraZeneca).

[0009] This invention found in in vitro and in vivo experiments that TEVs can promote the phagocytosis and degradation of PD-L1 antibodies by macrophages, while pericidatinib can effectively inhibit the proportion of macrophages in the liver and peripheral blood of tumor-bearing mice. To investigate the in vivo anti-tumor effect of this drug, two tumor models (prostate cancer and colon cancer) were used. It was found that while pericidatinib alone had a certain inhibitory effect on tumor growth in tumor-bearing mouse models, the difference was not significant. However, when used in combination with PD-L1 antibodies, it greatly enhanced the therapeutic effect of PD-L1 antibodies and also significantly inhibited prostate cancer that was previously insensitive to PD-L1 antibody treatment. Further analysis using proximity-linked assays (PLA) revealed that in tumor-bearing mouse models, compared with the PD-L1 antibody monotherapy group, the proportion of antibody-bound tumor cells in tumor tissue was significantly increased in the group treated with pericidatinib in combination with PD-L1 antibodies.

[0010] This invention has shown that the combination of pericidatinib and PD-L1 antibody has a good inhibitory effect on various tumor cells and can be used to prepare anti-tumor drugs. Pericidatinib has already been used clinically as an anti-tumor drug and has a promising application prospect. Moreover, it can be used as an adjuvant drug to improve the therapeutic effect of PD-L1 antibody drugs. Attached Figure Description

[0011] Figure 1The figure shows the results of TEVs promoting the phagocytosis and degradation of PD-L1 antibodies by macrophages. In figure a, TEVs affect the phagocytosis of PD-L1 antibodies by macrophages in vivo; in figure b, TEVs affect the colocalization of PD-L1 antibodies with lysosomes in macrophages.

[0012] Figure 2 The image shows the results of detecting the macrophage deletion effect of pericidatinib in mice.

[0013] Figure 3 The figure shows the results of detecting the effect of combined treatment with pecildatinib and PD-L1 antibody in a tumor-bearing mouse model. Figure a shows the effect of combined treatment with pecildatinib and PD-L1 antibody on the change in tumor volume of MC38 tumors; figure b shows the effect of combined treatment with pecildatinib and PD-L1 antibody on the change in tumor volume of TRAMP-C2 tumors.

[0014] Figure 4 The image shows the results of detecting the binding of PD-L1 antibodies to tumor tissue and TEVs after treatment with pericidatinib. Figure a shows the changes in the binding of EVs-TT to PD-L1 antibodies after combined treatment with pericidatinib and PD-L1 antibodies; figure b shows the changes in the binding of PD-L1 antibodies to tumor cells on tumor tissue after combined treatment with pericidatinib and PD-L1 antibodies. Detailed Implementation

[0015] Male C57BL / 6 (6-8 weeks old) mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd., and all mice were bred in SPF-grade facilities. The mouse colon cancer cell line MC38 and prostate cancer cell line TRAMP-C2 were purchased from the American Type Culture Collection (ATCC).

[0016] Example 1

[0017] The effects of TEVs on the distribution and degradation of PD-L1 antibodies are detailed below:

[0018] (1) Preparation of cellular EVs

[0019] Fetal bovine serum (FBS) was centrifuged at 120,000 g for 10 h or overnight to remove interference from EVs in subsequent cell culture supernatants. FBS was then added to DMEM medium at a final concentration of 10% (v / v) to culture cells. After experimental manipulation, MC38 and TRAMP-C2 cells were grown to approximately 90% confluence. The supernatant was collected and centrifuged at 4 °C using differential centrifugation: 300 g for 10 min, 2,000 g for 10 min to remove cell debris and apoptotic bodies, and 10,000 g for 30 min to remove microvesicles. Next, the supernatant from high-speed centrifugation was filtered through a 0.22 μm PVDF filter and then centrifuged at 120,000 g for 70 min at 4 °C using an SW 32Ti horizontal rotor to deposit EVs. The preliminarily obtained EVs were resuspended in pre-chilled PBS and washed again by centrifugation at 120,000 g for 70 min. Finally, the precipitated EVs were resuspended in an appropriate amount of sterile PBS, aliquoted, and stored at -80°C. The protein concentration of the EVs was measured using the BCA protein assay kit according to the kit instructions.

[0020] (2) Flow cytometry analysis of the effect of TEVs on phagocytosis of PD-L1 antibody by mouse macrophages

