CN106692967A - Application of cyclic dinucleotide cGAMP combined PD1 antibody (Nivolumab) in preparation of antitumor drugs - Google Patents

Abstract

The invention relates to the technical field of biological medicines, and relates to application of cyclic dinucleotide cGAMP combined PD1 antibody (Nivolumab) in preparation of antitumor combined drugs. Studies indicate that the cGAMP combined Nivolumab can significantly inhibit the growth of a variety of tumor cells, has obvious anti-tumor effect, and can be used for preparing the antitumor combined drugs. A C57BL / 6 mouse subcutaneous transplantation tumor model shows that the cGAMP combined Nivolumab has obvious inhibition effect on adenocarcinoma cell line MC38, lung cancer cell line LLC, breast cancer cell line EO771, melanoma cell line B16-F10 and colon cancer cells MC38, and the obvious inhibition effect is superior to single drug use effect, so that the cGAMP can be combined with the Nivolumab for the treatment of tumors.

Description

Ring dinucleotides cGAMP Joint PD1 Antibody（ Nivolumab ）Application in antineoplastic is prepared

Technical field

The invention belongs to biomedicine technical field, and in particular to a kind of ring dinucleotides（cGAMP）Applications of the joint Nivolumab in antitumor and in antineoplastic is prepared.

Background technology

Tumour, is currently the major disease of a class serious harm human life and health, and it is embodied in cell hyperproliferation and disdifferentiation, and is difficult to suppress.According to WHO scholarly forecasts, to the year two thousand twenty, the tumor invasion number of population in the world is up to 20,000,000 people, and death toll is up to 12,000,000 people, therefore, tumour will constitute the threat of most serious to human survival as the big killer of this century mankind first.In China, the morbidity and mortality including lung cancer, colorectal cancer, stomach cancer, liver cancer etc. occupy the prostatitis of all kinds of malignant tumours, according to (2012 China's tumour registration annual reports that national tumour Register issues》Statistics, annual kainogenesis tumor cases are about 3,120,000, average daily 8 550 people, and the whole nation is per minute to have 6 people to be diagnosed as cancer.From the point of view of disease, lung cancer, stomach cancer, the knot/carcinoma of the rectum, liver cancer and the cancer of the esophagus occupy first five position of national malignant tumor morbidity.With the increase year by year of Cancer Mortality and the death rate, treating malignant tumor demand is increasing.Aging, fresh water food pollution, environmental pollution in particular with Chinese population etc., Chinese malignant tumor patient number will continue to increase.

At present, chemotherapy is one of conventional effective ways for the treatment of tumour.The mechanism of action of classic chemotherapy medicine is mainly the synthesis for preventing DNA (DNA), ribonucleic acid (RNA) or protein, or directly these macromoleculars are had an effect, so as to suppress the division growth of tumour cell, is allowed to dead.Some medicines can also be balanced by changing hormone in vivo and suppress tumour growth.Current antineoplastic has developed to 6 major classes：1. antimetabolite；2. alkylating agent；3. cytotoxin class antibiotic；4. plant alkaloid and other crude drugs；5. antitumor steroids；6. platinum class and other antineoplastics.With the transformation and the discovery of some new antineoplastic target spots of clinical treatment pattern, the research and development of field of antineoplastic medicaments there occurs great variety：For the mechanism of action of medicine, the targeted drug exploitation of non-cytotoxicity class is turned to from traditional nonspecific cytotoxic drug.In the antineoplastics of FDA approvals in 2012, small molecule tyrosine kinase inhibitors (TKI) turn into the most popular series antineoplastic medicament of research and development, the TKI (accounting for 3/4) of multiple target spots is especially acted on, by the TKI of June U.S. FDA approval in 2013 up to 18 kinds.Additionally, other focus mechanism of action medicines include immunostimulant, AI, cell cycle inhibitor, immunodepressant and stimulant, kinases inhibitor etc..In recent years, more and more immunomodulators are used for clinic, and for oncotherapy.

