CN113599527A

CN113599527A - Application of APOE inhibitor and PD-1 monoclonal antibody in preparation of medicine for treating digestive tract tumor

Info

Publication number: CN113599527A
Application number: CN202110995835.5A
Authority: CN
Inventors: 王学浩; 唐薇薇; 刘力; 惠冰清; 卢琛; 荣大伟
Original assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Current assignee: Jiangsu Province Hospital First Affiliated Hospital With Nanjing Medical University
Priority date: 2021-08-27
Filing date: 2021-08-27
Publication date: 2021-11-05
Anticipated expiration: 2041-08-27
Also published as: CN113599527B

Abstract

The invention discloses an application of APOE inhibitor and PD-1 monoclonal antibody in preparation of a medicament for treating digestive tract tumor, belonging to the technical field of medicines. The inventor researches and finds that compared with the prior art that the PD-1 monoclonal antibody is used for treating the digestive tract tumor, the APOE inhibitor and the PD-1 monoclonal antibody can be used together to further inhibit the digestive tract tumor and improve the treatment efficiency, and especially for patients with low treatment sensitivity of the PD-1 monoclonal antibody, the APOE inhibitor and the PD-1 monoclonal antibody have important significance for treating the digestive tract tumor.

Description

Application of APOE inhibitor and PD-1 monoclonal antibody in preparation of medicine for treating digestive tract tumor

Technical Field

The invention relates to an application of APOE inhibitor and PD-1 monoclonal antibody in preparation of a medicament for treating digestive tract tumor, belonging to the technical field of medicines.

Background

Digestive tract tumors are one of the most common malignant tumors in China, the incidence rate of the digestive tract tumors is increased year by year, the onset age of the digestive tract tumors also tends to be younger, and the health and the life of people are seriously threatened. Because of the specificity and complexity of the etiology and pathogenesis of the digestive tract tumor, the clinical cure rate is low, and the effective rate of targeted therapy and immunotherapy is about 20%, so that the improvement of the effective rate is extremely important.

At present, the effective rate of the PD-1 monoclonal antibody for treating the digestive tract tumor is 15-20 percent, and the effective rate is lower. The anti-PD-1, anti-PD-L1 and anti-PD-L2 antibodies all function depending on their expression on the cell surface. Antibodies do not work well for tumors that express low and no corresponding substances. Studies have shown that at least 70% of cancer patients respond poorly to PD-1 antibody therapy. Therefore, single drug therapy does not meet the clinical needs and combination therapy is considered to improve the efficacy.

In recent years, immune checkpoint combination therapies have found widespread use. Although these immune checkpoint inhibitors improve the efficacy of treatment in cancer patients, previous clinical trial data suggest that immune checkpoint treatment does not achieve the expected positive response rate, and many of the previously proposed hypotheses are consequently clinically questioned. Nevertheless, a large number of immune checkpoint antibody combination therapies do achieve superior efficacy in controlling disease progression in certain advanced cancers. With the intensive study of the mechanisms of primary and acquired tolerance of immune checkpoint inhibitors, combination treatment regimens will be applied in more clinical trials. In addition to the combination therapy with CTLA-4 and PD-1 antibodies, the combination therapy further comprises: immune interference factors of the tumor microenvironment such as toll-like receptor antagonists, oncolytic viruses, antibodies blocking T cell rejection, suppressive immune cells such as regulatory T cells (Tregs) or macrophage M. These immune factor combination therapies enhance the tumor cell clearance of T cells on the one hand and increase the T cell number around tumor tissues on the other hand, thereby synergizing immune checkpoint antibodies to inhibit tumor progression. In addition, the combined application of other treatment modes such as radiotherapy, chemotherapy, tumor targeted therapy, cancer vaccine and the like also shows that the activity of immune factors in the tumor microenvironment can be regulated and controlled, and the immune factors and the immune check points generate synergistic action, so that the effect of enhancing the anti-tumor curative effect is achieved. However, there is no systematic study on the mechanism of combination therapy. Further elaborating the intrinsic molecular mechanism, is important for improving the effective rate of tumor immunotherapy.

Disclosure of Invention

The invention aims to solve the problem of low effective rate of the PD-1 monoclonal antibody for treating the digestive tract tumor in the prior art, provides the application of the combination of the APOE inhibitor and the PD-1 monoclonal antibody in preparing the medicament for treating the digestive tract tumor, and tests prove that the combination of the APOE inhibitor and the PD-1 monoclonal antibody can further inhibit the digestive tract tumor and improve the treatment effective rate.

Technical scheme

The APOE inhibitor and the PD-1 monoclonal antibody are combined to be applied to the preparation of the medicine for treating the digestive tract tumor.

Further, the APOE inhibitor is COG133 TFA. COG133TFA is a fragment of an apolipoprotein E (APOE) peptide. COG133TFA competes with APOE holoprotein for binding to LDL receptors, and has potent anti-inflammatory and neuroprotective effects. The inventor researches to find that the APOE inhibitor can activate the immunogenicity of cancer patients, enhance the expression of PD-1 in CD8+ T cells, and enhance the sensitivity of the medicine by combining the PD-1 inhibitor.

