CN113750215B - Combination Drugs Used to Treat Cancer - Google Patents

Abstract

The invention discloses a combined medicine for treating tumors, and aims to provide a treatment scheme for treating tumor patients by applying immunotherapy aiming at activating tumor microenvironment and relieving immunosuppression; the technical point is that the carfilzomib and the PD1 pathway inhibitor are used for combined treatment of a tumor patient, wherein the PD1 pathway inhibitor is a PD1 inhibitor or a PDL1 inhibitor. Wherein: the PD1 inhibitor is an inhibitory antibody (PD 1-anti, PD1-Ab for short) or a small molecule inhibitor of PD1, and the PDL1 inhibitor is an inhibitory antibody or a PDL1 small molecule inhibitor; belongs to the field of medical biotechnology.

Description

Combination Drugs Used to Treat Cancer

technical field

The invention relates to a combination drug for treating tumors, belonging to the technical field of medicine.

Background technique

Cancer is the second leading cause of death among patients, at least 10 million people die from cancer every year, and about 17% of the deaths worldwide are caused by cancer. According to the latest statistics in 2020, 1.8 million lung cancer patients, 935,000 colon and rectal cancer patients, and 830,000 liver cancer patients died. At present, cancer treatment methods mainly include surgical resection, chemotherapy, radiotherapy, immunotherapy and targeted therapy, but advanced patients have lost the opportunity for surgical treatment, and chemotherapy and radiotherapy cannot meet the treatment needs of advanced cancer patients. Tumor targeted therapy plays a very important role in the clinical treatment of cancer. For example, gefitinib and erlotinib can prolong the treatment of cancers driven by EGFR gene mutations and prolong the survival of patients to a certain extent. However, some patients are not sensitive to targeted therapy, or after a short period of treatment, it becomes effective and develops resistance to the drug in the later stage. It is an urgent task in the clinical practice of tumors to develop new and effective treatments.

Since 2013, cancer immunotherapy research has made significant progress and has become an indispensable treatment for advanced cancer treatment. Unlike traditional cancer treatments, inhibitors of the immune checkpoint programmed death receptor 1 (programmed death 1, PD1) can block the binding of PD1 to programmed death ligand 1 (programmed death-ligand 1, PDL1), thereby releasing The inhibitory effect on effector T cells and memory T cells, while down-regulating the differentiation of regulatory T lymphocytes and exhausted T cells, and then using the body's own immune system to play a role in killing tumors. It is noteworthy that PD-1/PDL1 inhibitors have been successfully applied in the clinical treatment of tumors, successfully prolonging the survival of some cancer patients and providing more treatment options for advanced cancer patients.

However, the tumor microenvironment is complex and various immune processes interact with each other, which makes the clinical transformation of lung cancer immunotherapy face many problems. For example, only about 20% of lung cancer patients respond clinically to PD-1 antibody inhibitors. Therefore, synergists of PD-1 drugs are urgently needed clinically.

Studies in recent years have found that 50% of the total weight of tumor tissue is macrophages. These macrophages secrete IL-10, TGF-β, Arginase, and express molecules such as CD206 and CD163 on the surface. They are called tumor-associated macrophages. (TAM, tumor associated macrophage). TAM secretes anti-inflammatory and cancer-promoting cytokines, inhibits the function of T cells, prevents T cells from attacking tumor cells, and forms an immunosuppressive tumor microenvironment, thereby limiting the efficacy of immune checkpoint PD1 inhibitors, and ultimately leading to the growth of tumor cells , transfer and spread. At present, antibodies against CFS1R are used to kill TAMs in tumors in clinical practice, but the therapeutic effect of this drug in most tumor patients is not obvious.

In contrast to TAM, macrophages can be induced to differentiate into M1 macrophages expressing factors such as IL-1β and IL-6 under the action of specific cytokines, which can inhibit tumor growth. Therefore, the development of drugs that can target TAMs in the tumor microenvironment and differentiate them into M1 macrophages to relieve tumor heterogeneity and treat patients with PD1 inhibitors is in high clinical demand.

Contents of the invention

Based on the current prominent problem that some cancer patients are resistant to targeted therapy and have little response to immune checkpoint inhibitor immunotherapy, the purpose of the present invention is to provide a combination therapy for activating the tumor microenvironment and treating tumor patients. drug.

For this reason, the technical scheme that the present invention proposes is such:

A combination drug for the treatment of cancer, mainly composed of proteasome inhibitors, especially carfilzomib (Carfilzomib, referred to as: Carf.) and PD1 pathway inhibitors, PD1 pathway inhibitors are PD1 inhibitors or PDL1 inhibitors agent. Wherein: the PD1 inhibitor is an inhibitory PD1 antibody (PD1-antibody, PD1-Ab for short) or a small molecule inhibitor of PD1, and the PDL1 inhibitor is an inhibitory PDL1 antibody or a small molecule inhibitor of PDL1.

Among them: carfilzomib, a proteasome inhibitor, molecular formula: C40H57N5O7, molecular weight: 719.91g/mol, is currently considered to inhibit the function of proteasome, induce the activation of tumor cell apoptosis pathway, and then lead to tumor cell apoptosis. The PD1 pathway inhibitor is a PD1 inhibitor or a PDL1 inhibitor, wherein: the PD1 inhibitor is an inhibitory PD1 antibody (PD1-antibody, referred to as: PD1-Ab) or a small molecule inhibitor of PD1, and the PDL1 inhibitor is an inhibitory PDL1 antibody Or small molecule inhibitors of PDL1, they activate the immune system and relieve the inhibitory function of T cells, thereby treating tumors. Among them, inhibitory PD1 antibody or PDL1 antibody can recognize PD1 protein or PDL1 protein, and block the interaction of PD1-PDL1, thereby releasing immunosuppression; small molecule inhibitors of PD1 or PDL1 can inhibit PD-1 or PDL1 respectively. The function of PDL1, and then promote the function of activated T cells.

