CN113750215B

CN113750215B - Combination for the treatment of tumors

Info

Publication number: CN113750215B
Application number: CN202111125450.XA
Authority: CN
Inventors: 陈良; 周倩; 梁锦霞
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2023-06-23
Anticipated expiration: 2041-09-24
Also published as: CN113750215A

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 for the treatment of tumors

Technical Field

The invention relates to a combined medicine for treating tumors, and belongs to the technical field of medicines.

Background

Cancer is the second leading cause of death in patients, with at least 1 million people dying from cancer annually, with about 17% of all deaths worldwide being caused by cancer. According to the latest statistics in 2020, 180 ten thousand lung cancer patients, 93.5 ten thousand colorectal cancer patients and rectal cancer patients, 83 ten thousand liver cancer patients die, and the like. At present, the treatment means of the cancer mainly comprise surgical excision, chemotherapy, radiotherapy, immunotherapy, targeted therapy and the like, but patients in the late stage lose the opportunity of surgical treatment, and the chemotherapy and the radiotherapy cannot meet the treatment requirements of the patients in the late stage cancer. Targeted treatment of tumors plays a very important role in clinical treatment of cancer, for example gefitinib and erlotinib can prolong the treatment of EGFR gene mutation-driven cancer, and to a certain extent, the survival of patients. However, some patients are also insensitive to targeted therapy, or after a short treatment, begin to be effective until they become resistant to the drug at a later stage. Development of new and effective treatments is an urgent task in the clinic of tumors.

Since 2013, cancer immunotherapy research has made significant progress, and has become an indispensable treatment for advanced cancer. Unlike traditional cancer treatment approaches, inhibitors of immune checkpoint programmed death receptor 1 (pd1) can block the binding of PD1 to programmed death ligand 1 (pdl 1), thereby relieving the inhibition of effector T cells and memory T cells, and simultaneously down regulating the differentiation of regulatory T lymphocytes and depleting T cells, thereby exerting a killing effect on tumors by the immune system of the body itself. Of interest are: the PD-1/PDL1 inhibitor is successfully applied to clinical treatment of tumors, so that the survival time of some cancer patients is successfully prolonged, and more treatment options are provided for advanced cancer patients.

However, the tumor microenvironment is intricate and various immune processes are mutually affected, so that immunotherapy of lung cancer faces a plurality of problems in clinical transformation. For example, only about 20% of lung cancer patients respond clinically to PD-1 antibody inhibitors. Therefore, there is a great clinical need for potentiators of PD-1 drugs.

Recent studies have found that 50% of the total weight in tumor tissue is macrophages, which secrete IL-10, TGF-beta, arginase, and surface-expressed molecules such as CD206, CD163, and the like, referred to as tumor associated macrophages (TAM, tumor associated macrophage). TAM inhibits the function of T cells by secreting anti-inflammatory and anticancer cytokines, prevents the T cells from attacking tumor cells, forms an immunosuppressive tumor microenvironment, further limits the curative effect of the immune checkpoint PD1 inhibitor, and finally leads to the growth, metastasis and diffusion of tumor cells. At present, antibodies of CFS1R are used for killing TAM in tumors in clinic, but the therapeutic effect of the drug in most tumor patients is not obvious.

In contrast to TAM, macrophages can induce differentiation into M1 type macrophages expressing factors such as IL-1 beta and IL-6 under the action of specific cytokines, and the macrophages can inhibit tumor growth. Therefore, there is a great clinical need to develop drugs that target TAM in the tumor microenvironment and differentiate it into M1-type macrophage transformation, and to treat patients with inhibitors of PD1 in combination with the release of tumor heterogeneity.

Disclosure of Invention

Based on the outstanding problem that some cancer patients are resistant to targeted therapy and do not respond well to immune checkpoint inhibitor immunotherapy at present, the invention aims to provide a drug for combined therapy of activating tumor microenvironment and treating tumor patients.

Therefore, the technical scheme provided by the invention is as follows:

a combination for the treatment of cancer, consisting essentially of a proteasome inhibitor, in particular Carfilzomib (carf.) and a PD1 pathway inhibitor, which is a PD1 inhibitor or an inhibitor of PDL 1. Wherein: the PD1 inhibitor is an inhibitory type PD1 antibody (PD 1-anti, PD1-Ab for short) or a small molecule inhibitor of PD1, and the PDL1 inhibitor is an inhibitory type PDL1 antibody or a PDL1 small molecule inhibitor.

Wherein: carfilzomib, proteasome inhibitor, molecular formula: C40H57N5O7, molecular weight: 719.91g/mol, it is currently believed that activation of tumor cell apoptosis pathways is induced primarily by inhibition of proteasome function, thereby leading to tumor cell apoptosis. The PD1 pathway inhibitor is a PD1 inhibitor or a PDL1 inhibitor, wherein: PD1 inhibitor is an inhibitory PD1 antibody (PD 1-anti, PD1-Ab for short) or a small molecule inhibitor of PD1, and PDL1 inhibitor is an inhibitory PDL1 antibody or a small molecule inhibitor of PDL1, which can release the inhibition function on T cells by activating the immune system, thereby treating tumors. Wherein, the inhibition type PD1 antibody or PDL1 antibody can recognize PD1 protein or PDL1 protein and block the interaction of PD1-PDL1 so as to relieve immunosuppression; small molecule inhibitors of PD1 or PDL1 act by inhibiting the function of PD-1 or PDL1, respectively, thereby promoting activated T cells.

