Use of methotrexate and its pharmaceutical compositions in tumor immunotherapy

Abstract

This invention provides the use of methotrexate and pharmaceutical compositions thereof in tumor immunotherapy. This invention verifies the pharmacological activity of methotrexate (MTX) and its analogue raltitrexed as ENPP1 inhibitors, and also verifies the role of methotrexate in enhancing the efficacy of radiation therapy. Furthermore, it verifies that methotrexate can inhibit tumor resistance to PD-1 monoclonal antibodies. The combination of MTX and PD-1 monoclonal antibodies enhances the infiltration of immune cells into mouse tumor tissue, increases the number of active cytotoxic T cells, and reduces the number of exhausted T cells.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically, it relates to the use of methotrexate and its pharmaceutical compositions in tumor immunotherapy. Background Technology

[0002] Methotrexate (MTX) and its analogues aminopterin (AP), raltitrexed (RTD), and pemetrexed (PTD) inhibit tumor growth by antagonizing folic acid metabolism, interfering with DNA and RNA synthesis, and are widely used in clinical practice for a variety of malignant tumors, including acute leukemia[1], malignant lymphoma[2], breast cancer[3], and head and neck tumors[4].

[0003] Another important clinical application of methotrexate is as an immunosuppressant for the treatment of rheumatoid arthritis [5]. There are various theories about its mechanism of action. The most widely accepted theory is that methotrexate metabolite polyglutamate indirectly inhibits the activity of AMP deaminase and adenosine deaminase, increases the release of extracellular adenosine, and adenosine binds to cell surface receptors to inhibit immune and inflammatory responses [6]. Some studies have also reported that although methotrexate cannot directly induce T cell apoptosis, it can reverse T cell resistance to apoptosis [7] and inhibit the persistently activated NF-κB pathway in T cells of patients with rheumatoid arthritis [8], thereby reducing immune responses. Although many biologics are used to treat rheumatoid arthritis and other forms of inflammatory arthritis, low-dose methotrexate treatment remains the gold standard for the treatment of rheumatoid arthritis.

[0004] Stimulator of interferon genes (STING) is a transmembrane protein located on the endoplasmic reticulum and plays an important role in the innate immune signaling pathway [9]. Tumor cell-derived DNA can activate cyclic GMP-AMP synthase (cGAS) to synthesize the second messenger cGAMP. cGAMP binds to and activates STING, which then activates kinases TBK1 and IKK, ultimately leading to the release of type I interferons (IFNs) and pro-inflammatory cytokines [10,11]. Type I interferons can promote tumor antigen presentation and activate tumor-killing CD8 T cells, thereby initiating an adaptive immune response against tumors

[12] . Several small molecule STING agonists have been reported to induce anti-tumor immune responses in mice and cause persistent tumor regression [13-15].

[0005] ENPP1 (Ectonucleotide pyrophosphatase phosphodiesterase 1) is a phosphodiesterase located outside the cell membrane that can catalyze the hydrolysis of ATP or GTP into AMP or GMP, while generating inorganic pyrophosphate. Inorganic pyrophosphate inhibits bone and cartilage mineralization, and overexpression of ENPP1 can cause diseases such as pyrophosphate arthropathy and aortic valve calcification

[16] .

[0006] Recent studies have reported that ENPP1 can also hydrolyze cGAMP to negatively regulate the cGAS-STING signaling pathway

[17] , playing an important role in tumor immunity. ENPP1 on the surface of tumor cells hydrolyzes cGAMP to produce AMP, which is then dephosphorylated by CD73 to produce adenosine. Adenosine binds to adenosine receptors on the cell surface to exert an immunosuppressive effect, jointly promoting the immune escape of tumor cells [18,19]. Compared with normal tissues, ENPP1 is highly expressed in breast cancer

[18] , lung cancer

[19] and ovarian cancer

[20] cells, which provides the possibility of developing ENPP1 inhibitors to selectively activate the STING pathway in the tumor microenvironment, avoiding the excessive immune activation caused by systemic administration of STING agonists.