Furthermore, in the medicine for treating the digestive tract tumor, the APOE inhibitor and the PD-1 monoclonal antibody are main active ingredients.

The invention has the beneficial effects that: compared with the prior art that the PD-1 monoclonal antibody is used for treating the digestive tract tumor, the APOE inhibitor and the PD-1 monoclonal antibody can be used for further inhibiting the digestive tract tumor, improving the treatment efficiency, and particularly aiming at a patient with low treatment sensitivity of the PD-1 monoclonal antibody. The method has important significance for treating digestive tract tumors.

Drawings

FIG. 1 shows the test results of the APOE inhibitor and PD-1 monoclonal antibody alone or in combination for treating mice of a model of subcutaneous tumor of liver cancer;

FIG. 2 shows the test results of APOE inhibitor and PD-1 monoclonal antibody alone or in combination for treating subcutaneous tumor model mice with intestinal cancer;

FIG. 3 shows the results of a test on mice model of subcutaneous carcinoma model treated with APOE inhibitor and PD-1 monoclonal antibody alone or in combination.

Detailed Description

The technical solution of the present invention is further explained with reference to the accompanying drawings and specific embodiments. In the examples below, COG133TFA was purchased from sigmaldrich; PD-1 mAb was purchased from Bioxcell.

Example 1

Establishing wild mouse C57BL/6 model of subcutaneous liver cancer, intestinal cancer and gastric cancer transplantation tumor

C57BL/6 mice (purchased from Witonglihua), 4 weeks old, cell strains (MC 38-intestinal cancer, Hepa 1-6-liver cancer, MFC-gastric cancer, all purchased from Shanghai cell biology institute of Chinese academy of sciences), and subcutaneous liver cancer, intestinal cancer and gastric cancer transplantation tumor models are established by injecting at groin. The specific method comprises the following steps: selecting cells (MC 38-intestinal cancer, Hepa 1-6-liver cancer or MFC-stomach cancer cells) with good growth state, pouring out the culture solution in the bottle, adding 2ml of D-Hanks solution, gently shaking and then pouring out, adding 3ml of digestive juice mixed (1: 1) by 0.25% of pancreatin and O.02% of EDTA into the bottle, gently shaking the culture bottle to enable the digestive juice to flow over all the cell surfaces, and digesting for 3 minutes at room temperature and 25 ℃. When the digestion was carried out under an inverted microscope, the cytoplasm retracted and the intercellular spaces increased, the digestion solution was poured off, and 3ml of the culture solution was added to the flask to terminate the digestion. Repeatedly blowing and beating the cells on the bottle wall by using a suction pipe until all the cells are flushed down, slightly blowing, beating and uniformly mixing to form a cell suspension according to the proportion of 1: 2, distributing, inoculating into two culture bottles, supplementing the culture solution to 5ml in each bottle, and continuing to culture. The culture was terminated when the cell adherence was 80% as observed under an inverted microscope. Digesting with 0.25% pancreatin at room temperature, removing cell wall, adding 2ml RPMI-1640 culture solution to terminate digestionAnd (4) transforming. Collecting cell suspension in 50ml centrifuge tube, centrifuging at 800 rpm for 8min, discarding supernatant, resuspending with RPMI-1640 culture medium, gently blowing and beating into single cell suspension, sucking 9 microliters of cell suspension in EP tube, adding 1 microliter trypan blue, mixing, placing 10 microliters of suspension on blood cell counting plate, counting under inverted microscope, and adjusting cell suspension density to lxl 0% if cell activity reaches more than 95%⁷One/ml for standby. Disinfecting the skin of the right inguinal region of the mouse, collecting cells in logarithmic growth phase, and adjusting the cell concentration to lxl0⁷Each ml, 0.2ml (2X 10) was aspirated by syringe⁶One) cell suspension was inoculated subcutaneously in the right groin of mice and the injection site was pressed with a cotton swab for several seconds after injection. After inoculation, the animals were returned to the rearing room for further rearing and examined daily for tumor growth. Obtaining the liver cancer, intestinal cancer or gastric cancer transplantation tumor model mouse.

Example 2

APOE inhibitor and PD-1 monoclonal antibody combined treatment digestive tract tumor

1. Taking liver cancer transplantation tumor model mice, dividing the mice into four groups, and setting the mice into a control group, a PD1 monoclonal antibody single-use group (alpha PD-1), a COG133TFA single-use group (alpha APOE), a PD1 monoclonal antibody and COG133TFA combined group (alpha APOE + alpha PD-1), wherein each group comprises 4 mice, the four groups of mice are respectively treated according to corresponding groups, wherein the PD1 monoclonal antibody single-use group comprises: injecting 6.6mg/kg in the abdominal cavity on the eighth day, and injecting every three days later; COG133TFA alone group: injecting 1mg/kg of the abdominal cavity on the next day, and injecting every five days later; PD1 mab and COG133TFA combination: the COG133TFA1mg/kg is intraperitoneally injected on the next day, the COG133TFA1mg/kg is injected once every five days later, the PD1 monoclonal antibody 6.6mg/kg is intraperitoneally injected on the eighth day, and the COG133TFA 1/kg is injected once every three days later; control group: injecting 100/of PBS into the abdominal cavity on the next day, and injecting every five days; the activity, spirit, diet and other conditions of the mice were observed daily before and after the experiment. The tumor length a (mm) and the tumor length b (mm) were measured with a vernier caliper every 4 days, and the tumor volume (V) of the mice was calculated according to the following formula: ab ═ V²And/2, the tumor growth curve is plotted. Mice were sacrificed 20 days after dosing by cervical dislocation. The test results are shown in FIG. 1.

