Method for producing live cell vaccine for breast cancer prevention

FIELD: medicine.

SUBSTANCE: murine benign spontaneous breast tumour cells are cultivated. At first, the cultivation procedure is in vitro in the DMEM/RPMI-1640 medium with 10 % of bovine embryo serum up to 20 passages. Then, in vivo up to 22 passages. Then, once again in vitro up to 25 passages. Then, 10 thousand-1 million produced cells are introduced in a connective tissue capsule formed in the Balb/c mice after the introduction of a biocompatible gel. The tumour growth is enabled. The encapsulated tumour is aseptically removed and trypsinised to produce primary epithelial cell cultures. The produced cells concentrated 10 thousand-1 million are introduced into the capsule subcutaneously gel-formed in the Balb/c mice. The tumour growth is enabled for vaccination. In the in vitro culture, the cells are transplanted once a week while being disseminated in the ratio 1:3 - 1:4, with a monolayer twice washed with EDTA solution and removed with 0.25% trypsin during 2-3 min at room temperature, and the trypsinisation process is terminated by adding 10 % of fetal serum "к.р.с.".

EFFECT: invention allows producing an anticancer vaccine application of which is based on a principle of transplantation of a live pathological material to a person from an animal with similar disease.

3 dwg

 

The invention relates to immunology and represents a way to obtain transplantable lines of tumor cells of the mouse with the properties of a vaccine against adenocarcinoma of the mammary glands. Being a benign tumor of mice of Balb/c derived cell line intended for transplantation in syngeneic animals only under condition of cell injection in a pre-established connection woven capsule. If xenotransplantation man thus obtained cell line, called BBCV, can be proposed as a safe immunogenic drug for the prevention of adenocarcinoma of the breast in women (Line BBCV deposited in the special collection of cell cultures vertebrate Russian cell culture collection and maintained in a Bank of cell cultures, MNIMA, see Appendix, Passport cell line BBCV).

The level of technology.

The basis of the invention is a known vaccine Genera for humans is similar to the pathological disease process, but limited to the injection site and creating immunity against Angeleno related pathological beginning.

Immunoprophylaxis in Oncology is currently being developed in several directions. The most attractive for clinicians is a surgical prevention of metastasis and generator is Itachi neoplastic process. The drugs used in this area, referred to as vaccines, in the sense that they act as active specific stimulators of antitumor immunity. In a sense, their impact should be more therapeutic than preventive, what fundamentally distinguishes the majority of developing cancer vaccines from classical preventive vaccines, successfully applied in infectious pathology. Prevention of the disease by artificial reproduction is similar, but a benign process, was the initial vaccination, which sir Edward Jenner introduced in medical practice for more than 200 years ago. Inoculation of cowpox, vaccinia, vacca - cow (lat.), on Generro start the fight with the virus of smallpox long before the word virus and descriptions of smallpox

Infection common to man and animals (zoonoses), and the potential risk of new diseases in the human infection with animal viruses prevented the attempts of repetition schemes Jenner - application of the pathogen of the animal as a vaccine against human disease. The first successful pair of this kind - the smallpox virus cows for smallpox was almost the only one. But this approach allowed us to eliminate smallpox as a disease all over the world. When creating the image is etenia also taken into account the known from the prior art using Angeleno modified cellular vaccines of cancer cells for cancer treatment US 7094603, C12N 15/83, 08.22.2003 /1/.

The essence of the invention.

The result, which directed the invention is the creation of an anti-cancer vaccine, which is based on the principle of vaccinations person living pathological material from animals with similar disease. When implementing the inventive method received the vaccine, representing a transplantable tumor line cells of the mouse with the properties of a vaccine against adenocarcinoma of the mammary glands. With the principles that enables the accumulation of maple cell lines described, for example, in US 4003789, C12N 5/06, 18.01.1977 /2, 4/, and the principles, confirming the possibility of using an inert gel to create a connective tissue capsule, described in US 6972194, AK 9/00, 06.12.2005 /3/.

The basis of the invention is the use of benign tumors of mice Balb/c cell line, transplantable when transplanted in syngeneic animals only under condition of cell injection in a pre-established soedinitelnotkannoy capsule. Since the cell line can be cultured indefinitely in vitro in a nutrient medium, DMEM/RPMI-1640 with 10% fetal bovine serum, allowing for the accumulation of production quantities of standard vaccine material.