Furthermore, the combination for treating tumors mainly comprises a proteasome inhibitor, particularly carfilzomib and a PD1 pathway inhibitor.

Further, the PD1 pathway inhibitor is a PD1 inhibitor or a PDL1 inhibitor, which is a combination for treating tumors.

Furthermore, the PD1 inhibitor is an inhibitory PD1 antibody or a small molecule inhibitor.

Furthermore, the PDL1 inhibitor is an inhibitory type PDL1 antibody or a small molecule inhibitor.

Furthermore, the above combined medicine for treating tumor, wherein the mass ratio of the carfilzomib to the inhibitory type PD1 antibody is 3: 10.

Furthermore, the above combined medicine for treating tumor, wherein the mass ratio of carfilzomib to the small-molecule inhibitor PD1 is 3: 20.

Furthermore, the above combined medicine for treating tumor, wherein the mass ratio of the carfilzomib to the small molecule inhibitor PDL1 is 1: 10.

Furthermore, the above combination for treating tumor, wherein the mass ratio of carfilzomib to PDL1 inhibitory antibody is 1: 4.

Furthermore, the administration amount of the carfilzomib is 3 mg/kg/time, and the administration amount of the inhibitory type PD1 antibody is 10 mg/kg/time.

Furthermore, the dose of the carfilzomib is 3 mg/kg/time, and the dose of the PD1 small molecule inhibitor is 20 mg/kg/time.

Furthermore, the combined medicine for treating tumors is characterized in that the administration dose of carfilzomib is 3 mg/kg/time, and the administration dose of PDL1 antibody is 12 mg/kg/time.

Furthermore, the dosage of the carfilzomib is 3 mg/kg/time, and the dosage of the PDL1 small molecule inhibitor is 30 mg/kg/time.

Furthermore, the frequency of administration of the carfilzomib for the combination for treating tumors is as follows: the PD1 antibody was administered 3 times per week with a frequency of 1 times per day of 2.

Furthermore, the frequency of administration of the carfilzomib for the combination for treating tumors is as follows: the administration was 3 times per week, and the frequency of administration of the PD1 small molecule inhibitor was 1 time per day.

Furthermore, the frequency of administration of the carfilzomib for the combination for treating tumors is as follows: the PDL1 antibody was administered 3 times per week 1 time per day 2.

Furthermore, the frequency of administration of the carfilzomib for the combination for treating tumors is as follows: the administration was 3 times per week, with a PDL1 small molecule inhibitor administration frequency of 1 time per day.

Furthermore, the combination for treating tumors is characterized in that the carfilzomib is one of the proteasome inhibitors, but not limited to the carfilzomib.

Furthermore, the combination for treating tumors is characterized in that the tumors are lung cancer, but not limited to lung cancer.

Further, the above combination for treating tumors including carcinoma in situ and metastatic.

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

1. because TAM eliminates the response of anti-tumor T cells by over-expressing immune checkpoint ligands (such as PD-L1, PD-L2, siglec-15, etc.), and also secretes anti-inflammatory, pro-cancerous growth factors (TGFb, IL-10, etc.) or metabolically converts the tumor microenvironment, thereby nourishing the tumor cells, inhibiting the function of the T cells, limiting the efficacy and scope of immunotherapy, and ultimately leading to the growth, metastasis and spread of the tumor cells. Therefore, the technical scheme provided by the application constructs a cell model for inducing macrophages to become TAM in vitro, and utilizes the model to evaluate the influence of the proteasome inhibitor carfilzomib on the TAM phenotype. The invention discovers that the proteasome inhibitor carfilzomib can convert the cancer-promoting TAM into an anticancer M1 type macrophage in vitro and promote the division and proliferation of T cells. In vivo, the proteasome inhibitor carfilzomib can also induce TAM to become M1 type macrophages, increase infiltration and activation of T cells, and further improve the tumor inhibition type immune microenvironment.

In order to further verify the effect of proteasome on tumor treatment, the applicant utilized a dual-gene EGFR gene mutant primary mouse lung cancer model (CC 10RTTA/Tet-EGFR-T790M/DEL19, mouse model: EGFR-TD for short) and TC-1 xenograft lung cancer model to verify the effect of proteasome inhibitor carfilzomib, PD1 pathway inhibitor and proteasome inhibitor carfilzomib combined PD1 pathway inhibitor on primary lung cancer and lung cancer transplantation tumor treatment of mice. In small animal models, including lung cancer transplantation tumor models and primary tumor models of mice, it was found that the single drug proteasome inhibitor carfilzomib has a certain therapeutic effect on lung cancer of mice, the single drug PD1-Ab has substantially no effect, and the single drug PD1 small molecule inhibitor has substantially no effect, and when the proteasome inhibitor carfilzomib and the PD1 pathway inhibitor are combined, tumors of mice are almost completely resolved, including TC-1 transplantation tumor models of mice and EGFR-TD primary lung cancer models. The combined treatment of the proteasome inhibitor carfilzomib and the PD1 pathway inhibitor achieves the aim of curing the tumor.

Drawings

Figure 1 is that carfilzomib can significantly induce M2-type macrophages to turn into M1-type macrophages in vitro.

