CN113925850B

CN113925850B - Use of D-mannose in combination with PD-1 antibodies in the preparation of a medicament for the treatment of triple negative breast cancer

Info

Publication number: CN113925850B
Application number: CN202111223061.0A
Authority: CN
Inventors: 吕雷; 张若男
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2023-07-14
Anticipated expiration: 2041-10-20
Also published as: CN113925850A

Abstract

The invention belongs to the field of tumor treatment, and in particular relates to an application of D-mannose in promoting immunotherapy and radiotherapy of triple negative breast cancer. According to the invention, through cell and animal experiments, the effect of D-mannose is very obvious in promoting the curative effects of immunotherapy and radiotherapy of triple negative breast cancer: in mice, compared with a control group, the single oral administration of D-mannose achieves the same curative effect as PD-1 antibody treatment, and the combination of D-mannose and PD-1 antibody remarkably improves the effect of PD-1 antibody treatment and prolongs the survival period of mice. In mice, the oral administration of D-mannose alone achieved almost the same therapeutic effect as ionizing radiation, and the combination of D-mannose and radiotherapy inhibited tumor growth more significantly than the control group. The technical scheme disclosed by the invention shows that the D-mannose or the drug combination thereof has wide application prospect in the aspect of preparing the drug for treating the triple negative breast cancer, and particularly has remarkable effect on the condition of ineffective or drug resistance of immunotherapy and radiotherapy compared with the prior art.

Description

Use of D-mannose in combination with PD-1 antibodies in the preparation of a medicament for the treatment of triple negative breast cancer

Technical Field

The invention belongs to the field of tumor treatment, and in particular relates to an application of D-mannose in promoting immunotherapy and radiotherapy of triple negative breast cancer.

Background

Breast cancer is the most common type of cancer in women, and most early non-metastatic breast cancers (about 70-80%) are curable. Triple Negative Breast Cancer (TNBC) accounts for about 15-20% of all breast cancers and is characterized by the lack of Estrogen Receptor (ER), progestogen Receptor (PR) and human epidermal growth factor 2 (HER 2) molecular markers, with the worst prognosis and highest risk of distant recurrence within the first 3 to 5 years after diagnosis. The therapeutic strategies for TNBC were very limited, and until 2019, TNBC treatment was limited to chemotherapy. Immunotherapy in combination with chemotherapy has also been applied to treat TNBC for the last two years, showing good application prospects, but there are still a large number of TNBC patients that do not respond to immunotherapy. In addition, radiotherapy also shows a certain effect on local treatment of breast cancer, but TNBC has drug resistance to radiotherapy, and the local recurrence rate after radiotherapy is not reduced. Therefore, the treatment of TNBC remains a clinical challenge, and there is an urgent need to find effective therapeutic agents.

Disclosure of Invention

The invention aims to provide a new strategy for treating Triple Negative Breast Cancer (TNBC), and discovers that oral D-mannose has obvious inhibition effect on TNBC of mice and can promote the curative effects of TNBC immunotherapy and radiotherapy. The method is simple to operate and low in cost.

The invention provides an application of D-mannose in promoting the curative effects of immunotherapy and radiotherapy of triple negative breast cancer.

The invention provides an application of D-mannose in preparing a medicament for treating triple negative breast cancer.

The invention discloses an application of D-mannose in preparation of an immune medicament for treating triple negative breast cancer.

The invention discloses an application of D-mannose in preparing a medicine for treating triple negative breast cancer radiotherapy.

The invention discloses an application of D-mannose combined with a PD-1 antibody in preparing a medicament for treating triple negative breast cancer.

The invention discloses an application of D-mannose in preparation of a PD-L1 inhibitor.

The invention discloses an application of D-mannose in preparation of a PD-L1 degradation agent.

The invention discloses an application of D-mannose in the preparation of a T cell activator.

The invention discloses a pharmaceutical composition for treating triple negative breast cancer, which is characterized by comprising D-mannose and a pharmaceutically acceptable carrier.