[0007] References:

[0008] 1. Yoon, S.-A., et al., Influence of reduced folate carrier and dihydrofolate reductase genes on methotrexate-induced cytotoxicity. Cancerresearch and treatment, 2010.42(3):p.163-171.

[0009] 2. Kim, A., et al., A combination of methotrexate and irradiationpromotes cell death in NK / T-cell lymphoma cells via down-regulation of NF-κB signaling. Leukemia Research, 2012.36(3):p.350-357.

[0010] 3.Shakeran,Z.,et al.,Biodegradable nanocarriers based on chitosan-modified mesoporous silica nanoparticles for delivery of methotrexate forapplication in breast cancer treatment.2021.118:p.111526.

[0011] 4.Ham,J.C.,et al.,Methotrexate plus or minus cetuximab as first-linetreatment in arecurrent or metastatic(R / M)squamous cell carcinoma populationof the head and neck(SCCHN),unfit for cisplatin combination treatment,a phaseIb-randomized phase II study Commence.2020.42(5):p.828-838.

[0012] 5.Cronstein,B.N.and T.M.Aune,Methotrexate and its mechanisms ofaction in inflammatory arthritis.Nature Reviews Rheumatology,2020.16(3):p.145-154.

[0013] 6.Montesinos,M.C.,et al.,The antiinflammatory mechanism ofmethotrexate depends on extracellular conversion of adenine nucleotides toadenosine by ecto-5′-nucleotidase:findings in a study of ecto-5′-nucleotidasegene–deficient mice.Arthritis Rheum,2007.56(5):p.1440-1445.

[0014] 7.Spurlock III,C.F.,et al.,Increased sensitivity to apoptosis inducedby methotrexate is mediated by JNK.Arthritis&Rheumatism,2011.63(9):p.2606-2616.

[0015] 8.Spurlock III,C.F.,et al.,Methotrexate-mediated inhibition ofnuclear factorκB activation by distinct pathways in T cells and fibroblast-like synoviocytes.Rheumatology,2015.54(1):p.178-187.

[0016] 9.Shang,G.,et al.,Cryo-EM structures of STING reveal its mechanism ofactivation by cyclic GMP–AMP.Nature,2019.

[0017] 10.Woo,S.-R.,et al.,STING-dependent cytosolic DNA sensing mediatesinnate immune recognition of immunogenic tumors.2014.41(5):p.830-842.

[0018] 11.Zhang,X.,et al.,Cyclic GMP-AMP containing mixed phosphodiesterlinkages is an endogenous high-affinity ligand for STING.Mol Cell,2013.51(2):p.226-35.

[0019] 12.Diamond,M.S.,et al.,Type I interferon is selectively required bydendritic cells for immune rejection of tumors.2011.208(10):p.1989-2003.

[0020] 13.Ramanjulu,J.M.,et al.,Design of amidobenzimidazole STING receptoragonists with systemic activity.Nature,2018.564(7736):p.439-443.

[0021] 14.Chin,E.N.,et al.,Antitumor activity of a systemic STING-activatingnon-nucleotide cGAMP mimetic.Science,2020.369(6506):p.993.

[0022] 15.Pan,B.-S.,et al.,An orally available non-nucleotide STING agonistwith antitumor activity.Science,2020.369(6506):p.eaba6098.

[0023] 16.Onyedibe,K.I.,M.Wang,and H.O.Sintim,ENPP1,an Old Enzyme with NewFunctions,and Small Molecule Inhibitors—A STING in the Tale ofENPP1.Molecules,2019.24(22).

[0024] 17.Carozza,J.A.,et al.,Extracellular cGAMP is a cancer-cell-producedimmunotransmitter involved in radiation-induced anticancer immunity.NatureCancer,2020.1(2):p.184-196.

[0025] 18. Lau, WM, et al., Enpp1: a potential facilitator of breast cancerbone metastasis. 2013.8(7): p.e66752.

[0026] 19. Hu, M., et al., Dysregulated ENPP1 increases the malignancy of human lung cancer by inducing epithelial-mesenchymal transition phenotypes and stem cell features. 2019.9(1):p.134.

