Application of celecoxib in preparation of tumor immunotherapy medicine

Abstract

The invention relates to the technical field of biological medicines, in particular to application of celecoxib in preparation of a tumor immunotherapy drug, and provides a mechanism that the celecoxib promotes PD-L1 instability through deglycosylation so as to enhance immune response. The invention aims to provide the application of the celecoxib in preparation of the tumor immunotherapy medicine, which proves that the celecoxib promotes PD-L1 instability, improves immune response and realizes in-vitro and survival synergistic anti-tumor effect through deglycosylation, and provides a new treatment method and a solid theoretical basis for combined immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the use of celecoxib in the preparation of tumor immunotherapy drugs. The invention also proposes a mechanism by which celecoxib promotes PD-L1 instability through deglycosylation to enhance immune response. Background Art

[0002] In recent years, the development of cancer immunotherapy has partially improved the prognosis of patients with non-small cell lung cancer (NSCLC) by activating immune cells to enhance anti-tumor immune responses. Programmed cell death-1 (PD-1) is a T-cell co-inhibitory receptor with ligand specificity for PD-ligand 1 (PD-L1) and PD-L2. Programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) are well-known immune checkpoint inhibitors. When PD-1 binds to PD-L1, it inhibits T-cell activation, immune tolerance, and tumor proliferation. Immune checkpoint inhibitors can competitively bind to PD-1 or PD-L1, thereby overcoming tumor immune escape, reawakening immune cells, and eliminating cancer cells. Due to its low toxicity and significant therapeutic effects, tumor immunotherapy has gradually become one of the main anti-tumor treatments and is considered the fourth treatment option after surgery, radiotherapy, and chemotherapy. Although immunotherapy has made breakthrough progress in many tumors, the efficacy of immunotherapy alone is only about 20%, so combined treatment strategies based on immunotherapy have been studied in a variety of tumors.

[0003] Currently, several immunotherapy combination strategies are in clinical trials. Combining immunotherapy with chemotherapy has demonstrated synergistic effects by influencing the tumor microenvironment and activating anti-tumor immune responses. Similarly, combining immunotherapy with radiotherapy can synergistically combat tumors by promoting the release of tumor-associated antigens, maturation of dendritic cells, and expression of MHC1. Radiotherapy also produces a variety of chemokines that can improve the tumor's immunosuppressive microenvironment and enhance T cell immune responses. Combining immunotherapy with anti-angiogenic drugs and tyrosine kinase inhibitors has also demonstrated synergistic effects. Anti-angiogenic drugs enhance antigen presentation by dendritic cells, promoting T cell activation. Simultaneously, they normalize tumor vasculature, promoting T cell trafficking and infiltration into the tumor area, leading to an increased immune response within the tumor microenvironment. Furthermore, combining immunotherapy with other immunotherapies has demonstrated potent tumor-killing effects with specific mechanisms. CTLA-4 is highly expressed on Treg cells and activated T cells. Antibodies to CTLA-4 can block T cell inhibitory signals, promoting rapid T cell proliferation and rapidly initiating an immune response. PD-L1 antibodies block the binding of PD-1 to tumor cells in the tumor microenvironment, reversing tumor immune escape and reactivating T cell recognition function. A comprehensive understanding of the interactions between targeted drugs with different mechanisms and immunotherapy drugs is crucial for the development and integration of immunotherapy-based treatment strategies, which will help explore the optimal treatment approach and provide long-term clinical benefits for cancer patients. Summary of the Invention

[0004] The purpose of the present invention is to provide a use of celecoxib in the preparation of tumor immunotherapy drugs, confirm that celecoxib promotes PD-L1 instability through deglycosylation, improves immune response, achieves synergistic anti-tumor effects in vitro and in vivo, and provides a new treatment method and a solid theoretical basis for combined immunotherapy.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention discloses an application of celecoxib in the preparation of a tumor immunotherapy drug.

