CN115381822A - Medicine for treating lung cancer based on immune response activation and pharmaceutical application - Google Patents
Medicine for treating lung cancer based on immune response activation and pharmaceutical application Download PDFInfo
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Abstract
The invention discloses a medicine for treating lung cancer based on immune response activation and pharmaceutical application thereof, wherein the medicine can cause a mouse body to carry out immunotherapy on the lung cancer by 40mg/kg of intraperitoneal injection. In vitro experiments show that compared with a control group, the killing capacity of OT-1 mouse spleen-derived cytotoxic T cells to A549-OVA lung cancer cells can be remarkably enhanced by BCP16671 treatment, and the killing capacity is caused by activating T cell co-stimulatory molecules, promoting secretion of T cells INF-gamma, IL2, perforin and the like, and enhancing T cell effector capacity. Similarly, in an animal model, the BCP16671 can inhibit the growth of A549-OVA lung cancer cell transplanted tumor after the cytotoxic T cells are returned and inhibit the progress of CC10RTTA-EGFRDEl cancer driving gene mutation in situ lung cancer, thereby achieving the purpose of treating the lung cancer of the type.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a medicine for treating lung cancer based on immune response activation and pharmaceutical application.
Background
Lung cancer is one of the highest incidence and mortality cancers worldwide. The current immunotherapy clinically applied to lung cancer treatment is a new therapy which is different from main treatment modes such as operation, radiotherapy, chemotherapy, targeted therapy and the like, and the effective treatment basis is to recover the normal anti-tumor immune response of an organism by inducing, stimulating and enhancing the autoimmune system of a human body so as to control and eliminate lung cancer.
Immune cells are known to be an important cell group in the tumor microenvironment, which is involved in the immune defense against tumors. Fully activated T cells in the immune system secrete cytokines directly or indirectly through other effector cells to attack and destroy the identified tumor cells, inhibiting tumor development. However, T cell activation requires recognition of the antigen peptide-MHC I/II complex on Antigen Presenting Cells (APCs) by the T Cell Receptor (TCR), and stimulation of cell surface costimulatory (costimulatory) signals, both of which are not preferred. Therefore, in tumor patients, drugs which effectively act on costimulatory molecules, such as CD27, CD137, OX40, and activate signals thereof can cause immune response of the body, and can effectively inhibit tumors. However, the discovery of drugs that can activate the co-stimulatory molecule signal is limited so far, and most of the research focuses on finding agonist antibodies of co-stimulatory molecules, such as CD27 agonist antibody, CD137 agonist antibody, etc., which have been proved to be effective in promoting T cell activation to achieve anti-tumor therapeutic effect.
Disclosure of Invention
Based on the lack of small molecule drugs that are effective in activating the signals of costimulatory molecules, the present invention aims to provide a compound for treating lung cancer by activating costimulatory signaling molecules, thereby eliciting an immune response. The invention screens a small molecule compound library by constructing a T cell killing system in vitro, detects T cell secretion factors by a flow cytometer, and evaluates the treatment effect of the compound on lung cancer by utilizing a mouse subcutaneous lung cancer cell transplantation tumor animal model and a CC10RTTA-EGFR Del cancer driving gene mutated transgenic mouse lung cancer orthotopic tumor model.
The purpose of the invention is realized by the following technical scheme:
application of BCP16671 shown in formula I in preparation of medicine for treating lung cancer is provided.
Preferably, the medicament for treating lung cancer is a medicament for activating immune response to treat lung cancer.
Preferably, the medicament comprises BCP16671.
Preferably, the lung cancer includes carcinoma in situ and carcinoma in transplant.
Preferably, the lung cancer is a549-OVA lung cancer cell transplant tumor or a lung cancer with CC10RTTA-EGFR Del cancer driver gene mutation.
Preferably, the lung cancer is in situ lung cancer driven by tumor transplantation based on in vitro cultured lung cancer cells or overexpression of the 19 th exon deleted oncogene EGFR induced by Dox.
Preferably, the medicament comprises at least one carrier; the carrier is at least one of a sustained release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.
Preferably, the medicament is an injection, a tablet, a granule or a capsule.
The invention finds that BCP16671 is a co-stimulatory molecule activating compound, and CTLs secrete cytokines such as INF-gamma, IL2, perforin and the like by activating T cells, so that the capacity of CTLs for killing target cells is improved, and the growth of lung cancer is inhibited. These studies suggest the potential ability of compounds with the small molecular structure of BCP16671 to treat lung cancer.