Wherein A: carfilzomib and other proteasome inhibitors (bortezomib and MLN 9708) promote M2-type macrophages to express the markers IL-1β, IL-6 and TNF- α of M1 type, while inhibiting the expression of the transcriptional levels of the M2-type markers IL-10 and TGF- β; carfilzomib promotes M2-type macrophages to secrete M1-type markers IL-1 β and IL-6; c, carfilzomib can promote M2 type macrophages to express M1 type markers CD80 on the surface of a cell membrane (left graph), and the right graph is statistics of CD80 changes; carfilzomib inhibits M2-type macrophages from expressing the marker CD206 of M2 type on the cell membrane surface (left panel), right panel is statistics of CD206 changes; e, carfilzomib can promote M2 type macrophages to phagocytose tumor cells (left image), and the right image is statistics of phagocytic efficiency change.

Figure 2 is that carfilzomib can significantly enhance M2-type macrophage induced T cell division in vitro.

Wherein A: carfilzomib can significantly enhance division of M2-type macrophages on CD8T cells in vitro (left panel), right panel is statistics of CD8T cell changes; carfilzomib significantly enhanced the division of CD4T cells by M2-type macrophages in vitro (left panel), right panel is a statistic of CD4T cell changes.

Figure 3 is that carfilzomib can significantly induce M2-type macrophages to M1-type macrophages in the tumor microenvironment in vivo and increase infiltration and activation of T cells.

Wherein: a: carfilzomib can significantly induce M2-type macrophages in tumor microenvironment to M1-type macrophages in vivo (left), right statistics of CD80 and CD206 changes; carfilzomib can significantly induce infiltration and activation of CD8T cells in tumor microenvironment in vivo (left), right statistics of CD8 and CD69 changes.

Figure 4 is that carfilzomib slightly inhibited growth of lung cancer transplants in mice.

Wherein: a: evaluation graph of carfilzomib on growth inhibition of mouse subcutaneous transplantation tumor; b, a tumor weight statistical chart; c: statistical graphs of growth curves of mice transplanted tumors after carfilzomib treatment.

FIG. 5 shows that PD1-Ab has substantially no inhibitory effect on the growth of a transplanted tumor for lung cancer in mice.

Wherein A: evaluation graph of the effect of subcutaneous engraftment tumor growth in mice; b, a tumor weight statistical chart; c: statistical graphs of growth curves of mice transplanted tumors following PD1-Ab treatment.

FIG. 6 shows that the carfilzomib-PD 1-Ab combined treatment can remarkably inhibit the growth of lung cancer transplantation tumor of mice and greatly enhance the treatment effect of the PD1-Ab on solid tumors of the mice.

Wherein A: an evaluation chart for the growth inhibition of subcutaneous transplantation tumor of mice; b, a tumor weight statistical chart; c: statistical graphs of growth curves of mice transplanted tumors following carfilzomib combination PD1-Ab treatment.

Figure 7 is that MB can slightly inhibit the growth of lung cancer transplants in mice.

Wherein: a: evaluation graph of MB versus mouse subcutaneous engraftment tumor growth inhibition; b, a tumor weight statistical chart; c: statistical graphs of growth curves of mice transplanted tumors after MB treatment.

Figure 8 is a graph showing that the carfilzomib-MB combined therapy can significantly inhibit growth of lung cancer transplantation tumor in mice, and greatly enhance therapeutic effects of PD1 small molecule inhibitors on solid tumors in mice.

Figure 9 is that carfilzomib potentiates the therapeutic effect of PD1 inhibitors (antibodies to PD1 or small molecule inhibitors) on lung cancer transplants in mice.

Wherein A: summary of statistical graphs of carfilzomib-PD 1-Ab versus weight of transplanted tumors; summarizing statistical graphs of growth curves of the graft tumors of the carfilzomib combined PD 1-Ab; c: summary of statistical plots of carfilzomib combined PD1 small molecule inhibitor MB versus weight of transplanted tumors; statistical graphs of the growth curves of the graft tumors of carfilzomib combined with the PD1 small molecule inhibitor MB.

Figure 10 is that carfilzomib can significantly enhance treatment of primary lung cancer in mice with PD1-Ab.

Wherein A: CT effect patterns of the synergistic treatment of carfilzomib and PD1-Ab on the inhibition of lung cancer primary tumors are evaluated in a CC10-RTTA/EGFR-TD transgenic mouse model; b, a statistical graph of relative infiltration areas of tumors in lung tissues before and after treatment of the mice; c: paraffin section HE staining (hematoxylin-eosin staining) and Ki67 staining evaluation plots of lung tissue after two weeks of drug treatment; two weeks after drug treatment, paraffin sections of lung tissue were HE stained (hematoxylin-eosin stained) versus tumor area statistics (left) and Ki67 positive cytostatistics (right).

Figure 11 is that carfilzomib can significantly enhance treatment of EGFR-TD mice primary lung cancer with PD1 small molecule inhibitor MB.

Wherein A: CT effect patterns of inhibition of the combined MB synergistic treatment of the carfilzomib on the primary tumor of the lung cancer are evaluated in a CC10-RTTA/EGFR-TD transgenic mouse model; b, a statistical graph of relative infiltration areas of tumors in lung tissues before and after treatment of the mice; c: evaluation chart of paraffin section HE staining (hematoxylin-eosin staining) of lung tissue after two weeks of drug treatment; two weeks after drug treatment, paraffin sections of lung tissue were HE stained (hematoxylin-eosin stained) versus tumor area statistics.

Detailed Description

The present invention will be described in further detail with reference to examples of experiments and drawings, but embodiments of the present invention are not limited thereto.