Microorganism and viral DNA can secrete the induction strong immune response in Inner sources by stimulating interferon in the mammalian cell of infection.Endoplasmic reticulum（ER）Receptor protein（STING）Immune response to cytoplasmic DNA is required factor.It has recently been demonstrated that cyclisation cGMP-AMP dinucleotides synzyme（cGAS）Under the activation condition after combining DNA, the synthesis of cGAMP is endogenously catalyzed.CGAMP is a kind of cytoplasmic DNA sensor, and it stimulates the sensing of INF- β as second messenger by STING, mediates the activation of TBK1 and IRF-3, and then start the transcription of INF- β genes.Report, recombinates cGAS catalytic cyclization cGMP-AMP dinucleotides GAMP under DNA conjugation conditions recently.CGAS combinations 18bp The crystal structure of the compound of dsDNA has also been reported, and researchs of the cGAMP in terms of antiviral immunity has been found to.CGAMP combination STING, activate transcription factor IRF3 and produce IFN-β.By STING paths, cGAMP can be antitumor for activating immune system by as immunomodulator.

The PD1 antibody of Mei-Shi Guibao companies exploitation when Nivolumab is hundred, and FDA approval listings were obtained in 2014, it has been applied to treat melanoma and breast cancer at present.PD1 is the acceptor of cell surface, after PD1 is combined with PDL1, can suppress the activation of T cell, so as to lower Human immune responses process, promotes the identification of neoplastic cells escape immune t-cell.Nivolumab can combine PD1, and blocking PD1 is combined with PDL1, and promotes effector T cell to activate, so that killing tumor cell, by the immunization route, treats malignant tumour.At present, Nivolumab antibody authorizes breakthrough medicine by FDA, and the PD1 antibody has started to other antibody alone or in combination（Such as CTLA4 antibody）Deng for clinical treatment kinds cancer.

At present, ring dinucleotides cGAMP and PD1-PDL1 interaction inhibiting antibody is still used in combination the relevant report for the treatment of tumour without document report.

The content of the invention

It is an object of the invention to provide applications of the cGAMP joints Nivolumab in antitumor.Experimental study of the present invention shows that cGAMP joints Nivolumab can significantly inhibit the growth of kinds of tumor cells, with obvious antitumor action, can be used to prepare antineoplastic.In the present invention, the tumour includes but is not limited to gland cancer, lung cancer, colon cancer, breast cancer, melanoma etc..

Specific embodiment

Present disclosure is illustrated below by embodiment.In the present invention, it, in order to preferably illustrate the present invention, is not for limiting the scope of the present invention that embodiments discussed below is.

Embodiment 1 ： cGAMP Preparation

cGAMP （Cyclisation-GMP-AMP）By literature method under the activation condition after combining DNA, by being cyclized cGMP-AMP dinucleotides synzyme（cGAS）Catalyze and synthesize.Purity is more than 98%.（Pingwei Li, et al., Immunity, 2013, 39(6), 1019-1031.）

Embodiment 2 ： Nivolumab Preparation

Nivolumab 5C4 variable regions are hybrid antibody with mouse IgG2b constant domains, and the gene is synthesized and be cloned into pTT3 plasmids by Nanjing Genscript Biotechnology Co., Ltd..The big extraction reagent kit of plasmid, Protein A chromatographic columns, molecular sieve and Endotoxin removal post, endotoxin measurement kit are purchased from common therapy or Nanjing Genscript Biotechnology Co., Ltd..

Expression Nivolumab 5C4 are transiently transfected in 293F cells.By Protein A posts, molecular sieve and Endotoxin removal post（Purchased from common therapy）After purification, purity is 98%, and endotoxin is less than 1 EU/ml.Protein dissolution is preserved in phosphate buffer after freezing.

Embodiment 3 ：CGAMP combines for antitumor animal experiment with Nivolumab

The inhibitory action grown to animal subcutaneous transplantation knurl using mice with tumor model inspection cGAMP and Nivolumab combined with antineoplastic and the toxic action to animal.

Test medicine

Title：CGAMP, Nivolumab

Proterties：White powder

Solvent：Physiological saline.

Compound method：The solution of required concentration is configured to normal saline solution before use.