[0007] The celecoxib specifically inhibits cyclooxygenase-2 (COX-2), and inhibits the production of inflammatory prostaglandins by inhibiting cyclooxygenase-2 (COX-2), thereby achieving anti-inflammatory, analgesic and antipyretic effects; COX2 expression is related to the occurrence and development of various malignant tumors; PD-L1 is a transmembrane protein, and TNF, IFN-r, IL-6, IL-10 and granulocyte-stimulating factor can upregulate the expression of PD-L1 in different cells; post-translational modification PTMs of PD-L1 is an important regulatory mechanism, which regulates the immunosuppressive function of tumor patients through glycosylation, phosphorylation, ubiquitination, ubiquitination and acetylation. Deglycosylated PD-L1 increases instability and shortens half-life. Cancer cells with glycosylated PD-L1 are resistant to T cell killing, while cancer cells with deglycosylated PD-L1 are very sensitive to T cell killing.

[0008] By detecting the expression level of cyclooxygenase-2 (COX-2) in patients, it can be used as a biomarker for evaluating the efficacy and prognosis of tumor immunotherapy, or as a tumor resistance marker, and used to construct a tumor immunotherapy efficacy and prognosis evaluation model, or to construct a tumor resistance evaluation model.

[0009] The tumors include but are not limited to esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumor, melanoma, and breast cancer.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] Celecoxib specifically inhibits cyclooxygenase-2 (COX-2), suppressing the production of inflammatory prostaglandins by inhibiting COX-2, resulting in anti-inflammatory, analgesic, and antipyretic effects. COX2 expression is associated with the development and progression of various malignancies. PD-L1 is a transmembrane protein. We have found that TNF, IFN-γ, IL-6, IL-10, and granulocyte-stimulating factor can upregulate PD-L1 expression in various cell types. Post-translational modifications (PTMs) of PD-L1 are an important regulatory mechanism, primarily modulating the immunosuppressive function of cancer patients through glycosylation, phosphorylation, ubiquitination, ubiquitinylation, and acetylation. Previous studies have shown that glycosylated PD-L1 is the most stable form, while deglycosylated PD-L1 increases instability and shortens its half-life. Cancer cells expressing glycosylated PD-L1 are resistant to T cell cytotoxicity, whereas cancer cells expressing deglycosylated PD-L1 are highly sensitive to T cell cytotoxicity.

[0012] In the present invention, we demonstrated that celecoxib promotes PD-L1 instability through deglycosylation, enhances immune response, and achieves synergistic anti-tumor effects in vitro and in vivo, providing a new treatment method and a solid theoretical basis for combined immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The figure shows the effect of different concentrations of CCB on PD-L1 expression in lung cancer cell lines H460 and PC9 detected by WB.

[0014] Figure 2 Schematic diagram of glycosylation detection showing that CCB treatment leads to partial deglycosylation of PD-L1.

[0015] Figure 3 Schematic diagram of WB detection of COX2 and PD-L1 expression levels after DMSO and CCB treatment.

[0016] Figure 4 Schematic diagram of H460 and PC9 cells treated with CCB for 4, 8, 12, and 24 hours. DETAILED DESCRIPTION

[0017] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0018] Cell culture

[0019] Cell culture and treatment

[0020] All cell lines used were obtained from the American Type Culture Collection (Manassas, VA, USA) and independently confirmed by STR DNA fingerprinting at the MD Anderson Cancer Center in Texas and tested negative for mycoplasma. Human breast cancer cell lines and mouse cancer cell lines were cultured in DMEM / F12, DMEM, and MEM, respectively, supplemented with 10% fetal bovine serum and 1% antibiotic cocktail. Human lung cancer cell lines and RKO were cultured in RPMI-1640 and MEM, respectively, supplemented with 10% fetal bovine serum and 1% antibiotic cocktail. Metformin was added to complete culture medium at the indicated concentrations and times.