Line BBCV was the floor of the Jena from benign spontaneous mammary tumor mouse Balb/c hybridoma laboratory of biotechnology, NIIME and cultivated for 7 years in vitro and in vivo. Cells BBCV were cultured in vitro for 20 passages, then through passage 22 in vivo in mice of Balb/c and then were cultured in vitro 25 passages. The cultural medium is DMEM/RPMI-1640 with 10% fetal serum CRS the cultivation Temperature +37°C. Cells transplanted 1 week, Russia 1:3-1:4. The monolayer washed twice with a solution of EDTA and filmed 0.25% trypsin solution for 2-3 min at room temperature.

Transplantation of the tumor was not possible with the introduction 1.0 million cells intravenously, intraperitoneally or subcutaneously with syngeneic mice of Balb/c. Animals remained healthy during the observation period of twelve months.

It was found that the line BBCV causes benign solid tumor when administered 10 thousand - 1.0 million cells in the connective tissue capsule formed from mice of Balb/c mice after injection of biocompatible gel (Matrigel, Silicone, Agarose and Polyacrylamide gel shown in US 6972194, AK 9/00, 06.12.2005 /3/). This cell line also does not cause tumors in subcutaneous and intraperitoneal injection of 100 thousand cells of mice lines SN, DVA and C57Black/6.

Under cultivation in vivo encapsulated tumor aseptically removed and subjected trypsinization by the standard method of obtaining primary epithelial cell cultures: 2.5% trypsin, 20 min, 37°C. Trypsinization was stopped by adding 10% fetal serum CRS Glue the key besieged by centrifugation at 1500 rpm, resuspendable in medium RPMI-1640 to a concentration of 1 million/ml and was administered at a dose of 10 thousand to 1 million in a capsule formed by the gel under the skin of mice of Balb/c. Tumor growth begins after 1-2 weeks.

Property immunogenicity was shown as follows.

When allogeneic transplantation 10 million cells in the connective capsule mice lines C57Black/6 cells BBCV do not cause cancer, but generate protective immunity that protects mice from the development of syngeneic adenocarcinoma CA-755, introduced subcutaneously at a dose of 100 million cells. Figure 1 presents the total results of the experiment to estimate wakciniruemogo effect cells BBCV for mice C57Black/6. Three groups of 4 mice were immunized by injecting the cells in soedinitelnotkannye capsules formed under the skin in the shoulder prior to the introduction of 0.5 ml of one of the following inert gels: 2% polyacrylamide, 2% agarose and liquid silicone (filler breast implants), 4 animals in each gel. All gels were evaluated for ability to form connective tissue capsule and to support the growth of cells BBCV in syngeneic Balb/c mice in a separate experiment. Gels were equally effective in maintaining growth of syngeneic tumors reached a size of 4-6 cm220-30 days after the adoption of the cells BBCV mice Balb/c mice. The mice of C57Black/6 cells BBCV did not cause the image is of tumors and one month after their introduction, vaccinated thus mice were injected 100 million cells syngeneic adenocarcinoma CA-755 (5, 6). 10 control animals tumor appeared on 6-7 day and has evolved to a state incompatible with life to 14-15 days. In vaccinated animals, the tumor appeared on 5-7 days later and never appeared in 20% of mice (figure 1).

Subcutaneous injection of allogeneic cells BBCV the mice C57Black/6, without using a connective tissue capsule, did not cause formation in animals protective immunity against syngeneic tumor CA-755.

When used for transplantation of syngeneic adenocarcinoma of the lower dose of cells CA-755 effect of vaccination with allogeneic cells BBCV reached 100%, while lengthening the life of the animals in the control up to 45 days (figure 2).

From the experiments it follows that the benign tumor line BBCV, for syngeneic mice of Balb/c can serve as a preventive allogeneic vaccine adenocarcinoma CA-755 in mice of C57Black/6.

Vaccination of animals cells BBCV led to an increase in their serum antibodies to the tumor cells of the mammary glands of mouse CA-755 and human MCF-7 in vitro.

Studies have shown that breast adenocarcinoma BBCV is transplanted tumor, for syngeneic mice of Balb/c provided transplantation of tumor cells to connect latkany capsule and, as a result, it was concluded that the cell line BBCV can be used as a living allogeneic vaccine adenocarcinoma mice.

