WO2011111898A1

WO2011111898A1 - Method for preparing mycobacterial cell wall skeleton

Info

Publication number: WO2011111898A1
Application number: PCT/KR2010/002678
Authority: WO
Inventors: Tae-Hyun Paik
Original assignee: Tae-Hyun Paik
Priority date: 2010-03-09
Filing date: 2010-04-28
Publication date: 2011-09-15
Also published as: KR101096577B1; KR20110101547A

Abstract

The present invention relates to a method of preparing mycobacterial CWS from mycobacterial cells through deproteination and delipidation processes using a surfactant. More specifically, the invention relates to a method for preparing mycobacterial CWS, the method comprising the steps of: (A) adding a surfactant to killed mycobacterial cells and heat-treating the cells in the presence of the surfactant, thereby delipidating and deproteinating the cells; and (B) washing the delipidized and deproteinized mycobacterial cells. According to the invention, mycobacterial CWS can be economically mass-produced by a simple process without using enzyme. Also, the mycobacterial CWS prepared according to the invention can be stably stored as a homogenized suspension without forming an aggregate, and thus can be more efficiently used in anticancer immunotherapy or adjuvant therapy.

Description

METHOD FOR PREPARING MYCOBACTERIAL CELL WALL SKELETON

The present invention relates to a method capable of economically mass-producing mycobacterial cell-wall skeleton.

Generally, the cell walls of mycobacteria strongly stimulate host inflammatory responses, leading to granuloma formation, upregulation of antigen presentation and inflammatory cytokines, and subsequent increases in immune responses. Mycobacterium bovis bacillus Calmette-Guerin (BCG), the current live attenuated vaccine against tuberculosis, has been administered to more than three billion people worldwide since 1921, and its safety has been proved. Up to now, BCG has been studied not only as a tuberculosis vaccine, but also as an anticancer immunotherapeutic agent against a variety of malignant tumors including bladder cancer.

The cell wall of mycobacteria contains about 60% complex lipids on a dry weight basis and has a structure in which peptidoglycan, arabinogalactan, mycolic acid, lipoarabinomanan (LAM), superficial lipids and mycosides are deposited to a thickness of about 20 nm (see FIG. 1). Among the mycobacterial cell-wall components, the cell-wall skeleton (hereinafter referred to as "CWS" refers to insoluble particles excluding soluble components (e.g., free proteins, nucleic acids and lipids) of the cells (J. Nat. Cancer Inst., 52, 95-101 (1974)) and is composed of a complex of peptidoglycan, arabinogalactan and mycolic acid.

The CWS of BCG is composed of immunoactive microparticles and is known as a potent adjuvant that stimulates innate immunity. Also, it exhibits anticancer immune effects by inducing the proliferation of cytotoxic T lymphocyte (CTL) and the activation of natural killer (NK) cells. Since its clinical utility as an anticancer immunotherapeutic agent was described by Yamamura et al. in 1970s (Ann N Y Acad Sci 277: 209-227), it has been introduced as new immunotherapy in the treatment of all cancers, including lung cancer, gastric cancer, intestinal cancer, breast cancer, tongue cancer, laryngeal cancer, acute myelogenous leukemia, pancreatic cancer and ovarian cancer.

Up to now, BCG-CWS anticancer immunotherapy has been used only as adjuvant therapy in a state of impaired immunity after surgery, chemotherapy or radiotherapy, but it has recently been suggested to perform BCG-CWS anticancer immunotherapy from the initial stage of cancer diagnosis. This is because extensive damage to the immune system, which occurs in chemotherapy or radiotherapy, results in severe immune impairment, such that the immune boosting effect of BCG-CWS cannot be sufficiently secured. Indeed, it was reported that a single therapy of BCG-CWS showed a much better anticancer immunotherapeutic effect than a combination therapy of chemotherapy and BCG-CWS immunotherapy.

For BCG-CWS immunotherapy, intracutaneous injection of 10-200 ㎍ (dry weight) of BCG-CWS should be repeated several tens of times at intervals of 1 week to 1 month. This injection therapy has an advantage in that a constant dose of CWS can be administered; however, if the repeated injection of CWS is required, it is very complicated and inconvenient and causes patients to suffer from pain. Also, because immune cells are localized to the injected skin site, local skin reactions such as skin redness, induration, blisters and ulcers can occur, and in severe cases, swelling of local lymph nodes can also occur. Particularly, scars remain in the injected area, and thus if the repeated injection of CWS places a significant burden on patients.

