METHOD FOR THE PRODUCTION OF MATURE DENDRITIC CELLS IN VITRO USING A BACTERIAL LYSATE

Abstract

The invention is in the field of cell biology and immunology. The invention provides methods for the production of a vaccine for cancer and infectious diseases. More in particular it provides a method for the improved production of mature dendritic cells. This improved production includes the use of a bacterial lysate produced by a new method.

Claims

1. Method producing mature dendritic cells in vitro or ex vivo, wherein immature dendritic cells are contacted with a composition comprising growth factors and a bacterial lysate, characterized in that the bacterial lysate is obtainable by a method comprising the steps of: a) providing a suspension comprising bacteria, b) sonicating the suspension followed by c) heat treatment of the suspension, and optionally isolating mature dendritic cells from the composition.

2. Method according to claim 1 wherein the growth factors are selected from the group consisting of interferon gamma (IFN-gamma), the pattern recognition receptor-ligand (PRR-ligand) CL075 and the PRR-ligand poly I:C.

3. Method according to claim 1, wherein the bacteria are Gram-negative bacteria or bacteria containing endotoxin or OmpA.

4. Method according to claim 3 wherein the bacteria are selected from the group consisting of Pseudomonas, Klebsiella, Proteus, Salmonella, Providencia, Escherichia, Morganella, Aeromonas, and Citrobacter.

5. Method according to claim 3 wherein the bacteria are from the family of Enterobacteriaceae preferably from the genus Klebsiella, such as Klebsiella pneumoniae.

6. Method according to claim 1, wherein the heat treatment includes a treatment at a temperature above 100° C.

7. Method according to claim 6 wherein the heat treatment results in a sterile composition.

8. Method for preparing a bacterial lysate, comprising the steps of: a) providing a suspension comprising bacteria, b) sonicating the suspension followed by c) heat treatment of the suspension, thereby obtaining a bacterial lysate capable of increasing the production of IL-12 by dendritic cells when contacted in vitro with said dendritic cells, wherein the increase of the production of IL-12 is higher than the increase when the lysate is prepared by a method wherein the bacterial suspension in step a) is first subjected to heat treatment (step c)) followed by a sonication, step b).

9. Method according to claim 8 wherein the growth factors are selected from the group consisting of interferon gamma (IFN-gamma), the pattern recognition receptor-ligand (PRR-ligand) CL075 and the PRR-ligand poly I:C.

10. Method according to claim 8 wherein the bacteria are Gram-negative bacteria or bacteria containing endotoxin or OmpA.

11. Method according to claim 10 wherein the bacteria are selected from the group consisting of Pseudomonas, Klebsiella, Proteus, Salmonella, Providencia, Escherichia, Morganella, Aeromonas, and Citrobacter.

12. Method according to claim 10 wherein the bacteria are from the family of Enterobacteriaceae preferably from the genus Klebsiella, such as Klebsiella pneumoniae.

13. Method according to claim 8, wherein the heat treatment includes a treatment at a temperature above 100° C.

14. Method according to claim 13 wherein the heat treatment results in a sterile suspension.

15. Method according to claim 8, wherein the bacterial lysate is capable of increasing the production of IL-12 by dendritic cells when contacted in vitro with said dendritic cells, wherein the increase of the production of IL-12 is higher than the increase when the lysate is prepared by a method wherein the bacterial suspension is first heat-treated followed by a sonication step.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 shows the normalized IL-12 production by mature DC, 20 hours after induction of maturation with different preparations of membrane fragments.

DETAILED DESCRIPTION OF THE INVENTION

[0034] The invention is concerned with a method for the improved production of dendritic cells and compositions useful in such a method. The model system that we employed was as follows. At day 0, PBMC were isolated from fresh blood, followed by isolation of CD14 positive cells (monocytes) from these PBMCs using CD14 beads and CliniMACS columns. CD14 positive cells were put into culture and differentiated into immature DC (iDC) using appropriate cytokines, in particular IL-4 and GM-CSF. At day 3, the culture medium was refreshed by performing a half medium exchange using differentiation medium containing IL-4 and GM-CSF. At day 6, iDC were matured into mDC by incubating them for 4 hours at 37° C. and 5% CO2 using different maturation cocktails containing IL-4, GM-CSF, IFN-y and CL075. To this mixture also bacterial membrane fragments were added, in particular membrane fragments obtained from Klebsiella pneumoniae (KpMF).