The invention discloses a pharmaceutical composition for treating triple negative breast cancer, which is characterized by comprising D-mannose, a PD-1 antibody and a pharmaceutically acceptable carrier.

The dosage form of the pharmaceutical composition can be tablets, capsules, powder, granules or oral liquid.

In some embodiments, D-mannose was found in experiments to be capable of degrading PD-L1 in breast cancer cells and exosomes.

In some embodiments, D-mannose has been found in vitro experiments to promote T cell activation in vitro, promoting T cell killing of triple negative breast cancer cells.

In some embodiments, D-mannose was found to be effective in inhibiting the growth of triple negative breast cancer in mice by oral administration, and PD-1 antibody in combination was found to significantly promote the effect of immunotherapy.

In some embodiments, D-mannose has been found to inhibit PD-L1-mediated DNA damage repair in vitro experiments, increasing sensitivity of triple negative breast cancer cells to ionizing radiation in vitro.

In some embodiments, D-mannose is effective to inhibit growth of triple negative breast cancer in mice by oral administration, and to promote DNA damage in triple negative breast cancer tumors when combined with ionizing radiation therapy, to promote therapeutic efficacy of radiation therapy.

Compared with the prior art, the invention has the following advantages:

1) Promoting immunotherapy of triple negative breast cancer: in mice, compared with a control group, the D-mannose alone achieves the same curative effect as PD-1 antibody treatment, the combination of the D-mannose and the PD-1 antibody remarkably improves the effect of PD-1 antibody treatment, prolongs the survival period of the mice, and the mice treated by the PD-1 antibody all die on the 41 st day after tumor inoculation, while the mice treated by the D-mannose and the PD-1 antibody still have 40% survival on the 65 th day. (FIG. 3.A, B, C)

2) Promoting radiation therapy of triple negative breast cancer: in mice, compared with the control group, the single oral administration of D-mannose almost achieves the same curative effect as that of ionizing radiation, and the combination of D-mannose and radiotherapy can remarkably inhibit the growth of tumor and remarkably reduce the volume of tumor. (FIGS. 5.A, B, C)

3) Animal experiments prove that the above therapeutic effect can be achieved by oral administration of D-mannose.

Drawings

FIG. 1 is a graph showing the effect of D-mannose degradation on PD-L1 proteins in triple negative breast cancer cells and in exosomes.

FIG. 2 is a graph showing the effect of D-mannose in promoting T cell killing of triple negative breast cancer cells in vitro.

FIG. 3 shows that D-mannose is effective in inhibiting the growth of triple negative breast cancer in mice; the combined use of D-mannose and PD-1 antibodies promotes the effect of PD-1 antibody immunotherapy.

FIG. 4 shows that D-mannose enhances sensitivity of triple negative breast cancer cells to ionizing radiation in vitro.

Figure 5 shows that D-mannose is effective in inhibiting the growth of triple negative breast cancer in mice and in combination with ionizing radiation to promote the efficacy of radiation therapy.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

The PD-1 antibody described in the examples is InVivoMAb anti-mouse PD-1 (CD 279), which is a PD-1 antibody commonly used in the art. Other PD-1 antibodies may be used in combination with D-mannose according to clinical or experimental needs by those skilled in the art.

EXAMPLE 1D-mannose degradation of PD-L1 protein in triple negative breast cancer cells and exosomes

The expression level of PD-L1 in the cells was detected by Western blot after 24, 48, 72 hours, respectively, by adding 100mM D-mannose treatment to the medium of human breast cancer MDA-MB-231 cells. And collecting cell culture supernatant after 72 hours of treatment to separate exosomes, and detecting the expression level of PD-L1 in the exosomes by Western blot. The results are shown in FIG. 1.

