Vaccine for tumor immunotherapy

Abstract

The present invention relates to a vaccine comprising dendritic cells and bacterial ghosts for tumor immunotherapy.

Claims

1. A composition comprising: (i) antigen-presenting cells; (ii) at least one tumor cell lysate; and, (iii) bacterial ghosts from E. coli Nissle 1917.

2. The composition of claim 1 for use as vaccine for tumor immunotherapy.

3. The composition of claim 1, wherein the antigen-presenting cells comprise monocytes.

4. The composition of claim 1, wherein the antigen-presenting cells comprise dendritic cells.

5. The composition of claim 1, wherein the bacterial ghosts are obtained from bacterial cells comprising a gene encoding a lytic protein.

6. The composition of claim 1, wherein the bacterial ghosts have been treated with β-propiolactone.

7. The composition of claim 1, wherein the bacterial ghosts are loaded with an activator or silencer of the immune system, a pharmaceutical agent and/or DNA.

8. The composition of claim 1, wherein the bacterial ghosts carry recombinant tumor-associated antigens.

9. The composition of claim 8, wherein the bacterial ghosts are loaded with tumor-associated antigens.

10. The composition of claim 8, wherein the bacterial ghosts are derived from bacteria recombinantly expressing tumor-associated antigens.

11. The composition of claim 1, comprising from 1 to 10,000, or from 10 to 1,000 bacterial ghosts per antigen-presenting cell.

12. The composition of claim 1, comprising 1 to 10 million antigen-presenting cells per dose.

13. The composition of claim 1, wherein the antigen-presenting cells induce tumor-antigen recognition and tumor-specific killing by T cells.

14. The composition of claim 1, wherein antigen-presenting cells are autologous antigen-presenting cells.

15. The composition of claim 1, further comprising a pharmaceutically acceptable carrier or excipient.

16. A therapeutic vaccine comprising: (i) antigen-presenting cells; (ii) at least one tumor cell lysate; (iii) bacterial ghosts from E. coli Nissle 1917; and (iv) a pharmaceutically acceptable carrier or excipient.

17. A method of treating cancer in a patient comprising: administering to the patient a therapeutic vaccine composition comprising (i) antigen-presenting cells; (ii) at least one tumor cell lysate; (iii) bacterial ghosts from E. coli Nissle 1917; and (iv) a pharmaceutically acceptable carrier or excipient, in an amount effective to treat the cancer in the patient.

18. The method of claim 17, wherein the therapeutic vaccine composition is prepared by a process comprising a step of incubating the antigen-presenting cells with the at least one tumor cell lysate in presence of the bacterial ghosts for 10 minutes to 12 hours.

19. The method of claim 18, wherein the antigen-presenting cells are dendritic cells.

20. The method of claim 19, wherein after the incubation the dendritic cells exhibit an increase in one or more of levels of cytokine release, expression of CD83, expression of HLA-DR, expression of CD80, expression of CD86 and capacity to stimulate proliferation of autologous CD4.sup.+ and CD8.sup.+ T-cells, in comparison with a composition prepared by a process comprising a step of incubating the dendritic cells with the at least one tumor cell lysate without the presence of the bacterial ghosts.

21. The composition of claim 1, wherein the composition is prepared by a process comprising a step of incubating the antigen-presenting cells with the at least one tumor cell lysate in presence of the bacterial ghosts for 10 minutes to 12 hours.

22. The composition of claim 1, wherein the antigen-presenting cells are dendritic cells.

23. The composition of claim 22, wherein the composition is prepared by a process comprising a step of incubating the dendritic cells with the at least one tumor cell lysate in presence of the bacterial ghosts for 10 minutes to 12 hours.

24. The composition of claim 23, wherein after the incubation the dendritic cells exhibit an increase in one or more of levels of cytokine release, expression of CD83, expression of HLA-DR, expression of CD80, expression of CD86 and capacity to stimulate proliferation of autologous CD4.sup.+ and CD8.sup.+ T-cells, in comparison with a composition prepared by a process comprising a step of incubating the dendritic cells with the at least one tumor cell lysate without the presence of the bacterial ghosts.