In the following experimental examples, carfilzomib (Carfilzomib), bortezomib (Bortezomib), MLN9708 (i Sha Zuo m) are proteasome inhibitors; PD1-Ab (inhibitory type PD1 antibody), methylene blue (MB, small molecule inhibitor of PD 1) is the inhibitor of PD1 pathway, which can block PD1 signal pathway, relieve immunosuppression and activate immune system.

Example 1

The invention provides a combined medicine for treating tumors, which is prepared from carfilzomib and a PD1 antibody according to a mass ratio of 3: 10.

More specifically, the carfilzomib was administered at a dose of 3 mg/kg/time, and the inhibitory type PD1 antibody was administered at a dose of 10 mg/kg/time. The administration frequency of the carfilzomib is as follows: the PD1 antibody was administered 3 times per week with a frequency of 1 times per day of 2.

Example 2

The invention provides a combined medicine for treating tumors, which is prepared from carfilzomib and a PD1 small molecule inhibitor according to a mass ratio of 3: 20.

More specifically, the dosage of carfilzomib is 3 mg/kg/time, the dosage of the PD1 small molecule inhibitor is 20 mg/kg/time, and the dosage frequency of the carfilzomib is as follows: the administration was 3 times per week, and the frequency of administration of the PD1 small molecule inhibitor was 1 time per day.

Example 3

The invention provides a combined medicine for treating tumors, which is prepared from carfilzomib and a PDL1 small molecule inhibitor according to a mass ratio of 1: 10.

More specifically, the carfilzomib dose is 3 mg/kg/time, and the PDL1 antibody dose is 12 mg/kg/time; the administration frequency of the carfilzomib is as follows: the PDL1 antibody was administered 3 times per week 1 time per day 2.

Example 4

The invention provides a combined medicine for treating tumors, which is prepared from carfilzomib and PDL1 inhibitory antibodies according to the mass ratio of 1: 4.

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

Experimental example 1

Carfilzomib can significantly induce M2-type macrophages to convert to M1-type macrophages in vitro.

1. Experimental method

(A) Human peripheral blood mononuclear cell PBMCs were obtained: the peripheral blood of a person is extracted into a heparin anticoagulation tube, after the peripheral blood is gently shaken, the equal volume of PBS is added to dilute the blood, ficoll (lymphocyte separation liquid, the volume of the Ficoll using amount and the volume of the blood before dilution are 1:1) is added into a new centrifuge tube in advance, the centrifuge tube is inclined at 45 degrees, and the diluted blood is slowly added onto the Ficoll along the tube wall. Centrifugation at 2000rpm at room temperature for 20 minutes, after centrifugation, the plasma layer, mononuclear cell layer (cloud layer), stratified liquid layer, red blood cell and granulocyte layer were separated from top to bottom. Gently aspirate the cloud and transfer to a new centrifuge tube, add PBS (at least 5 times the volume of the aspirated cloud) and centrifuge at 2000rpm for 10 minutes, repeating 2-3 times.

(B) Induction of differentiation of PBMCs into mature macrophages: PBMCs were isolated, centrifuged at 220RCF for 8 min at 4℃and resuspended in PBS for cell counting. After re-centrifugation, PBMCs were resuspended in DMEM complete medium (containing 10% FBS and 1% penicillin streptomycin) at a density of 5X 10≡6 cells/ml. The cell dilutions were added to 24-well cell culture plates, 1ml per well, and incubated in an incubator for 1-2 hours. Serum-free DMEM was then used twice to wash off non-adherent cells (leaving monocytes), and finally 1ml DMEM complete medium containing M-CSF (50 ng/ml) was added to each well to culture for 7 days, and after induction to form mature macrophages, IL-4 (20 ng/ml) was added to induce for 24 hours, and macrophage differentiation to M2 type macrophages was induced.

(C) Drug-treated macrophages: PBMCs-derived macrophages were divided into five groups, namely a Mock control group, an IL-4 (20 ng/ml) treated group, and an IL-4 pretreatment 24 hours followed by Carfilzomib, bortezomib, MLN9708 treated group, respectively.

(D) Real-time fluorescent quantitative PCR detects expression of M1 or M2 macrophage markers: the cells in method (C) were collected for extraction of total RNA and reverse transcription into cDNA after 6 hours of drug treatment (Carfilzomib (1. Mu.M), bortezomib (1. Mu.M), MLN9708 (2. Mu.M)), followed by detection of the expression levels of the M1 type macrophage marker (IL-1. Beta., IL-6, TNFα) and the M2 type macrophage marker (IL-10, TGF-. Beta.).

(E) Enzyme-linked immunosorbent assay (ELISA) secretion of cytokines IL-1 beta and IL-6: cells in method (C) were treated with drug (Carfilzomib (500 nM), bortezomib (500 nM), MLN9708 (500 nM)) for 24 hours, cell culture supernatants were collected, and after filtration of cell debris, used in ELISA to detect IL-1. Beta. And IL-6 secretion. Specific experimental methods are described with reference to the kit (Novus Biologicals, VAL601& VAL 604).

(F) Flow cytometry detects the expression of M1 or M2 type macrophage surface membrane protein: cells in method (C) were treated with drug (Carfilzomib (500 nM), bortezomib (500 nM), MLN9708 (500 nM)) for 12hours, gently scraped with a cell scraper, collected by centrifugation, washed once with PBS, added with an equal amount of pre-diluted flow-through antibody per group of samples, incubated for 15min on ice in the absence of light, pre-cooled washed twice with PBS (1300 rpm was used to centrifuge for 3min to discard supernatant), resuspended with an appropriate amount of PBS, and then FACS detected for CD11B ⁺ CD80 in cells ⁺ Or CD206 ⁺ Proportion of cells.