[0035] Immediately after maturation, mDC were transfected with eGFP mRNA using the BioRad Gene pulser electroporation system. After maturation and electroporation, a cell count was performed using Via-1 cassettes and NucleoCounter followed by phenotype determination of mDC by FACS staining (CD80, CD83, CD86, HLA-DR and CD11c). 2.10.sup.6 DC were transferred to a new 24-wells plate and incubated for 16 hours at 37° C. and 5% C02. Then, the production of IL-10 and IL-12 was determined in the supernatant by Cytometric Bead Array while mDC were checked for eGFP expression by FACS.

[0036] A maturation cocktail successfully used to stimulate iDC to develop into mature DCs comprises IL-4, GM-CSF in combination with IFN-gamma, CL075 and a bacterial lysate termed KpMF (EP 2847321 B1). Such a bacterial lysate is commonly produced by sonication of isolated bacterial cells thereby lysing the cells and obtaining a lysate comprising membrane fragments. We now found that the bacterial lysate can even be further improved if it is prepared in a different way than so far described.

[0037] The present document describes a way to make a novel bacterial lysate that can successfully be used to further improve the maturation of immature dendritic cells (iDC). Traditionally, KpMF is prepared in a process wherein a bacterial suspension is subjected to sonication (51), sometimes preceded by heat treatment such as a process described in U.S. Pat. 7,033,591 B1.

[0038] We prepared four different batches of bacterial membrane fragments

TABLE-US-00001 Four different compositions comprising membrane fragments Sample # Treatment 1 Sonication 2 Heat treatment 3 First heat treatment, followed by sonication 4 First sonication, followed by heat treatment.

[0039] These 4 samples were used in 4 different maturation cocktails. The data showed that all cells were DC, being highly positive for CD11c and HLA-DR. Maturation of DC is shown by 2-3 times upregulation of the median value for costimulatory markers CD80, CD83 and CD86. There were no phenotypical differences observed between the samples prepared with the different membrane fragment samples #1 - #4.

[0040] Four preparations of mDCs were prepared, using KpMF that was subjected to sonication alone (preparation #1) to heat treatment alone (preparation #2), to a process of first heat treatment followed by sonication (preparation #3) and a preparation that was first subjected to sonication followed by heat treatment (preparation #4). immature DCs that were not subjected to the maturation cocktail served as the negative control.

[0041] Surprisingly, we found that the membrane preparation #4, which was first sonicated followed by a heat inactivation step, yielded a 3 times higher IL-12 production as compared to the prior art preparation #3 (heat inactivation followed by sonication) and was therefore a much more potent maturation inducer (FIG. 1). We also observed that the use of a maturation cocktail comprising preparation #4 yielded a 5 times higher IL-12 production as compared to a process wherein a maturation cocktail according to the prior art (preparation #1, positive control in FIG. 1) was used..

[0042] The invention therefore relates to a method for the production of mature dendritic cells in vitro or ex vivo, wherein immature dendritic cells are contacted with a composition comprising growth factors and a bacterial lysate, characterized in that the bacterial lysate is obtained by a method comprising the steps of: [0043] a) providing a suspension comprising bacteria, [0044] b) sonicating the suspension followed by [0045] c) heat treatment of the suspension. Mature dendritic cells may then optionally be isolated from the composition.

[0046] The skilled person is well aware of the kind of growth factors that may be used for the maturation of immature DC into mature DC. In a particularly advantageous embodiment of the invention growth factors are selected from the group consisting of interferon-gamma (IFN-gamma), the pattern recognition receptor-ligand (PRR-ligand) CL075 and the PRR-ligand poly I:C.

[0047] When the experiments were repeated with bacterial lysates obtained from commonly isolated Gram-negative organisms including Pseudomonas, Klebsiella, Proteus, Salmonella, Providencia, Escherichia, Morganella, Aeromonas, and Citrobacter, essentially the same results were obtained. We conclude that the source and kind of bacteria is not critical and that all kinds of bacteria may be used to produce the membrane fragments as described herein. In a particularly advantageous embodiment, the bacteria are Gram-negative bacteria or bacteria containing endotoxin or OmpA, which are potential triggers to Toll like receptors. Particularly preferred are bacteria from the family of Enterobacteriaceae, even more preferably from the genus Klebsiella, such as Klebsiella pneumoniae.