Oral administration of BCG-CWS can solve the above-described problems of intacutaneous injection, and thus can be more conveniently and widely used for anticancer immunotherapy. However, in the oral administration method, because the in vivo absorption rate of CWS through the intestinal mucosa is very lower than that in the injection therapy, the single oral dose (dry weight) of CWS should be increased to a unit of 5-10 ㎎ (generally 0.05-0.1 mg for intracutaneous injection) and it is required to orally administer CWS everyday for a period ranging from 6 months to 1 year or more. For this reason, CWS is required in an amount which is at least several hundred times more than that for injection therapy. However, it is practically difficult to perform the oral administration of CWS, because a method capable of producing a large amount of CWS has not yet been developed.

Akira et al. reported a method of preparing BCG-CWS by deproteinizing BCG cells by sequential treatment with proteases, trypsin-chymotrypsin and pronase, and then subjecting the deproteinized cells to a complicated delipidation process using various organic solvents (US 6,593,096).

Wataru et al. improved the delipidation process and reported a method of preparing BCG-CWS, comprising the steps of: (A) disrupting killed BCG cells and collecting the cell walls; (B) deproteinating the cell walls by sequential treatment with benzonase and pronase; (C) treating the deproteinated cell walls with a surfactant (1% Triton X-100) under heating; and (D) washing the treated cell walls with an organic solvent and drying the washed cell walls (Japanese Patent JP 2008214266 A). Through this method, Wataru et al. produced about 10 g of CWS from 190 g of BCG cell-wall pellets obtained by disrupting 600 g of killed BCG cells, but the method of Wataru et al. has problems in that the process is lengthy due to the time of enzyme treatment and that the production efficiency is low because several steps are carried out. In addition, CWS is needs to be effectively dispersed for use in adjuvant therapy or anticancer immunotherapy, but CWS obtained by the method of Wataru et al. is a very hard aggregate. For this reason, there is an additional problem in that a process of either crushing the CWS particles by milling or dissolving the CWS particles by oil before the use thereof is required.

As described above, in order to use CWS as a medical material, a method capable of mass-producing CWS is required. However, it is difficult for currently known technology to achieve the mass production of CWS. Therefore, there is a need to develop a method which can mass-produce CWS by a simpler process in an inexpensive way.

The present invention has been made in view of the above-described problems occurring in the prior art, and it is an object of the present invention to provide a process capable of economically mass-producing mycobacterial CWS.

Another object of the present invention is to provide a method for preparing mycobacterial CWS, which enables a homogenized suspension to be easily prepared.

To achieve the above objects, the present invention provides a method of preparing mycobacterial CWS from mycobacterial cells through deproteination and delipidation processes using a surfactant. More specifically, the present invention provides a method for preparing mycobacterial CWS, the method comprising the steps of: (A) adding a surfactant to killed mycobacterial cells and heat-treating the cells in the presence of the surfactant, thereby delipidating and deproteinating the cells; and (B) washing the delipidized and deproteinized mycobacterial cells.

As used herein, the term "Mycobacteria" refers to bacteria belonging to the genus Mycobacterium. Although Examples of the present invention illustrated only the preparation of the CWS of Mycobacterium bovis BCG as an example of mycobacteria, it is to be understood that the present invention can also be applied to all other mycobacteria, because mycobacteria have the same CWS structure.

Killed mycobacterial cells which are used as a starting material in the present invention are known in the art and can be easily prepared by any person skilled in the art by using a known method. For example, killed mycobacterial cells can be obtained by culturing mycobacterial cells in Sauton medium, centrifuging the cultured cells and heating the centrifuged cells, but the scope of the present invention is not limited thereto (J. Nat. Cancer Inst., 52, 95-101 (1974)).

