METHOD FOR PREPARING DENDRITIC CELL LOADED WITH ANTIGEN

Abstract

The present invention provides a method for preparing dendritic cell loaded with antigen, the method comprising the steps of adding serum-free cell culture medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and inter-leukin (IL)-4 into mononuclear cells, culturing in an incubator at 37° C. under 5% carbon dioxide for 5 days, adding target antigen wrapped cationic liposome and culturing for 8-24 hours to obtain target antigen loaded dendritic cell.

Claims

1. A preparation method of dendritic cells loaded with antigens efficiently, which is characterized in that: the steps thereof are adding serum-free cell culture medium containing GM-CSF and IL-4 into mononuclear cells, and placing in an incubator of 37° C., with 5% of CO2 for culture; after 5 days, adding cationic liposomes that encapsulate target antigen to culture for 8-24 hours, and then dendritic cells loaded with the target antigen can be obtained.

2. The preparation method of dendritic cells loaded with antigens efficiently of claim 1, characterized in that: the cationic liposome is a mannose-modified cationic liposome complex.

3. The preparation method of dendritic cells loaded with antigens efficiently of claim 1, characterized in that: the cationic liposome is obtained by coupling a mannose or a mannoside to a polyethylene glycol derivatized phospholipid to obtain a mannose-modified polyethylene glycol derivatized phospholipid; dissolving a cationic lipid and the mannose-modified polyethylene glycol derivatized phospholipid into a mixed solvent of chloroform and methanol respectively, after mixing to obtain a mixture liquid; rotarily evaporating the mixture liquid with a steady nitrogen stream or an inert gas stream so as to form a uniform film; adding a PBS buffer solution containing tumor antigen after vacuum drying and placing at 4° C. for sonicating to hydrate; obtaining the cationic liposome after extruding through film; wherein the loading amount of tumor antigen is 1-500 g antigen/mol liposome.

4. The preparation method of dendritic cells loaded with antigens efficiently of claim 3, characterized in that: the molar number ratio range of the cationic lipid to the mannose-modified polyethylene glycol derivatized phospholipid is 1:1 to 1:10.

5. The preparation method of dendritic cells loaded with antigens efficiently of claim 3 characterized in that: the cationic lipid is any one of didecyldimethylammonium bromide, dioleoyltrimethylammoniumpropane, dioleoylpropyltrimethylammonium chloride, 3-(N-(N′, N′-dimethylaminoethane) carbamoyl) cholesterol and dioleyl ether phosphatidylcholine.

6. The preparation method of dendritic cells loaded with antigens efficiently of claim 1, characterized in that: the target antigen is one or more tumor antigen protein or polypeptide having different epitopes.

7. The preparation method of dendritic cells loaded with antigens efficiently of claim 6, characterized in that: the antigen may be selected from the group consisting of tumor cell lysate, autologous or allogeneic tumor antigen protein, polypeptide or protein product of genetic engineering and synthetic antigen polypeptide.

8. The preparation method of dendritic cells loaded with antigens efficiently of claim 6, characterized in that: the antigen is HBsAg antigen, tumor tissue antigens, electroneutral polypeptide antigen, electronegative polypeptide antigen survivin or OVA antigen protein.

9. The preparation method of dendritic cells loaded with antigens efficiently of claim 1, characterized in that: the DC cells include peripheral blood mononuclear cell induced DC cells, hematopoietic stem cell and umbilical cord blood stem cell induced DC cells.

10. The preparation method of dendritic cells loaded with antigens efficiently of claim 1, characterized in that: human peripheral blood mononuclear cells are suspended in basal medium, and then inoculated into cell culture plates for attachment culture for 1-2 hours at 37° C. in incubator; after non-attached cells are removed, a serum-free cell culture medium containing GM-CSF and IL-4 is added into the attached cells to culture at 37° C., with 5% of CO2, under saturated humidity for carrying out the induction of DC cells; on the third day, DC cell culture medium is supplemented to DC cell culture plate in half amount; on the fifth day, liposome-encapsulated antigen is added to DC cells, cultured for 8-24 hours, wherein the adjustment dose of the antigen is 1-50 ug/ml, and DC cells loaded with tumor antigens are obtained; preferably, the serum-free cell culture medium contains 25-500 ng/ml of GM-CSF and 5-100 ng/m1 of IL-4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG. 1 is a structural schematic view of nanovaccine which uses mannose-modified cationic liposomes as the carrier.