[0027] 20. Wang, H., et al., High expression of ENPP1 in high-grade serousovarian carcinoma predicts poor prognosis and as a molecular therapy target. 2021.16(2):p.e0245733. Summary of the Invention

[0028] One technical objective of this invention is to develop a novel ENPP1 inhibitor.

[0029] Another technical objective of this invention is to provide a pharmaceutical composition for the treatment of tumors.

[0030] Another technical objective of this invention is to provide the use of methotrexate in the preparation of tumor radiotherapy enhancers.

[0031] Therefore, on the one hand, the present invention provides the use of methotrexate and raltitrexed in the preparation of ENPP1 inhibitors.

[0032] In a specific implementation, the ENPP1 inhibitor can be used for the treatment of tumors.

[0033] In a specific embodiment, the tumor is selected from breast cancer, lung cancer, ovarian cancer, and colorectal cancer.

[0034] On the other hand, the present invention provides the use of methotrexate in the preparation of tumor radiation therapy enhancers.

[0035] In a specific embodiment, the tumor is selected from breast cancer, lung cancer, ovarian cancer, and colorectal cancer.

[0036] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of methotrexate and a PD-1 monoclonal antibody, and optionally pharmaceutically acceptable excipients.

[0037] In a specific embodiment, the weight ratio of methotrexate to PD-1 monoclonal antibody in the pharmaceutical composition is 1:0.1 to 1, preferably 1:0.5.

[0038] In another aspect, the present invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating or preventing tumors.

[0039] In a specific embodiment, the tumor is selected from breast cancer, lung cancer, ovarian cancer, and colorectal cancer.

[0040] Beneficial effects

[0041] This invention uses a self-developed SSGCN model to predict seven compounds that may be active against ENPP1. Then, through biochemical (enzyme activity assays) and biophysical (protein thermal migration, NMR, isothermal titration calorimetry) methods, methotrexate (MTX) was confirmed as an inhibitor of ENPP1. Further cell and animal experiments revealed that methotrexate (MTX) and its analogues, aminopterin and raltitrexed, can inhibit ENPP1 activity, thereby inhibiting cGAMP hydrolysis and enhancing the activity of the STING signaling pathway. Simultaneously, due to the reduced hydrolysis of cGAMP and ATP, leading to a decrease in AMP sources, MTX and its analogues can also reduce adenosine levels in the tumor microenvironment. Furthermore, MTX and its analogues can cause genomic damage in cancer cells such as breast cancer and colon cancer, releasing double-stranded DNA, thereby activating the cGAS-STING signaling pathway in cancer cells and releasing cGAMP, type I interferon, and pro-inflammatory cytokines. The inventors have also discovered that MTX and its analogues can enhance the sensitivity of tumors to radiation therapy, improve the efficacy of PD-1 monoclonal antibodies in sensitive tumor models, and induce anti-tumor immune responses in tumors that are insensitive to PD-1 monoclonal antibodies.

[0042] In summary, the present invention contributes to the prior art in the following aspects:

[0043] 1. Based on an artificial intelligence-assisted drug design method, this invention has discovered that MTX and its analogues aminopterin and raltitrexed are potent ENPP1 inhibitors;

[0044] 2. This invention elucidates that MTX activates the cGAS-STING signaling pathway in tumor cells within the tumor microenvironment, while simultaneously inhibiting cGAMP and ATP hydrolysis and reducing adenosine levels, thereby exerting a combined anti-tumor immune effect.

[0045] 3. The inventors have discovered that MTX can enhance the sensitivity of tumors to radiation therapy;

[0046] 4. The inventors also discovered that the combination of MTX and PD-1 monoclonal antibody can restore the response of PD-1 resistant tumors. Attached Figure Description

[0047] Figure 1 The results showed that MTX and its analogues (RTD, PTD) as well as the positive compound E1 directly bound to ENPP1, inhibiting its enzyme activity.