Further, the above-mentioned combined drugs for treating tumors mainly consist of proteasome inhibitors, especially carfilzomib and PD1 pathway inhibitors.

Further, in the aforementioned combination drug for treating tumors, the PD1 pathway inhibitor is a PD1 inhibitor or a PDL1 inhibitor.

Further, in the aforementioned combination drug for treating tumors, the PD1 inhibitor is an inhibitory PD1 antibody or a small molecule inhibitor.

Further, in the aforementioned combination drug for treating tumors, the PDL1 inhibitor is an inhibitory PDL1 antibody or a small molecule inhibitor.

Further, in the aforementioned combination drug for treating tumors, the carfilzomib and inhibitory PD1 antibody are composed at a mass ratio of 3:10.

Further, in the aforementioned combination drug for treating tumors, the carfilzomib and PD1 small molecule inhibitors are composed at a mass ratio of 3:20.

Further, in the aforementioned combination drug for treating tumors, the carfilzomib and PDL1 small molecule inhibitor are composed at a mass ratio of 1:10.

Further, in the aforementioned combined drug for treating tumors, the carfilzomib and PDL1 inhibitory antibody are composed at a mass ratio of 1:4.

Further, for the aforementioned combination drug for treating tumors, the dosage of carfilzomib is 3 mg/kg/time, and the dosage of inhibitory PD1 antibody is 10 mg/kg/time.

Further, in the aforementioned combination drug for treating tumors, the dosage of carfilzomib is 3 mg/kg/time, and the dosage of PD1 small molecule inhibitor is 20 mg/kg/time.

Further, for the aforementioned combination drug for treating tumors, the dosage of carfilzomib is 3 mg/kg/time, and the dosage of PDL1 antibody is 12 mg/kg/time.

Further, for the aforementioned combination drug for treating tumors, the dosage of carfilzomib is 3 mg/kg/time, and the dosage of PDL1 small molecule inhibitor is 30 mg/kg/time.

Further, for the aforementioned combination drug for treating tumors, the administration frequency of carfilzomib is: 3 times a week, and the administration frequency of PD1 antibody is once every 2 days.

Further, for the aforementioned combination drug for treating tumors, the administration frequency of carfilzomib is: 3 times a week, and the administration frequency of PD1 small molecule inhibitor is 1 time a day.

Further, for the aforementioned combined drug for treating tumors, the administration frequency of carfilzomib is: 3 times a week, and the administration frequency of PDL1 antibody is once every 2 days.

Further, for the aforementioned combination drug for treating tumors, the administration frequency of carfilzomib is: three times a week, and the administration frequency of the PDL1 small molecule inhibitor is once a day.

Further, the aforementioned combination drug for treating tumors is characterized in that the carfilzomib is a kind of proteasome inhibitor, but not limited to carfilzomib.

Further, the above-mentioned combination drug for treating tumors is characterized in that the tumor is lung cancer, but not limited to lung cancer.

Further, the above-mentioned combination drug for treating tumors includes carcinoma in situ and metastatic carcinoma.

Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

1. Because TAM eliminates the response of anti-tumor T cells by overexpressing immune checkpoint ligands (such as PD-L1, PD-L2 and Siglec-15, etc.), and also secretes anti-inflammatory and cancer-promoting growth factors ( TGFb, IL-10, etc.) or metabolically transform the tumor microenvironment, thereby nourishing tumor cells, inhibiting the function of T cells, limiting the efficacy and scope of immunotherapy, and ultimately leading to the growth, metastasis and spread of tumor cells. Therefore, the technical solution provided by the present application constructs a cell model that induces macrophages to become TAM in vitro, and uses this model to evaluate the effect of the proteasome inhibitor carfilzomib on the phenotype of TAM. The research of the present invention finds that the proteasome inhibitor carfilzomib can convert cancer-promoting TAMs into anti-cancer M1 macrophages in vitro, and promote the division and proliferation of T cells. In vivo, the proteasome inhibitor carfilzomib can also induce TAMs to become M1 macrophages, increase the infiltration and activation of T cells, and improve the tumor suppressive immune microenvironment.

In order to further verify the therapeutic effect of proteasomes on tumors, the applicant used a double-gene EGFR gene mutation primary mouse lung cancer model (CC10RTTA/Tet-EGFR-T790M/DEL19, mouse model abbreviation: EGFR-TD) and TC-1 Xenograft lung cancer model to verify the therapeutic effect of proteasome inhibitor carfilzomib, PD1 pathway inhibitor, and proteasome inhibitor carfilzomib combined with PD1 pathway inhibitor on primary lung cancer and lung cancer xenografts in mice. In small animal models, including mouse lung cancer xenograft tumor models and primary tumor models, it was found that the single-drug proteasome inhibitor carfilzomib has a certain therapeutic effect on mouse lung cancer, and the single-drug PD1-Ab has basically no effect. Single-drug PD1 small molecule inhibitors have basically no effect. When combined with proteasome inhibitor carfilzomib and PD1 pathway inhibitors, the tumors in mice almost all disappeared, including the TC-1 xenograft tumor model and EGFR-TD in mice Primary lung cancer model. Combination therapy of proteasome inhibitor carfilzomib and PD1 pathway inhibitor has achieved the goal of curing this type of tumor.

Description of drawings

Figure 1 shows that carfilzomib can significantly induce the transformation of M2 macrophages into M1 macrophages in vitro.