Description

(1) The invention is further described by the enclosed Figures and the following Examples.

(2) FIG. 1. The cell surface markers expression on DCs. Immature DCs were prepared by incubation of monocytes obtained from peripheral blood of normal healthy donors in CellGro® DC medium supplemented with IFN-α (3000 IU/mL) and rhGM-CSF (1000 IU/mL) for 3 days. Maturation markers of DCs were analyzed by multicolor flow cytometry 48 h after short (4 h) stimulation of immature DCs with tumor lysate and BGs from E. coli Nissle 1917 (10 and 100 BGs/1DC) in the presence of IFN-α and rhGM-CSF. Immature DCs incubated with IFN-α and rhGM-CSF and tumor lysate supplemented with Lipopolysaccharide (LPS) (200 ng/mL) or without extra maturation stimuli served as controls. The y-axis represents the percentage of cells expressing specific differentiation antigen. Data represent the mean±SD of 4 independent experiments performed using cells obtained from different donors.

(3) CD14 is an indicator of maturation of dendritic cells. While monocytes show CD14 expression, matured dendritic cells show no or little CD14 expression. CCR7 is a marker for attractants of the cells to the lymph node. As can be seen from FIG. 1, expression of marker CCR7 increases for dendritic cells incubated with bacterial ghosts showing their mobility.

(4) CD83 is a major marker of mature dendritic cells.

(5) CD80, CD86, CD1a, CD11c and HLA-DR are maturation markers for dendritic cells.

(6) FIG. 2. Migratory capacity of DCs after incubation with tumor lysate and LPS or short stimulation with BGs. Chemotaxes of distinct DC populations matured in in the presence of IFN-α, rhGM-CSF and tumor lysate supplemented with BGs from E. coli Nissle 1917 (10 and 100 BGs/1DC), LPS (200 ng/mL) or without extra maturation stimuli in response to CCL21 (chemokine eliciting its effects by binding to a cell surface chemokine receptor CCR7) were determined as the number of cells which migrated into the lower part of a transwell system containing different concentrations of CCL21 and counted by flow cytometer. Each bar represents the mean number of migrated cells±SD of 4 independent experiments performed using cells obtained from different normal healthy donors.

(7) FIG. 3. Cytokine profile of DCs matured in the presence of tumor lysate and LPS or short time incubation with BGs. Immature DCs were stimulated with IFN-α, rhGM-CSF and tumor lysate for short time (4 h) in the presence of pure LPS (200 ng/mL) or BGs from E. coli Nissle 1917 (10 and 100 BGs/1DC) prior to measuring cytokine released into supernatants after 24 h and 48 h incubations. Cells incubated without additional stimuli served as negative control. The levels of cytokines released from DCs were measured using FACSArray Bioanalyzer. Data represent the mean±SD of 4 independent experiments performed using cells obtained from various normal healthy donors. P values <0.05 were considered significant and are indicated with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).

(8) IL-12 and IFN-γ are major activation markers for differentiation of T lymphocytes into Th1 type lymphocytes.

(9) IL-23 (the growth and stabilization factor) and IL-6 (the differentiation factor) are cytokines involved in the development of Th17 lymphocytes. FIG. 3 shows that T helper cells are induced by compositions according to the invention.

(10) IL-1β and TNF-α are pro-inflammatory markers.

(11) IL-10 is an anti-inflammatory cytokine.

(12) IL-2 is a T cell growth factor.

(13) FIG. 4. Allogeneic (A) and autologous (B) immunostimulatory capacities of analysed DCs. Short time incubation of immature DCs with IFN-α, rhGM-CSF, tumor lysate and BGs (4 h) significantly enhanced capacity of DCs to stimulate proliferation of autologous T cells compared to DCs matured with IFN-α, rhGM-CSF and tumor lysate supplemented with pure LPS or without extra maturation stimuli. Autologous and allogeneic immunostimulatory capacities of analyzed DC populations were determined after 6 days of incubation in the presence of fluorescence-labeled (CFSE-labeled) allogenic or autologous T cells at the ratio DCs:T cells—1:10. Stimulated cells were stained after incubation with a panel of monoclonal antibodies (anti-CD3, anti-CD4, and anti-CD8) and proliferation of both autologous and allogeneic T cells was determined by multicolor flow cytometry. Values were calculated as percentage of T cells proliferated spontaneously subtracted from the percentage of cells proliferated after stimulation with distinct populations of DCs. T cells incubated with phytohemaglutinin (PHA) (5 μg/ml) served as positive control. Data represent the mean±SD of 4 independent experiments performed using cells obtained from different donors. P values <0.05 were considered significant and are indicated with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).