[0048] A. Inhibition rate of the compound on human and mouse ENPP1 enzyme activity; B. Increase in protein thermal stability value by the compound; C. One-dimensional nuclear magnetic resonance experiments show that MTX and RTD directly bind to human ENPP1 protein.

[0049] Figure 2 : Displays the KD value of MTX and its analogues (RTD, PTD) combined with ENPP1.

[0050] Figure 3 The results showed that MTX and its analogues inhibited the hydrolysis of extracellular cGAMP and enhanced the STING signaling pathway.

[0051] AB. Changes in extracellular (A) and intracellular (B) cGAMP concentrations over time under different compound combinations; CD. Changes in IFNB1 and CXCL10 gene expression levels over time under different compound combinations; E. Effects of MTX, RTD, PTD, and STING positive agonist cGAMP treatment alone on IFNB1 expression in cells; F. Western blot analysis of expression levels of key STING pathway activation proteins pSTING, pTBK1, and pIRF3 after MTX treatment.

[0052] Figure 4 This study showed that MTX and its analogues activate the cGAS-STING signaling pathway in tumor cells.

[0053] AC. MTX, RTD, and PTD increased the transcription levels of Ifnb1, Cxcl10, and Il6 in tumor cells; DF. Blocking the function of cGAS, STING, or TBK1, MTX, RTD, and PTD could no longer promote Ifnb1 transcription; GH. MTX, RTD, and PTD caused the accumulation of cytoplasmic dsDNA.

[0054] Figure 5 The results showed that MTX enhanced the therapeutic effect of tumor radiation.

[0055] The combined use of AB.MTX and radiation therapy resulted in tumor regression in mice (A) and prolonged mouse survival (B); the combined use of CF.MTX and radiation enhanced the infiltration of immune cells into mouse tumor tissue and increased the number of cytotoxic T cells (CD8+) and dendritic cells (DCs, CD11c+), P<0.05, **P<0.01, ***P<0.001, ns, no significant difference.

[0056] Figure 6 The results showed that MTX enhanced the anti-tumor effect of PD-1 monoclonal antibody.

[0057] AB.MTX enhanced the inhibitory effect of PD-1 monoclonal antibody on MC-38 tumor growth, and the combination of the two prolonged the survival time of mice; CE.MTX combined with PD-1 monoclonal antibody increased the infiltration of immune cells in mouse tumor tissue, increased the number of cytotoxic T cells (CD8+), and decreased the number of exhausted T cells (PD1+CD8+), P<0.05, **P<0.01, ***P<0.001, ns, no significant difference.

[0058] Figure 7 The results showed that the combination of MTX and PD-1 monoclonal antibody overcame the resistance of 4T-1 tumor cells to PD-1 monoclonal antibody.

[0059] The combination of AB.MTX and PD-1 monoclonal antibody overcame the resistance of 4T-1 tumors to PD-1 monoclonal antibody and prolonged the survival time of mice. The combination of CE.MTX and PD-1 monoclonal antibody enhanced the infiltration of immune cells in mouse tumor tissue, increased the number of active cytotoxic T cells (GzmB+CD8+), and reduced the number of exhausted T cells (PD1+CD8+). P<0.05, **P<0.01, ***P<0.001, ns, no significant difference. Detailed Implementation

[0060] the term:

[0061] Methotrexate analogs: Methotrexate analogs mentioned in this application may include raltitrexed (RTD) and pemetrexed (PTD).

[0062] The following embodiments are provided only to enable those skilled in the art to better understand the technical content of the present invention, and are not intended to limit the scope of the present invention.

[0063] Example 1: Molecular-level confirmation of the inhibitory activity of MTX and its analogues RTD and PTD on ENPP1.

[0064] 1.1 Effects of MTX and its analogues RTD and PTD on the activity of recombinant human and mouse ENPP1 enzymes.

[0065] Experimental methods:

[0066] Enzyme activity assays were performed using recombinant human and mouse ENPP1 proteins with p-Nph-5'-TMP as the substrate. The reaction system contained 50 mM Tris-HCl (pH 8.5), 130 mM NaCl, 1 mM CaCl2, 5 mM KCl, 1 nM human or mouse ENPP1 protein, and different concentrations of the compound. Finally, 1 mM p-Nph-5'-TMP was used to initiate the reaction. The enzyme activity process was characterized by monitoring the absorbance change of the product p-nitrophenol at 37 °C.