Among them, A: Carfilzomib and other proteasome inhibitors (bortezomib and MLN9708) promote the expression of M1 markers IL-1β, IL-6 and TNF-α in M2 macrophages, while inhibiting M2 markers The transcriptional expression of IL-10 and TGF-β; B: Carfilzomib can promote the secretion of M1 markers IL-1β and IL-6 from M2 macrophages; C: Carfilzomib can promote the secretion of M2 macrophages Macrophages express M1-type marker CD80 on the cell membrane surface (left figure), and the right figure shows the statistics of CD80 changes; D: Carfilzomib inhibits M2-type macrophages from expressing M2-type marker CD206 on the cell membrane surface (left figure Figure), the right figure is the statistics of CD206 changes; E: Carfilzomib can promote the phagocytosis of tumor cells by M2 macrophages (left figure), and the right figure is the statistics of the changes in phagocytosis efficiency.

Figure 2 shows that carfilzomib can significantly enhance the division of T cells induced by M2 macrophages in vitro.

Among them, A: Carfilzomib can significantly enhance the division of M2 macrophages to CD8 T cells in vitro (left figure), and the right figure shows the statistics of CD8 T cell changes; B: Carfilzomib can significantly enhance M2 macrophages in vitro The division of CD4 T cells by type macrophages (left picture), and the right picture shows the statistics of changes in CD4 T cells.

Figure 3 shows that carfilzomib can significantly induce the transformation of M2 macrophages in the tumor microenvironment into M1 macrophages in vivo, and increase the infiltration and activation of T cells.

Among them: A: Carfilzomib can significantly induce M2 macrophages in the tumor microenvironment to transform into M1 macrophages in vivo (left), and the right is the statistics of CD80 and CD206 changes; B: Carfilzomib can The infiltration and activation of CD8 T cells in the tumor microenvironment were significantly induced in vivo (left), and the statistics of CD8 and CD69 changes were shown on the right.

Figure 4 shows that carfilzomib can slightly inhibit the growth of lung cancer xenografts in mice.

Among them: A: Carfilzomib's evaluation chart of growth inhibition of subcutaneous transplanted tumors in mice; B: Statistical chart of tumor weight; C: Statistical chart of growth curve of mouse transplanted tumors after carfilzomib treatment.

Figure 5 shows that PD1-Ab has basically no inhibitory effect on the growth of transplanted tumors of lung cancer in mice.

Among them, A: the evaluation chart of the effect on the growth of the subcutaneous transplanted tumor in mice; B: the statistical chart of tumor weight; C: the statistical chart of the growth curve of the transplanted tumor in mice after PD1-Ab treatment.

Figure 6 shows that carfilzomib combined with PD1-Ab treatment can significantly inhibit the growth of lung cancer xenografts in mice, and greatly enhance the therapeutic effect of PD1-Ab on solid tumors in mice.

Among them, A: the evaluation chart of the growth inhibition of subcutaneous transplanted tumors in mice; B: the statistical chart of tumor weight; C: the statistical chart of the growth curve of mouse transplanted tumors after carfilzomib combined with PD1-Ab treatment.

Figure 7 shows that MB can slightly inhibit the growth of lung cancer xenografts in mice.

Among them: A: evaluation chart of MB on growth inhibition of mouse subcutaneous transplanted tumor; B: statistical chart of tumor weight; C: statistical chart of growth curve of mouse transplanted tumor after MB treatment.

Figure 8 shows that carfilzomib combined with MB treatment can significantly inhibit the growth of transplanted tumors of lung cancer in mice, and greatly enhance the therapeutic effect of PD1 small molecule inhibitors on solid tumors in mice.

Among them, A: the evaluation chart of the growth inhibition of subcutaneous transplanted tumors in mice; B: the statistical chart of tumor weight; C: the statistical chart of the growth curve of mouse transplanted tumors after carfilzomib combined with PD1-Ab treatment.

Figure 9 shows that carfilzomib can enhance the therapeutic effect of PD1 inhibitors (PD1 antibodies or small molecule inhibitors) on mouse lung cancer xenografts.

Among them, A: summary statistics of carfilzomib combined with PD1-Ab on transplanted tumor weight; B: summary statistics of carfilzomib combined with PD1-Ab on growth curve of transplanted tumors; C: summary of carfilzomib combined with PD1 small Statistical graph summary of molecular inhibitor MB on transplanted tumor weight; D: Statistical graph of carfilzomib combined with PD1 small molecule inhibitor MB on transplanted tumor growth curve.

Figure 10 shows that carfilzomib can significantly improve the treatment of primary lung cancer in mice by PD1-Ab.

Among them, A: CT effect of evaluating carfilzomib combined with PD1-Ab synergistic therapy on primary lung tumor inhibition in CC10-RTTA/EGFR-TD transgenic mouse model; B: Relative tumor infiltration in lung tissue of mice before and after treatment Area statistical chart; C: HE staining (hematoxylin-eosin staining) and Ki67 staining evaluation chart of paraffin section of lung tissue after two weeks of drug treatment; D: HE staining of paraffin section of lung tissue after two weeks of drug treatment (Hematoxylin-eosin staining) Statistical graph of relative tumor area (left) and statistical graph of Ki67-positive cells (right).

Figure 11 shows that carfilzomib can significantly improve the treatment of PD1 small molecule inhibitor MB on primary lung cancer of EGFR-TD mice.

Among them, A: CT effects of carfilzomib combined with MB synergistic therapy on the inhibition of primary lung cancer tumors in the CC10-RTTA/EGFR-TD transgenic mouse model; B: statistics of the relative tumor infiltration area in the lung tissue of mice before and after treatment Figure; C: HE staining (hematoxylin-eosin staining) evaluation chart of paraffin section of lung tissue after two weeks of drug treatment; D: after two weeks of drug treatment, HE staining of paraffin section of lung tissue (hematoxylin-eosin staining) Statistical graph of eosin staining) relative to tumor area.

