AU2020103625A4

AU2020103625A4 - Applications of bacillus calmette-guerin (bcg) polysaccharide and nucleic acid in in-vitro culture of cytokine-induced-killer (cik) cells and preparation of antitumor drugs

Info

Publication number: AU2020103625A4
Application number: AU2020103625A
Authority: AU
Inventors: Chun Chen; Yaobiao Huang; Minghao Lu
Original assignee: China Jiliang University
Current assignee: China Jiliang University
Priority date: 2020-11-24
Filing date: 2020-11-24
Publication date: 2021-02-04
Anticipated expiration: 2028-11-24

Abstract

Austracy The present invention relates to applications of bacillus calmette-guerin (BCG) polysaccharide and nucleic acid in in-vitro culture of cytokine-induced-killer (CIK) cells and preparation of antitumor drugs. According to the applications, the BCG polysaccharide and nucleic acid is added into a culture system for in-vitro culture of the CIK cells. The CIK cells cultured by the BCG polysaccharide and nucleic acid have an obvious proliferation-inducing effect; CD3 and CD56 double positives of the cells are significantly increased; and the CIK cells subjected to induced culture have a significant killing effect on tumor growth. Therefore, the CIK cells have extremely high clinical application values, and particularly provide a new way to develop tumor therapy drugs. 1 Drawings of Description 6.0 > 5.5C Culture medium containing 35 pg/mL of BCG polysaccharide oA Culture medium containing 3.5 tg/mL of BCG polysaccharide 0Culture medium containing 0.35 g/mL of BCG polysaccharide Culture medium containing no BCG polysaccharide 4.5 1 Id 2d 3d 4d 5d 6d 7d FIG. 1 1

Description

Austracy

The present invention relates to applications of bacillus calmette-guerin (BCG) polysaccharide and nucleic acid in in-vitro culture of cytokine-induced-killer (CIK) cells and preparation of antitumor drugs. According to the applications, the BCG polysaccharide and nucleic acid is added into a culture system for in-vitro culture of the CIK cells. The CIK cells cultured by the BCG polysaccharide and nucleic acid have an obvious proliferation-inducing effect; CD 3 and CD 56 double positives of the cells are significantly increased; and the CIK cells subjected to induced culture have a significant killing effect on tumor growth. Therefore, the CIK cells have extremely high clinical application values, and particularly provide a new way to develop tumor therapy drugs.

Drawings of Description

6.0

> 5.5C

Culture medium containing 35pg/mL of BCG polysaccharide oA Culture medium containing 3.5 tg/mL of BCG polysaccharide

Culture medium containing 0.35 g/mL of BCG polysaccharide Culture medium containing no BCG polysaccharide 4.5 1 Id 2d 3d 4d 5d 6d 7d

FIG. 1

Description

APPLICATIONS OF BACILLUS CALMETTE-GUERIN (BCG) POLYSACCHARIDE AND NUCLEIC ACID IN IN-VITRO CULTURE OF CYTOKINE-INDUCED-KILLER (CIK) CELLS AND PREPARATION OF ANTITUMOR DRUGS

Technical Field

The present invention relates to the technical field of traditional Chinese medicine applications, and particularly relates to applications of bacillus calmette-guerin (BCG) polysaccharide and nucleic acid in in-vitro culture of cytokine-induced-killer (CIK) cells and preparation of antitumor drugs.

Background

Adoptive cellular immunotherapy is a current research focus, and is a biotherapy method that infuses autologous or allogeneic immunologic effector cells activated in vitro to patients, kills in-vivo tumor cells of the patients and repairs and improves the immune system of the body. Cytokine-induced-killer (CIK) cells are a category of heterogeneous cells that are induced by multiple cytokines together and can simultaneously express two membrane protein molecules, i.e., CD 3 and CD5 6 heterocysts. The CIK cells have restricted tumor killing characteristics of non-major histocompatibility complexes (MHCs) of natural killer cells, also have strong antitumor activity of T-lymphocytes, and have important clinical application values of immunotherapy. Therefore, large-scale in-vitro proliferation of the CIK cells, i.e., increase of an in-vitro proliferation rate of the CIK cells and killability to tumor cells, is the key to large-scale use of the CIK cells in immunotherapy. Bacillus calmette-guerin (BCG) mainly includes components as follows: lipids, polysaccharides, proteins and nucleic acids, wherein the polysaccharides (including lipids) and nucleic acids play an important physiological role. Major components of cell walls of the BCG are composed of

Description

polysaccharides (mainly including peptidoglycan, arabinogalactan and mycolic acid) and lipids. Researches show that, a large quantity of ligands of Toll receptors (TLR) that can be bound with humans and animals exist on the BCG cell walls and other organelles, and these ligands may be at least bound with 5 kinds of Toll receptors (such as TLR1, TLR2, TLR4, TLR6 and TLR9). After the ligands are bound with the Toll receptors, maturation of dendritic cells may be promoted, and secretion of IL-12 is induced. Moreover, the IL-12 can secrete IFN-y and promote transformation from ThO cells to Th1 cell subsets, thereby producing ThI-dominant nonspecific immune response.