[0056] FIG. 2 is a view illustrating the preparation of mannose-modified cationic liposome and a characterization diagram thereof.

[0057] FIG. 3 is an efficiency diagram of dendritic cell efficiently taking electroneutral polypeptide antigen loaded by a cationic liposome.

[0058] FIG. 4 is an efficiency diagram of dendritic cell efficiently taking electronegative polypeptide antigen loaded by a cationic liposome.

[0059] FIG. 5 is a diagram indicating the influence of the percentage of DSPE-PEG-Mannose on the efficiency of dendritic cells loaded with tumor antigens.

DETAILED EMBODIMENT OF THE INVENTION

[0060] Embodiments of the present invention will be described in detail below. The example of the embodiment is shown in the figures, wherein the same or similar reference number indicates the same or similar element or elements having the same or similar function throughout. The following embodiments described with reference to the figures are exemplary and are intended to illustrate the invention, but should not be construed as limiting the present invention. Embodiments which are not described with the specific technique or conditions are carried out in accordance with the technique or conditions described in the literature in the art or in accordance with the product instructions. All reagents or equipments, of which the manufacturers are not indicated, are conventional products that can be commercially available

EXAMPLE 1

[0061] A isothiocyano-α-D-mannopyranose (i.e., α-D-Mannopyranosylphenyl isothiocyanate, Sigma-Aldrich) is reacted with NH.sub.2-PEG-DSPE under basic conditions at room temperature for 24 hours, so that the isothiocyano of mannose and the amino group of PEG are reacted to form a thiourea bond, resulting mannosylated PEG-DSPE (DSPE-PEG-Man, see FIG. 2A). NMR spectrum shows that a peak (f peak) at 7.25 ppm is the benzene ring of the isothiocyano mannopyranose, indicating the mannose is labeled successfully to DSPE-PEG (see FIG. 2B).

[0062] The DOTAP and the above DSPE-PEG-Man are dissolved respectively in a mixed solvent of chloroform and methanol of a volume ratio of 2:1 (DOTAP: DSPE-PEG-Man=9:1, mol/mol), and the resulting mixture liquid after mixing the both is placed in a round flask. The mixture liquid is vortically dried with a steady nitrogen stream, so as to form a uniform film, and after drying in vacuo overnight, a PBS buffer solution of antigen protein HBsAg (100 ug antigen/1 umol liposomes/ml, calculated by the feed ratio) is added to place to hydrate at 4° C. on the next day, then a cationic liposome which encapsulates HBsAg is obtained by sonicating the bath for 10 minutes. Concanavalin A (Con A) aggregation tests indicated that a mannose-modified cationic liposome (LP-man) is co-incubated with Con A, and the particle size of the liposome may increase dramatically. On the contrary, an unmodified liposomes (LP) is co-incubated with Con A, and its particle size did not change. This result further confirms that the surface of the liposome is modified with mannose residues (see FIG. 2C).

[0063] Human peripheral blood mononuclear cells are suspended in basal medium (10.sup.6 cells/ml), and then inoculated into cell culture plates for attachment culture for 1-2 hours at 37° C. in incubator; after non-attached cells are removed, a serum-free cell culture medium containing GM-CSF (25-500 ng/m1) and IL-4 (5-100 ng/m1) is added into the attached cells to culture at 37° C., with 5% of CO.sub.2, under saturated humidity for carrying out the induction of DC cells; on the third day, DC cell culture medium is supplemented to DC cell culture plate in half amount; on the fifth day the above-described cationic liposome encapsulating HBsAg (5 ug antigen/50 nmol liposome/ml) is cultured for 8-24 hours, to obtain DC cells loaded with tumor antigen.