As the surfactant in step (A) of the method of the present invention, a nonionic surfactant and/or an anionic surfactant is preferably used. It is generally known that the nonionic surfactant is more effective for delipidation and the anionic surfactant is more effective for deproteination. For this reason, in the heat treatment in step (A), the nonionic surfactant and the anionic surfactant are preferably used together. More preferably, these surfactants are sequentially used. Herein, although it is more efficient to treat the cells with the nonionic surfactant before after treatment with the anionic surfactant, treatment of the cells with the anionic surfactant may be performed before treatment with the nonionic surfactant. Namely, the order of treatment with the surfactants is not critical for the present invention.

Moreover, in order to more efficiently remove lipids and proteins from the cells by the surfactants, a sonication process may be carried out in addition to the heat treatment in step (A). The sonication may be carried out before or after the heat treatment.

As the nonionic surfactant, either one of octyl phenol ethoxylate derivatives like Triton X-100 of Triton X series, or nonyl phenoxylpolyethoxylethanol (NP-40) may be used, and as the anionic surfactant, either sodium dodecyl sulfate (SDS) or sodium lauryl ether sulfate (SLES) may be used, but the scope of the present invention is not limited thereto. The concentration of the nonionic and/or anionic surfactant can be suitably selected depending on the kind, treatment temperature, amount, pH and ionic strength of surfactant used and the kind of additive used, but the surfactants are preferably used as 0.5-5% (w/v) solutions. If the concentration of the surfactants is too low, the surfactants will have insufficient delipidation and deproteination effects, and even if the final concentration of the surfactants is higher than the critical micelle concentration (CMC), the delipidation and deproteination effects of the surfactants will not be improved, and the concentration of surfactant residues can be increased. The surfactant solution is preferably used in an amount 4-30 times (w/w) the amount of wet-state mycobacterial cells. If the amount of the surfactant solution is too small, the sufficient delipidation and deproteination of the cells will not be achieved, and if the amount of the solution is too much, the production efficiency will be reduced. For this reason, the amount of the surfactant solution is preferably suitably adjusted in the above-described range. In the case in which the purity of CWS is to be increased by increasing the degree of delipidation and deproteination in the process of treatment with the surfactants, or in the case in which the amount of surfactants used is small due to problems associated with equipment used, treatment with the surfactants may be repeated.

Heat treatment of the surfactants is performed by heating them to a temperature ranging from 70℃ to the boiling point thereof. The heat-treatment temperature is too low, delipidation and deproteination reactions will not efficiently occur, and thus the delipidation and deproteination of the cells can be incomplete or the heating time can be increased. When the surfactants are heat-treated at a temperature ranging from 70℃ to the boiling point thereof, the heat treatment is preferably carried out for 2-18 hours.

The surfactants remain in the mycobacterial CWS prepared by delipidating and deproteinating the mycobacterial cells by heat treatment in the presence of the surfactants. Thus, step (B) is a step of washing out the surfactants remaining in the mycobacterial CWS obtained by delipidation and deproteination. The washing step is preferably performed by (1) washing the CWS with acetone, and then (2) re-washing the CWS with a 10-20% (v/v) aqueous solution of at least one alcohol selected from the group consisting of methanol, ethanol, propanol, butanol and benzyl alcohol. The washing solvent is preferably used in an amount 5-30 times (v/w) the wet weight of the mycobacterial CWS obtained in step (A), but the amount of washing solvent used can be suitably adjusted depending on the wetness of the CWS. Like the process of treatment with the surfactant, the process of washing with acetone or an aqueous alcohol solution may be repeated. In addition, the CWS may be pre-washed with an aqueous alcohol solution before washing with acetone.

In the present invention, after treatment with the surfactants and washing, the mycobacterial cells or CWSs are collected by a conventional centrifugation process. The CWSs prepared according to the present invention may be prepared in solid form as described in Examples, but if the CWSs are dried to form a solid, they will aggregate with each other in an aqueous solution or a hydrophilic solution due to the hydrophobic property of the CWS, and thus there will be significant difficulty in preparing a homogenous suspension from the CWS. To solve this problem, the mycobacterial CWS is not dried after the washing step and is preferably suspended and stored in a C₁-C₄ alcohol (e.g., methanol, ethanol, propanol or butanol) or benzyl alcohol before use. In an experiment conducted by the present inventors, when the CWSs were suspended in one of the above-mentioned alcohols, they were maintained at a stable state without forming an aggregate for at least two years at -20℃, even if the concentration of the CWSs was 10% (dry weight: 100 mg/ml).

