CN112656941A - Application of anti-CD 47 monoclonal antibody in preparation of anti-adriamycin-resistant acute lymphocytic leukemia cell medicine - Google Patents
Application of anti-CD 47 monoclonal antibody in preparation of anti-adriamycin-resistant acute lymphocytic leukemia cell medicine Download PDFInfo
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Abstract
The invention discloses an application of an anti-CD 47 monoclonal antibody in resisting adriamycin-resistant acute lymphocytic leukemia cells, and relates to an activity of an anti-CD 47 monoclonal antibody in treating adriamycin-resistant acute lymphocytic leukemia. PBMC-derived macrophages were prepared from healthy donors. anti-CD 47 monoclonal antibody-mediated phagocytosis of cells was detected by antibody-dependent cell-mediated phagocytosis (ADCP). By constructing an adriamycin-resistant acute lymphoblastic leukemia cell strain Nalm6-Luc/ADR subcutaneous transplantation tumor mouse model and a systemic xenotransplantation mouse model, an anti-CD 47 monoclonal antibody acts on a model mouse, and the effect of the anti-CD 47 monoclonal antibody on treating adriamycin-resistant acute lymphoblastic leukemia is observed.
Description
Technical Field
The novel application relates to the field of medicines, in particular to application of an anti-CD 47 monoclonal antibody in resisting adriamycin-resistant acute lymphoblastic leukemia.
Background
Acute Lymphoblastic Leukemia (ALL) is a group of highly heterogeneous hematological tumors in which malignant clonal proliferation and differentiation of hematopoietic stem/progenitor cells is hindered, accounting for about 20% of acute leukemia in adults and 80-85% of acute leukemia in children. Despite intensive studies on the biological properties of leukemia, there are still limited advances currently made in the treatment of ALL. Although the first remission rate of induction treatment can reach 89% -90%, the disease-free survival rate of 3-5 years is only 10% -30%, some patients still have no effect on induction chemotherapy or relapse after Complete Remission (CR), and finally develop into relapse/refractory (R/R) ALL. At present, R/R ALL has no unified standard treatment scheme, and the curative effect and prognosis are poor, so that the R/R ALL is a great challenge of current clinical treatment and a great problem to be solved in ALL research.
Chemotherapy drug resistance refers to the phenomenon of insensitivity or resistance of leukemia cells to drugs. The primary or secondary drug resistance of leukemia cells to chemotherapeutic drugs is an important factor causing poor treatment effect of adult ALL, high recurrence rate and poor prognosis, and is considered to be a main reason for ineffective leukemia treatment. Therefore, the search of the molecular marker related to chemotherapy resistance and the reversal of chemotherapy resistance are key factors for practically improving the clinical treatment effect of acute lymphocytic leukemia.
CD47 (also called integrin, ovarian cancer marker) is an immune checkpoint molecule expressed on the surface of various tumor cells and acting on macrophages and DC cells, and transmits a "Don ́ t eat me" signal to macrophages by binding to sirpa on the surfaces of the macrophages, thereby achieving immune escape. Blocking the CD47-SIRP alpha channel can promote phagocytosis of tumor cells by macrophages, improve the immune presentation of tumor specific antigens, activate the killing of specific T cells on the tumor cells, and achieve the effects of activating innate immunity and acquired immune response at the same time. The research shows that the monoclonal antibody (Mab) has obvious improvement effect on treating tumors, especially cancers with drug-resistant risk factors by combining standard chemotherapy. The anti-CD 47 monoclonal antibody is reported to be effective to treat leukemia stem cell THP-1 alone or in combination with cytarabine (Ara-C), and in addition, the anti-CD 47 monoclonal antibody is reported to have the potential to treat hepatocellular carcinoma (HCC) liver cancer alone or in combination with sorafenib standard chemotherapy.
At present, the research on ADM resistant hematologic malignancy treated by the anti-CD 47 monoclonal antibody has not been reported. This study demonstrates that the anti-CD 47 monoclonal antibody is effective in treating B-ALL resistant cells, Nalm6-Luc-ADR cells, which are potentially resistant to doxorubicin.
Disclosure of Invention
The invention aims to provide application of an anti-CD 47 monoclonal antibody to acute lymphocytic leukemia.
The technical scheme adopted by the invention is as follows: an application of the anti-CD 47 monoclonal antibody in preparing the medicines for treating the acute lymphoblastic leukemia with adriamycin resistance. Specifically, Peripheral Blood Mononuclear Cells (PBMCs) were first prepared from healthy donors from normal healthy donors at the institute for blood research in the department of fujia medical university subsidiary. anti-CD 47 monoclonal antibody-mediated phagocytosis of cells was detected by antibody-dependent cell-mediated phagocytosis (ADCP). By constructing an adriamycin-resistant acute lymphoblastic leukemia cell strain Nalm6-Luc/ADR subcutaneous transplantation tumor mouse model and a systemic xenotransplantation mouse model, an anti-CD 47 monoclonal antibody acts on a model mouse, and the effect of the anti-adriamycin-resistant acute lymphoblastic leukemia cell in an anti-CD 47 monoclonal antibody is observed.