EXAMPLE 2

[0064] 4-iosthiocyanophenyl-A-D-mannoside is coupled to DSPE-PEG.sub.2000 in covalent manner to obtain mannoside-modified DSPE-PEG.sub.2000 (DSPE-PEG-Man). The DSPE-PEG-Man and DOTAP are dissolved respectively in a mixed solvent of chloroform and methanol of a volume ratio of 2:1, the ratio of the molar number of N-2 amido glucosamine modified DSPE-PEG.sub.2000 to the total molar number of DOTAP is 1:19, and the resulting mixture liquid after mixing the both is placed in a round flask. The mixture liquid is rotarily evaporated with a steady nitrogen stream, so as to form a uniform film. After drying in vacuo overnight, the human hepatocarcinoma cell HepG2 is suspended in PBS buffer solution. After repeatedly freezing (−80° C.) and thawing (70° C.) to disrupt the cells, tumor cell lysate containing tumor antigen is obtained from the supernatant by centrifuging. A certain amount of the tumor cell lysate (0.25 g antigen protein/mol liposome) is added into a phospholipid membrane and to place to hydrate at 4° C., and then the bath is sonicated for 10 minutes, followed by extrusion of a polycarbonate film twice to obtain a cation liposome encapsulating tumor antigens. Mononuclear cells are sampled and separated from human peripheral blood and the DC cell culture medium containing GM-CSF (100 ng/ml) and IL-4 (25 ng/ml) is added, and then placed in an incubator at 37° C., with 5% of CO.sub.2 to culture. 5 days later, the cation liposome encapsulating tumor tissue antigens is added to culture for 8-24 hours, and then the dendritic cells loaded with tumor tissue antigens can be obtained.

EXAMPLE 3

[0065] One end of the DSPE-PEG is modified with succinimide (DSPE-PEG-NHS), and then is condensed with 4-aminophenyl-D-mannoside via the amino group and succinimide to form a ester, so as to obtain a mannosyl PEG (DSPE-PEG-Man). The DOTAP and DSPE-PEG-Man are dissolved respectively in a mixed solvent of chloroform and methanol of a volume ratio of 2:1. Then the resulting mixture liquid obtained by mixing the DOTAP and the DSPE-PEG-Man in a molar ratio of 9:1 is placed in a round flask. The mixture liquid is rotarily evaporated with a steady nitrogen stream, so as to form a uniform film, and after drying in vacuo overnight, a PBS buffer solution (0.1 mg protein antigen/mol liposome) containing an electroneutral survivin polypeptide labeled with FITC (survivin-FITC, the sequence of survivin see SEQ ID NO: 1, PI=8.1, MW=3610.19) is added on the next day. And placing to hydrate at 4° C., then a survivin-FITC cationic liposome is obtained by sonicating the bath for 10 minutes followed by extrusion of a polycarbonate film twice. Mononuclear cells are sampled and separated from human peripheral blood and the DC serum-free medium is added, and then the cytokine GM-CSF (100 ng/ml) and IL-4 (25 ng/ml) are added into the medium, and then the medium is placed in an incubator at 37° C., with 5% of CO.sub.2 to culture. The dendritic cells are collected after 5 days; then a nanoliposome encapsulating an electroneutral polypeptide antigen is added and adjusted the concentration to 2.5 μg polypeptide/ml to culture for another 2 hours, and a dendritic cell efficiently loaded with antigen can be obtained as shown in FIG. 3. As can be seen from the figure, according to confocal and flow results, it indicates that the uptake efficiency of DC to the cationic liposome which encapsulates electroneutral polypeptide antigen survivin-FITC is better than that of DC to free electroneutral polypeptide antigen.