[0021] plasmids

[0022] As previously described, we constructed a PD-L1 / Flag-hPD-L1 binary vector to downregulate endogenous PD-L1 expression (Li et al., 2016). Briefly, pGIPZ-sh(h)PD-L1 clone #5 (30-UTR region (h)PD-L1, Thermo Scientific, Pennsylvania) and (h)-PD-L1WT (shRNA / ORF Core Facility MD Anderson Cancer Center, Houston, TX, USA) were generated by deleting the GFP sequence from pGIPZ shPD-L1 clone #5 by digestion with Cla1 and Nhe1 restriction enzymes and inserting Flag-(h)PD-L1WT. pGIPZ-sh(h)PD-L1-P1-195A, S195D were obtained by direct mutagenesis.

[0023] pGIPZ-sh(h)PD-L1-S195D and 4NQ. pGIPZ-sh(m)PD-L1 clone #3 (Thermo Scientific, Pittsburgh, USA) and Flag-PD-L1WT (shRNA / ORF Core Facility, MD Anderson Cancer Center, USA) were used to construct pGIPZ-sh(m)PD-L1-(m)PD-L1 and served as templates for pGIPZ-sh(m)PD-L1 / Flag-(m)PD-L1 S194A and S194E. pPET21a His-PD-L1WT was constructed by inserting the extracellular domain of PD-L1 (aa 19238) after digestion with NDEE1 and Xho1. pPET21a His-PD-L1 S195A was generated by site-directed mutagenesis of pPET21a His-PD-L1 WT. All plasmids were confirmed by restriction digestion and DNA sequencing.

[0024] Generation of PD-L1 stable and knockout (KO) cells

[0025] Using the pGIPZ-shPD-L1 / Flag-PD-L1 dual expression construct, we established MDA-MB-231 and BT-549 stable cell lines expressing Flag-PD-L1 WT, S195A, S195E, or 4NQ mutants, with endogenous PD-L1 knockout. Human PD-L1 KO MDA-MB-231 cells were generated using the Pdcd-L1KO MDA-MB-231 vector (Pdcd-1L1 CRISPR / sc-401140, Santa Cruz, Dallas, USA). To generate AMPKa KO MDA-MB231, we used the pLentiCRISPRv2 vector to target three different regions of AMPKa 1 (NM_006251.5) and AMPKa 2 (NM_006252.3), respectively. The target sequences are as follows:

[0026] AMPKA1-1 (5 to 27); AMPKA1-2 (4008 to 4030); AMPKA1-3 (1325 to 1347) AMPKA2-1

[0027] (79 to 101)AMPKAA2-2(117 to 139)AMPKa2-3(1647 to 1669).

[0028] To package lentivirus, two packaging plasmids (VSV-G and deltaKO dual expression or LentiCRISPR) were transfected into HEK293T cells. The medium was changed 24 hours after transfection, and the supernatant was collected twice every 24 hours. The supernatant after centrifugation was filtered through a 0.45 mm filter. MDA-MB-231 and BT-549 cells (30% confluence) were added to the polyvinylpyrrolidone lentivirus medium (10 mg / mL; EMD Millipore, MA, USA). After 48 hours of infection, the cells were subjected to puromycin selection.

[0029] s.1. Transfection of siRNA and expression vector

[0030] Commercial siRNAs were used to knock down SEL1L (#1SASI_Hs01_00233054 and #2SASI_Hs01_00233056; Sigma-Aldrich), ERLEC1 (#1SASI_Hs02_00329444 and #2SASI_Hs02_00329447; Sigma-Aldrich), OS9 (#1SASI_Hs01_00097558 and #2SASI_Hs01_00097559; Sigma-Aldrich), and ERLEC1 (#1SASI_Hs02_00329444 and #2SASI_Hs02_00329447; Sigma-Aldrich).

