Bacteriologically-modified whole-cell tumor vaccine and method of making same

Abstract

Disclosed are a bacteriologically-modified whole-cell tumor vaccine and a method of making the same. The method includes: lysing bacteria at logarithmic growth phase to obtain a bacterial lysate; mixing the bacterial lysate with an excessive amino compound solution to aminate the bacterial lysate in the presence of EDC; mixing the aminated bacterial lysate with the tumor cells for a certain period of time to produce bacteriologically-modified tumor cells; and inactivating the bacteriologically-modified tumor cells to produce the bacteriologically-modified whole-cell tumor vaccine. The bacteriologically-modified whole-cell tumor vaccine has been demonstrated to have desirable therapeutic effect in tumor model mice.

Claims

1. A method of preparing a bacteriologically-modified whole-cell tumor vaccine, comprising: (1) mixing an amino compound with a bacterial lysate under neutral or weakly alkaline conditions to allow an amino group positively charged in the amino compound to react with a carboxyl group of a protein of the bacterial lysate to aminate the bacterial lysate; (2) mixing the aminated bacterial lysate prepared in step (1) with tumor cells under gentle shaking to allow the aminated bacterial lysate to be bound to a surface of the tumor cells through electrostatic attraction to produce bacteriologically-modified tumor cells; and (3) inactivating the bacteriologically-modified tumor cells to produce the bacteriologically-modified whole-cell tumor vaccine.

2. The method of claim 1, wherein a solution of the aminated bacterial lysate is prepared through steps of: (1-1) culturing bacteria; collecting the bacteria followed by freezing-thawing and ultrasonication to produce a supernatant as the bacterial lysate; and lyophilizing the bacterial lysate; (1-2) mixing the amino compound with the lyophilized bacterial lysate in ultrapure water; wherein the amino compound is selected from the group consisting of ethylenediamine, N,N-dimethylethylenediamine, polyethyleneimine and a combination thereof; (1-3) adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to the reaction mixture; and stirring the reaction mixture at 4° C. with fresh EDC added at an interval; (1-4) dialyzing the reaction mixture in ultrapure water to remove the free amino compound and EDC to produce a solution of the aminated bacterial lysate; and (1-5) adjusting the solution of the aminated bacterial lysate to pH 7-9.

3. The method of claim 2, wherein the amino compound is ethylenediamine; and in step (1-3), the stirring is performed for 48-52 h.

4. The method of claim 1, wherein the bacterial lysate is prepared through steps of: culturing bacteria to a logarithmic growth phase; and harvesting the bacteria; freezing the bacteria in liquid nitrogen at −160° C. for more than half an hour and thawing the frozen bacterium completely in a water bath at 37° C.; and repeating the freezing and thawing three times; rupturing the thawed bacteria under ultrasonication followed by centrifugation at 10,000 rpm and 4° C. for 10-12 min to collect a supernatant as the bacterial lysate; and freezing the bacterial lysate at −80° C. followed by lyophilization to produce bacterial lysate powder.

5. The method of claim 1, wherein the bacteriologically-modified tumor cells are prepared through steps of: culturing the tumor cells in vitro to logarithmic growth phase; harvesting and counting the tumor cells; adjusting the tumor cells with PBS to produce a tumor cell suspension having a content of 1×10.sup.6 cells/100 μL; and mixing 50 μL of the tumor cell suspension with 50 μL of the solution of the aminated bacterial lysate uniformly; and shaking the reaction mixture gently at room temperature for 30 min to produce the bacteriologically-modified tumor cells.

6. The method of claim 1, wherein the bacterial lysate is derived from bacteria selected from the group consisting of E. coli, Mycobacterium tuberculosis, Staphylococcus, Pseudomonas and Klebsiella pneumoniae.

7. The method of claim 1, wherein the inactivation is performed using an X-ray irradiator at an irradiation intensity of 80-100 Gy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 schematically shows the preparation of bacteriologically-modified tumor cells of the invention.

(2) FIG. 2A is a confocal micrograph showing the FITC-labeled aminated bacterial lysate (green fluorescence); FIG. 2B is a confocal micrograph showing the tumor cells bacteriologically-modified with the FITC-labeled aminated bacterial lysate with nuclei further stained with DAPI (blue fluorescence); where FIG. 2A and FIG. 2B share the same vision field; and FIG. 2C shows the superimposition of FIGS. 2A and 2B.

(3) FIG. 3A shows mice in the group vaccinated with the bacteriologically-modified tumor cells according to an embodiment of the invention, where tumors of 3 of the 5 mice from the treatment group completely or almost completely disappear, and the other two mice have a tumor significantly smaller in size than the mice in the control; and FIG. 3B shows the control group in which the mice were not treated.

DETAILED DESCRIPTION OF EMBODIMENTS

(4) The invention will be further described below with reference to the embodiments, but these embodiments are not intended to limit the invention. Various modifications and changes made based on the content disclosed herein should fall within the scope of the invention.