Compared with the prior art, the invention has the following advantages and effects:
the application discovers through a great deal of creative work: the BCP16671 small molecule can effectively activate T cells and play an important role in inhibiting lung cancer by activating T cell surface costimulatory molecules. In vitro experiments, the BCP16671 micromolecules cause the secretion of INF-gamma, IL2 and perforin by Cytotoxic T Cells (CTLs) to be increased, and the capability of the CTLs in killing target cells is effectively enhanced. Similarly, in an animal model, the BCP16671 small molecule can obviously inhibit the growth of A549-OVA lung cancer cell transplantation tumor of a mice transfused with CTLs, the lung cancer tumor of a CC10RTTA-EGFR Del cancer driving gene mutated transgenic mouse is obviously reduced under the action of the BCP16671 small molecule, and the small molecule achieves the purpose of treating the lung cancer.
Drawings
In order to clearly illustrate the specific embodiments of the present invention and certain detection techniques employed in the experiments, embodiments and techniques employed are described below, primarily by way of the accompanying drawings.
FIG. 1: BCP16671 activates T cell effect pattern through costimulator; jurkat NFAT luciferase, jurkat NFAT luciferase OE-CD27, jurkat NFAT luciferase OE-CD137 and Jurkat NFAT luciferase OE-OX40 cells were treated with Vehicle or BCP16671 and assayed for T cell activation following co-stimulatory factor overexpression.
FIG. 2: evaluation chart of promoting CTLs secretion cell factor by BCP 16671; wherein A is a flow result of cytokines INF-gamma, IL2 and perforin secreted by CTLs after being treated by Vehicle or BCP16671 under the stimulation of A549-OVA lung cancer cells; b is the percentage statistics of cytokine INF-gamma, IL2 and perforin secreted by CTLs treated by Vehicle or BCP16671 under the stimulation of A549-OVA lung cancer cells.
FIG. 3: BCP16671 promotes CTLs to kill A549-OVA lung cancer cell flow evaluation chart; wherein, A is a flow detection apoptosis evaluation graph of A549-OVA lung cancer cells marked by Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) after the Vehicle or BCP16671 treats CTLs; b is CFSE marked apoptosis A549-OVA lung cancer cell percentage statistical chart.
FIG. 4 is a schematic view of: BCP16671 evaluation chart of growth inhibition of subcutaneous transplanted tumor of B6 mice for reinfused CTLs; wherein A is the recorded result of the weight change of a B6 mouse treated by Vehicle or BCP16671 and returned to CTLs for 14 days; b is the recorded result of the change of the volume of the B6 mice transplanted with the Venhicle or BCP16671 to treat the reinfused CTLs for 14 days; c is a comparison graph of tumor size taken 14 days after Vehicle or BCP16671 treatment of B6 mice transfused with CTLs; d is a statistical plot of the tumor weight taken 14 days after Vehicle or BCP16671 treatment of B6 mice reinfused with CTLs.
FIG. 5 is a schematic view of: a diagram for evaluating the inhibition effect of the shown drug on primary lung cancer tumor in a CC10RTTA-EGFR Del lung cancer transgenic mouse model; wherein A is a CT image of the lung of the mouse before and after the Vehicle or BCP16671 treated mouse; b is a quantitative plot of the change in lung tumor burden in mice two weeks after treatment with Vehicle or BCP16671.
Detailed description of the preferred embodiments
The detailed description of the present invention is explained by the examples, and the partial solutions in the described examples belong to some of the examples of the present invention, and the examples obtained by the skilled person without inventive efforts belong to the scope of the present invention, except that the technology used for detection does not limit the present invention in any way.
In the following examples, the Vehicle group was a solvent-treated group in which BCP16671 was dissolved, and the BCP16671 group was a T cell costimulatory molecule-activating compound-treated group found in the present invention.
Example 1
BCP16671 activates T cells via costimulators
Jurkat NFAT luciferase cells are T cells engineered based on Jurkat and contain a luciferase gene driven by the NFAT promoter. T cell activation expresses a series of transcription factors which can be effectively combined on NFAT promoter to drive gene expression behind the NFAT promoter, therefore, lucifer reading value is increased and Jurkat NFAT luciferase cell activation can be reflected. Jurkat NFAT luciferase OE-CD27 cells, jurkat NFAT luciferase OE-CD137 cells, and Jurkat NFAT luciferase OE-OX40 cells are cells that overexpress the costimulators CD27, CD137, and OX40 on the basis of Jurkat NFAT luciferase cells, which can be used to determine whether a drug activates T cells via the costimulators.