(G) Flow cytometry detects phagocytic capacity of macrophages: cells in method (C) were starved for 2hours after 12hours of drug treatment (Carfilzomib (500 nM), bortezomib (500 nM), MLN9708 (500 nM)) and incubated with L1210-GFP target cells in serum-free medium for 2 hours. Washing with PBS for 2-3 times to thoroughly wash unbound target cells, then gently scraping with a cell scraper and collecting cells, centrifuging at 1300rpm for 3min, discarding supernatant, washing with PBS once, adding equal amount of pre-diluted CD11B streaming antibody (1:100) coupled with APC fluorescence to each group of samples, incubating for 15min on ice in the absence of light, pre-cooling with PBS for two times, adding PBS to resuspend cells, and detecting CD11B by flow cytometry ⁺ The percentage of GFP positive cells in the cell population was used to characterize the phagocytic efficiency of macrophages.

2. Experimental results and analysis

In PBMCs-derived macrophages, real-time fluorescent quantitative PCR assays found that the addition of Carfilzomib, bortezomib or MLN9708 to IL-4-induced M2 macrophages significantly upregulated the mRNA levels of the M1 macrophage markers IL-1 beta, IL-6, TNFα, and down-regulated the mRNA levels of the M2 macrophage markers IL-10, TGF-beta, as compared to IL-4 alone (i.e., M2 macrophages) (FIG. 1A). ELISA detection revealed that the addition of PBMCs-derived macrophages after treatment with Carfilzomib on the basis of IL-4-induced M2-type macrophages significantly increased the pro-inflammatory cytokines IL-1. Beta. And IL-6 secreted from the macrophages compared to the IL-4 alone treatment group (FIG. 1B). By flow-through assays, it was found that CD80 was produced by adding additional Carfilzomib-treated PBMCs-derived macrophages to IL-4-induced M2-type macrophages as compared to the IL-4 alone-treated group ⁺ The proportion of cells was significantly increased (FIG. 1C), in contrast to significant inhibition of IL-4 promoted CD206 following Carfilzomib treatment ⁺ Cell proliferation (FIG. 1D) indicates that Carfilzomib significantly promoted M1-type macrophage surface membrane protein expression and inhibited M2-type macrophage surface membrane protein expression. Meanwhile, in phagocytosis experiments, carfilzomib increased the proportion of GFP-positive macrophages, indicating that it promoted the phagocytic efficiency of macrophages (fig. 1E).

Experimental example 2

Carfilzomib can significantly enhance M2-type macrophage induced T cell division in vitro.

1. Experimental method

(A) Bone marrow cells were isolated from mice: taking adult mice about 8 weeks old, killing the mice when cervical dislocation occurs, soaking in 75% ethanol for sterilization for 2min, taking out the mice, dissecting and separating the rear legs of the mice, removing the fur and most of the muscles, soaking in 75% ethanol for 10min, taking out the leg bones, placing in PBS, washing with PBS for 2-3 times, and removing residual ethanol. The femur and tibia are separated by scissors and forceps, and then the articular cartilage at one end of the femur and tibia is torn off, the cross section is exposed, and the other end is cut off by scissors. The PBS containing 1% serum and 1% diabody prepared in advance was aspirated by a 10ml syringe, and bone marrow cells were washed out of the leg cavity and collected in a centrifuge tube. Centrifuging at 1500rpm for 3min, discarding supernatant, adding erythrocyte lysate (#NH4CL 2009, TBD) at 5 times of the cell amount, blowing to resuspension cells, lysing for 5min, adding 10 times of PBS for dilution, centrifuging at 1500rpm for 3min, and discarding supernatant. After adding PBS for resuspension, the crushed tissue blocks are removed by filtering with a 40 μm cell screen, and the supernatant is discarded after centrifugation at 1500rpm for 3 min. And (3) centrifuging for 3min at 1500rpm with PBS (phosphate buffer solution) for 1-2 times, and fully washing out residual erythrocyte lysate to obtain the bone marrow cell suspension of the mice.

(B) Inducing differentiation of mouse bone marrow cells into mature macrophages: adding RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibody to resuspend isolated mouse bone marrow cells, counting, adding 1×10 ⁷ The individual cells were seeded in 10cm dishes and then fed with the supernatant secreted by L929 cells (containing growth factor G-MCSF) at 20% -30% of the total volume, and after 3-5 days, the cells differentiated into mature bone marrow-derived macrophages (BMDM).

(C) Drug treatment BMDMs: BMDMs were divided into three groups, namely a Mock control group, an IL-4 (20 ng/ml) treated group, and an IL-4 (20 ng/ml) pre-treatment 12hours followed by Carfilzomib (500 nM) treated group.

(D) Spleen lymphocytes were isolated: transgenic mice from OT-I or OT-II TCR (CD 8 of OT-I mice ⁺ T cells can specifically recognize OVA _257-264 Peptide fragment, CD4 of OT-II mice ⁺ T cells can specifically recognize OVA _323-339 Peptide fragment), grinding the spleen tissue by using a cell screen, collecting the cells ground from the tissue, lysing the erythrocytes and washing the erythrocytes according to the method (A), thereby obtaining the mouse spleen cell suspension. Then 5. Mu.M CFSE was added and stained at room temperature in the dark for 5-10min, and the complete medium was added and washed 2 more times to terminate the staining and remove unbound CFSE fluorochrome.