[0031] SASI_Hs01_00097559, Sigma-Aldrich) and HRD1 (SYVN1) (Signal Silencing SYVN1 siRNA I#13204; Cell Signaling). siRNAs were transfected into MDA-MB-231 naive cells and MDA-MB-231 PD-L1 / S195E stable cells using an electroporator (Nuclear Vector II; Amaxa Biosystems) according to the manufacturer's instructions. pCMVHA-ubiquitin (WT) and pCMVHA-ubiquitin (K48R) were transfected into MDA-MB-231 PD-L1 WT, S195A, S195E, or mock stable cells (15338100; Life Technologies, Grand Island, NY, USA).

[0032] Deglycosylation assay

[0033] A431 cells were lysed with ice-cold BlastR lysis buffer containing NEM, TSA, Na₃VO₄, and protease inhibitors. Lysates were subjected to IP using ubiquitin affinity beads (UBA01) or control beads (CUB02). Elution was performed with alternative elution buffer (50 mM Tris pH 8.0, 0.2% SDS, 0.1% Tween-20) for 15 minutes at 25°C. Immunoprecipitation and WCL samples were deglycosylated using PNGase F from NEB (NEB, MA) with the following modifications: 0.2 μl of PNGase F per reaction, incubation time at 37°C for 30 minutes. In addition, additional NP-40 was used during the SDS neutralization step to account for the additional 0.2% SDS in the elution step. Finally, samples were run on SDS-PAGE, transferred to PVDF, and analyzed by Western immunoblotting.

[0034] Glycosidase deglycosylation assay

[0035] Cells were lysed in RIPA buffer (150 mM sodium chloride, 50 mM Tris [pH 7.5], 1% Nonidet P-40, and a protease inhibitor cocktail) and sonicated using a vibrating cell sonicator. PNGase F (NEB Inc., P0704) treatment was performed according to the manufacturer's instructions with slight modifications. 5-20 mg of cell lysate was combined with 1 ml of 103 glycoprotein denaturation buffer and water to create a total reaction volume of 10 ml. The mixture was heated at 100°C for 10 minutes, chilled on ice, and 2 ml of 103 glycoprotein buffer 2, 2 ml of 10% Nonidet P-40, and 6 ml of water were added to create a total reaction volume of 20 ml. The denaturation mixture was incubated overnight at 37°C without or with 1 ml of PNGase F to maintain a final glycerol concentration of 5%, and subjected to IB analysis with the indicated antibodies.

[0036] COX2 knockout

[0037] Step 1: Cas9 stable cell line

[0038] Follow our laboratory's protocol and select an appropriate concentration of antibiotics for the Cas9 plasmid before virus production.

[0039] Step 2 - sgRNA (target gene)

[0040] Follow the instructions for 2000 or 3000. After that, seed the cells into a 96-well plate to select individual clones for verification and selection of qualified clones. (For a 96-well plate, only 80-100 cells need to be seeded).

[0041] Construction of PD-L1 mutant plasmid

[0042] The plasmid is 3*Flag-CMV-PD-L1

[0043] result

[0044] 1. CCBs induce PD-L1 in a time- and dose-dependent manner

[0045] Figure 1 Western blot was used to detect the effects of different concentrations of CCB on PD-L1 expression in lung cancer cell lines H460 and PC9. Figure 1 As shown in A, in both cell lines, the PD-L1 main band decreased significantly when CCB concentrations ranged from 0 to 30 μM. In addition, the expression of COX2 and apoptosis protein PARP increased ( Figure 1 A).

[0046] In addition, we performed WB to explore whether CCB induced the cleavage of PD-L1 in a time-dependent manner. We treated H460 cells with CCB for 4, 8, 12, and 24 hours. As the treatment time increased, the expression level of cleaved PD-L1 increased ( Figure 1 B). Interestingly, in H460, the expression of CCB was also upregulated ( Figure 1 B). Consistent results were obtained in PC9 cell line ( Figure 1 C).