Detailed ways

The present invention will be further described in detail below in conjunction with experimental examples and accompanying drawings, but the implementation of the present invention is not limited thereto.

In the following experimental examples, Carfilzomib (carfilzomib), Bortezomib (bortezomib), and MLN9708 (ixazomib) are all proteasome inhibitors; PD1-Ab (inhibitory PD1 antibody), methylene blue (abbreviation: MB, PD1 Small molecule inhibitors) are inhibitors of the PD1 pathway, which can block the signaling pathway of PD1, relieve immunosuppression, and activate the immune system.

Example 1

A combined drug for treating tumors provided by the present invention is composed of carfilzomib and PD1 antibody at a mass ratio of 3:10.

More specifically, the carfilzomib dosage is 3 mg/kg/time, and the inhibitory PD1 antibody dosage is 10 mg/kg/time. The administration frequency of carfilzomib is: 3 times a week, and the administration frequency of PD1 antibody is once every 2 days.

Example 2

A combination drug for treating tumors provided by the present invention is composed of carfilzomib and PD1 small molecule inhibitor according to the mass ratio of 3:20.

More specifically, the dosage of carfilzomib is 3 mg/kg/time, the dosage of PD1 small molecule inhibitor is 20 mg/kg/time, and the frequency of administration of carfilzomib is: every The administration was given three times a week, and the administration frequency of the PD1 small molecule inhibitor was once a day.

Example 3

The combined drug for treating tumors provided by the present invention is composed of carfilzomib and PDL1 small molecule inhibitor according to the mass ratio of 1:10.

More specifically, the dosage of carfilzomib is 3 mg/kg/time, and the dosage of PDL1 antibody is 12 mg/kg/time; the frequency of administration of carfilzomib is: 3 mg/kg/time per week. The frequency of PDL1 antibody administration was once every 2 days.

Example 4

A combination drug for treating tumors provided by the invention is composed of carfilzomib and PDL1 inhibitory antibody at a mass ratio of 1:4.

More specifically, the dosage of carfilzomib is 3 mg/kg/time, and the dosage of PDL1 small molecule inhibitor is 30 mg/kg/time. The administration frequency of carfilzomib is: 3 times a week, and the administration frequency of PDL1 small molecule inhibitor is 1 time a day.

Experimental example 1

Carfilzomib can significantly induce the transformation of M2 macrophages into M1 macrophages in vitro.

1. Experimental method

(A) Obtaining PBMCs from human peripheral blood mononuclear cells: extract human peripheral blood into a heparin anticoagulant tube, shake it gently, add an equal volume of PBS to dilute the blood, and mix Ficoll (lymphocyte separation medium, the amount of Ficoll with The volume of blood before dilution is 1:1) into a new centrifuge tube in advance, tilt the centrifuge tube at 45 degrees, and slowly add the diluted blood onto the Ficoll along the tube wall. Centrifuge at room temperature at 2000rpm for 20 minutes. After centrifugation, it will be divided into plasma layer, mononuclear cell layer (cloud layer), stratified liquid layer, red blood cell layer and granulocyte layer from top to bottom after centrifugation. Gently suck out the cloud layer and transfer it to a new centrifuge tube, then add PBS (at least 5 times the volume of the suctioned cloud layer), centrifuge at 2000rpm for 10 minutes, repeat 2-3 times.

(B) Inducing PBMCs to differentiate into mature macrophages: the isolated PBMCs were centrifuged at 220 RCF for 8 minutes at 4°C, resuspended in PBS, and counted. After centrifugation again, PBMCs were resuspended in DMEM complete medium (containing 10% FBS and 1% penicillin and streptomycin) at a density of 5×10^6 cells/ml. Add the cell dilution solution to a 24-well cell culture plate, add 1ml to each well, and culture in an incubator for 1-2 hours. Then use serum-free DMEM twice to wash away unattached cells (leaving monocytes), and finally add 1 ml of DMEM complete medium containing M-CSF (50 ng/ml) to each well and culture for 7 days to induce the formation of After mature macrophages, IL-4 (20ng/ml) was added for 24 hours to induce macrophages to differentiate into M2 macrophages.

(C) Drug-treated macrophages: PBMCs-derived macrophages were divided into five groups, namely Mock control group, IL-4 (20ng/ml) treatment group, and IL-4 pretreatment for 24 hours before adding Carfilzomib, Bortezomib, MLN9708 treatment group.

(D) Detection of expression of M1 or M2 macrophage markers by real-time fluorescent quantitative PCR: cells in method (C) were treated with drugs (Carfilzomib (1 μM), Bortezomib (1 μM), MLN9708 (2 μM)) for 6 hours, Collected for total RNA extraction and reverse transcription into cDNA, followed by detection of M1 macrophage markers (IL-1β, IL-6, TNFα) and M2 macrophage markers (IL-10, TGF-β) of expression.

(E) Enzyme-linked immunosorbent assay (ELISA) secretion of cytokines IL-1β and IL-6: method (C) cells in drug treatment (Carfilzomib (500nM), Bortezomib (500nM), MLN9708 (500nM)) After 24 hours, the cell culture supernatant was collected, and after filtering the cell debris, it was used for ELISA to detect the secretion of IL-1β and IL-6. For the specific experimental method, refer to the description of the kit (Novus Biologicals, VAL601 & VAL604).