Test drug concentrations：cGAMP: 5mg/ml、10mg/ml、20mg/ml; Nivolumab, 10mg/ml。

Experimental animal

Kind, strain, sex, body weight, source, the quality certification

C57BL/6 mouse, female, body weight 20g, 8 week old, are purchased from Shanghai Slac Experimental Animal Co., Ltd.'s [Quality of Experimental Animals quality certification number by SPF grades：SCXK （Shanghai）2007-0005 ] 。

Rearing conditions

All mouse, freely look for food and drink water, in room temperature（23±2）DEG C, raise in army medical university of Chinese People's Liberation Army Experimental Animal Center.Feed and water are processed through autoclaving, and all experimentss feeding process is SPF grades.

Dosage is set

CGAMP intravenous injection into mice, sets 3 dosage groups：5 mg/kg, 10 mg/kg, 20 mg/kg

Experimental control

Negative control：Normal saline solution

Positive control：Nivolumab, dosage 10mg/kg

Medication

Method of administration：Tail vein injection is administered

Nivolumab administration number of times：Every 4 days 1 time, it is administered 4 times.

Administered volume：1ml/kg

CGAMP administration number of times：Once a day, continuous 14 days

Every group of number of animals：10

Tumor cell line

Adenocarcinoma cell strain MC38, lung cancer cell line LLC, breast carcinoma cell strain EO771, melanoma cells line B16-F10, colon cancer cell MC38, are purchased from Chinese Academy of Sciences's cell bank or ATCC companies of the U.S..

Experiment key step

1 The foundation of lotus knurl mouse model and intervention

6 kinds of subcutaneous transplantation knurl models are prepared respectively：Adenocarcinoma cell strain MC38, lung cancer cell line LLC, breast carcinoma cell strain EO771, melanoma cells line B16-F10, colon cancer cell MC38, and observe the effect that cGAMP combines Nivolumab.

Adenocarcinoma cell strain MC38, lung cancer cell line LLC, breast carcinoma cell strain EO771, melanoma cells line B16-F10, colon cancer cell MC38 culture, passage.Cell is collected in the cell log phase, it is 5.0 × 10 to make concentration⁶0.2 ml cell suspensions (cell number is 1.0 × 106 /) are injected in every milliliter of cell suspension, C57BL/6 mouse right fores oxter, and tumour is long to diameter about 3-6 mm, tumorigenesis success within 7-10 days or so.

It is divided into 5 groups, every group of 10 mouse at random：

Negative control group（Intravenous injection physiological saline group）

Nivolumab administration groups (vein note 10mg/kg groups)

CGAMP low dosage joints group (the intravenous injection mg/kg of cGAMP 5 add the mg/kg of Nivolumab 10)

(the intravenous injection mg/kg of cGAMP 10 add Nivolumab to cGAMP middle dose groups 10 mg/kg)

CGAMP group high doses group (the intravenous injection mg/kg of cGAMP 20 add the mg/kg of Nivolumab 10).

CGAMP is administered once daily, successive administration 14 days.Nivolumab is administered once for every 4 days, totally 4 times.After 14 days, put to death mouse and claim tumor weight, tumour inhibiting rate=[the average average knurl weight of knurl weight/negative control group of 1- experimental groups)] × 100%.

2 Statistical analysis

Data are represented with x ± s, processed using SPSS10.0 softwares, using one-way analysis of variance（one-way ANOVA）The conspicuousness of each group knurl weight difference, significance a=0.05 are compared in inspection.

Experimental result

Subcutaneous transplantation knurl model is successfully prepared after mouse hypodermic inoculation tumour cell, cGAMP joints Nivolumab can substantially suppress tumour growth, and the knurl weight after being administered 14 days is substantially less than Nivolumab control groups（P<0.05 , P<0.01）, show that cGAMP joint Nivolumab can combine Nivolumab and use better than exclusive use Nivolumab, therefore cGAMP, and with notable antitumor action.Concrete outcome is shown in Table 1- tables 5.