Example 2D-mannose promotes killing of triple negative breast cancer cells by T cells in vitro.

a) In vitro culture and activation of cd8+ T cells. 10ml of fresh blood was obtained from healthy adult volunteers, and human Peripheral Blood Mononuclear Cells (PBMC) were isolated. PBMC were incubated in ImmunoCurt-XF T cell expansion medium containing IL-2 (10 ng/mL) and activated with ImmunoCult Human CD/CD 28/CD 2T cell activator (25. Mu.g/mL) for the first 3 days of incubation. Activated human cd8+ T cells were obtained after 7 days of culture. As shown in fig. 2A.

b) D-mannose treats tumor cells. Tumor cells MDA-MB-231 were divided into two groups, control cells were cultured in normal DMEM medium, and treated cells were cultured in 100mM D-mannose-containing DMEM medium. Two groups of cells were grown for 72 hours, digested with trypsin and counted, and plated uniformly into 24-well plates, 2 wells each.

c) Co-culture of CD8+ T cells with tumor cells. The activated CD8+ T cells are prepared according to tumor cells: t cell = 1:10 were added to tumor cell culture plates and co-cultured with DMEM/F12 medium containing CD3 antibody (100 ng/mL) and IL-2 (10 ng/mL).

d) The viability of tumor cells was examined. After 48 hours of co-culture of cd8+ T cells with tumor cells, the plates were blotted and washed with PBS to remove cell debris. The surviving tumor cells in the cell culture plate are stained with crystal violet staining solution, decolorized with 33% (m/v) acetic acid solution, and finally the absorbance of the decolorized solution at 570nm is detected. The results are shown in FIGS. 2B and C.

Example 3D-mannose is effective in inhibiting the growth of triple negative breast cancer in mice; the combined use of D-mannose and PD-1 antibodies promotes the effect of PD-1 antibody immunotherapy.

a) Construction of a mouse triple negative breast cancer model. Will be 1X10 ⁵ Three negative breast cancer cells of 4T1 mice were injected into mammary fat pads of female BALB/c mice, and after about 6 days, solid breast tumors developed.

b) D-mannose is administered orally. From day 6 to day 24 after inoculation of tumor cells, 10% (w/v) of D-mannose was added to the drinking water of the mice of the group to which D-mannose was administered, and the mice were subjected to gastric lavage with 20% (w/v) of the drinking water every other two days, 200. Mu.l each time.

c) PD-1 antibodies were administered by intraperitoneal injection. Mice were intraperitoneally injected with PD-1 antibody 100 μg each time every two days from day 6 to day 24 after tumor cell inoculation.

d) Detection of tumor volume. From the inoculation of the tumor, the length and width of the tumor at the mammary gland site of the mice were measured with vernier calipers every two days, and the volume was calculated. The results are shown in FIG. 3.

Example 4D-mannose enhanced sensitivity of triple negative breast cancer cells to ionizing radiation in vitro.

A100 mM D-mannose treatment was added to the medium of human breast cancer MDA-MB-231 cells. After 72 hours, the control and treatment cells were subjected to Ionizing Radiation (IR) treatment at a dose of 3Gy. Control and treatment cells were then individually counted and plated into 6-well plates, 1000 cells per well, and the number of clone formations was observed and counted. The results are shown in FIG. 4.

Example 5D-mannose is effective in inhibiting growth of triple negative breast cancer in mice and in promoting therapeutic efficacy of radiation therapy in combination with ionizing radiation.

b) D-mannose is administered orally. From day 6 to day 21 after inoculation of tumor cells, 10% (w/v) of D-mannose was added to the drinking water of the D-mannose administration group mice, and the mice were further gastrected with 20% (w/v) of D-mannose-containing drinking water every day, 200. Mu.l each time.

c) Mice were treated with radiation. On days 7 and 14 after inoculation of tumor cells, mice were subjected to radiation therapy with Ionizing Radiation (IR) at 5Gy doses.

d) Detection of tumor volume. From the inoculation of the tumor, the length and width of the tumor at the mammary gland site of the mice were measured with vernier calipers every two days, and the volume was calculated. The results are shown in FIG. 5.

Claims

Use of d-mannose in combination with a PD-1 antibody for the manufacture of a medicament for the treatment of triple negative breast cancer; the D-mannose is prepared into oral liquid.