(F) Flow cytometry detection of M1 or M2 macrophage surface membrane protein expression: cells in method (C) were treated with drugs (Carfilzomib (500nM), Bortezomib (500nM), MLN9708 (500nM)) for 12 hours, Gently scrape the cells with a cell scraper and collect them by centrifugation. After washing once with PBS, add an equal amount of pre-diluted flow cytometry antibody to each group of samples, incubate on ice in the dark for 15 minutes, and wash twice with pre-cooled PBS (centrifuge at 1300rpm for 3 minutes) Discard the supernatant), add an appropriate amount of PBS to resuspend the cells, and detect the proportion of CD80 ⁺ or CD206 ⁺ cells in CD11B ⁺ cells by FACS.

(G) Flow cytometry detects the phagocytic ability of macrophages: the cells in the method (C) are starved for 2 hours after drug treatment (Carfilzomib (500nM), Bortezomib (500nM), MLN9708 (500nM)) for 12 hours. Incubate with L1210-GFP target cells for 2 hours in serum-free medium. Wash with PBS 2-3 times to thoroughly wash away unbound target cells, then scrape gently with a cell scraper and collect the cells, centrifuge at 1300rpm for 3 minutes, discard the supernatant, wash with PBS once, add an equal amount of pre-diluted samples to each group A good CD11B flow cytometry antibody coupled with APC fluorescence (1:100), incubated on ice in the dark for 15 minutes, washed twice with pre-cooled PBS, resuspended cells in PBS, and detected the CD11B ⁺ cell population by flow cytometry The percentage of GFP-positive cells was used to characterize the phagocytosis efficiency of macrophages.

2. Experimental results and analysis

In macrophages derived from PBMCs, real-time fluorescent quantitative PCR detection found that compared with IL-4 alone treatment (ie, M2 macrophages), adding Carfilzomib on the basis of IL-4-induced M2 macrophages, Bortezomib or MLN9708 treatment can significantly up-regulate the mRNA levels of M1 macrophage markers IL-1β, IL-6, and TNFα, while down-regulate the mRNA levels of M2 macrophage markers IL-10 and TGF-β (Fig. 1A). ELISA detection found that, compared with IL-4 alone treatment group, on the basis of IL-4-induced M2 macrophages plus Carfilzomib treatment, PBMCs-derived macrophages could secrete pro-inflammatory cytokines IL-1β and IL-6 were significantly increased (Fig. 1B). Through flow cytometry, it was found that compared with IL-4 alone treatment group, on the basis of IL-4-induced M2 macrophages, plus Carfilzomib-treated macrophages derived from PBMCs, the proportion of CD80 ⁺ cells could be significantly increased. increased (Fig. 1C), on the contrary, Carfilzomib treatment significantly inhibited the increase of CD206 ⁺ cells promoted by IL-4 (Fig. 1D), indicating that Carfilzomib can significantly promote the expression of surface membrane proteins of M1 macrophages and inhibit the expression of M2 macrophages Cell surface membrane protein expression. At the same time, in the phagocytosis experiment, Carfilzomib can increase the proportion of GFP-positive macrophages, indicating that it can promote the phagocytosis efficiency of macrophages (Fig. 1E).

Experimental example 2

Carfilzomib can significantly enhance the division of T cells induced by M2 macrophages in vitro.

1. Experimental method

(A) Isolation of bone marrow cells from mice: Take adult mice about 8 weeks old, kill them by cervical dislocation, soak them in 75% ethanol for 2 minutes, take them out, dissect the hind legs of the mice, remove The coat and most of the muscles were soaked in 75% ethanol for 10 minutes, then the leg bones were taken out, placed in PBS, and rinsed with PBS 2-3 times to remove residual ethanol. Use scissors and tweezers to separate the femur and tibia, and then peel off the articular cartilage at one end of the femur and tibia to expose the cross section, and cut the other end with scissors. Use a 10ml syringe to draw the PBS containing 1% serum and 1% double antibody prepared in advance, wash the bone marrow cells out of the bone cavity of the leg, and collect them into a centrifuge tube. Centrifuge at 1500rpm for 3min, discard the supernatant, add erythrocyte lysate (#NH4CL2009, TBD) to resuspend the cells according to 5 times the volume of the cells, blow and resuspend the cells, lyse for 5min, add 10 times the volume of PBS to dilute, centrifuge at 1500rpm for 3min, discard the supernatant. After adding PBS to resuspend, filter with a 40 μm cell mesh to remove broken tissue pieces, centrifuge at 1500 rpm for 3 min, and discard the supernatant. Then centrifuge with PBS1500rpm for 3min and wash 1-2 times to fully wash away the residual erythrocyte lysate to obtain the mouse bone marrow cell suspension.

(B) Inducing mouse bone marrow cells to differentiate into mature macrophages: add RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibody to resuspend the isolated mouse bone marrow cells, count them, and divide 1×10 ⁷ cells were seeded in a 10cm cell dish, and then 20%-30% of the total volume was added to the supernatant secreted by L929 cells (containing growth factor G-MCSF). After 3-5 days, the cells will differentiate into mature bone marrow. macrophages (BMDM).

(C) Drug treatment of BMDMs: BMDMs were divided into three groups, namely Mock control group, IL-4 (20ng/ml) treatment group, and IL-4 (20ng/ml) pretreatment for 12hours plus Carfilzomib (500nM) treatment group .

(D) Isolation of spleen lymphocytes: from OT-I or OT-II TCR transgenic mice (CD8 ⁺ T cells of OT-I mice can specifically recognize OVA _257-264 peptide, CD4 ⁺ T cells of OT-II mice The cells can specifically recognize the OVA _323-339 peptide) to separate the spleen tissue, grind it with a cell mesh, collect the cells ground from the tissue, and lyse the red blood cells and wash them according to the method (A) to obtain Mouse spleen cell suspension. Then add 5 μM CFSE and stain at room temperature for 5-10 minutes in the dark, then add complete medium and wash twice to terminate the staining and remove unbound CFSE fluorescent dye.