(5) FIG. 1 schematically shows the preparation of bacteriologically-modified tumor cells of the invention, specifically, an excessive positively-charged amino compound (such as ethylenediamine) is mixed with a bacterial lysate in the presence of EDC to allow the amino compound to react with the protein of the bacterial lysate to render the bacterial lysate positively-charged. Then the bacterial lysate is bound to the tumor cells through electrostatic attraction to produce the bacteriologically-modified tumor cells, which are inactivated to be used as the tumor vaccine.

(6) 1. Preparation of Bacterial Lysate

(7) (1) Preparation of bacteria medium (E. coli)

(8) A liquid LB medium was specifically prepared as follows. 5 g of yeast extract, 10 g of tryptone and 10 g of NaCl were added to a vessel, to which 900 mL of deionized water was added. The mixture was adjusted to pH 7.2-7.4 with 1 mol/L NaOH, added with deionized water to 1 L and sterilized at 121° C. and 0.1-0.15 MPa for 20 min. For a solid LB medium, 15 g of agar was additionally added and the mixture was sterilized under high pressure and poured into special petri dishes for use.

(9) (2) Culturing and Harvesting of Bacteria (E. coli JM 109)

(10) E. coli JM 109 stored in glycerol at −80° C. was transferred to a 0° C. ice bath, and a small amount of frozen bacterial solution was picked up with a disinfected toothpick or an inoculation loop to be uniformly dispersed onto the solid LB medium. Then the medium was cultured at 37° C. in an incubator for 8-12 h to allow the single colony to appear. On the next day, a single colony was picked with a disinfected toothpick or an inoculation loop and added to 30 mL of liquid LB medium. The liquid medium was cultured in a shaker at 37° C. and 225 rpm for 12-16 h. Then the bacterial suspension was added to fresh liquid LB medium in a volume ratio of 1:100, and the fresh medium was cultured in a shaker at 37° C. and 225 rpm to an OD value of 0.5-0.6 (cultured for about 2.5 h) and centrifuged at 4° C. and 5000×g for 5 min. The supernatant was discarded and the bacterial cells were collected.

(11) (3) Preparation of E. coli Lysate

(12) The bacterial cells obtained above were washed twice with PBS pre-cooled at 4° C. for more than 5 h and then washed once with ultrapure water at 4° C. The cells were resuspended with ultrapure water having a volume 1/10 of the original medium at 4° C., frozen at −160° C. in liquid nitrogen for more than half an hour and thawed completely in a 37° C. water bath. The process of freezing and thawing was repeated three times, and the thawed cells were ruptured by ultrasonication. During the ultrasonication, the container containing the bacterial suspension was placed in cool water (4° C.) and the probe should be placed ⅓ below the liquid surface. The ultrasonication was performed intermittently at a power of 30%, specifically, the probe was operated for 3 s and then stopped for 5 s. The ultrasonication was performed three times each for 10 min, and there was an interval of 10 min between adjacent two ultrasonic treatments. Then the bacterial suspension was centrifuged at 4° C. and 10,000 rpm for 10 min to collect a supernatant as a bacterial lysate. The bacterial lysate was immediately frozen at −80° C. and lyophilized using a lyophilizer (Labconco FreeZonc®4.5 Freeze Dryer). The lyophilized lysate was stored at −20° C. for use.

(13) 2. Amination of Bacterial Lysate

(14) (1) Preparation of Solutions

(15) 10 mg of the lyophilized lysate was dissolved completely in 1 mL of ultrapure water at 4° C. to produce a first solution. 10 μL of ethylenediamine (purity >99.5%) was mixed uniformly with 0.5 mL of ultrapure water at 4° C., adjusted to pH 8-9 with hydrochloric acid (12 mol/L) or sodium hydroxide (1 mol/L) and added with ultrapure water at 4° C. to 1 mL to produce a second solution. 5 mg of EDC was dissolved in 125 μL of ultrapure water at 4° C. to produce a third solution. The above solutions were prepared based on 10 mg of the lyophilized lysate, and can be accordingly adjusted according to the amount of lyophilized lysate.

(16) (2) Amination of Bacterial Lysate

(17) The first solution was added to a flask and slightly stirred using a magnetic stirrer (set to 2-3 level, about 200-400 rpm). Then the second solution was slowly added to the first solution. After the first solution and the second solution were completely mixed, the third solution was slowly added, and the reaction mixture was stirred at 4° C. for 48 h to produce an aminated bacterial lysate, where the third solution was additionally added every other 8-12 h, each for 125 μL, to enable the EDC to persistently work.