[0048] The heat treatment step is intended to sterilize the composition. That may be achieved by conventional ways, well known in the art. For practical reasons, the heat treatment includes heating the composition to a temperature above 100° C. for an appropriate amount of time. The skilled person is well aware of methods suitable for determining whether a composition is sterile, for instance by plating the composition on a rich medium agar plate. Particularly preferred are even higher temperatures because they allow the procedure to be much quicker, such as temperatures above 110° C. Even more preferred are again higher temperatures, such as above 120° C., such as 121° C. Therefore, in a preferred embodiment, the invention relates to a method for the production of a suspension as described herein, which is sterile. Heat treatment also includes autoclaving.

[0049] The term “autoclaving” is used herein to indicate a process wherein a composition is heated to a certain temperature (typically 121° C.) for a certain amount of time (typically 15 minutes) in order to obtain a sterile composition. This process is usually performed under elevated pressure, i.e. above 1 bar.

[0050] The invention also relates to a method for the preparation of a bacterial lysate, comprising the steps of a) providing a suspension comprising bacteria, b) sonicating the suspension followed by c) heat treatment of the suspension, thereby obtaining a bacterial lysate capable of increasing the production of IL-12 by dendritic cells when the suspension is contacted in vitro with said dendritic cells, wherein the increase of the production of IL-12 is higher than the increase when the lysate is prepared by a method wherein the bacterial suspension in step a) above is first subjected to heat treatment followed by a sonication step.

[0051] For the same reasons as described above, the invention also relates to such a method wherein the growth factors are selected from the group consisting of interferon-gamma (IFN-gamma, the pattern recognition receptor-ligand (PRR-ligand) CL075 and the PRR-ligand poly I:C. The bacteria are advantageously Gram-negative bacteria or bacteria containing endotoxin or OmpA. Even more preferred, the bacteria are from the family of Enterobacteriaceae preferably from the genus Klebsiella, such as Klebsiella pneumoniae.

[0052] The term “sonication” or “sonicated” is used herein to indicate a process wherein a liquid, suspension or composition is exposed or subjected to high frequency sound waves, also known as ultrasound, in such a way that the components of the liquid, suspension or composition physically disintegrate or are disrupted.

[0053] The heat treatment may include a treatment at a temperature above 100° C., such as above 110° C., such as above 120° C., such as 121° C. This treatment preferably results in a sterile suspension.

[0054] In a further embodiment, the invention relates to a bacterial lysate obtainable by a method as described herein, capable of increasing the production of IL-12 by dendritic cells when contacted in vitro with said dendritic cells, wherein the increase of the production of IL-12 is higher than the increase when the lysate is prepared by a method wherein the bacterial suspension is first heat-treated followed by a sonication step.

Legend to the Figure

[0055] Mean of normalized values of IL-2 production by maturating DCs in the presence of 5 different maturation cocktails comprising 5 different preparations of bacterial membrane fragments (KpMF).

EXAMPLES

Example 1: Experimental Procedures

Preparation of Bacterial Lysate

[0056] A stock Klebsiella pneumoniae vial is thawed and a droplet from the Klebsiella pneumoniae stock is added to 6 mL Nutrient Broth using an inoculation loop. This culture is incubated for 7 hours at 35 ± 2° C. on a rocking platform. Two mL bacterial suspension is added to 150 mL Nutrient Broth and incubated shaking for at least 16 hours at 35 ± 2° C. The bacterial suspension is pelleted by centrifugation at 2000 g for 15 minutes at 20 ± 3° C. in 50 ml tubes. After discarding the supernatant by decanting, the cell pellets are resuspended in 50 mL PBS by vortexing and pipetting up and down. This step is repeated 3 times. Finally, the pellets are resuspended in 750 .Math.L PBS and combined in one 15 mL tube. Again, the suspension is aliquoted as 750 .Math.L bacterial suspension in Eppendorf tubes, followed by pelleting the suspension by centrifugation at 14000 g for 15 minutes at RT. Pellets were frozen at -80° C. ± 10° C. and kept for at least 4 hours.

