WO2017045595A1 - Application of cyclic gmp-amp (cgamp) in combination with bevacizumab in tumor resistance - Google Patents

Abstract

Application of cyclic GMP-AMP (cGAMP) in combination with bevacizumab in tumor resistance. A research shows that combination of cGAMP and bevacizumab can significantly inhibit growth of multiple tumor cells, has an obvious tumor resistance effect, and can be used for preparing antitumor drugs. A model of subcutaneously transplantable tumors in a C57BL/6 mouse shows that the combination of the cGAMP and the bevacizumab has significant inhibition effects on an adenocarcinoma cell line MC38, a lung cancer cell line LLC, melanoma cell lines B16-F10, and a colon cancer cell MC38, and the effects are better than a single medication effect. Therefore, the combination of the cGAMP and the bevacizumab can be used for treating tumors.

Description

Application of cyclic dinucleotide cGAMP combined with bevacizumab in anti-tumor Technical field

The invention belongs to the technical field of biomedicine, and particularly relates to the application of a cyclic dinucleotide (cGAMP) combined with bevacizumab in antitumor and in preparing antitumor drugs.

Background technique

Tumors are currently a major class of diseases that seriously endanger human health. They are characterized by abnormal cell proliferation and differentiation, and are difficult to suppress. According to WHO experts, by 2020, the number of cancers in the global population will reach 20 million, and the death toll will reach 12 million. Therefore, cancer will become the biggest killer of human beings in this century, posing the most serious threat to human survival. . In China, the incidence and mortality rates of lung cancer, colorectal cancer, stomach cancer, liver cancer, etc. are among the highest in all kinds of malignant tumors. According to the National Cancer Registry (2012 China Cancer Registration Annual Report), new tumor cases occur every year. About 3.12 million cases, an average of 8550 people per day, 6 people per minute in the country were diagnosed with cancer. From the disease, lung cancer, stomach cancer, colorectal cancer, liver cancer and esophageal cancer, the top five in the country's malignant tumors As the incidence and mortality of malignant tumors increase year by year, the demand for treatment of malignant tumors is increasing. Especially with the aging of China's population, freshwater food pollution, environmental pollution, etc., the number of patients with malignant tumors in China will continue to increase. .

Currently, chemotherapy is one of the effective methods for treating tumors. The mechanism of action of traditional chemotherapeutic drugs is mainly to prevent the synthesis of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or protein, or directly affect these macromolecules, thereby inhibiting the proliferation and death of tumor cells. Some drugs can also inhibit tumor growth by altering the hormone balance in the body. At present, anti-tumor drugs have been developed into 6 categories: 1 antimetabolites; 2 alkylating agents; 3 cytotoxic antibiotics; 4 plant alkaloids and other natural drugs; 5 anti-tumor hormones; 6 platinum and other anti-tumor drugs . With the transformation of clinical treatment models and the discovery of some new anti-tumor drug targets, the development of anti-tumor drugs has undergone tremendous changes: from the traditional non-specific cytotoxic drugs to the mechanism of action of drugs Non-cytotoxic targeted drug development. Among the anti-tumor drugs approved by the FDA in 2012, small molecule tyrosine kinase inhibitors (TKI) have become the most popular class of anti-tumor drugs, especially TKI (about 3/4) acting on multiple targets. As of June 2013, the US FDA approved 18 TKIs. In addition, other hot-acting mechanisms include immunostimulants, angiogenesis inhibitors, cell cycle inhibitors, immunosuppressants and stimulators, protein kinase inhibitors, and the like. In recent years, more and more immunomodulators have been used clinically and for tumor therapy.

Microbial and viral DNA can induce an endogenous potent immune response by stimulating interferon secretion in infected mammalian cells. The immune response of the endoplasmic reticulum (ER) receptor protein (STING) to cytoplasmic DNA is an essential factor. Recent studies have shown that cyclized cGMP-AMP dinucleotide synthetase (cGAS) endogenously catalyzes the synthesis of cGAMP under the activation conditions following DNA binding. cGAMP is a cytoplasmic DNA sensor that acts as a second messenger to stimulate INF-β induction by STING, mediates the activation of TBK1 and IRF-3, and then initiates transcription of the INF-β gene. It has recently been reported that recombinant cGAS catalyzes the cyclization of cGMP-AMP dinucleotide GAMP under DNA binding conditions. The crystal structure of a complex of cGAS binding to 18 bp dsDNA has also been reported, and studies on antiviral immunity of cGAMP have been confirmed. cGAMP binds to STING, activates the transcription factor IRF3 and produces beta interferon. Through the STING pathway, cGAMP can be used as an immunomodulator to activate the immune system against tumors.