Table 1 cGAMP Joint Nivolumab To cell line MC38 The inhibition of gland cancer

（N=10, mean ± SD）

Group Average knurl weight（ g ） Average tumour inhibiting rate（ % ）

Negative control group 2.120 ± 0.226（g） -

Nivolumab groups 0.892 ± 0.312（g） 58.0

CGAMP joint Nivoluma low dose groups 0.742 ± 0.261（g）* 65.0

CGAMP joint Nivoluma middle dose groups 0.671 ± 0.166（g）** 68.3

cGAMP Joint Nivoluma High dose group 0.439±0.101 （ g ） ** 79.3

Note：*P<0.05 vs Nivolumab groups；**P<0.01 vs Nivolumab groups.

Table 2 cGAMP Joint Nivolumab To cell LLC The inhibition of lung cancer

（N=10, mean ± SD）

Group Average knurl weight（ g ） Average tumour inhibiting rate（ % ）

Negative control group 2.802 ± 0.111（g） -

Nivolumab groups 0.919 ± 0.143 （g） 57.2

CGAMP joint Nivoluma low dose groups 0.799 ± 0.392（g）** 71.5

CGAMP joint Nivoluma middle dose groups 0.612 ± 0.126（g）** 78.2

cGAMP Joint Nivoluma High dose group 0.509±0.213 （ g ） ** 81.9

Note：*P<0.05 vs Nivolumab groups；**P<0.01 vs Nivolumab groups.

Table 3 cGAMP Joint Nivolumab To cell EO771 The inhibition of breast cancer

（N=10, mean ± SD）

Group Average knurl weight（ g ） Average tumour inhibiting rate（ % ）

Negative control group 2.610 ± 0.328（g） -

Nivolumab groups 0.640 ± 0.134（g） 75.5

CGAMP joint Nivoluma low dose groups 0.656 ± 0.277（g） 75.0

CGAMP joint Nivoluma middle dose groups 0.541 ± 0.134（g）** 79.3

cGAMP Joint Nivoluma High dose group 0.485±0.139 （ g ） ** 81.4

Note：*P<0.05 vs Nivolumab groups；**P<0.01 vs Nivolumab groups.

Table 4 cGAMP Joint Nivolumab To cell B16-F10 The inhibition of melanoma

（N=10, mean ± SD）

Group Average knurl weight（ g ） Average tumour inhibiting rate（ % ）

Negative control group 2.72 ± 0.116（g） -

Nivolumab groups 0.901 ± 0.198（g） 66.9

CGAMP joint Nivoluma low dose groups 0.721 ± 0.161（g）** 73.5

CGAMP joint Nivoluma middle dose groups 0.634 ± 0.172（g）** 76.7

cGAMP Joint Nivoluma High dose group 0.568±0.133 （ g ） ** 79.1

Note：*P<0.05 vs Nivolumab groups；**P<0.01 vs Nivolumab groups.

Table 5 cGAMP Joint Nivolumab To cell MC38 The inhibition of colon cancer

（N=10, mean ± SD）

Group Average knurl weight（ g ） Average tumour inhibiting rate（ % ）

Negative control group 2.001 ± 0.325（g） -

Nivolumab groups 1.261 ± 0.431（g） 37.0

CGAMP joint Nivoluma low dose groups 0.821 ± 0.267（g）** 59.0

CGAMP joint Nivoluma middle dose groups 0.579 ± 0.240（g）** 71.1

cGAMP Joint Nivoluma High dose group 0.414±0.198 （ g ） ** 79.3

Note：*P<0.05 vs Nivolumab groups；**P<0.01 vs Nivolumab groups.

Claims (3)

1. applications of the ring dinucleotides cGAMP joints Nivolumab in antitumor.

2. Ring dinucleotides cGAMP combines applications of the Nivolumab in antineoplastic combination medicine is prepared, the ring dinucleotides united preparation of cGAMP and Nivolumab that it is prepared into comprising the different size ring united unit formulation of dinucleotides cGAMP and Nivolumab and pharmaceutically acceptable carrier.

3. routinely pharmacy is made various formulations to combination medicine according to claim 2, described formulation including tablet, capsule, granule, supensoid agent, emulsion, solution, syrup or injection etc. in one or more, take oral or injection（One or more in including intravenous injection, drip-feed, intramuscular injection or hypodermic injection etc.）One or more method of administration in carries out prevention, protection or the treatment of tumour and its directly related disease.