1. Experimental methods
(1) The experimental design was divided into 4 groups, jurkat NFAT luciferase cell group, jurkat NFAT luciferase OE-CD27 cell group, jurkat NFAT luciferase OE-CD137 cell group and Jurkat NFAT luciferase OE-OX40 cell group, the cell number was calculated, 10 ten thousand cells per well were plated into 96 well plates, CD3 antibody was added to a final concentration of 1 μ g/ml to stimulate T cell first messengers, and 5 co was added 2 The culture was carried out in an incubator at 37 ℃ for 6 hours.
(2) Adding BCP16671 (shown in formula one below) separately to each cell group to a final concentration of 4 μ M, or adding an equal volume of solvent, 5% 2 The lucifer reading was measured after 6 hours of incubation in an incubator at 37 ℃. The chemical name of BCP16671 is (E) -N-benzyl-3- (6-bromopyridine-2-yl) -2-cyanoacrylamide, which can be purchased from Shanghai Van Bio-technology Limited (CAS 857064-40-5), and related research work reports related to the function of T cells activated by small molecular compound BCP16671 are not available at present.
2. Results and analysis of the experiments
As a result of lucifer reading value detection, the addition of BCP16671 is found to significantly increase the reading value of the lucifer of the cells overexpressing the co-stimulators CD27, CD137 and OX40 Jurkat NFAT luciferase, i.e., the T cells overexpressing the co-stimulators are activated under the action of BCP16671 (shown in FIG. 1).
Example 2
BCP16671 promotes CTLs to secrete cytokines
CTLs come from OT-1 mice, and CTLs in OT-1 mice can specifically recognize chicken Ovalbumin (OVA), so that immune cells separated from spleens of OT-1 mice are activated after being stimulated by OVA peptide, and CTLs secrete cytokines to show the phenotype of activated T cells. A549-OVA lung cancer cell is lung cancer A549 cell expressing OVA peptide, which can be recognized and killed by OT-1 mouse CTLs.
1. Experimental methods
(1) Obtaining CTLs: selecting an OT-1 mouse with the age of 8 weeks, killing the spleen, preparing a single cell suspension, adding 5ml of erythrocyte lysate, cracking for 5min at room temperature, centrifuging for 3min at 1600rpm, reserving precipitated cells, culturing for 1 day by using an OVA peptide 1640 culture medium containing 10% of serum, 1% o of beta mercaptoethanol, 1% o of IL2 and 2 mu g/ml, removing the OVA peptide, and culturing for 1 day by using an OVA peptide 1640 culture medium containing 10% of serum, 1% o of beta mercaptoethanol and 1% o of IL 2.
(2) Detection of CTLs cytokine secretion: the CTLs obtained and cultured for 2 days were subjected to centrifugal counting, 20 ten thousand cells were mixed with A549-OVA lung cancer cells after 10 ten thousand 2.5. Mu.M CFSE staining for 1min, cultured in 1 well of a 12-well plate, BCP16671 was added to a final concentration of 4. Mu.M, an equal volume of solvent was added to the control group, and 5% CO was determined 2 Culturing in 37 deg.C incubator for 12 hr, collecting all cells, fixing with 1ml 4% paraformaldehyde at 4 deg.C overnight, washing with PBS for 2 times, dividing cells into 4 parts, respectively staining with PE-isotype, PE-INF-gamma, PE-IL2, and PE-perforin flow type antibody on ice for 20min, and determining PE based on cell PE-isotype staining result + Cell population, flow assay FITC - PE + The proportion of the cell population.
2. Results and analysis of the experiments
(1) It was found by flow cytometry that BCP16671 treatment significantly increased secretion of cytokines such as INF-gamma, IL2 and perforin by CTLs (FIG. 2A).
(2) By analyzing FITC - PE + Cell population ratio, quantification of the ratio of flow-cytokine-positive CTLs it was found that BCP16671 treatment promoted secretion of cytokines such as CTLs INF- γ, IL2, and perforin (fig. 2B).