Vascular endothelial growth factor, also known as VEGF. The VEGF protein was successfully purified and identified by scientists from two biotechnology companies in the United States in 1989, and the gene sequence was cloned and determined, demonstrating that VPF and VEGF are the same protein encoded by the same gene. VEGF has six isoforms: VEGF-A, -B, -C, -D, and -E; its molecular weight ranges from 35 to 44 kDa, and each haplotype specifically interacts with three "vascular endothelial growth." A specific combination of factor receptors (VEGFR-1, -2, and -3) is combined. VEGF is a highly conserved homodimeric glycoprotein. Two single chains each having a molecular weight of 24 kDa form a dimer with a disulfide bond. The monomer decomposed by VEGF is inactive, and removal of the N2 glycosylation has no effect on biological effects, but may play a role in cell secretion. Due to different splicing patterns of mRNA, at least five protein forms such as VEGF121, VEGF145, VEGF165, VEGF185 and VEGF206 are produced. VEGF121, VEGF145 and VEGF165 are secreted soluble proteins, which can directly act on vascular endothelial cells to promote vascular endothelial cell proliferation. Increase vascular permeability. In 1990, Dr. Folkman of Harvard University in the United States proposed the famous Folkman theory that tumor tissue growth must rely on neovascularization to provide sufficient oxygen and nutrients to maintain. It is considered to be the basis for clinical application of VEGF.

Bevacizumab (trade name: Avastin) is a VEGF monoclonal antibody that inhibits vascular endothelial growth factor and is used in the treatment of various metastatic cancers. It is a popular drug for cancer treatment in Roche. Avastin’s 2009 sales reached $5.9 billion. Bevacizumab is a drug that blocks angiogenesis. It blocks the blood supply to tumors by inhibiting the action of vascular endothelial growth factor, inhibits the spread of tumors in the body, and enhances the effect of chemotherapy. The drug is approved by the US Drug Administration for the treatment of lung, colon and rectal cancer and is approved for breast cancer treatment in Europe.

Summary of the invention

It is an object of the present invention to provide an anti-tumor application of cGAMP in combination with bevacizumab.

The experimental study of the present invention shows that cGAMP combined with bevacizumab can significantly inhibit the growth of a variety of tumor cells, has obvious anti-tumor effect, and is superior to bevacizumab alone in treating tumors, so cGAMP can be used for preparation of anti-tumor. The drug or combination is used to treat the tumor.

In the present invention, the tumor includes, but is not limited to, adenocarcinoma, lung cancer, colon cancer, melanoma, and the like.

detailed description

The contents of the present invention will be specifically described below by way of examples. In the present invention, the following examples are presented to better illustrate the invention and are not intended to limit the scope of the invention.

Example 1: Preparation of cGAMP

cGAMP (cyclized-GMP-AMP) was synthesized by cyclized cGMP-AMP dinucleotide synthetase (cGAS) under the activation conditions of DNA binding according to literature methods. The purity is above 98%. (Ping weiLi, et al., Immunity, 2013, 39(6), 1019-1031.)

Example 2: Preparation of bevacizumab

The bevacizumab variable region was fused to the murine IgG2b constant region as a hybrid antibody, which was synthesized by Nanjing Kingsray and cloned into the pTT3 plasmid. The plasmid extraction kit, the Protein A column, the molecular sieve, and the endotoxin-purifying column and the endotoxin assay kit were purchased from General Medical or Nanjing Kingsray.

The 293F cells were transiently transfected with bevacizumab and purified by Protein A column, molecular sieve and endotoxin-clearing column. The purity was 90%, the endotoxin was less than 1 EU/ml, the protein was dissolved in PBS, and stored after lyophilization.

Example 3: Anti-tumor effect of cGAMP (cyclized-GMP-AMP) The tumor-bearing mouse model was used to examine the inhibitory effect of cGAMP on the growth of subcutaneous xenografts in animals.

Test drug

Name: cGAMP

Traits: white powder

Solvent: normal saline.

Preparation method: Prepare a solution of the desired concentration with a physiological saline solution before use.

Test drug concentration: 5 mg/ml, 10 mg/ml, 20 mg/ml. .

animal

Species, strains, gender, weight, source, certificate

C57BL/6 mice, female, weighing 20g, 8 weeks old, SPF grade, purchased from Shanghai Slack Laboratory Animals Co., Ltd. [Experimental Animal Quality Certificate No.: SCXK (Shanghai) 2007-0005].

Breeding conditions

All C57BL/6 mice were given free access to food and water and were housed at the Experimental Animal Center of a military medical university of the People's Liberation Army at room temperature (23 ± 2) °C. Both the feed and the water were autoclaved, and all the experimental feeding processes were SPF grade.