(14) FIG. 5. Recognition of tumor cells and cytotoxic effects of allogenic (A) and autologous (B) T cells induced by DCs loaded with tumor lysate after short time stimulation with BGs. Allogenic or autologous T cells were incubated for 6 days in the presence of tumor cell lysate (T98G cells) loaded DCs pre-stimulated with pure LPS (200 ng/mL), short time incubation (4 h) with BGs from E. coli Nissle 1917 (10 and 100 BGs/1DC) or without extra maturation stimuli. Subsequently, stimulated T cells were added to fresh fluorescence-labeled (CFSE-labeled) T98G tumor cells at the Effector:Target ratio 10:1. Specific lysis of tumor cells was determined 24 h after mutual co-incubation by flow cytometry. Lysis of tumor cells after incubation with 10% Et-OH served as positive control (100%). Tumor cells incubated without the presence of pre-stimulated effector cells served as negative control for spontaneous cell death. Data represent the mean±SD of 4 independent experiments performed using cells obtained from different donors. P values <0.05 were considered significant and are indicated with asterisks (*, P<0.05; **, P<0.01; ***, P<0.001).

(15) In particular, the results of treatment of T98G tumor cells show a considerable enhancement for DCs+BGs compared to DCs alone or DCs+LPS.

(16) In all experiments shown in FIGS. 1 to 5 human dendritic cells are included.

EXAMPLES

Example 1

Bacterial Ghosts

(17) Bacterial ghosts are prepared in accordance with Patent Application No. PCT/EP2009/000272.

Example 2

Tumor Lysate Preparation

(18) Tumor tissue obtained from a cancer patient or tissue culture plates (flasks) are stored in tubes filled with NaCl (0.9% sodium chloride in water for injection “Fresenius”). The tube with tumor tissue or cells are stored at +4° C. and processed up to 3 days after the surgery. The tube is irradiated (120 Gy) before processing of tissue. The donors (patients) have to be screened for sexually-transmitted diseases (STD), e.g. HIV, HCV, HBV, syphilis, and patients with positive detection of any of the mentioned diseases are excluded. Tumor tissue obtained from the patient is transferred to a sterile Petri dish filled with 5-10 ml HBSS (Hanks' Balanced Salt Solution; Lonza, No.: 10-547F or 10-527F; manufactured in accordance with cGMP regulations). Both necrotic and connective tissues are removed by scalpel and tweezers. Remaining tumor tissue is cut into small pieces of approximately 5 mm and ground by sterile syringe plunger. The obtained cell suspension is additionally homogenized by passing the suspension through a 20 G (0.9 mm) needle attached to a sterile syringe several times until a homogenized suspension is obtained. The cell suspension is subsequently filtered through a nylon strainer (100 μm) and collected in a sterile 50 ml tube. The Petri dish is washed with remaining HBSS, filtered through a nylon strainer and combined with filtered cells. The cell suspension is spun down at 1600 RPM for 7 min at +4° C. The supernatant is quickly and carefully decanted and a pellet is resuspended in 2 ml of CellGro® culture medium. Single cell suspension is equally divided into microtubes. The microtubes are placed into liquid N.sub.2 or a mixture of dry ice and methanol for approximately 3 minutes. Subsequently, frozen cells are thawed at room temperature (RT) for 15-30 minutes until the cell pellet becomes completely melted (cells should not be kept at RT too long in order to prevent degradation of tumor cell proteins). The freezing-thawing procedure should be repeated 5 times. The cell lysate is then sonicated in an ultrasonic bath for 5 minutes. Cell debris together with cell lysate is collected in one tube, spun down at 10000-15000 RPM for 10 min, followed by quick and careful collection of the supernatant (cell lysates) using a fine needle separating off the pellet. Cell lysates should not be filtered and all steps have to be done under sterile conditions. Non-filtered cell lysates are aliquoted and stored at −80° C. until further use. The cell lysate is diluted with 10×DPBS (Dulbecco's Phosphate Buffered Saline, 10×PBS, Lonza, No.:17-515F). A spectrophotometer is used for determination of protein concentration.