Summary

By utilizing the great advantage of BCG polysaccharide and nucleic acid in organic combination with CIK cell immunotherapy in tumor therapy, a process system of the BCG polysaccharide and nucleic acid for promoting large-scale proliferation of immune cells and increasing killability is built, thereby indirectly improving living quality of tumor patients and prolonging lifetime. A purpose of the present invention is to provide applications of an optimal extracting solution concentration of BCG polysaccharide and nucleic acid in an aspect of increasing a proliferation rate of CIK cells, CD 3 and CD5 6 double positives and tumor killability. The present invention provides a BCG polysaccharide and nucleic acid for promoting proliferation and differentiation of CIK cells. The BCG polysaccharide and nucleic acid is prepared by BCG thallus breakage and hot phenol treatment. The present invention provides a CIK cell culture medium, including a basal culture medium and BCG polysaccharide and nucleic acid. Further, content of the BCG polysaccharide and nucleic acid in a culture medium is 0.35-35 pg/mL. Preferably, the content of the BCG polysaccharide and nucleic acid in the culture medium is 35 pg/mL.

Description

Further, the basal culture medium is selected from an RPM1640, a DMEM or an MEM. Further, the culture medium further includes IFN-y and CD 3 monoclonal antibodies. The present invention provides a culture method of CIK cells, including the following steps: (1) preparing starter cells of CIK cells; (2) preparing a culture medium containing BCG polysaccharide and nucleic acid; adding the starter cells of the step (1); and culturing the cells, thereby obtaining the CIK cells. Further, content of the BCG polysaccharide and nucleic acid in a culture medium is 0.35-35 pg/mL. Preferably, the content of the BCG polysaccharide and nucleic acid in the culture medium is 35 pg/mL. Further, the culture medium further includes IFN-y and CD 3 monoclonal antibodies. Further, the method in the step (2) includes: culturing the cells in a 5% CO2 incubator at 37°C for 7 d; and periodically replacing 1/2 of corresponding culture solution. The present invention further provides an application of BCG polysaccharide and nucleic acid in promotion of proliferation and differentiation of CIK cells. The present invention further provides an application of CIK cells prepared by a preparation method of the BCG polysaccharide and nucleic acid in preparation of tumor therapy drugs. Further, the preparation method of the BCG polysaccharide and nucleic acid includes the following steps: 1) thallus culture: dissolving the bacteria seeds, stored at low temperature in liquid, at a room temperature; inoculating the bacteria seeds to a potato Sauton's medium; and continuously culturing the bacteria seeds at 37°C for 14-20 days;

Description

2) thallus collection: collecting pellicle in a culture flask after the thallus grows to a logarithmic phase; adding an appropriate amount of deionized distilled water for washing; and pressing the pellicle to be dry for later use; 3) thallus breaking and hot phenol treatment: adding the deionized distilled water into the collected thallus according to a ratio of 10:1; breaking the thallus by a tissue mashing homogenizer (12000 rpm/min) for 3 times by 3 min each time; mashing the thallus; adding hot phenol (30-100°C) which is 0.5-2.0 times of the volume of the broken bacterial suspension; and maintaining the temperature for 30 minutes to 1 hour while low-speed stirring; 4) extraction of a mixture of BCG polysaccharide and nucleic acid: naturally precipitating the mixed solution treated with the hot phenol for 1-10 days; sucking the supernatant; centrifuging the solution by a tube centrifuge at a high speed; and filtering the supernatant by a 0.45pm sterile filter so as to obtain the mixture of BCG polysaccharide and nucleic acid; 5) adding medicinal ethanol into the collected extracting solution of BCG polysaccharide and nucleic acid for performing alcohol precipitation at alcohol content of 70-75%; collecting a precipitate within 4 days after natural precipitation; fully and uniformly stirring the precipitate with absolute ethyl alcohol; centrifugally washing the precipitate for 3 times; drying the precipitate in a vacuum drier at 50°C after the precipitate is centrifugally washed with diethyl ether for 3 times, thereby obtaining the dry matter, i.e., the extract of BCG polysaccharide and nucleic acid. The present invention has beneficial effects as follows: (1) compared with other chemically induced proliferation factors, the BCG polysaccharide and nucleic acid in the present invention meets cell culture requirements, that is, has no toxic or side effect to the human body; thus, the proliferation-induced CIK cells can be directly applied to clinical tests; (2) compared with corresponding polysaccharide stimulating factors, the polysaccharide extraction method in the