Example 3
BCP16671 promotes CTLs to kill A549-OVA lung cancer cells
1. Experimental methods
(1) Obtaining CTLs: selecting an OT-1 mouse with the age of 8 weeks, killing and taking the spleen, preparing a single cell suspension, adding 5ml of erythrocyte lysate, cracking for 5min at room temperature, centrifuging for 3min at 1600rpm, leaving precipitated cells, culturing for 1 day by using an OVA peptide 1640 culture medium containing 10% of serum, 1 thousandth of beta mercaptoethanol, 1 thousandth of IL2 and 2 mu g/ml, removing the OVA peptide, and culturing for 1 day by using an OVA peptide 1640 culture medium containing 10% of serum, 1 thousandth of beta mercaptoethanol and 1 thousandth of IL2 for 1 day 1640.
(2) CTLs kill A549-OVA tumor cells: the CTLs obtained and cultured for 2 days were counted centrifugally, 4 ten thousand cells were taken to be mixed with A549-OVA lung cancer cells after 2 ten thousand 2.5. Mu.M CFSE staining for 1min, cultured in 1 well of a 96-well plate, BCP16671 was added to a final concentration of 4. Mu.M, an equal volume of solvent was added to a control group, and 5% CO was determined 2 Culturing in 37 deg.C incubator for 6 hr, collecting all cells, and performing flow-type identification of FITC after PI staining + PI + The proportion of the cell population.
2. Results and analysis of the experiments
(1) The apoptosis ratio of A549-OVA lung cancer cells is obviously increased by the BCP16671 treatment detected by flow cytometry (figure 3A).
(2) By analysis of FITC + PI + Cell population ratio, quantification of apoptosis ratio of lung cancer cells of flow type A549-OVA, and finding that the treatment of BCP16671 promotes CTLs to kill the lung cancer cells of A549-OVA (figure 3B).
Example 4
BCP16671 effectively inhibits the growth of subcutaneous A549-OVA lung cancer transplantable tumor of B6 mice which receive back transfusion of CTLs
The transplanted tumor treatment model can conveniently observe and measure the size change of tumor growth in the evaluation of the drug effect, has a single system and can clearly explain the problem.
1. Experimental methods
(1) About 8 weeks of B6 mice were inoculated subcutaneously on the right side with 500 ten thousand A549-OVA lung cancer cells, and the mice were normally fed for 3-4 days before reinfusion of activated CTLs.
(2) Obtaining CTLs: selecting an OT-1 mouse with the age of 8 weeks, killing the spleen, preparing a single cell suspension, adding 5ml of erythrocyte lysate, cracking for 5min at room temperature, centrifuging for 3min at 1600rpm, reserving precipitated cells, culturing for 1 day by using an OVA peptide 1640 culture medium containing 10% of serum, 1% o of beta mercaptoethanol, 1% o of IL2 and 2 mu g/ml, removing the OVA peptide, and culturing for 1 day by using an OVA peptide 1640 culture medium containing 10% of serum, 1% o of beta mercaptoethanol and 1% o of IL 2.
(3) The number of CTLs was counted, and 200 million CTLs were returned per tail vein of mice into the transplanted tumor mouse model.
(4) A549-OVA transplantation tumor model mice after transfusion of CTLs are randomly divided into 2 groups, 6 mice in each group are respectively injected with 40mg/kg BCP16671 ul or an equal volume of solvent intraperitoneally, the weight of the mice is recorded every day, the size of the transplantation tumor is measured, and the transplantation tumor of the mice is dissected and weighed to take a picture after 14 days.
2. Results and analysis of the experiments
(1) After 14 days of treatment, no significant change in body weight was found for BCP16671 treatment (fig. 4A).
(2) Tumor growth curves plotted from daily measured tumor size data can see the growth of mouse transplantable tumors after BCP16671 inhibition of reinfused CTLs (fig. 4B).
(3) The transplanted tumors were dissected after 14 days and found to be significantly smaller in the BCP16671 treated group than in the control group (fig. 4C).
(4) The dissected graft tumor after 14 days showed that the BCP16671 treated group was significantly lighter in weight than the control group (fig. 4D), indicating that BCP16671 was effective in inhibiting the growth of subcutaneous graft tumors in mice by CTLs.