The principal possibility of preventive vaccination when breast cancer xenogeneic tumor cells, was shown in an experiment by vaccination of mice with human adenocarcinoma MCF-7. In this experiment, four mice 57l/6, with pre-formed under the skin connective tissue capsule, were introduced cells MCF-7 grown in vitro. Cells were besieged by centrifugation and injected into the capsule in a volume of 0.25 ml dose of 10 million cells per mouse. After 30 days, four control and four vaccinated mice under the skin was introduced on 10 million cells syngeneic adenocarcinoma CA-755 (figure 3).

The effect of xenogeneic vaccination appeared later, at 8-10 days, the appearance of tumors in vaccinated animals and reliable increase their life expectancy by 14 days. Although all vaccinated human adenocarcinoma mice eventually developed a tumor, CA-755, the protective effect of vaccination was reliable. At the time of loss of control mice all vaccinated mice were alive and tumor started to grow.

Low efficiency xenogeneic vaccination on the model of MCF-7 - Sa can be explained in erwou the that used human adenocarcinoma is a cell culture with a long history of life in vitro, had much change in comparison with the tumor in vivo. Tumor mouse and man, growing in the same conditions in vivo may be more antigenic relationship and, therefore, can serve as a more effective xenogeneic vaccine.

If xenotransplantation human cell line BBCV can be proposed as a safe immunogenic drug for the prevention of adenocarcinoma of the breast in women.

Implementation of the claimed method of producing a living cell vaccine has created a line of mouse cells, does not cause tumors in syngeneic mice when the usual methods of transplantation. This line of mouse cells, transplanted as a benign tumor, not spreads, provided the introduction of a connective tissue capsule of syngeneic mice. This line of mouse cells, causing protective immunity (vaccine), can be transplanted in the connective tissue capsule of allogeneic and xenogeneic recipients. While allogeneic recipient is any mice of Balb/c, a xenogeneic recipient is the man.

Literature

1. US 7034603, A61K 38/16, 08.22.2003.

2. US 4003789, C12N 5/06, 18.01.1977.

3. US 6972194, A61K 9/00, 06.12.2005.

4. A.D.Borowsky, R.Namba, L.J.T.Young, K.W.Hunter, J.G.Hadgson, C.G.Tepper, E.T.McGoldrick W.J.Muller, R.D.Cardiff, J.P.Gregg. Syngeneic mouse mammary carcinoma cell lines: Two closely related cell lines with divergent metastatic behavior. Clinical & Experimental Metastasis. 22, 47-58, 2005.

5. Liautaud-Roger F., Desoize Century, Mili, H., Y. Carpenter, Delvicourt C., Conix P. Serum enzymes and triglycerides in mice with a mammary tumor (Ca-755 adenocarcinoma), Pathol.Biol. (Paris), 1987, Oct, 35(8): 1119-22.

6. Niimura, K., Furisho T., Fujii M., Takahashi N., Matsunaga K., Sugita n, Toshikumi C., Kawai Y. Tumor and tissue distribution of 1251 - labeled natural and anti-tumor antibodies in murine experimental tumor systems. Anticancer Res. 1988, jul-Aug, 8(4); 589-93.

Application: Passport cell line BBCV in 3 liters

The method of obtaining a living cell vaccine for the prevention of breast cancer, comprising culturing cells deprecating spontaneous mammary tumor mouse:
in vitro in DMEM/RPMI-1640 with 10% fetal bovine serum to 20 passages;
in vivo to 22 passages;
in vitro up to 25 passages;
enter 10 thousand-1 million received cells in the connective tissue capsule formed in mice of Balb/c after the introduction of biocompatible gel;
provide tumor growth;
encapsulated tumor aseptically remove and expose trypsinization accessing primary epithelial cell cultures;
the obtained cells at a concentration of 10 thousand-1 million injected into the capsule formed by the gel under the skin of mice of Balb/c;
provide tumor growth intended for vaccination;
when cultivated in vitro cell perejivaut 1 time per week, the race is the first in a ratio of 1:3 - 1:4, twice, rinsing the monolayer with a solution of EDTA and relieving him of 0.25%trypsin solution for 2-3 min at room temperature, and trypsinization stopped by the addition of 10% fetal serum CRS



 

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