Description

present invention is simplified and advanced in dosage form; and the polysaccharide extracting solution is obvious in effect, clear in component and controllable in quality; and (3) the BCG polysaccharide and nucleic acid in the present invention has an effect of significantly increasing the proliferation rate of the CIK cells; the CD 3 and CD5 6 double positives are significantly increased; and the killability of the activated cells is obviously higher than that of control treatment of the CIK cells.

Description of Drawings Fig. 1 is a trend chart of proliferation of cells induced by BCG polysaccharide and nucleic acid at different concentrations; and Fig. 2 shows killability evaluation of CIK cells induced by BCG polysaccharide and nucleic acid at an optimal concentration to tumors.

Detailed Description

The present invention will be described in detail below in combination with specific drawings. Only some of specific embodiments are listed without departing from the spirit of the present invention, but shall not exclude other specific embodiments generated by combining the prior art and the present invention by those skilled in the art, or other specific embodiments generated by utilizing existing clinical traditional Chinese medicine injection. Preparation of major materials: A preparation method of a BCG polysaccharide and nucleic acid having a significant effect of increasing proliferation and differentiation of CIK cells includes the following steps: (1) the bacteria seeds stored at low temperature in liquid were dissolved at a room temperature; the bacteria seeds were inoculated to a potato Sauton's medium; and the bacteria seeds were continuously cultured for 14-20 days;

Description

(2) pellicle was collected after the thallus grew to a logarithmic phase; and the pellicle was washed and pressed to dry; (3) the collected thallus was broken by a tissue mashing homogenizer; and the thallus was subjected to thermal insulation with hot phenol while low-speed stirring; (4) the mixed solution treated with the hot phenol was naturally precipitated; the supernatant was sucked; the solution was filtered and sterilized after centrifugation so as to obtain a mixture of BCG polysaccharide and nucleic acid; (5) the collected extracting solution of BCG polysaccharide and nucleic acid was subjected to alcohol precipitation; a precipitate was collected within 4 days after natural precipitation; the precipitate was fully and uniformly stirred with absolute ethyl alcohol, centrifugally washed, and dried in a vacuum drier, thereby obtaining the dry matter, i.e., the extract of BCG polysaccharide and nucleic acid; (6) the BCG polysaccharide and nucleic acid which was dried in vacuum was precisely weighed and dissolved with water; and the volume was fixed, thereby obtaining the prepared solution, i.e., the solution of BCG polysaccharide and nucleic acid. Embodiment 1 Culture and harvest of BCG 1. Thallus culture: the bacteria seeds stored at low temperature in liquid (BCG strain D2PB302 for Chinese BCG preparation, China National Institute for the Control of Pharmaceutical and Biological Products) were dissolved at room temperature; the bacteria seeds were inoculated to a potato Sauton's medium; and the bacteria seeds were continuously cultured at 37°C for 14-20 days; wherein a preparation method of the potato Sauton's medium may include the following steps: (1) one fresh potato was cleaned, punctured into a cylinder by a puncture and then cut into an inclined plane of 4 cm long; the inclined plane of the potato was washed with flowing drinking water for an hour; the inclined plane block of the

Description

potato was washed with purified water and washed with the Sauton's medium; and ml of the Sauton's medium was taken and added into 100 ml large sterilized tube; (2) the washed inclined plane of the potato was put into the large sterilized tube filled with the Sauton's medium; (3) the inclined plane of the potato was sterilized under an atmospheric pressure of 0.11 MPa at 121°C for 20 minutes, and then cooled to a room temperature for inoculation. Preparation proportions of the Sauton's medium were as follows: Every 1000 ml of the medium included: 0.5 g of magnesium sulfate, 1.04 g of dipotassium phosphate; 8.0 g of monosodium glutamate, 2.0 g of citric acid; 60 ml of glycerin, 0.5 ml of 10% ammonium ferric citrate. The purified water was added into 1000 ml of ammonia liquor to regulate the pH value to be about 8.0. 2. Thallus collection: pellicle in a culture flask was collected after the thallus grew to a logarithmic phase; an appropriate amount of deionized distilled water was added for washing the pellicle; and the pellicle was pressed to be dry and weighed. Embodiment 2 Preparation of mixture of BCG polysaccharide and nucleic acid 1. Thallus breaking and hot phenol treatment: the deionized distilled water was added into the collected thallus according to a ratio of 10:1; the thallus was broken by a tissue mashing homogenizer (12000 rpm/min) for 3 times by 3 min each time; the thallus was mashed; hot phenol (30-100°C) which was 0.5-2.0 times of the volume of the broken bacterial suspension was added; and the temperature was maintained for 30-60 minutes while low-speed stirring;