[0021] Normal C57BL / 6 mice were injected intravenously with 10 μg of PD-L1 antibody (BioLegend, Cat#124302) labeled with Alexa Fluor 680 (Thermo Fisher Scientific, Cat#A20173). Simultaneously, another group of mice were injected intravenously with 20 μg of MC38-EVs. Peripheral blood, liver, and spleen tissues were collected from the mice to be tested 24 h later. The liver and spleen tissues were removed, minced, resuspended in serum-free 1640 medium, and incubated at 2 mg / ml on a 360° shaker. -1 Type I collagenase, 2mg / ml -1 Type IV collagenase, 0.2 mg / ml -1Tissue blocks were digested with DNase I, and the digestion process was terminated 1 hour later with medium containing 10% FBS (v / v). The digested tissue solution was filtered through a 200-mesh sieve to obtain a single-cell suspension. Peripheral blood and tissue digested cell suspensions were treated with erythrocyte lysis buffer to remove erythrocytes, followed by Fixed Viability Dye staining to eliminate the influence of dead cells on the experimental results. The suspensions were then incubated with membrane surface antibodies (anti-CD45 (BioLegend, Cat#103126), anti-CD11b (BioLegend, Cat#101207), anti-F4 / 80 (Invitrogen, Cat#MF48004-3)) at 4°C for 30 min. After washing with PBS, the samples were directly analyzed using flow cytometry. Samples were collected on a CytoFlex or Novocyte flow cytometer and analyzed using FlowJo software.

[0022] (3) TEVs promote the degradation of PD-L1 antibodies by mouse macrophages

[0023] Normal C57BL / 6 mouse peritoneal macrophages were harvested, treated with erythrocyte lysis buffer, and seeded into 24-well plates with small round glass slides. After culturing for 24 h, labeled αPD-L1 or TEV-bound αPD-L1 was added and co-incubated for 2 h. After washing three times with PBS, the cells were fixed with pre-chilled methanol at -20°C for 10 min, followed by infiltration with 0.1% Triton X-100 at room temperature for 10 min. After blocking with 5% BSA (v / v) and 3% goat serum (v / v) in PBS, the cells were incubated overnight with LAMP1 antibody (Abcam, Cat#ab208943) in blocking buffer at 4°C. The next day, after washing three times with PBS, the cells were incubated with anti-mouse IgG DyLight 488 (Multi Sciences Biotech, Cat#70-GAM4882) at room temperature for 30 min, and then washed with PBS. Finally, the cell nuclei were stained with DAPI (Thermo Fisher Scientific, Cat#S36973). The stained sections were observed using an Olympus IX83-FV3000 confocal microscope (Olympus).

[0024] Experimental results showed that MC38-EVs significantly increased the F4 / 80 ratio of peripheral blood monocytes, liver, and spleen. + Phagocytosis of αPD-L1 by macrophages ( Figure 1 .a). Simultaneously, immunofluorescence results showed that the PD-L1 antibody binding to MC38-EVs had greater co-localization with lysosomes ( ). Figure 1(b) These results indicate that TEVs can promote the phagocytosis and degradation of PD-L1 antibodies by macrophages, and the deletion of macrophages may increase the duration of antibody residence in vivo, thereby improving efficacy.

[0025] Example 2

[0026] Detection of the macrophage deletion effect of percidatinib in mice. To investigate the effect of percidatinib on macrophages in vivo, male SPF grade C57BL / 6 mice were used as experimental subjects. Mice were intraperitoneally injected with 20 μg of PLX3397 (MedChemExpress, Cat#HY-16749), once every 2 days, for a total of 3 injections. 24 hours after the last injection, monocytes in the blood and macrophages in the liver were analyzed by flow cytometry (the tissue cell extraction procedure was the same as in Example 1). Afterwards, Fixed Viability Dye staining was used to eliminate the influence of dead cells on the experimental results. The cells were then incubated with membrane surface antibodies (anti-CD45 (BioLegend, Cat#103126), anti-CD11b (BioLegend, Cat#101207), anti-F4 / 80 (Invitrogen, Cat#MF48004-3)) at 4°C for 30 min, washed with PBS, and directly analyzed by flow cytometry. Samples were collected on a CytoFlex or Novocyte flow cytometer and analyzed using FlowJo software.

[0027] Experimental results showed that PLX3397 significantly reduced the number of mononuclear macrophages in peripheral blood and liver of mice. Figure 2 This suggests that PLX3397 may be used in combination with PD-L1 antibodies to reduce the phagocytosis and degradation of PD-L1 antibodies by deleting macrophages.

[0028] Example 3

[0029] Percidatinib inhibits tumor growth mainly by selectively inhibiting the internal tandem repeats of CSF-1R, c-KIT proto-oncogene receptor tyrosine kinase, and Fms-like tyrosine kinase-3 genes. Meanwhile, PD-L1 antibodies can specifically block the binding of PD-1 to PD-L1, terminating the PD-1 immunosuppressive signaling caused by the interaction between PD-1 and PD-L1 in T cells.

[0030] Our study found that percidatinib can reduce the number of macrophages in mice. To verify whether the combination therapy of percidatinib and PD-L1 antibody can improve the anti-tumor efficacy, colon cancer cells (MC38) and prostate cancer cells (TRAMP-C2) were subcutaneously inoculated into C57BL / 6 mice. When the tumor volume reached 100 mmHg...3 Each patient was randomly divided into four groups. The treatment groups received PLX3397, PD-L1 antibody, or a combination of PLX3397 and PD-L1 antibody, respectively. The control groups received either water for injection or normal saline. PLX3397 was administered intraperitoneally every two days at a dose of 1 mg / kg. -1 PD-L1 antibody administered via tail vein injection, once every two days, at a dose of 0.5 mg / kg. -1 Record the tumor volume each time.