The invention has the beneficial effects that:
in vitro and in vivo experiments show that the anti-CD 47 monoclonal antibody has high anti-cancer activity and obvious inhibition effect on drug-resistant acute lymphocytic leukemia cells. The adriamycin-resistant Nalm6-Luc/ADR cells can achieve the effect of phagocytizing tumor cells through the phagocytosis of antibody-mediated cells in vitro, can effectively inhibit the tumor invasion and growth of a whole body xenograft mouse model and a subcutaneous transplantation tumor mouse model in vivo, and can inhibit the expression of MDR1 gene and protein related to drug resistance. This will help in developing clinical trials for acute lymphocytic leukemia resistance and relapsing cases.
Drawings
FIG. 1 is a fluorescent microscope for observing antibody-dependent cell-mediated phagocytosis (ADCP) according to the present invention, in which the upper panel is a fluorescent field and the lower panel is a bright field. A is macrophage phagocytosis of tumor cells indicated at arrow in the presence of anti-CD 47 monoclonal antibody; b is that in the absence of anti-CD 47 monoclonal antibody, almost all tumor cells were not phagocytosed.
FIG. 2 is a graph showing the in vivo effect of the anti-CD 47 monoclonal antibody of the present invention against an Adriamycin-resistant acute lymphoblastic leukemia whole body mouse model. In the figure, Nalm6-Luc/ADR cells expressing luciferase were injected into mice via tail vein. In the figure, the upper panel is a PBS-treated group (control group), and the lower panel is an anti-CD 47 monoclonal antibody-treated group (experimental group). In the figure, A is IVIS bioluminescence imaging before treatment initiation. In the figure, B represents that the mice receive treatment for 14 days, the experimental group receives 100 mug/day of the anti-CD 47 monoclonal antibody, and the control group receives equal dose of PBS injection.
FIG. 3 is a graph showing that the anti-CD 47 monoclonal antibody treatment group has significant effect and significant difference (p < 0.001) compared with the control group.
FIG. 4 is a graph showing the in vivo effect of the anti-CD 47 monoclonal antibody of the present invention against an Adriamycin-resistant acute lymphoblastic leukemia subcutaneous graft tumor mouse model. The right hind limb of the mouse was injected with Nalm6-Luc/ADR expressing luciferase, and in the figure, the upper panel is a PBS-treated group (control group) and the lower panel is an anti-CD 47 monoclonal antibody-treated group (experimental group). In the figure, A is IVIS bioluminescence imaging before treatment initiation. In the figure, B is two post-treatment IVIS bioluminescence imaging sets.
Detailed Description
In order to make the content of the present application easier to understand, the technical solutions of the present application will be further described below with reference to the specific embodiments, but the present application is not limited thereto.
Example 1: in vitro detection of antibody-dependent cell-mediated phagocytosis (ADCP)
PBMC-derived macrophages were prepared from healthy donors and harvested by trypsin/EDTA (Gibco) incubation for 3-5min and gently scraping. 5.5X 104Individual macrophages were in 24 well plates per well plate. Tumor cells were labeled with the standard dye 3 μ M carboxyfluorosuccinimide ester (CFSE) according to standard protocols. The macrophage is incubated in serum-free IMDM for 2-4 h and then added with 2.5 multiplied by 105cfse labeled live tumor cells. anti-CD 47 monoclonal antibody (Bio X Cell, USA) was added at 10-15. mu.g/mL and incubated at 37 ℃ for 2-4 hours. After incubation, the wells were washed thoroughly 3-5 times with PBS, followed by imaging with a fluorescence microscope. The phagocytic index was calculated from the number of effector cells (macrophages) phagocytosing CFSE positive tumor cells. The result analysis of a fluorescence microscope shows that ADCP macrophages mediated by the anti-CD 47 monoclonal antibody can phagocytose ADM drug-resistant Nalm6-Luc/ADR cells. The results indicate that human ALL cells can be cleared by sirpa protein interaction blocking macrophage phagocytosis induced by anti-CD 47 monoclonal antibodies. As shown in fig. 1.