Sonication

[0057] Each cell pellet was thawed at room temperature and resuspended in 2.0 ml sonication buffer (5 mM Na.sub.2EDTA / pH 7.0 ± 0.3) in a 15 ml tube. The tube was placed in a lab cooler during sonication. The sonotrode (Hielscher Ultrasonics Sonotrode S26D7 Titanium 7 mm) was set at Amplitude 100% (180 .Math.m) and Pulse control mode 0.9 and attached to an Ultrasonic Processor UP200Ht. The solution was sonicated on ice for at least 2 times, more preferably 5, 6, 7, 8 ,9 times or even more preferably 10 times, each time with 10 pulses more preferably 15 or 20 pulses and even more preferably 25 pulses. Each time for at least 10 seconds, or 20 seconds or more preferably 30 seconds. Also, each pulse may last 10 minutes, more preferably 5 minutes or even more preferably for 1 minute.

[0058] The solution was then centrifuged for 15 minutes at 14000 RCF in sterilized micro centrifuge tubes. Afterwards the supernatant was collected for further steps. A sample was taken of the collected supernatant to measure the concentration at A280 using a spectrophotometer.

[0059] The final concentration of the supernatant was adjusted with phosphate buffered saline (PBS) to the desired concentration based on the A280 result where ABS280 of 1 corresponds to 1 mg /mL cell membrane lysate (containing sugars, proteins and lipids). Typically, the A280 after sonication of a sample was between 1 and 1.5 AU which corresponds to 20 - 30 mg/ml lysate. The A280 of the final membrane product was typically between 0.7 and 0.85 AU, which corresponds to 3.5 - 4.5 mg/ml lysate after a dilution of 5 times.

Sterilization

[0060] Filled Sterilized Crimp Vials 1.5 mL (VWR Collection) from each lysate were sterilized in an autoclave. Program for 2 L liquid material was used (121° C. for 15 min). Vials were placed in a tray so they are kept upright during the run. Vials were labelled after sterilization and stored at -80° C.

Example 2: Process for the Preparation of Mature Dendritic Cells (mDC)

[0061] At day 0, PBMC were isolated from fresh blood, followed by isolation of CD14 positive cells (monocytes) from these PBMCs using CD14 beads and CliniMACS columns. CD14 positive cells were put into culture and differentiated into immature DC (iDC) using IL-4 and GM-CSF. At day 3, the culture medium was refreshed by performing a half medium exchange using differentiation medium containing IL-4 and GM-CSF. At day 6, iDC were matured into mDC by incubating them for 4 hours at 37° C. and 5% CO2 using different maturation cocktails containing IL-4, GM-CSF, IFN-gamma, CL075 and 3 different stocks of KpMF. Immediately after maturation, mDC were transfected with eGFP mRNA using the BioRad Gene pulser electroporation system. After maturation and electroporation, a cell count was performed using Via-1 cassettes and NucleoCounter followed by phenotype determination of mDC by FACS staining (CD80, CD83, CD86, HLA-DR and CD11c). 2E6 DC were transferred to a new 24-wells plate and incubated for 16 hours at 37° C. and 5% CO2. Then, the production of IL-12 was determined in the supernatant by Cytometric Bead Array while mDC were checked for eGFP expression by FACS.

Legend to the Figure

[0062] FIG. 1: The figure shows the normalized IL-12 production by mature DC, 20 hours after induction of maturation with different preparations of membrane fragments (table 1). Monocytes were positively selected by CD14 from peripheral blood as described. Cells were subsequently cultured for 6 days in GM-CSF and IL-4 to obtained differentiated immature DCs. One sample was kept as immature DC by no addition of maturation cocktail (negative control). Mature dendritic cells were obtained by adding a maturation cocktail of IFN-gamma and CL075 at proper concentrations per 2E6 DC/well. Different preparations of membrane fragments were finally added to this mixture and DC were allowed to mature for 4 hours after which the cells were washed and allowed to further mature for 20 hours. A previous preparation of membrane fragments (KpMF), that only had been sonicated, was used as a positive control. Supernatant was taken from all cultures and 50 microliter per sample was subjected to Cytometric Bead Array analysis specifically for IL-12 on a FACS cytometer.

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