[0067] Experimental results:

[0068] like Figure 1 As shown in Figure A, MTX and RTD significantly inhibited human ENPP1 enzyme activity, IC50 50 The concentrations were 0.16 μM and 0.67 μM, respectively; MTX also showed a strong inhibitory effect on mouse ENPP1, with an IC50 concentration of 0.16 μM and 0.67 μM. 50 The concentration was 0.57 μM; PTD showed no activity.

[0069] 1.2 Effects of MTX and its analogues RTD and PTD on the thermal stability of recombinant human and mouse ENPP1 proteins.

[0070] Experimental methods:

[0071] The effect of compounds on the thermal stability of proteins was evaluated using a protein thermal migration assay. A 20 μL reaction mixture was prepared by adding 1.25 μM of mouse and human ENPP1 protein, 5xSYPRO Orange dye (Sigma S5692), and different concentrations of the analyte compounds to white 96-well plates. Detection was performed on a bio-rad CFX Connect instrument, with the heating temperature linearly increased from 25 °C to 95 °C, and temperature and fluorescence intensity were recorded. The temperature-fluorescence data were analyzed using CFX Manager, and the solubility temperature (Tm) of ENPP1 protein was obtained by fitting the data. A DMSO group was used as a control to analyze the effect of different compounds on the Tm value of ENPP1 protein.

[0072] Experimental results: such as Figure 1 As shown in B, both MTX and RTD can directly bind to mouse and human ENPP1 proteins, increasing their Tm values; PTD has virtually no effect.

[0073] 1.3 The direct binding of MTX and its analogues RTD and PTD to human ENPP1 protein was determined by combining saturated transfer difference spectroscopy (STD) and T1ρ nuclear magnetic resonance.

[0074] Experimental methods:

[0075] Saturated transfer difference (STD) and T1p NMR experiments were performed on an NMR spectrometer equipped with a cryogenic cooling probe at a temperature of 25°C. STD and T1p spectra were acquired.

[0076] Experimental results:

[0077] like Figure 1 As shown in C, MTX and RTD bind directly to the human ENPP1 protein.

[0078] 1.4 Isothermal titration calorimetry (ITC) was used to determine the binding dissociation constants (KL) of MTX and its analogues RTD and PTD with recombinant human ENPP1 protein. D ).

[0079] Experimental methods:

[0080] The purified human ENPP1 protein and the test compound were dialyzed into ITC buffer (20 mM Tris, 150 mM NaCl) to final concentrations of 20 μM and 200 μM, respectively. The protein was titrated using an ITC 200 calorimeter (General Electric Co.), with real-time monitoring of calorific changes and determination of enthalpy change (ΔH). K was calculated based on the concentration at titration equilibrium. D value.

[0081] Experimental results:

[0082] like Figure 2 As shown, MTX and RTD bind directly to human ENPP1, K D The values ​​were 0.27 μM and 0.38 μM, respectively; PTD had no effect.

[0083] Example 2: MTX and its analogues RTD inhibit the hydrolysis of extracellular cGAMP and enhance the STING signaling pathway.

[0084] 2.1 Effects of MTX and its analogue RTD on extracellular and intracellular cGAMP concentrations.

[0085] Experimental methods:

[0086] Two hours prior to incubation, THP-1 cells were treated with 10 μM of the compound or DMSO. Then, 500 nM of cGAMP was added to the culture medium. After incubation for 6, 12, 24, and 48 hours, the culture medium supernatant and cells were collected, and the extracellular and intracellular cGAMP concentrations were determined using a kit.

[0087] Experimental results:

[0088] like Figure 3As shown in AB, MTX and RTD can inhibit the hydrolysis of extracellular cGAMP, thereby maintaining high concentrations of cGAMP both extracellularly and intracellularly for extended periods.