EXAMPLE 4

[0066] 4-iosthiocyanophenyl-A-D-mannoside is coupled to DSPE-PEG.sub.2000 in covalent manner to obtain mannoside-modified DSPE-PEG.sub.2000 (DSPE-PEG-Man). The DOTAP and DSPE-PEG-Man are dissolved respectively in a mixed solvent of chloroform and methanol of a volume ratio of 2:1. Then the resulting mixture liquid obtained by mixing the DOTAP and the DSPE-PEG-Man in a molar ratio of 19:1 is placed in a round flask. The mixture liquid is rotarily evaporated with a steady nitrogen stream, so as to form a uniform film, and after drying in vacuo overnight, a PBS buffer solution (0.1 mg protein antigen/mol liposome) containing an electronegative polypeptide antigen survivin-FITC, (the sequence of survivin see SEQ ID NO: 1, PI=3.84, MW=3593.96) is added on the next day. And placing to hydrate at 4° C., then a cationic liposome which encapsulates electronegative polypeptide antigen survivin-FITC is obtained by sonicating the bath for 10 minutes followed by extrusion of a polycarbonate film twice. Mononuclear cells are sampled and separated from human peripheral blood and the DC serum-free medium is added, and then the cytokine GM-CSF (100 ng/ml) and IL-4 (25 ng/ml) are added into the medium, and then the medium is placed in an incubator at 37° C., with 5% of CO.sub.2 to culture. The dendritic cells are collected after 5 days; then a nanoliposome encapsulating an electronegative polypeptide antigen is added and to adjust the concentration to 2.5 μg polypeptide/ml to culture for another 2 hours, and then a dendritic cell efficiently loaded with antigen can be obtained, as shown in FIG. 4. As can be seen from FIG. 4, according to confocal and flow results, it indicates that the uptake efficiency of DC to the nanoliposome which encapsulates electronegative polypeptide antigen is better than that of DC to free electronegative polypeptide antigen.

EXAMPLE 5

[0067] 4-iosthiocyanophenyl-A-D-mannoside is coupled to DSPE-PEG.sub.2000 in covalent manner to obtain mannoside-modified DSPE-PEG.sub.2000 (DSPE-PEG-Man). The resulting mixture liquid obtained by mixing the cationic lipid DOTAP and the DSPE-PEG-Man in molar ratios of 9:1, 19:1 and 99:1 is placed in a round flask, so that the molar percentage of DSPE-PEG-Man in the mixture is 1%, 5% and 10%. The mixture is rotarily evaporated with a steady nitrogen stream, so as to form a uniform film, and after drying in vacuo overnight, a PBS buffer solution (0.1 mg protein antigen/mol liposome) containing antigen proteins OVA-FITC is added on the next day. And placing to hydrate at 4° C., then a cationic liposome which encapsulates antigen proteins ovalbumin (OVA-FITC) labeled with fluorescein is obtained by sonicating the bath for 10 minutes followed by extrusion of a polycarbonate film twice. Mononuclear cells are sampled and separated from human peripheral blood and the DC serum-free medium is added, and then the cytokine GM-CSF and IL-4 are added into the medium, and then the medium is placed in an incubator at 37° C., with 5% of CO.sub.2 to culture. The dendritic cells are collected after 5 days; then nanoliposome encapsulating an antigen protein OVA-FITC is added and to adjust the concentration to 2.5 OVA/ml to culture for another 2 hours. Result of Flow cytometry is shown in FIG. 5, and it can be seen from FIG. 5 that as the percentage of DSPE-PEG-Mannose increasing (from 1%, 5% to 10%), the effect of liposome on the efficiency of dendritic cells loading with antigen is more and more significant

[0068] Although the above description has been illustrated and described embodiments of the present invention, it is understood that the above-described embodiments are illustrative and are not to be construed as limiting the present invention. One of ordinary skill in the art may make modifications, substitutions and variations to the above-described embodiments within the scope of the present invention, without departing from the principles and spirit of the invention.