(E) OVA _257-264 peptide or OVA _323-339 peptide (Ovalbumin referred to as OVA, chicken ovalbumin, OVA injected into mice will trigger an immune response) was transferred to BMDMs treated by method (C) at a concentration of 10 μg/mL , and incubated for 1 hour. Then the BMDMs were fully washed with normal temperature PBS to remove unbound OVA, and then incubated with CFSE-labeled OT-I or OT-II cells respectively. In the environment where macrophages and T cells are co-incubated, 10ng/ml IL-2 (PeproTech) and β-mercaptoethanol should be added to the medium to maintain the survival of T cells. The CD8 or CD4 antibody was used to stain OT-I or OT-II cells. The staining method was referred to the method (F) in Experimental Example 1. Finally, the change of CFSE fluorescence intensity was detected by flow cytometry.

2. Experimental results and analysis

From the results of flow cytometry, it was found that compared with the macrophages treated with IL-4 alone (ie, M2 macrophages), adding Carfilzomib-induced macrophages on the basis of IL-4 induction for 12 hours could make The CFSE fluorescence of T cells co-incubated with it was weakened (Figure 2), indicating that carfilzomib enhanced the antigen presentation ability of macrophages, enabling them to present the captured OVA antigen to T cells that can specifically recognize, thereby promoting Dividing capacity of CD8 ⁺ and CD4 ⁺ T cells.

Experimental example 3

Carfilzomib can significantly induce the transformation of M2 macrophages in the tumor microenvironment into M1 macrophages in vivo, and increase the infiltration and activation of T cells.

1. Experimental method

(A) Formation of tumor in situ of lung cancer in transgenic mice induced with CC10RTTA-EGFR cancer driver gene mutation: In this experiment, the deletion of exon 19 of the EGFR gene and the mutation of threonine at position 790 to formazan were used Thionine transgenic mice (hereinafter referred to as TD mice) were used for drug treatment. When the transgenic TD mice were four weeks old, they were fed with doxycycline grains, and after three months of feeding with doxycycline grains, tumors could form in the lungs of TD mice.

(B) Drug-treated mice: Lung cancer mice were randomly divided into a control group (Vehicle) and a Carfilzomib treatment group (Carfil.) (3 mg/kg dose was administered by tail vein injection each time). A two-week dosing treatment was carried out simultaneously.

(C) Preparation of single cell suspension from mouse lung tissue: After two weeks of treatment, the mice were euthanized, and the mice were dissected to separate the lung tissue from the thorax. Lung tissue isolated from mice was then ground on a cell mesh. Collect the cell suspension and use the erythrocyte lysate to destroy the erythrocytes, filter the lysed cell suspension with a cell mesh, and then add PBS to wash 2-3 times to prepare a single cell suspension.

(D) Changes of immune cells infiltrated in the lung tissue of lung cancer mice detected by flow cytometry: CD45-FITC (BioLegend, 103108), CD11B-APC (eBioscience, 17-0112-81), F4 coupled with different fluoresceins /80-BV650 (BD Biosciences, 743280) and CD80-PE (Biolegend, 104707) or CD206-PE (Biolegend, 12-2061-80) antibodies were mixed according to the recommended ratio (in this example, different flow antibodies were diluted in PBS containing 1% BSA at a dilution ratio of 1:100), and this antibody combination was used to analyze the infiltrating macrophages in mouse lung tissue; or CD45-FITC, CD3-APC-cy ^TM 7 ( BD Biosciences, 557596), CD8a-PerCP-Cyanin5.5 (eBioscience, 45-0081-82), CD69-PE (BioLegend, 104507) antibodies were mixed according to the recommended ratio (in this experimental example, CD45, CD3, CD8a were mixed according to 1:100 dilution, CD69 was diluted 1:50 in PBS containing 1% BSA), this antibody combination was used to analyze the infiltrating CD8 ⁺ T cells in mouse lung tissue. The dyeing step refers to the method (F) in Experimental Example 1, and then the flow detection is performed.

2. Experimental results and analysis

Flow cytometry detection of infiltrating lymphocytes in lung tissue of TD lung cancer mice found that compared with Vehicle group mice, there were more CD80-positive macrophages and CD206-positive macrophages in the lung tissue of Carfilzomib-treated mice It was also found that there were more CD8 ⁺ T cells in the lung tissue of Carfilzomib-treated mice, and the expression of CD69 in CD8 ⁺ T cells was increased (Figure 3B). It shows that the treatment of EGFR-TD mutant lung cancer mice with Carfilzomib can promote the infiltration of M1 macrophages in the tumor focus, reduce the infiltration of M2 macrophages, and can also promote the infiltration of CD8+ T cells in the tumor focus. infiltration and activation.

Experimental example 4

Carfilzomib can slightly inhibit the growth of transplanted tumors of lung cancer cells in mice.

1. Experimental method

(A) Establishment of mouse xenograft tumor model: resuspend the prepared mouse lung cancer cell TC-1 in a mixture of PBS and Matrigel (CORNING, 356237) at a ratio of 1:1, so that every 100 μL of the mixture Containing 5×10^6 cells, the tumor cell gel mixture was implanted into the flank of 6-week-old C57BL/6 mice, and a volume of 100 μL of cell suspension was injected into each implantation point.

(B) Drug treatment: when the tumor volume reached about 80 mm ³ , the mice were randomly divided into a control group (vehicle) and a Carfilzomib treatment group, and the mice in the two groups were given drugs simultaneously.

(C) After 2 weeks of treatment, the tumor-bearing mice were euthanized, the transplanted tumors were dissected, and the tumor weights were recorded and photographed.