Example 3

Culture of Monocyte-Derived Dendritic Cells for Anti-Tumor Vaccine Preparation

(19) Monocytes obtained from peripheral blood of patients are isolated either by elutriation (Elutra Cell Separation System, CaridianBCT Europe NV/SA) or magnetic separation (CliniMACS, Miltenyi Biotec GmbH). GM-CSF is dissolved in CellGro® culture medium to obtain a final concentration of 1×10.sup.5 IU/ml and aliquoted into microtubes (700 μl per tube) and stored at −80° C. IFN-α (Roche, ROFERON-A 12×10.sup.6 IU/ml) is aliquoted into microtubes (17.5 μl per tube) and stored between +2° C. and +8° C. Cryopreservation freeze media CryoStor CS2 or CryoStor CS5 are aliquoted under sterile conditions into microtubes (1.5 ml per tube). Separated monocytes are resuspended in culture medium and added into a culture flask, approximately 167×10.sup.6 monocytes in 70 ml of culture medium. Approximately 150 μg of tumor lysate is required for one culture flask or tumor lysate from cells corresponding to a number of tumor cells 3 times that of immature DCs (monocytes). Monocyte cell suspension from culture flasks is centrifugated at 1500 RM for 10 min at RT. After decantation of supernatant the cell pellet is resuspended in a small volume of CellGro® culture medium and completed with CellGro® culture medium up to 35 ml. The cell suspension is transferred to a culture flask. GM-CSF (700 μl/7×10.sup.4 IU) and IFN-α (17.5 μl/2.1×10.sup.6 IU) in 35 ml of CellGro® culture medium are added to the monocyte cell suspension. The contents of the culture flask are carefully mixed by gently moving the flask from side to side. The cells are incubated in a 5% CO.sub.2 humidified incubator at +37° C. for 3 days.

(20) The prepared cell suspension of immature DCs is collected and distributed into 3 sterile 50 ml tubes. Fresh CellGro® culture medium (5 ml) is added meanwhile into each culture flask to avoid death of cells attached to the surface of culture flask. The cell suspension is spun down at 1500 RPM for 10 minutes at RT. Supernatant is quickly and carefully decanted and the cell pellets are resuspended in 2 ml of CellGro® culture medium and the cells from the culture flasks are transferred to 1 tube. Each of the tubes used for spinning of cells is gently rinsed with 4 ml of CellGro® culture medium and the content is transferred to the tube with collected cells. Tumor lysate (5 ml) obtained from tumor tissue or tumor cell lines (ratio of tumor cells:DCs=3:1; 501×10.sup.6:167×10.sup.6) is mixed with 167×10.sup.8 bacterial ghosts prepared from E. coli Nissle 1917 (ratio of BGs:DCs=100:1), vortexed thoroughly and incubated with gentle shaking at RT for 60 min. The mix of BGs and tumor cell lysate supplemented with GM-CSF (25 μl/2.5×10.sup.4 IU) and IFN-α (6.25 μl/7.5×10.sup.4 IU) is added to the DC suspension. Subsequently, the cell suspension with all reagents is transferred to a culture flask. The tube which contained the cell suspension is rinsed with 5 ml of CellGro® culture medium and the medium is transferred to the culture flask and carefully mixed with gentle shaking from side to side. The cells are incubated in a 5% CO.sub.2 humidified incubator at +37° C. for 4 hours to allow internalization of BGs and tumor lysate and to start the maturation process. After 4 h of incubation, the cells are transferred to a 50 ml tube and the culture flask is gently rinsed with CellGro® culture medium. The medium used for rinsing of flasks is transferred to the tube with cell suspension. Fresh CellGro® culture medium (5 ml) is added meanwhile into the culture flask to avoid death of cells attached to the surface of the culture flask. The cell suspension is spun down at 1500 RPM for 10 minutes at RT. The supernatant is quickly and carefully decanted and cell pellets are resuspended in 20 ml of fresh CellGro® culture medium supplemented with GM-CSF (25 μl/2.5×10.sup.4 IU) and IFN-α (6.25 μl/7.5×10.sup.4 IU). The cell suspension is transferred into the original culture flask which is carefully and gently shaken from side to side, and the cells are incubated in a 5% CO.sub.2 humidified incubator at +37° C. for additional 6 h.