Example 2: in vivo Effect of anti-CD 47 monoclonal antibody against Adriamycin-resistant acute lymphoblastic leukemia Whole body mouse model
A3-4-week-old NOD/SCID mouse is selected, and a whole-body B-ALL drug-resistant mouse model is constructed by using an adriamycin-resistant acute lymphoblastic leukemia cell strain Nalm 6-Luc/ADR. A luciferase-expressing doxorubicin-resistant human B-ALL cell line Nalm6-Luc/ADR was first isolated at the logarithmic growth phase. Count 3X 104The individual cells are injected into NOD/SCID mice through tail veins, after the mouse model is successfully constructed, the cells are divided into a PBS control group and an anti-CD 47 monoclonal antibody treatment group, each group comprises 10 cells, the amount of the anti-CD 47 monoclonal antibody is 100 ug/cell, and the control group is treated by PBS. Dosing was 3 days with 14 days observation time. The mouse experiments were performed according to the animal experimental guidelines of the university of fujian medicine. The IVIS bioluminescent imaging system regularly detects mouse tumor invasion.
The results suggest that IVIS bioluminescence imaging shows that each group of mice showed luciferase positivity prior to the start of treatment. After 14 days, IVIS bioluminescence imaging shows that fluorescence signals of mice in an anti-CD 47 monoclonal antibody treatment group basically disappear, and compared with a control group, the treatment effect is obvious, and the difference has obvious statistical significance (p is less than 0.001). As shown in fig. 2 and 3.
Example 3: in vivo effect of anti-CD 47 monoclonal antibody on Adriamycin-resistant acute lymphoblastic leukemia subcutaneous transplantation tumor mouse model
A3-4-week-old NOD/SCID mouse is selected, and an adriamycin-resistant acute lymphoblastic leukemia cell strain Nalm6-Luc/ADR is used for constructing a subcutaneous transplantation tumor B-ALL drug-resistant mouse model. The luciferase-expressing doxorubicin-resistant human B-ALL cell line Nalm6-Luc/ADR was isolated at logarithmic growth phase and counted at 5X 106The cells are injected subcutaneously at the back side of the right hind leg, and after the mouse model of the subcutaneous transplantation tumor is successfully constructed, the cells are divided into a PBS negative control group and an anti-CD 47 monoclonal antibody treatment group, 5 cells are in each group, the dosage of the anti-CD 47 monoclonal antibody is 100 ug/cell, the administration is carried out for 3 days, the growth condition of the tumor body is observed, and the cells are generated by IVISAnd recording by an object luminescence imaging system.
Results suggest that before treatment, IVIS bioluminescence imaging shows that all groups of mice show positive luciferase, and all groups of mice have uniform tumor body size and no significant difference (p is more than 0.05). After 14 days, IVIS bioluminescence imaging shows that the fluorescence signals of mice in the anti-CD 47 monoclonal antibody treatment group and subcutaneous transplantation tumors basically disappear, and compared with a control group, the treatment effect is obvious, and the difference has obvious statistical significance (p is less than 0.01). As shown in fig. 4.
In conclusion, in vitro and in vivo experiments show that the anti-CD 47 monoclonal antibody has high anticancer activity, can achieve the effect of phagocytizing tumor cells by antibody-mediated phagocytosis of adriamycin-resistant Nalm6-Luc/ADR cells in vitro, can effectively inhibit tumor invasion and growth of a whole-body xenograft mouse model and a subcutaneous transplantation tumor mouse model in vivo, and can inhibit the expression of drug-resistant related MDR1 genes and proteins. The result shows that the anti-CD 47 monoclonal antibody has obvious inhibiting effect on the acute lymphoblastic leukemia with adriamycin resistance, and is helpful for developing clinical tests aiming at the drug resistance and relapse cases of the acute lymphoblastic leukemia. The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. An application of the anti-CD 47 monoclonal antibody in preparing the medicines for treating the acute lymphoblastic leukemia with adriamycin resistance.
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Citations (2)
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| US20160177276A1 (en) * | 2014-08-15 | 2016-06-23 | Merck Patent Gmbh | Sirp-alpha immunoglobulin fusion proteins |
| WO2020206033A1 (en) * | 2019-04-02 | 2020-10-08 | Kenjockety Biotechnology, Inc. | Efflux pump-cancer antigen multi-specific antibodies and compositions, reagents, kits and methods related thereto |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160177276A1 (en) * | 2014-08-15 | 2016-06-23 | Merck Patent Gmbh | Sirp-alpha immunoglobulin fusion proteins |
| WO2020206033A1 (en) * | 2019-04-02 | 2020-10-08 | Kenjockety Biotechnology, Inc. | Efflux pump-cancer antigen multi-specific antibodies and compositions, reagents, kits and methods related thereto |
| CN113692413A (en) * | 2019-04-02 | 2021-11-23 | 肯乔克蒂生物技术股份有限公司 | Efflux pump-cancer antigen multispecific antibodies and compositions, reagents, kits and methods related thereto |
Non-Patent Citations (2)
| Title |
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| 朱孔黎等: "CD47分子在抗肿瘤免疫中的应用", 《中国新药与临床杂志》 * |
| 王颖超等: "抗CD47抗体联合阿糖胞苷靶向治疗NOD/SCID小鼠单核细胞白血病的研究", 《中国当代儿科杂志》 * |
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