2. Experimental results and analysis

Compared with the control group (vehicle), the growth of xenograft tumors in mice treated with Carfilzomib was inhibited, and the tumor volume and weight were smaller (Figure 4). Therefore, Carfilzomib has a slight inhibitory effect on the growth of subcutaneous xenografts of lung cancer cells TC-1 in mice.

Experimental example 5

Inhibitory PD1-Ab has little effect on the growth of mouse lung cancer cell xenografts.

1. Experimental method

The experimental method refers to Experimental Example 4, the difference is that PD1-Ab is used instead of Carfilzomib, and intraperitoneal injection is performed, and the dose of each administration is 10 mg/kg.

2. Experimental results and analysis

The growth trend of the xenograft tumor in the mice treated with PD1-Ab, the volume and weight of the tumor were not compared with those of the control group.

Significant difference (Figure 5). Therefore, PD1-Ab blocking therapy has no significant inhibitory effect on the growth of mouse lung cancer cell TC-1 subcutaneously transplanted tumors.

Experimental example 6

Carfilzomib combined with inhibitory PD1-Ab therapy can significantly inhibit the growth of transplanted tumors of lung cancer cells in mice, and greatly enhance the therapeutic effect of PD1-Ab on solid tumors in mice.

1. Experimental method

The experimental method refers to Experimental Example 4, the difference is that PD1-Ab is added for combined administration.

2. Experimental results and analysis

Compared with the control group (vehicle), the transplanted tumor volume and weight of the mice treated with Carfilzomib combined with PD1-Ab were significantly higher.

become smaller and slower in growth rate (Figure 6). Therefore, carfilzomib combined with PD1-Ab treatment can significantly inhibit the growth of mouse lung cancer cell TC-1 subcutaneously transplanted tumors, indicating that carfilzomib can enhance the therapeutic effect of PD1-Ab on mouse solid tumors.

Experimental example 7

PD1 Pathway Inhibitors: Small molecule inhibitors of PD1 (MB) can slightly inhibit the growth of lung cancer cell xenografts in mice.

1. Experimental method

The experimental method refers to Experimental Example 4. The difference is that MB is used instead of Carfilzomib for intragastric administration, and the dose for each administration is 30mg/kg.

2. Experimental results and analysis

Compared with the control group (vehicle), the transplanted tumor volume and weight of the mice in the MB treatment group were slightly smaller, and the growth rate was slightly slower ( FIG. 7 ). Therefore, treatment with a small molecule inhibitor of PD1 (MB) slightly inhibited the growth of mouse lung cancer cell xenografts TC-1 subcutaneously.

Experimental example 8

Carfilzomib combined with MB treatment can significantly inhibit the growth of transplanted tumors of lung cancer cells in mice, and greatly enhance the therapeutic effect of PD1 small molecule inhibitors on solid tumors in mice.

1. Experimental method

The experimental method refers to Experimental Example 4, the difference is that the PD1 small molecule inhibitor (MB) is added for combined administration.

2. Experimental results and analysis

Compared with the control group, the transplanted tumor volume and weight of the mice treated with Carfilzomib combined with MB were significantly

become smaller and grow slower (Figure 8). Therefore, carfilzomib combined with MB treatment can significantly inhibit the growth of mouse lung cancer cell TC-1 subcutaneously transplanted tumors, indicating that carfilzomib can enhance the therapeutic effect of PD1 small molecule inhibitor MB on mouse solid tumors.

Experimental example 9

Carfilzomib can enhance the therapeutic effect of PD1 pathway inhibitors on mouse lung cancer cell xenografts.

1. Experimental method

The experimental method is combined with experimental examples 4, 5, and 6, or combined with experimental examples 4, 7, and 8.

2. Experimental results and analysis

Compared with the control group, PD1 antibody treatment alone had no significant inhibitory effect on the growth of transplanted tumors in mice, and carfilzomib alone had a partial inhibitory effect on the growth of transplanted tumors in mice, while carfilzomib and PD1 -Ab combined administration treatment can significantly inhibit the growth of mouse lung cancer cell TC-1 subcutaneously transplanted tumor (Figure 9A&B). Compared with the control group, the single treatment of PD1 small molecule inhibitor MB has a weak inhibitory effect on the growth of transplanted tumors in mice, while the combined administration of carfilzomib and MB can significantly inhibit the growth of mouse lung cancer cell TC. Growth of -1 subcutaneous xenografts (Fig. 9C&D).

Experiment 10

Carfilzomib can significantly improve the PD1-Ab treatment of primary lung cancer tumors in EGFR-TD transgenic mice.

The mouse xenograft tumor model can easily observe the growth of the tumor and monitor the size of the tumor in evaluating the therapeutic effect of the drug. The mouse xenograft tumor model has a single system and can clearly explain the problem. However, the xenograft tumor model in mice cannot well simulate the complicated process of lung cancer onset, carcinogenesis, and progression. Therefore, the ability to predict the therapeutic effect of the drug in the patient is poor. To this end, the present invention further utilizes the primary tumor model of lung cancer in transgenic mice with CC10RTTA-EGFR cancer driver gene mutation to evaluate the therapeutic effect of carfilzomib, PD1-Ab and MB on tumor in situ of lung cancer.

1. Experimental method

(A) Formation of lung cancer in situ tumors in transgenic mice induced with mutations in the EGFR-TD cancer driver gene: experimental approach

The method refers to the method (A) of Experimental Example 3. After feeding the mice with doxycycline for four weeks, the size and severity of the tumors were recorded by computer tomography (CT).

(B) Drug-treated mice: the mice with lung cancer were randomly divided into Vehicle group, PD1-Ab treatment group (peritoneal injection, each administration dose was 10 mg/kg), Carfilzomib treatment group (tail vein injection Administration, each administration dose is 3mg/kg) and Carfilzomib combined with PD1-Ab treatment group. Two weeks of drug treatment were performed simultaneously, and after two weeks of treatment, CT scans were performed on the mice again to monitor the changes in the lung tumors of the mice.