[0047] Furthermore, we aimed to determine whether another COX inhibitor could affect the PD-L1 expression pattern. We treated H460 and PC9 cells with DMSO, CCB, VCB, RCB, sulfide, indole, ibuprofen, and asphaltene overnight. PD-L1 and COX2 expression was then detected by Western blotting. The results were shown in Table 1. Figure 1 As shown in D, in lung cancer cells (H460), only CCB and sulfide can induce cleavage of PD-L1, while COX2 expression is increased. Other COX inhibitors did not induce cleavage of PD-L1. Compared with DMSO, many COX inhibitors can downregulate PD-L1 expression, while COX2 expression is reduced ( Figure 1 D).

[0048] 2. CCB-induced PD-L1 cleavage is caused by partial deglycosylation

[0049] We further used the CHX stability assay to examine the protein stability of PD-L1 after CCB treatment. In H460 cells, cells were treated with DMSO and CCB (30 μM) for 8 hours, followed by CHX 10 μg / ml treatment for 0, 2, 4, 6, 8, and 12 hours. Cells were collected for Western blotting. Image J was used for grayscale analysis. CCB treatment resulted in an increased degradation rate of PD-L1, which resulted in its degradation in H460 cells ( Figure 2 A) and PC9( Figure 2 B) The stability is significantly reduced.

[0050] 2DG and metformin are. Metformin ( Figure 2 C).

[0051] Glycosylation assay The effect of CCB on PD-L1 glycosylation In H460, PC9 and HCC827, glycosylation assay showed that CCB treatment resulted in partial deglycosylation of PD-L1. Flow cytometry assay of the effect of CCB on PD-L1 expression on the surface of tumor cells In lung cancer cells (H460 and PC9), cells were treated with IgG, DMSO, CCB and 2DG (as a positive control known to induce PD-L1 deglycosylation) ( Figure 1 F) After CCB treatment, the expression of PD-L1 on the cell membrane surface changed, which was consistent with the change trend after 2DG treatment.

[0052] 3. CCB-induced deglycosylation is independent of COX2

[0053] Based on the previous results, we wondered whether COX2 expression is crucial for PD-L1 deglycosylation. We densely transfected Si-COX2 in Calu1 and then used WB to detect the expression levels of COX2 and PD-L1 after DMSO and CCB treatment. Figure 3 As shown in A, downregulated COX2 did not affect the deglycosylation of PD-L1.

[0054] 4. The COX2 gene was knocked out using CRISPR-Cas9 technology, and a monoclonal cell line with gene knockout (KO) was selected for further culture and experiment. A COX2 KO cell line was constructed in the H460 cell line, and monoclonal cells with COX2 gene knockout were screened. The cells were treated with DMSO and CCB, respectively. After COX2 knockout, WB was used to detect the effect of CCB drug treatment on PD-L1 expression. PD-L1 still underwent partial deglycosylation, indicating that CCB-induced PD-L1 deglycosylation is not dependent on COX2 expression ( Figure 3 B). In addition, we also explored the expression of COX2 in PDL1 KO cell lines. In H460KO, COX2 expression could not be detected after DMSO and CCB treatment ( Figure 3 C), these data suggest that CCB-induced deglycosylation may be independent of COX2, whereas PDL1 expression may be linked to COX2 expression.

[0055] 4. CCB promotes DR5 cell death, and N200 N219 may be the main deglycosylation site

[0056] We then investigated how CCB induces partial deglycosylation of PD-L1 by examining pathway proteins after CCB treatment in a time-dependent manner.

[0057] H460 and PC9 cells were treated with CCB for 4, 8, 12, and 24 hours. PARP bands increased significantly with the extension of treatment time, and the expression of DR5 also increased significantly. Pathway proteins previously associated with PD-L1 deglycosylation, including AKT, p-AKT, GSK3, and p-GSK3, showed no significant changes ( Figure 4 A). This suggests that CCB-induced PDL1 deglycosylation is not caused by the previously validated signaling pathway. However, CCB may upregulate EGFR expression and reduce cyclin D1 and cyclin A ( Figure 4 B). Trail did not alter CCB-induced PDL1 deglycosylation ( Figure 4 C).