Fig. 1 shows the test results of treating the subcutaneous hepatoma tumor model mouse with the APOE inhibitor and the PD-1 monoclonal antibody alone or in combination, and it can be seen from fig. 1 that the COG133TFA or the PD1 monoclonal antibody alone can slow down the growth of hepatoma, and the PD1 monoclonal antibody has better effect than the COG133TFA in inhibiting cancer, but the combination of the two can significantly slow down the growth of hepatoma and even disappear.

2. Taking intestinal cancer transplantation tumor model mice, dividing the mice into four groups, and setting the mice as a control group, a PD1 monoclonal antibody single-use group, a COG133TFA single-use group, a PD1 monoclonal antibody and COG133TFA combined group, wherein each group comprises 4 mice, and the four groups of mice are respectively treated according to corresponding groups, wherein the PD1 monoclonal antibody single-use group comprises: injecting 6.6mg/kg in the abdominal cavity on the eighth day, and injecting every three days later; COG133TFA alone group: injecting 1mg/kg of the abdominal cavity on the next day, and injecting every five days later; PD1 mab and COG133TFA combination: the COG133TFA1mg/kg is intraperitoneally injected on the next day, the COG133TFA1mg/kg is injected once every five days later, the PD1 monoclonal antibody 6.6mg/kg is intraperitoneally injected on the eighth day, and the COG133TFA 1/kg is injected once every three days later; control group: injecting 100/of PBS into the abdominal cavity on the next day, and injecting every five days; the activity, spirit, diet and other conditions of the mice were observed daily before and after the experiment. The tumor length a (mm) and the tumor length b (mm) were measured with a vernier caliper every 4 days, and the tumor volume (V) of the mice was calculated according to the following formula: ab ═ V²And/2, the tumor growth curve is plotted. Mice were sacrificed 20 days after dosing by cervical dislocation. The test results are shown in FIG. 2.

Fig. 2 shows the test results of treating subcutaneous tumor model mice with intestinal cancer with APOE inhibitor and PD-1 monoclonal antibody alone or in combination, and it can be seen from fig. 2 that either APOE inhibitor COG133TFA or PD1 monoclonal antibody alone can slow down intestinal cancer growth, and PD1 monoclonal antibody has better cancer inhibition effect than APOE inhibitor COG133TFA, but both can significantly slow down intestinal cancer growth and even disappear.

3. Taking gastric cancer transplantation tumor model mice, dividing the mice into four groups, and setting the mice into a control group, a PD1 monoclonal antibody single-use group, a COG133TFA single-use group, a PD1 monoclonal antibody and COG133TFA combined group, wherein each group comprises 4 mice, and the four groups of mice are respectively treated according to corresponding groups, wherein the PD1 monoclonal antibody single-use group comprises: injecting 6.6mg/kg in the abdominal cavity on the eighth day, and injecting every three days later; COG133TFA alone group: injecting 1mg/kg of the abdominal cavity on the next day, and injecting every five days later; PD1 mab and COG133TFA combination: the following day, COG133 was intraperitoneally injectedTFA1mg/kg, which is injected once every five days later, PD1 monoclonal antibody 6.6mg/kg is injected intraperitoneally on the eighth day, and is injected once every three days later; control group: injecting 100/of PBS into the abdominal cavity on the next day, and injecting every five days; the activity, spirit, diet and other conditions of the mice were observed daily before and after the experiment. The tumor length a (mm) and the tumor length b (mm) were measured with a vernier caliper every 4 days, and the tumor volume (V) of the mice was calculated according to the following formula: ab ═ V²And/2, the tumor growth curve is plotted. Mice were sacrificed 20 days after dosing by cervical dislocation. The test results are shown in FIG. 3.

Fig. 3 shows the test results of the model mouse with the single or combined use of APOE inhibitor and PD-1 monoclonal antibody for treating subcutaneous carcinoma of stomach, and it can be seen from fig. 3 that either APOE inhibitor COG133TFA or PD1 monoclonal antibody can slow down the growth of stomach cancer, and PD1 monoclonal antibody has better effect than that of APOE inhibitor COG133TFA, but the combination of the two can significantly slow down the growth of stomach cancer.

Claims

The application of the APOE inhibitor and the PD-1 monoclonal antibody in preparing a medicament for treating digestive tract tumors.
2. The use of claim 1, wherein the APOE inhibitor is COG133 TFA.
3. The use as claimed in claim 1 or claim 2, wherein the medicament for the treatment of a tumour of the digestive tract comprises as active ingredients an APOE inhibitor and PD-1 mab.