(C) Perform digital statistics and analysis (ImageJ software) on the shadowed part (tumor) of the lungs of the CT scan mouse, to compare the Vehicle group, PD1-Ab monotherapy group, carfilzomib monotherapy group and carfil The curative effect of Zomi and PD1-Ab combination therapy group was evaluated.

(D) Preparation of paraffin sections of mouse lung tissue: euthanize the mice after the above treatment, dissect the mice to expose the chest cavity, and perfuse 10% formalin into the lungs from the trachea to make the lungs full Then separate the lung tissue from the thorax, soak it in 10% formalin fixative solution, place it on a shaker for 24 hours, take out the fixed lung tissue, separate the lung lobe and put it in the immunohistochemical clip , followed by immersion in 70% ethanol, 80% ethanol, 90% ethanol for 30 minutes. Then the lung tissue was immersed in anhydrous ethanol:xylene mixture (1:1), xylene Ⅰ, and xylene Ⅱ for 15 minutes, and then the tissue was placed in a paraffin embedding machine for embedding. The embedded paraffin tissue block is fixed on a microtome, sliced at a thickness of 3-5 microns, spread in warm water, pasted on a glass slide, and dried.

(E) Perform hematoxylin and eosin staining (HE staining) on paraffin sections: place the paraffin sections in an oven at 60°C for one hour, (a) dewax the tissue sections: xylene Ⅰ (5 minutes), xylene Ⅱ (5 minutes) minute). (b) Gradient rehydration of tissue slices: absolute ethanol I (1 minute), absolute ethanol II (1 minute), 90% ethanol (1 minute), 80% ethanol (1 minute), 70% ethanol (1 minute) minutes), soak in tap water (1 minute). (c) Stain the tissue slices: hematoxylin staining (8 minutes), tap water rinse (1 minute), 1% hydrochloric acid alcohol rinse 2-3 times, tap water rinse 1 minute, 1% ammonia water rinse twice, tap water rinse 1 minutes, eosin staining for 2 minutes, and tap water for 1 minute. (d) Gradient dehydration of the tissue slices: put the tissue slices in 75% ethanol, 85% ethanol, 95% ethanol, and absolute ethanol in sequence, wash twice each, and then dehydrate in absolute ethanol for 2 minutes. (e) Transparency treatment of tissue slices: Xylene I for 1 minute, Xylene II for 1 minute. (f) Finally, seal the slides with neutral gum, let the gum dry naturally, and then conduct pathological observation under the microscope.

(F) Perform digital statistics and analysis (ImageJ software) on the above-mentioned microscopically photographed tissue sections to compare the Vehicle group, carfilzomib monotherapy group, PD1-Ab monotherapy group, and carfilzomib and PD1- The efficacy of the drugs in the Ab combination therapy group was evaluated.

2. Experimental results and analysis

After two weeks of treatment, using CT to scan the lung tumors of mice in different treatment groups, it was found that compared with the mice in the Vehicle group (the tumor area increased by about 25% compared with that before treatment), the lung tumors of the mice in the PD1-Ab treatment group The tumor area did not shrink but slightly increased. The lung tumor area of the mice treated with Carfilzomib was reduced by about 70% compared with that before treatment, and the tumor area of the lung cancer mice in the Carfilzomib combined with PD1-Ab treatment group could further make the lung tumor regress (the tumor area of the lung cancer mice was less than About 90% reduction before treatment) (Figure 10A&B). And from the HE-stained photos of pathological sections, the results consistent with CT scans were also obtained (Fig. 10C&D). Therefore, the therapeutic effect of the orthotopic tumor model further demonstrates that Carfilzomib combined with PD1-Ab has a more significant therapeutic effect on CC10RTTA-EGFR mutant transgenic mouse lung cancer.

Experiment 11

Carfilzomib can significantly improve the treatment of PD1 small molecule inhibitor methylene blue (referred to as MB) on primary lung cancer tumors in EGFR-TD transgenic mice.

1. Experimental method

The experimental method refers to Experimental Example 10, the difference is that PD1-Ab is replaced by PD1 small molecule inhibitor MB.

2. Experimental results and analysis

After two weeks of treatment, using CT to scan the lung tumors of mice in different treatment groups, it was found that compared with the mice in the Vehicle group (the tumor area increased by about 25% compared with that before treatment), the tumor area in the lungs of the mice in the MB treatment group increased by about 25%. Without shrinkage, the lung tumor area of mice treated with Carfilzomib was reduced by about 70% compared with that before treatment, while the group treated with Carfilzomib combined with MB could further cause lung tumors to regress (the tumor area of mice with lung cancer was reduced by about 85% compared with that before treatment) ( Figure 11A & B). And from the HE stained photos of the pathological sections, the results consistent with the CT scan were also obtained (Fig. 11C&D). Therefore, the therapeutic effect of the orthotopic tumor model further illustrates that Carfilzomib combined with MB has a more significant therapeutic effect on CC10RTTA-EGFR mutant transgenic mouse lung cancer.

The above-mentioned experimental example is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned experimental example, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (2)

1. A combination for the treatment of a tumor, characterized in that it consists of carfilzomib and methylene blue; wherein, the mass ratio of the carfilzomib to the methylene blue is 3:10 or 3:20, a step of;

the tumor is in-situ lung cancer or metastatic lung cancer.

2. The combination for treating tumors of claim 1, which is characterized in that the dose of carfilzomib is 3 mg/kg/time and the dose of methylene blue is 10 mg/kg/time; or carfilzomib was administered at a dose of 3 mg/kg/time and methylene blue was administered at a dose of 20 mg/kg/time.

Images