Example 5
BCP16671 can effectively treat lung cancer orthotopic tumor of transgenic mice
Transplanted tumors cannot simulate the complex canceration and tumor formation process of primary lung cancer and the interaction between tumors and stromal cells, and have poor capability of predicting the treatment of drugs in patients. Therefore, the invention further utilizes a lung cancer orthotopic tumor model of a CC10RTTA-EGFR Del cancer driving gene mutated transgenic mouse to evaluate the treatment effect of the BCP16671. The CC10 protein is specifically expressed protein in lung, the Tet-on system is a relatively mature eukaryotic exogenous gene induction expression system, and the RTTA protein can be combined with a specific TRE sequence under the assistance of doxycycline (Dox), so that the transcriptional expression regulation of the target gene EGFR connected with a TRE element is realized.
1. Experimental method
(1) Inducing lung cancer in situ neoplasia of CC10RTTA-EGFR Del cancer driver gene mutant transgenic mice: in this example, CC10RTTA-EGFR Del cancer driver mutant transgenic mice were used for treatment. The transgenic mouse is a transgenic mouse with the deletion of the 19 th exon of the EGFR gene. The transgenic mice are bred by doxycycline DOX grains when the mice are old at four weeks, and the lung can form tumors after the mice are induced by the DOX grains for four weeks.
(2) Before treatment, the size and severity of the tumor was recorded by Computer Tomography (CT).
(3) Mice with lung cancer were divided into a Vehicle control group (i.p., administered once a day, 100. Mu.l/time), and a BCP16671 treatment group (i.p., administered once a day, administered at a dose of 40mg/kg, 100. Mu.l/time). The administration treatment is carried out for two weeks in the same step behavior period. And (5) recording the change condition of the tumor size by CT detection after treatment.
2. Results and analysis of the experiments
(1) After two weeks of treatment, lung tumors in mice from different treatment groups were scanned by CT and found to regress significantly in lung tumors from EGFR cancer-driving gene mutant transgenic mice treated with BCP16671 as compared to the control group (fig. 5A).
(2) The tumor burden decreased by approximately 30% to 40% after two weeks of BCP16671 treatment (fig. 5B), suggesting that BCP16671 has a significant therapeutic effect on mouse lung cancer orthotopic tumors.
Claims (8)
- Use of BCP16671 in the preparation of a medicament for the treatment of lung cancer.
- 2. Use according to claim 1, characterized in that: the medicine for treating lung cancer is a medicine for activating immune response to treat lung cancer.
- 3. A medicament for treating lung cancer based on activation of an immune response, characterized by: the medicine contains BCP16671.
- 4. The medicament for treating lung cancer based on activation of immune response according to claim 3, characterized in that: the lung cancer comprises orthotopic cancer and transplantation cancer.
- 5. The medicament for treating lung cancer based on activation of immune response according to claim 3, characterized in that: the lung cancer is A549-OVA lung cancer cell transplantation tumor or CC10RTTA-EGFR Del cancer driver gene mutation lung cancer.
- 6. The medicament for treating lung cancer based on activation of immune response according to claim 3, characterized in that: the lung cancer is in-situ lung cancer driven by in-vitro culture of a tumor transplanted by lung cancer cells or over-expression of an oncogene EGFR with 19 th exon deletion induced by Dox.
- 7. The medicament for treating lung cancer based on activation of immune response according to claim 3, characterized in that: the medicine at least comprises a carrier; the carrier is at least one of a sustained release agent, an excipient, a filler, an adhesive, a wetting agent, a disintegrating agent, an absorption enhancer, an adsorption carrier, a surfactant or a lubricant.
- 8. The medicament for treating lung cancer based on activation of immune response according to claim 3, characterized in that: the medicine is injection, tablet, granule or capsule.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1894215A (en) * | 2003-12-11 | 2007-01-10 | 德克萨斯州大学系统董事会 | Compounds for treatment of cell proliferative diseases |
| CN110664818A (en) * | 2019-10-14 | 2020-01-10 | 暨南大学 | Medicine for treating lung cancer |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1894215A (en) * | 2003-12-11 | 2007-01-10 | 德克萨斯州大学系统董事会 | Compounds for treatment of cell proliferative diseases |
| CN110664818A (en) * | 2019-10-14 | 2020-01-10 | 暨南大学 | Medicine for treating lung cancer |
Non-Patent Citations (1)
| Title |
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| 王华民等主编: "《临床免疫学》", 28 February 2010, 军事医学科学出版社, pages: 79 - 80 * |
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