[0089] 2.2 Effects of MTX and its analogue RTD on the transcriptional levels of IFNB1 and CXCL10 downstream of the STING pathway in THP-1 cells.

[0090] Experimental methods:

[0091] THP-1 cells were treated with 10 μM of the compound or DMSO 2 hours in advance, and then 500 nM of cGAMP was added to the culture medium. After incubation for 6, 12, 24 and 48 hours, the cells were collected and the changes in mRNA transcription levels of IFNB1 and CXCL10 were analyzed by RT-qPCR.

[0092] Experimental results:

[0093] like Figure 3 As shown in CE, MTX and RTD can enhance the transcription of IFNB1 and CXCL10 downstream of the STING pathway.

[0094] 2.3 Effects of MTX on STING protein levels and activation of key downstream proteins TBK1 and IRF3 in THP-1 cells.

[0095] Experimental methods:

[0096] Two hours beforehand, THP-1 cells were treated with 10 μM MTX or DMSO. Then, 500 nM cGAMP was added to the culture medium. After incubation for 8 hours, the cells were harvested and Western blot was used to detect the levels of STING protein and the levels of key downstream proteins of STING, TBK1, p-TBK1, IRF, and p-IRF3.

[0097] Experimental results:

[0098] like Figure 3 As shown in F, MTX increases the phosphorylation levels of TBK1 and IRF3 downstream of STING.

[0099] Example 3: MTX and its analogues activate the cGAS-STING signaling pathway in tumor cells.

[0100] 3.1 Effects of MTX and its analogues RTD and PTD on the expression of IFN-β, CXCL10 and IL-6 in tumor cells.

[0101] Experimental methods:

[0102] Tumor cells (4T-1, MC-38) were given 10 μM of the compound. After 24 hours, RNA was extracted from the cells, and RT-qPCR was used to analyze the changes in the mRNA transcription levels of Ifnb1, Cxcl10, and Il6.

[0103] Experimental results:

[0104] like Figure 4 As shown in AC, MTX, RTD, and PTD can all increase the transcription of Ifnb1, Cxcl10, and Il6 in tumor cells.

[0105] 3.2 Blocking the function of key STING pathway proteins cGAS, STING, and TBK1, and detecting the effects of MTX on the expression of IFN-β, CXCL10, and IL-6.

[0106] Experimental methods:

[0107] Two hours prior to treatment, 4T-1 cells were treated with cGAS, STING, and TBK1 inhibitors, followed by administration of 10 μM MTX, RTD, and PTD. The effects on the expression of IFN-β, CXCL10, and IL-6 were then examined.

[0108] Experimental results:

[0109] like Figure 4 As shown in DF, blocking cGAS, STING, or TBK1 function, MTX, RTD, and PTD can no longer promote Ifnb1 transcription.

[0110] 3.3 MTX and its analogues RTD and PTD cause damage to tumor cell dsDNA.

[0111] Experimental methods:

[0112] 4T-1 cells were given 10 μM of compounds (MTX, RTD, PTD). After 24 hours, the cells were collected, and the levels of H2A.X and λH2A.X proteins were analyzed by Western blot. Cytoplasmic dsDNA and λH2A.X were detected by immunofluorescence.

[0113] Experimental results:

[0114] like Figure 4 As shown in GH, MTX, RTD, and PTD all increased the protein level of λH2A.X, and dsDNA was detected in the cytoplasm.

[0115] Example 4: Effect of Methotrexate on the Therapeutic Effect of Tumor Radiotherapy

[0116] Experimental methods:

[0117] MTX inhibits extracellular cGAMP hydrolysis, while radiation therapy can cause DNA damage and activate cGAS to synthesize cGAMP, suggesting that MTX can be used in combination with radiation to enhance efficacy. Balb / c mice were inoculated with 4T-1 cells in their fat pads, and when the tumor volume reached 50 mm... 3 Mice were treated with 12 Gy of radiation and simultaneously injected intraperitoneally with 10 mg / kg of MTX or PTD daily for 7 consecutive days. Tumor volume changes were recorded. After the administration was completed, tumor tissue was harvested and flow cytometry was used to analyze the immune cell infiltration in the tumor tissue. A new batch of Balb / c mice were inoculated with 4T-1 cells into their fat pads using the same administration method. Tumor volume and time to natural death were recorded. According to IACUC animal welfare guidelines, if the tumor volume was greater than 2000 mm², the mice were considered dead. 3 Mice were considered dead and then euthanized. Survival curves were calculated using the Kaplan-Meier method, and the logrank method was used to analyze mouse survival time.