Dose setting

CGAMP was injected into the tail vein of mice, and 3 dose groups were set: 5 mg/kg, 10 mg/kg, 20 mg/kg.

Test control

Negative control: physiological saline solution

Positive control: bevacizumab at a dose of 10 mg/kg

Method of administration

Route of administration: intravenous administration

The number of bevacizumab administrations: once every 4 days, 4 times.

Dosing volume: 10ml/kg

cGAMP administration times: once a day for 14 consecutive days

Number of animals per group: 10

Tumor cell line

Adenocarcinoma cell line MC38, lung cancer cell line LLC, melanoma cell line B16-F10, colon cancer cell line MC38, purchased from the Chinese Academy of Sciences cell bank or ATCC.

Main steps of the test

1. Establishment and intervention of tumor-bearing mouse model

Six subcutaneous xenograft models were prepared: adenocarcinoma cell line MC38, lung cancer cell line LLC, melanoma cell line B16-F10, colon cancer cell line MC38, and the effect of cGAMP combined with bevacizumab was observed.

Adenocarcinoma cell line MC38, lung cancer cell line LLC, melanoma cell line B16-F10, colon cancer cell line MC38 were cultured and passaged. The cells were collected in the log phase of the cells to prepare a cell suspension at a concentration of (5.0×10 6 )/ml per ml, and the C57BL/6 mouse was injected into the right forelimb with a 0.2 ml cell suspension (the number of cells was 1.0×10 6 cells/cell). ), about 7-10 days, the tumor grows to a diameter of about 3-6 mm, and the tumor is successful.

Randomly divided into 5 groups, each group of 10 mice:

Negative control group (intravenous saline group)

Bevacizumab administration group (intravenous injection 10mg/kg group)

cGAMP low-dose combination group (intravenous cGAMP 5mg/kg plus bevacizumab 10mg/kg)

cGAMP medium dose combination group (intravenous cGAMP 10mg/kg plus bevacizumab 10mg/kg)

High-dose cGAMP combination group (intravenous cGAMP 20 mg/kg plus bevacizumab 10 mg/kg).

cGAMP was administered once a day for 14 days. Bevacizumab was administered once every 4 days for a total of 4 times. After 14 days, C57BL/6 mice were sacrificed and weighed, and the tumor inhibition rate = [1 - experimental group mean tumor weight / negative control group mean tumor weight]] × 100%.

2. Statistical analysis

Data were expressed as x±s, and were processed by SPSS10.0 software. The one-way ANOVA test was used to compare the significance of tumor weight difference in each group, and the significance level was a=0.05.

result

C57BL/6 mice were subcutaneously inoculated with tumor cells to prepare a subcutaneous xenograft model. cGAMP combined with bevacizumab significantly inhibited tumor growth. The tumor weight after 14 days of administration was significantly lower than that of bevacizumab control group (P). <0.05, P<0.01), indicating that cGAMP combined with bevacizumab Bevacizumab is used alone, so cGAMP can be used in combination with bevacizumab and has a significant anti-tumor effect. The specific results are shown in Tables 1 to 4.

Table 1 Inhibitory effect of cGAMP combined with bevacizumab on cell line MC38 adenocarcinoma (n=10, mean±SD)

Note: *P<0.05 vs bevacizumab group; **P<0.01 vs bevacizumab group.

Table 2 Inhibitory effect of cGAMP combined with bevacizumab on cell LLC lung cancer (n=10, mean±SD)

Note: *P<0.05 vs bevacizumab group; **P<0.01 vs bevacizumab group.

Table 3 Inhibitory effect of cGAMP combined with bevacizumab on cell B16-F10 melanoma (n=10, mean±SD)

Note: *P<0.05 vs bevacizumab group; **P<0.01 vs bevacizumab group.

Table 4 Inhibitory effect of cGAMP combined with bevacizumab on cell MC38 colon cancer (n=10, mean±SD)

Note: *P<0.05 vs bevacizumab group; **P<0.01 vs bevacizumab group.

Claims (5)

The application of cGAMP combined with bevacizumab in anti-tumor tumors. The application of cGAMP combined with bevacizumab in the preparation of antitumor drugs. The use of the cGAMP combined with bevacizumab according to claim 2 for the preparation of an antitumor drug, the tumor being adenocarcinoma, lung cancer, colon cancer, breast cancer, melanoma. Tumors were treated with cGAMP in combination with bevacizumab. The dosage form according to any one of claims 1 and 2, which is prepared by conventional pharmacy, including one of a tablet, a capsule, a granule, a suspension, an emulsion, a solution, a syrup or an injection. One or more of the administration of one or more of the oral or injection (including one or more of intravenous, intravenous, intramuscular or subcutaneous injections), etc., for the tumor and its directly related diseases Prevention, protection or treatment.