(18) (3) Preparation of a Solution of the Aminated Bacterial Lysate

(19) The aminated bacterial lysate was loaded in a dialysis bag (14K MWCO), and then dialyzed in 1 L of ultrapure water at 4° C. for 48 h to produce a solution of the aminated bacterial lysate, where the ultrapure water was replaced every other 8-12 h. The solution of the aminated bacterial lysate was adjusted to pH 8-9 with hydrochloric acid (12 mol/L) or sodium hydroxide (1 mol/L), where the solution of the aminated bacterial lysate was determined using a BCA kit for protein content and then adjusted with PBS to a protein content of 0.2 mg/100 μL.

(20) 3. Culturing and Harvesting of Tumor Cells

(21) Tumor cells were transferred from a liquid nitrogen tank immediately to a 37° C. water bath and shaken repeatedly for complete dispersion (about 1-2 min). The cell solution was centrifuged at 2,000 rpm for 5 min, and the supernatant was removed. The cells were added with a corresponding medium (such as DMEM and RPMI 1640), fetal bovine serum, antibiotics and growth factors (referring to the instruction of the tumor cells for details), mixed uniformly and added to a tissue culture flask. The tissue culture flask was cultured at 37° C. and 5% CO.sub.2, where the medium was replaced every other 1-2 days. When proliferating to occupy 80% of the space inside the culture flask, the cells were washed twice with PBS, digested with 0.25% trypsin and continuously cultured in another culture flask. After proliferating to the desired number, the cells were washed with PBS twice again, digested with 0.25% trypsin and centrifuged at 2,000 rpm for 5 min. The supernatant was discarded, and the cells were washed twice with PBS and counted. The tumor cells were adjusted to a content of 1×10.sup.6 cells/100 μL with PBS for subsequent use.

(22) 4. Preparation of Bacteriologically-Modified Whole-Cell Tumor Vaccine

(23) (1) Bacteriological modification of tumor cells 50 μL of the above tumor cell suspension (containing 0.5×10.sup.6 cells) and 50 μL of the solution of the aminated bacterial lysate (containing 0.1 mg of protein) were mixed uniformly and shaken gently at room temperature for 30 min to produce the bacteriologically-modified tumor cells (FIGS. 2A-2C). FIGS. 2A-2C show the primary features of the bacteriologically-modified tumor cells of the invention observed under a confocal microscope, where FIG. 2A shows the FITC-labeled aminated bacterial lysate (green fluorescence); FIG. 2B shows tumor cells bacteriologically-modified with the FITC-labeled aminated bacterial lysate with nuclei further stained with DAPI (blue fluorescence); FIG. 2A and FIG. 2B shares the same vision field; and FIG. 2C is the superimposition of FIGS. 2A and 2B and shows the relationship between the bacterial lysate (green fluorescence) and the nuclei (blue fluorescence). It can be confirmed by the figures that the aminated bacterial lysate was effectively bound to the surface of the tumor cells.

(24) (2) Inactivation of Bacteriologically-Modified Tumor Cells

(25) The above prepared bacteriologically-modified tumor cells were inactivated using an X-ray instrument (Rad Source RS2000 X-ray irradiator) at an irradiation intensity of 80-100 Gy to prepare the vaccine. Individual tumor model mice were vaccinated with 3×10.sup.5-5×10.sup.5 cells subcutaneously, intravascularly, intramuscularly or intraperitoneally.

(26) 5. Verification of Therapeutic Effect

(27) 10 female BALB/c mice, aged 6-8 weeks, were selected and averagely divided into two groups, i.e., treatment group and control group. The 10 mice were respectively injected subcutaneously with 2×10.sup.6 murine CT26 colon cancer cells at the lower left side of the back to establish a tumor model. 5 mice in the treatment group were further injected subcutaneously with 5×10.sup.5 of the inactivated bacteriologically-modified CT26 colon cancer cells (dissolved in 100 μL of PBS) at the upper right side of the back. The tumor growth in mice was monitored, and the mice were sacrificed 28 days after injection of tumor cells. The serum was collected and detected for the specific antibodies against the CT26 colon cancer cells, and the spleen lymphocytes were used as effector cells in the killing experiments of the CT26 colon cancer cells. It can be seen from the results that the tumor disappeared completely or almost completely in 3 of the 5 mice in the treatment group and the tumors in the other two mice were significantly smaller than those in the mice of the control group (as shown in FIGS. 3A-3B). Moreover, with regard to the mice from the treatment group, specific antibodies against the CT26 colon cancer cells were found in the serum thereof, and their lymphocytes had significant killing effect on the CT26 colon cancer cells. These experimental results demonstrated that the bacteriologically-modified CT26 whole-cell tumor vaccine can effectively induce the immune response specifically against the CT26 colon cancer cells, effectively treating the murine colon cancer.

(28) Described above are merely preferred embodiments of the invention, and these embodiments are not intended to limit the invention. Various modifications, changes and replacements made based on the content of the invention should fall within the scope of the invention.