[0118] Experimental results:

[0119] like Figure 5 As shown in AB, the combination of MTX and radiation induced tumor regression and prolonged survival in mice. Flow cytometry analysis results indicated that ( Figure 5 MTX, when combined with radiation, enhances the infiltration of immune cells into mouse tumor tissue and increases the number of cytotoxic T cells (CD8+) and dendritic cells (DCs, CD11c+).

[0120] Example 5: Effect of methotrexate combined with anti-PD1 monoclonal antibody on tumor growth

[0121] 5.1 Effect of methotrexate combined with anti-PD1 monoclonal antibody on MC-38 tumor growth.

[0122] Experimental methods:

[0123] C57 / BL6N mice were subcutaneously inoculated with MC-38 cells until the tumor volume reached 50 mm. 3 Mice were given 200 μg of PD-1 monoclonal antibody every 3 days for two consecutive days, and simultaneously began daily intraperitoneal injections of 10 mg / kg MTX or PTD for 7 consecutive days. Tumor volume changes were recorded. After the administration was completed, tumor tissue was harvested and immune cell infiltration was analyzed by flow cytometry. A new batch of C57 / BL6N mice were subcutaneously inoculated with MC-38 cells, administered via the same route, and tumor volume and time of natural death were recorded. According to IACUC animal welfare guidelines, if the tumor volume was greater than 2000 mm², the mice were considered dead. 3 Mice were considered dead and then euthanized. Survival curves were calculated using the Kaplan-Meier method, and the logrank method was used to analyze mouse survival time.

[0124] Experimental results:

[0125] like Figure 6 As shown in AB, MTX can enhance the inhibitory effect of PD-1 monoclonal antibody on MC-38 tumor growth, and the combination of the two prolongs the survival time of mice. Flow cytometry analysis showed that ( Figure 6 MTX combined with PD-1 monoclonal antibody enhanced the infiltration of immune cells in mouse tumor tissue, increased the number of cytotoxic T cells (CD8+), and reduced the number of exhausted T cells (PD1+CD8+).

[0126] 5.2 Effect of methotrexate combined with anti-PD1 monoclonal antibody on 4T-1 tumor growth.

[0127] Experimental methods:

[0128] Balb / c mice were inoculated with 4T-1 cells into their fat pads using the same administration method as above, and tumor volume changes were recorded. After administration, tumor tissue was harvested, and immune cell infiltration was analyzed by flow cytometry. A new batch of Balb / c mice were inoculated with 4T-1 cells into their fat pads using the same administration method, and tumor volume and time to natural death were recorded. According to IACUC animal welfare guidelines, if the tumor volume was greater than 2000 mm², the tumor was considered dead. 3 Mice were considered dead and then euthanized. Survival curves were calculated using the Kaplan-Meier method, and the logrank method was used to analyze mouse survival time.

[0129] Experimental results:

[0130] like Figure 7 As shown in AB, the combination of MTX and PD-1 monoclonal antibody overcame the resistance of 4T-1 tumors to PD-1 monoclonal antibody, and the combination prolonged the survival time of mice. Flow cytometry analysis showed that ( Figure 7 CE), the combination of MTX and PD-1 monoclonal antibody enhanced the infiltration of immune cells in mouse tumor tissue, increased the number of active cytotoxic T cells (GzmB+CD8+), and reduced the number of exhausted T cells (PD1+CD8+).

Claims

1. The use of methotrexate in the preparation of potentiators for tumor radiotherapy, among which, The tumor is breast cancer.

Images