(E) OVA of _257-264 Peptides or OVA _323-339 Peptides (ovvalbumin abbreviated as OVA, ovalbumin, injected into mice elicited an immune response) were transferred to BMDMs treated in method (C) at a concentration of 10 μg/mL and incubated for 1 hour. BMDMs were then washed extensively with room temperature PBS to remove unbound OVA and incubated with CFSE labeled OT-I or OT-II cells, respectively. In the environment where macrophages are incubated with T cells, 10ng/ml IL-2 (PeproTech) and beta-mercaptoethanol are added to the medium to maintain T cell survival, after 72 hours incubation, the OT-I or OT-II cells are stained with fluorescein-conjugated CD8 or CD4 antibody, the staining method is described in Experimental example 1 (F), and the change in fluorescence intensity of CFSE is detected by flow cytometry.

2. Experimental results and analysis

From the results of the flow assay, it was found that the addition of Carfilzomib-induced macrophages after 12hours of IL-4 induction resulted in reduced CFSE fluorescence in T cells incubated therewith (FIG. 2) compared to IL-4 alone-treated macrophages (i.e., M2-type macrophages), demonstrating that Carfilzomib enhanced antigen presenting ability of macrophages, enabling presentation of captured OVA antigen to specifically recognized T cells, thereby promoting CD8 ⁺ And CD4 ⁺ Division ability of T cells.

Experimental example 3

Carfilzomib can significantly induce M2-type macrophages in tumor microenvironment to turn into M1-type macrophages in vivo and increase infiltration and activation of T cells.

1. Experimental method

(A) Transgenic mouse lung carcinoma in situ tumor formation inducing CC10RTTA-EGFR cancer driving gene mutation: in this experimental example, a transgenic mouse (hereinafter abbreviated as TD mouse) in which the 19 th exon of EGFR gene is deleted and threonine 790 st is mutated into methionine was used for drug therapy. When the transgenic TD mice are four weeks old, the mice are fed with doxycycline grains, and after the doxycycline grains are fed for three months, the lung of the TD mice can form tumors.

(B) Drug-treated mice: lung cancer mice were randomized into control (Vehicle) and Carfilzomib-treated (carfi.) (administered by tail vein injection at 3mg/kg dose each time). The dosing treatment was performed simultaneously for two weeks.

(C) Preparation of single cell suspension from mouse lung tissue: two weeks after treatment, the mice were euthanized and dissected to separate lung tissue from the chest cavity. Lung tissue isolated from mice was then ground on a cell screen. The cell suspension was collected and the erythrocytes were destroyed using an erythrocyte lysate, and the cell suspension after the lysis was filtered with a cell screen, and then washed 2-3 times with PBS to prepare a single cell suspension.

(D) Flow cytometry detects changes in infiltrating immune cells in lung tissue of lung cancer mice: CD45-FITC (BioLegend, 103108), CD11B-APC (eBioscience, 17-0112-81), F4/80-BV650 (BD Biosciences, 743280) and CD80-PE (BioLegend, 104707) or CD206-PE (BioLegend, 12-2061-80) antibodies coupled to different luciferins were mixed in the recommended ratio (in this experimental example, the different flow antibodies were diluted in PBS containing 1% BSA at a 1:100 dilution ratio), and the antibody combination was analyzed for macrophages infiltrated in mouse lung tissue; or CD45-FITC, CD3-APC-cy ^TM 7 (BD Biosciences, 557596), CD8a-PerCP-Cyanin5.5 (ebiosciences, 45-0081-82), CD69-PE (BioLegend, 104507) antibodies were mixed in the recommended ratio (CD 45, CD3, CD8a were diluted 1:100 in this experimental example, CD69 was diluted 1:50 in PBS containing 1% BSA), and this antibody combination was used to wet CD8 in mouse lung tissue ⁺ T cells were analyzed. The staining procedure was as described in Experimental example 1, method (F), followed by flow-through detection.

2. Experimental results and analysis

Flow cytometry detection of TD lungThe infiltrating lymphocytes in lung tissue of cancer mice were found to be more CD80 positive macrophages and less CD206 positive macrophages in lung tissue of Carfilzomib-treated mice compared to Vehicle group mice (FIG. 3A), while CD8 was also found in lung tissue of Carfilzomib-treated mice ⁺ T cells will be more and CD8 ⁺ Expression of CD69 in T cells increased (fig. 3B). It is demonstrated that treatment of EGFR-TD mutated lung cancer mice with Carfilzomib promotes infiltration of M1 type macrophages in the tumor foci while reducing infiltration of M2 type macrophages, and also promotes infiltration and activation of CD8+ T cells in the tumor foci.

Experimental example 4

Carfilzomib slightly inhibits growth of lung cancer cell transplants in mice.

1. Experimental method

(A) Establishment of a mouse transplantation tumor model: prepared mouse lung cancer cells TC-1 were resuspended in a 1:1 ratio of PBS to Matrigel (CORNING, 356237) so that 5X 10-6 cells were contained per 100. Mu.L of the mix, tumor cell gel mixtures were implanted into the flanks of 6 week old C57BL/6 mice, and 100. Mu.L volumes of cell suspension were injected at each implantation point.