Description

2. Extraction of the mixture of BCG polysaccharide and nucleic acid: the mixed solution treated with the hot phenol was naturally precipitated for 1-10 days; the supernatant was sucked; the solution was centrifuged by a tube centrifuge at a high speed; and the supernatant was filtering by a 0.45pm sterile filter so as to obtain the mixture of BCG polysaccharide and nucleic acid; 3. Medicinal ethanol was added into the collected extracting solution of BCG polysaccharide and nucleic acid for performing alcohol precipitation at alcohol content of 70-75%; a precipitate was collected within 4 days after natural precipitation; the precipitate was fully and uniformly stirred with absolute ethyl alcohol; the precipitate was centrifugally washed for 3 times; the precipitate was dried in a 50°C vacuum drier after the precipitate was centrifugally washed with diethyl ether for 3 times, thereby obtaining the dry matter, i.e., the extract of BCG polysaccharide and nucleic acid; 4. 0.1 g of the BCG polysaccharide and nucleic acid dried in vacuum at 50°C was precisely weighed and dissolved with water; the volume was fixed in a 50 mL volumetric flask; the solution was transferred into a 1-25 mL volumetric flask by a pipette; water was added to fix the volume to 25 mL, wherein the prepared solution was the solution of BCG polysaccharide and nucleic acid. Embodiment 3 Screening of an optimal concentration of the BCG polysaccharide and nucleic acid by utilizing a cell proliferation rate According to a conventional CIK cell culture method, IFN-y (1000 U/mL) and a CD 3 monoclonal antibody (25 ng/mL) were added into aCO 2 incubator at 37°C for incubating for 24 h on Day 0, and the obtained CIK cells served as starter cells. An RPMI 1640 of BCG polysaccharide and nucleic acid (sterilized at a high temperature) containing 3 concentration gradients was respectively added into the starter cells and cultured in a 12-well plate, wherein the concentration gradients were 35 pg/mL, 3.5 pg/mL and 0.35 pg/mL (that is, every 1 mL of the culture

Description

system included 35 pg, 3.5 pg and 0.35 pg of the BCG polysaccharide and nucleic acid respectively); an RPMI 1640 containing no BCG polysaccharide and nucleic acid served as control and cultured in a 5%CO 2 incubator at 37°C for 7 d; and 1/2 of a corresponding culture solution was periodically replaced (e.g., cultured for 3 d). Cell concentrations were calculated every day, and a proliferation trend of the cells in each group was analyzed. The result was as shown in Fig. 1. It can be seen from comparative experimental results that, compared with the control group containing no BCG polysaccharide and nucleic acid, the experimental group had the advantage that the proliferation and differentiation rates of the CIK cells can be significantly increased after the 35 pg/mL, 3.5 pg/mL and 0.35 pg/mL of the BCG polysaccharide and nucleic acid were added. The optimal concentration of the BCG polysaccharide and nucleic acid was 35 pg/mL. Embodiment 4 Flow cytometry to detect cell phenotype at the optimal concentration The CIK starter cells obtained by the above method were respectively tested; groups A and B represented the RPMI 1640 of BCG polysaccharide and nucleic acid at the optimal concentration and the RPMI 1640 containing no BCG polysaccharide and nucleic acid (control); the cells were cultured in a 5% CO 2 incubator at 37°C, and 1/2 of the corresponding culture solution was periodically replaced within about 3 d; cell sap was uniformly mixed within 7 d after culture and taken out to be added into a 15 mL centrifuge tube; 10pl of CD 3FITC-H and CD 5 6PE-H monoclonal antibodies were respectively added within 15 min after centrifugation at 1500 rpm; the cells were incubated in the dark at a room temperature for 30 min; the cells were detected by flow cytometry; the cells were obtained and analyzed by Cellquest software; and the double positive of the cells induced by 35 pg/mL of BCG polysaccharide and nucleic acid was 73.9% and was