[0031] The results are as follows Figure 3 As shown, PD-L1 antibody significantly inhibited tumor growth in MC38 tumor-bearing mice. While PLX3397 monotherapy showed some inhibitory effect on tumor growth, it was not significant. However, the combined treatment of both was significantly superior to PD-L1 monotherapy. Figure 3 .a). Furthermore, the study found that TRAMP-C2 tumors are resistant to PD-L1 therapy, but combination therapy with PLX3397 and a PD-L1 antibody can effectively reverse this resistance. Figure 3 .b).

[0032] Example 4

[0033] This study investigated the binding of PD-L1 antibodies to tumor tissue and TEVs after percidatinib treatment in tumor-bearing mice. To further explore the binding of PD-L1 antibodies to TEVs and changes in PD-L1 antibody binding to tumor cells after macrophage deletion with percidatinib, tumors were extracted from MC38 tumor-bearing mice treated with different methods. EVs derived from the tumor tissue (EVs-TT) were extracted, and the binding of PD-L1 antibodies on EVs-TT was detected by flow cytometry. Changes in PD-L1 antibody binding in tumor cells within the tumor tissue were analyzed using PLA. The specific steps are as follows:

[0034] (1) Extraction of EVs-TT and flow cytometry detection of its binding with PD-L1 antibody

[0035] Tumor tissue was removed and minced, resuspended in serum-free 1640 medium, and then incubated on a 360° shaker at 2 mg / ml. -1 Type I collagenase, 2mg / ml -1 Type IV collagenase, 0.2 mg / ml -1Tissue blocks were digested with DNase I, and the digestion process was terminated 1 hour later with culture medium containing 10% FBS (v / v). The digested tissue solution was filtered through a 200-mesh filter to obtain a single-cell suspension. The suspension was then centrifuged at 4°C using differential centrifugation: 300g for 10 min, 2,000g for 10 min to remove cell debris and apoptotic bodies, and 10,000g for 30 min to remove microvesicles. Next, the supernatant from the high-speed centrifugation was filtered through a 0.22 μm PVDF filter and then ultracentrifuged at 120,000g for 70 min at 4°C using an SW 32Ti horizontal rotor to deposit EVs. The preliminarily obtained EVs were resuspended in pre-chilled PBS and ultracentrifuged again at 120,000g for 70 min to wash the sample. Finally, the precipitated EVs were resuspended in an appropriate amount of sterile PBS, and the protein concentration of the EVs was measured using a BCA protein assay kit.

[0036] 5 μg of EVs-TT was incubated with 4 μm latex microparticles at room temperature for 30 min. Then, EV-free FBS was added for blocking at room temperature for 30 min. Next, 1 ml of PBS was added, and the sample was washed twice at 4°C, 3500 × g, for 5 min. The pellet was resuspended in 100 μL of PBS, and anti-mouse IgG DyLight 488 was added and incubated at room temperature for 30 min. After washing with PBS, the sample was directly analyzed. Samples were collected using a CytoFlex or Novocyte flow cytometer and analyzed using FlowJo software.

[0037] (2) Detection of PLA binding to PD-L1 antibody in tumor tissue

[0038] Mouse tumor tissue sections underwent routine dewaxing and hydration, followed by antigen retrieval using 10 mM sodium citrate buffer (pH 6.0). After blocking with 1× blocking solution at 37°C for 1 h, the sections were incubated overnight at 4°C with αRat IgG2a (BioLegend, Cat#407502) and rabbit αPD-L1 (Abclonal, Cat#A1645). The next day, secondary antibody incubation, ligation, and amplification were performed according to the instructions of the Duolink In Situ PLA Detection Kit (Sigma–Aldrich, Cat#DUO92005, DUO92001). Finally, cell nuclei were stained with DAPI. The stained sections were observed using an Olympus IX83-FV3000 confocal microscope (Olympus). Positive staining areas were analyzed using ImageJ software.

[0039] The results showed that the amount of PD-L1 antibody bound to EVs-TT in tumor-bearing mice treated with a combination of PLX3397 and PD-L1 antibody was significantly lower than that in the treatment alone group. Figure 4 a). The proportion of tumor cells binding to PD-L1 antibodies in tumor tissue was significantly increased ( ). Figure 4 (b) These results further confirm that PLX3397 can increase the residence time of PD-L1 antibodies in vivo by deleting macrophages, thereby better exerting its anti-tumor effect. They also suggest the potential application of PLX3397 in combination with PD-L1 antibodies in the treatment of various tumors.

Claims

1. The application of the combination of pericidatinib and PD-L1 antibody drugs in the preparation of anti-tumor drugs, targeting prostate cancer.

2. Use according to claim 1, characterized in that, PD-L1 antibody drugs include atezolizumab, sugemalimab, envorimab, or durvalumab.

Images