(B) Drug treatment: when the tumor volume reaches 80mm ³ On the left and right, mice were randomized, one group was control group (vehicle), one group was Carfilzomib-treated group, and both groups of mice were dosed simultaneously.

(C) Tumor-bearing mice were euthanized 2 weeks after treatment, transplanted tumors were dissected apart, photographed and tumor weights recorded.

2. Experimental results and analysis

Compared to the control group (vehicle), the growth of the transplanted tumor was inhibited in the mice after Carfilzomib treatment, and the tumor volume and weight were smaller (fig. 4). Therefore, carfilzomib has a slight inhibitory effect on the growth of mouse lung cancer cell TC-1 subcutaneous transplantation tumor.

Experimental example 5

The inhibitory PD1-Ab has no substantial effect on the growth of mouse lung cancer cell transplants.

1. Experimental method

Experimental methods referring to Experimental example 4, except that PD1-Ab was used instead of Carfilzomib, the doses were 10mg/kg per administration by intraperitoneal injection.

2. Experimental results and analysis

The PD1-Ab treated mice had no tendency to graft tumor growth, no ratio of tumor volume to weight to control phase (vehicle)

Significant differences (fig. 5). So the PD1-Ab blocking treatment has no obvious inhibition effect on the growth of the cell TC-1 subcutaneous transplantation tumor of the lung cancer of the mice.

Experimental example 6

The carfilzomib combined inhibition type PD1-Ab treatment can obviously inhibit the growth of mouse lung cancer cell transplantation tumor, and greatly enhances the treatment effect of the PD1-Ab on mouse solid tumor.

1. Experimental method

Experimental procedure reference is made to experimental example 4, except that PD1-Ab is added for co-administration.

2. Experimental results and analysis

The transplanted tumor volume and weight of Carfilzomib-combined PD 1-Ab-treated mice were evident compared to control (vehicle)

And becomes smaller and the growth rate becomes slower (fig. 6). Therefore, the Carfilzomib combined with the PD1-Ab treatment can obviously inhibit the growth of mouse lung cancer cell TC-1 subcutaneous transplantation tumor, which indicates that the Carfilzomib can enhance the treatment effect of the PD1-Ab on the mouse solid tumor.

Experimental example 7

PD1 pathway inhibitor: small molecule inhibitors (MBs) of PD1 can slightly inhibit the growth of mouse lung cancer cell transplants.

1. Experimental method

Experimental methods reference Experimental example 4. Except that MB was used instead of Carfilzomib and administered by intragastric administration at a dose of 30mg/kg each time.

2. Experimental results and analysis

The transplanted tumor volume and weight of MB-treated mice were slightly smaller and growth rate slightly slower than control (vehicle) (fig. 7). Therefore, small molecule inhibitor (MB) treatment of PD1 has a slight inhibitory effect on the growth of mouse lung cancer cell TC-1 subcutaneous transplantation tumor.

Experimental example 8

The carfilzomib combined MB treatment can obviously inhibit the growth of the lung cancer cell transplantation tumor of the mice, and greatly improve the treatment effect of the PD1 small molecule inhibitor on the solid tumor of the mice.

1. Experimental method

Experimental procedure reference is made to experimental example 4, except that the PD1 small molecule inhibitor (MB) is added for co-administration.

2. Experimental results and analysis

The transplanted tumor volume and weight of the mice of the Carfilzomib combined MB treatment group are obvious compared with the control group

Smaller, the growth rate becomes slower (fig. 8). Therefore, the Carfilzomib combined MB treatment can obviously inhibit the growth of mouse lung cancer cell TC-1 subcutaneous transplantation tumor, which indicates that the Carfilzomib can enhance the therapeutic effect of PD1 small molecule inhibitor MB on mouse solid tumor.

Experimental example 9

Carfilzomib potentiates the therapeutic effect of PD1 pathway inhibitors on mouse lung cancer cell transplants.

1. Experimental method

Experimental methods are combined with experimental examples 4, 5, 6, or with experimental examples 4, 7, 8.

2. Experimental results and analysis

Compared with the control group, the antibody of PD1 has no obvious inhibition effect on the growth of the mouse transplanted tumor, the antibody of carfilzomib has partial inhibition effect on the growth of the mouse transplanted tumor, and the combined administration treatment of the carfilzomib and the PD1-Ab can remarkably inhibit the growth of the mouse lung cancer cell TC-1 subcutaneous transplanted tumor (figures 9A and B). Compared with the control group, the PD1 small molecule inhibitor MB singly has weak inhibition effect on the growth of mouse transplanted tumor, and the combined administration treatment of carfilzomib and MB can remarkably inhibit the growth of mouse lung cancer cell TC-1 subcutaneous transplanted tumor (figures 9C & D).

Experimental example 10

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

The mouse tumor implantation model can conveniently observe the growth of tumor and monitor the size of tumor in evaluating the therapeutic effect of the drug, and the mouse tumor implantation model has single system and can clearly describe the problems. However, the mouse tumor-transplantation model cannot well simulate the complicated onset, canceration, exacerbation and other processes of lung cancer. Thus, the ability to predict the therapeutic ability of a drug in a patient is poor. Therefore, the invention further utilizes the lung cancer primary tumor model of the transgenic mice with CC10RTTA-EGFR cancer driving gene mutation to evaluate the treatment effect of carfilzomib, PD1-Ab and MB on lung cancer in-situ tumors.