Description

increased by 3.8% compared with CD 3+CD5 6+double positive 70.1% in the control group. Embodiment 5 Killability determination of CIK cells induced at optimal concentration to tumor cells The cells were cultured with BCG polysaccharide and nucleic acid at an optimal concentration of the BCG polysaccharide and nucleic acid; cells that had been cultured in a culture flask for 7 d were sucked out and added into a 15 mL centrifuge tube; the supernatant was centrifugally removed; the CIK cells were eluted with 0.9% of NaCl twice; 0.8 mL of RPMI 1640 was added; the cells were counted to obtain a cell concentration; the supernatant in the tumor cell culture flask was removed; 1.5 mL of pancreatin was added for rinsing, and a rinsing solution was removed; then 1 mL of pancreatin was added and maintained at 37°C for 3 min; after tapping, 5 mL of preheated RPMI 1640 was added to terminate the reaction; the tumor cells were centrifugally collected at 1200 rpm for 5 min; 10 mL of the RPMI 1640 was added after elution twice; the cells were counted to obtain the cell concentration; the cells were diluted, and liquor was added in equal volume according to a cell number ratio (100 pl of CIK cell sap and 100 pl of tumor cell A549 liquor, totally 200 pl per well); positive control: 100 pl of tumor cell diluent + 100 pl of CIK cell sap, and negative control: 200 pl of CIK cell sap; after the liquor was added, the cells were cultured at aCO 2 concentration of 5% at 37°C for 12 hours; the well plate was taken out within 2 hours; 200 pl of liquid in each original well was sucked and cleaned with a 200 pl medium for 5 times; 100 pl of RPMI 1640 containing 10% of CCK8 was averagely added into each well for staining the cells; the cells were cultured at aCO 2 concentration of 5% at 37°C for 4 hours; the well plate was taken out and detected on a microplate reader, thereby obtaining the killability result of the CIK cells induced by BCG polysaccharide and nucleic acid to tumor cells. According to the results shown in Fig. 2, when an effect-to-target (E:T) ratio was 20:1, the killability of the CIK cells induced by

Description

BCG polysaccharide and nucleic acid to A549 tumor cells was up to 93.3%, and was obviously higher than that of the control group.

Claims

1. An application of bacillus calmette-guerin (BCG) polysaccharide and nucleic acid in in-vitro culture of cytokine-induced-killer (CIK) cells, wherein the BCG polysaccharide and nucleic acid is added into a culture system for in-vitro culture of the CIK cells.

2. The application according to claim 1, wherein the adding amount of the BCG polysaccharide and nucleic acid is 0.35-35 pg of BCG polysaccharide and nucleic acid in 1 ml of culture medium.

3. The application according to claim 2, wherein the adding amount of the BCG polysaccharide and nucleic acid is 35 pg of BCG polysaccharide and nucleic acid in 1 ml of culture medium.

4. A culture medium for in-vitro culture of CIK cells, comprising a basal culture medium, wherein the culture medium also comprises BCG polysaccharide and nucleic acid.

5. The culture medium according to claim 4, wherein the adding amount of the BCG polysaccharide and nucleic acid in the culture medium is 0.35-35 pg of BCG polysaccharide and nucleic acid in 1 ml of culture medium.

6. The culture medium according to claim 5, wherein the adding amount of the BCG polysaccharide and nucleic acid in the culture medium is 35 pg of BCG polysaccharide and nucleic acid in 1 ml of culture medium.

7. A method for in-vitro culture of CIK cells, comprising the following steps: (1) preparing starter cells of CIK cells; (2) preparing a culture medium containing BCG polysaccharide and nucleic acid; (3) adding the starter cells of CIK cells into the culture medium of the step (2); and culturing the cells, thereby obtaining the CIK cells.

8. The method according to claim 7, wherein the adding amount of the BCG polysaccharide and nucleic acid in the culture medium is 0.35-35 pg of BCG polysaccharide and nucleic acid in 1 ml of culture medium.

Claims

9. The method according to claim 8, wherein the adding amount of the BCG polysaccharide and nucleic acid in the culture medium is 35 pg of BCG polysaccharide and nucleic acid in 1 ml of culture medium.

10. An application of CIK cells in preparation of antitumor drugs, wherein the CIK cells are subjected to in-vitro culture; and the in-vitro culture system comprises the BCG polysaccharide and nucleic acid.