[0058] .EGF PDL1, r-INF on PD-L1 WB detection of the effect of CCB combined with EGF and r-INF on the glycosylation status of PD-L1 In lung cancer cells (H460), cells were treated with DMSO, CCB, r-INF, EGF, CCB+-INF and CCB+EGF. Both r-INF and EGF can upregulate the expression of PDL1. When r-INF and EGF cannot induce, CCB can induce partial deglycosylation of PD-L1. The combined induction of partial deglycosylation of PD-L1 by CCB and r-INF is more obvious than that by CCB alone, showing a synergistic effect. EGF has no significant effect on PD-L1 deglycosylation and obvious synergistic effect ( Figure 4 D).

[0059] The glycosylation sites of PDL1 include N35, N192, N200, and N219. We constructed mutant plasmids based on these four sites. In lung cancer cells (H460), after transient transfection with the constructed PD-L1 mutant plasmid, the cells were treated with CCB for 8 hours and then subjected to WB detection. The results showed that N200 and N219 may be the main sites of CCB-induced deglycosylation ( Figure 4 E).

[0060] 5.CCB enhanced its immunotherapeutic response in vivo.

[0061] Furthermore, we intend to elucidate the effects of CCB combined with PDL1 antibodies on tumor growth and immune responses.

[0062] We compared the effects of CCB, PDL1, and the synthesis of CCB and PDL1 on the growth of human lung cancer H1975 xenografts (Figure 2). Under the experimental conditions, the combination of CCB and PDL1 antibody did not reduce mouse body weight compared to the control group and CCB or PDL1 antibody, demonstrating the safety of the combination. Furthermore, flow cytometry was used to assess PDL1 expression and immune cytokines. Compared to the control and CCB-treated groups, PDL1 levels were significantly reduced in the PDL1 antibody and combination treatment groups. Compared to the control and monotherapy groups, the combination treatment group showed a decrease in microphages and M2 hyperplasia cells in tumors. Compared to the control and monotherapy groups, the combination treatment group showed an increase in M1 tumor stage and r-INF. Compared to the control and monotherapy groups, the combination treatment group showed an increase in spleen and PDL1 r-INF. Therefore, in vivo, CCB and PDL1 antibody effectively inhibited the growth of lung cancer xenografts, reduced microphages and M2 cells, and enhanced M1 cells and r-INF.

[0063] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the novel spirit and scope of the present invention. Such changes and improvements fall within the scope of the invention claimed. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. An application of celecoxib in the preparation of tumor immunotherapy drugs.

2. The use according to claim 1, characterized in that: The celecoxib specifically inhibits cyclooxygenase-2 (COX-2), and inhibits the production of inflammatory prostaglandins by inhibiting cyclooxygenase-2 (COX-2), thereby achieving anti-inflammatory, analgesic and antipyretic effects; the expression of COX2 is related to the occurrence and development of various malignant tumors; PD-L1 is a transmembrane protein, and TNF, IFN-r, IL-6, IL-10 and granulocyte stimulating factor can upregulate the expression of PD-L1 in different cells; the post-translational modification PTMs of PD-L1 is an important regulatory mechanism, which regulates the immunosuppressive function of tumor patients through glycosylation, phosphorylation, ubiquitination, ubiquitination and acetylation. Deglycosylated PD-L1 increases instability and shortens half-life. Cancer cells with glycosylated PD-L1 are resistant to T cell killing, while cancer cells with deglycosylated PD-L1 are very sensitive to T cell killing.

3. The use according to claim 2, characterized in that: The expression level of cyclooxygenase-2 (COX-2) in patients is detected as a biomarker for evaluating the efficacy and prognosis of tumor immunotherapy, or as a tumor resistance marker, and is used to construct a model for evaluating the efficacy and prognosis of tumor immunotherapy, or to construct a model for evaluating tumor resistance.

4. The use according to claim 1, characterized in that: The tumors include, but are not limited to, esophageal cancer, gastric cancer, colon cancer, rectal cancer, gastrointestinal stromal tumors, melanoma, and breast cancer.

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