Example 4

Storage of Anti-Tumor Vaccine Preparations

(21) A culture flask with stimulated DCs obtained in Example 3 is thoroughly shaken to release cells adhered to the flask walls. The cell suspension is completely transferred to a labeled 50 ml tube. The culture flask is rinsed with two additional volumes of the same HBSS (10 ml) used for tumor lysate preparation, if possible, and the whole volume of HBSS is transferred to the tube with cell suspension. The original culture flask is filled with 5 ml of HBSS and placed back into an incubator. The volume of cell suspension is filled up to 50 ml with HBSS and gently mixed by tube overturns. The cell suspension is spun down at 1500 RPM for 10 minutes at +4° C. The supernatant is quickly and carefully decanted and cell pellets are resuspended first in 5 ml HBSS, followed by addition of additional 45 ml of HBSS and mixed well by tube overturns. 10 μl of cell suspension is used to determine the cell number, using a Bürkner counting chamber. If the concentration of cell within the suspension is below 1.4×10.sup.6/ml, accutase should be used to release the remaining cells from culture flask, otherwise cell aliquots each containing of 5×10.sup.6 cells will be frozen for future administration to the patient. It is mandatory to make at least 6 aliquots for administration to the patient, one aliquot for quality control, one aliquot for immunomonitoring, one aliquot for testing of mycoplasma and three aliquots for arbitrage. The cell suspension is spun down at 1500 RPM for 10 minutes at +4° C. Cryotubes are transferred to a pre-cooled MiniCooler. The supernatant is quickly and carefully decanted; cell pellets are resuspended in cryopreservation freeze media CryoStor CS2 and transferred to cryotubes. Immediately after aliquoting of anti-tumor vaccine preparation, all cryotubes are placed in an isopropanol box at −80° C. After 24 h in −80° C., all frozen vaccine preparation is transferred to a Dewar container filled with liquid nitrogen specifically set for anti-tumor vaccine preparation purposes use only.

(22) TABLE-US-00001 TABLE 1 Standards for acceptability of anti-tumor vaccine preparations before administration to a patient. Test Criterion to pass quality control Sterility of anti-tumor vaccine preparation STERILE Detection of mycoplasma NEGATIVE Cell count (×10.sup.6) 1 × 10.sup.6 − 5 × 10.sup.6 Viability (%) 70-100% DCs purity (%) 70-100% Cell  CD3 (%) Total amount (CD3.sup.+ + CD19.sup.+) contamination CD19 (%) 0-30% DCs phenotype* CD80 (%) 60-100% CD86 (%) 60-100% MHC class II (%) 60-100% CD83 (%) 60-100% CD14 (%) 0-40% Production IL-12 (pg/ml) ≧100 pg/ml Allogenic MLR- DCs:PBMC s ≧30% activated Ratio 1:5  T-lymphocytes (%)** DCs:PBMC s ≧30% Ratio 1:10 DCs:PBMC s ≧15% Ratio 1:20 *at least 3 of 5 phenotypic markers should meet the criteria to pass quality control before administration of anti-tumor vaccine preparation to a patient **at least 2 of 3 examined ratios DCs:PBMCs in allogenic MLR tests should meet the criteria to pass quality control before administration of anti-tumor vaccine preparation to a patient