1. Experimental method

(A) Transgenic mouse lung carcinoma in situ tumor formation inducing EGFR-TD cancer driving gene mutation: experimental prescription

Method referring to experimental example 3 method (a), after the doxycycline-fed mice were kept four weeks, the size and severity of the tumor were recorded by computer tomography (Computer Tomography, CT).

(B) Drug-treated mice: mice with lung cancer were randomized into the Vehicle group, the PD1-Ab treatment group (10 mg/kg dose per intraperitoneal injection), the Carfilzomib treatment group (3 mg/kg dose per caudal intravenous injection), and the Carfilzomib-combined PD1-Ab treatment group. The administration treatment is carried out synchronously for two weeks, and after two weeks of treatment, the mice are scanned again by CT to monitor the change condition of lung tumors of the mice.

(C) The shadow (tumor) of the lung of the CT scanned mice was digitally counted and analyzed (imageJ software) to evaluate the efficacy of the drugs in the Vehicle group, the PD1-Ab single drug treatment group, the carfilzomib single drug treatment group, and the carfilzomib and PD1-Ab combination treatment group.

(D) Paraffin sections of mouse lung tissue were prepared: euthanized mice were subjected to the above treatment, the chest was anatomically exposed, 10% formalin was infused into the lungs from the trachea to inflate the lungs, then lung tissue was isolated from the chest, immersed in 10% formalin-fixed solution, fixed on a shaker for 24 hours, the fixed lung tissue was removed, and lung lobes were isolated and placed in an immunohistochemical clamp, sequentially immersed in 70% ethanol, 80% ethanol, 90% ethanol for 30 minutes. And then sequentially placing the lung tissues in absolute ethyl alcohol: the tissue is soaked in xylene mixed solution (1:1), xylene I and xylene II for 15 minutes, and then the tissue is placed in a paraffin embedding machine for embedding. The embedded paraffin tissue block is fixed on a slicing machine, slicing is carried out with the thickness of 3-5 microns, and the slice is spread in warm water, so that the slice is attached to a glass slide and dried.

(E) Paraffin sections were stained with hematoxylin eosin (HE staining): paraffin sections were left in an oven at 60 ℃ for hours, (a) deparaffinization of the tissue pieces: xylene I (5 minutes), xylene II (5 minutes). (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), tap water soak (1 minute). (c) staining the tissue pieces: hematoxylin staining (8 min), tap water rinsing (1 min), 1% hydrochloric acid alcohol rinsing 2-3 times, tap water rinsing 1 min, 1% ammonia rinsing twice, tap water rinsing 1 min, eosin staining 2min, tap water rinsing 1 min. (d) gradient dehydrating the tissue slice: sequentially adding 75% ethanol, 85% ethanol, 95% ethanol and anhydrous ethanol into tissue slices, washing for 2 times respectively, and dehydrating with anhydrous ethanol for 2 minutes. (e) transparent treatment of the tissue slice: xylene I for 1 minute and xylene II for 1 minute. (f) Finally, sealing with neutral gum, standing until the gum is naturally air-dried, and observing the pathology by a microscope.

(F) The microscopic tissue sections were subjected to digital statistics and analysis (ImageJ software) to evaluate the efficacy of the drugs in the Vehicle group, the carfilzomib single drug treatment group, the PD1-Ab single drug treatment group, and the carfilzomib and PD1-Ab combination treatment group.

2. Experimental results and analysis

After two weeks of treatment, mice from different treatment groups were scanned for lung tumors by CT and found that the lung tumor area was not reduced but slightly increased compared to Vehicle mice (approximately 25% increase in tumor area over pre-treatment), whereas Carfilzomib-treated mice had a reduction in lung tumor area of approximately 70% over pre-treatment, whereas Carfilzomib-combined PD 1-Ab-treated mice had a further regression of lung tumors (approximately 90% reduction in lung cancer mouse tumor area over pre-treatment) (FIGS. ten A & B). And results consistent with CT scan were also obtained from HE staining photographs of pathological sections (fig. 10c & d). The therapeutic effect of the orthotopic tumor model further demonstrates that the combination of Carfilzomib and PD1-Ab has more remarkable therapeutic effect on CC10RTTA-EGFR mutant transgenic mouse lung cancer.

Experimental example 11

Carfilzomib can significantly improve the treatment of primary tumors of lung cancer of EGFR-TD transgenic mice by a PD1 small molecule inhibitor methylene blue (MB for short).

1. Experimental method

Experimental procedure reference is made to experimental example 10, except that PD1-Ab is replaced with the PD1 small molecule inhibitor MB.

2. Experimental results and analysis

After two weeks of treatment, mice from different treatment groups were scanned for lung tumors and found that the MB treatment group had no reduction in lung tumor area compared to Vehicle group mice (approximately 25% increase in tumor area compared to pre-treatment), whereas the Carfilzomib treatment group had a reduction in lung tumor area of approximately 70% compared to pre-treatment, whereas the Carfilzomib combination MB treatment group had a further lung tumor regression (approximately 85% reduction in lung tumor area compared to pre-treatment) (fig. 11a & b). And results consistent with CT scan were also obtained from HE staining photographs of pathological sections (fig. 11c & d). The therapeutic effect of the orthotopic tumor model further demonstrates that the combination of Carfilzomib and MB has more remarkable therapeutic effect on CC10RTTA-EGFR mutated transgenic mouse lung cancer.

The above experimental examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above experimental examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and all the embodiments are included in the scope of the present invention.

Claims

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.