When the BCG-CWS prepared in Example of the present invention was used as an adjuvant in immune induction, it showed an antibody titer which was higher than that of alum which is currently used as an adjuvant, suggesting that the BCG-CWS may be an useful adjuvant for increasing immunogenicity of antigens.

As described above, according to the present invention, mycobacterial CWS can be economically mass-produced by a simple process without using enzyme.

Also, the mycobacterial CWS prepared according to the present invention can be stably stored as a homogenized suspension without forming an aggregate, and thus can be more efficiently used in anticancer immunotherapy or adjuvant therapy.

FIG. 1 is a schematic diagram showing the cell-wall structure of mycobacteria.

Hereinafter, the present invention will be described in further detail with reference to examples and the accompanying drawing. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention. Also, various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention.

Example 1: Preparation of BCG-CWS

BCG cells cultured in Sauton medium were killed, and then 2% Triton X-100 and 2% SDS were sequentially added thereto. Then, the cells were delipidated and deproteinated by heating and sonication, thereby obtaining BCG-CWS.

More specifically, Mycobacterium bovis BCG Pasteur-1173P2 was cultured in Sauton medium at 37℃ for 6 weeks, and then centrifuged (at 15,000 xg for 30 min) to collect the BCG cells. The collected cells were autoclaved at 121℃ for 15 min. To about 3,561 g (wet weight) of the autoclaved BCG cells, a 15-fold amount of 2% (w/v) Triton X-100 was added, and the cell suspension was sonicated with a VCX 750 ultrasonic processor (Sonics & Materials, Inc.) at 35 kHz and 60 W for 15 minutes. The sonicated suspension was incubated in a water bath at 100℃ for 9 hours, and then centrifuged (at 15,000 xg for 30 min) to collect 1,769.3 g (wet weight) of delipidated BCG cells. Then, to the collected BCG cells, a 15-fold amount of 2% SDS was added, and the cell suspension was sonicated (35 kHz and 60 W) for 5 minutes at room temperature according to the same process as described above, and then incubated in a water bath at 100℃ for 3 hours, followed by centrifugation, thereby obtaining 881.5 g (wet weight) of BCG-CWS pellets composed of peptidoglycan, arabinogalactan and mycolic acid.

The BCG-CWS pellets were washed sequentially with a 15-fold amount of acetone and a 20-fold amount of 10% 2-propanol. All the washing processes were carried out by adding the washing solvent to the pellets, shaking the suspension, and then centrifuging the suspension at 15,000 xg for 30 min to collect the BCG-CWS pellets. If the absorbance at 280 nm of the final supernatant was higher than 0.05, the pellets were additionally washed with a 10-fold amount of 10% 2-propanol, thus sufficiently removing the remaining SDS.

2-propanol was added to the above-prepared CWS pellets, and the pellet suspension was sufficiently stirred, thus obtaining a homogenized CWS suspension. 1 ml of the CWS suspension was dispensed into a 2-ml microtube, and then microfuged at 14,000 rpm for 10 minutes. The resulting CWS pellets were dried at 50℃ for 18 hours, and the dry weight thereof was measured, and then the total dry weight of the finally purified CWS was calculated. As a result, 107.2 g of BCG-CWS was obtained.

The finally purified BCG-CWS was suspended and stored in 2-propanol at -20℃ at a concentration of 50 mg/ml (dry weight) until use.

In order to compare the purity of the above-prepared BCG-CWS with the purity of CWS prepared according to the prior art and to verify and normalize the purity, the content of proteins in the BCG-CWS was measured with a bicinchoninic acid (BCA) protein assay kit (Pierce). The content of proteins in 1 mg of the BCG CWS was measured for five samples, and the measurements were averaged. As a result, the average content of proteins was 125.4 ㎍ which accounted for 12.5% of the dry weight of the BCG-CWS, which was similar to 12.3% reported by Azuma et al. (J. Nat. Cancer Inst., 52, 95-101 (1974)).

Example 2: Verification of efficacy of BCG-CWS as adjuvant

The adjuvant efficacy of the BCG-CWS prepared in Example 1 was verified.

1) Verification of efficacy as adjuvant against OVA antigen

As shown in Table 1 below, 6-week-old BALB/c mice were divided into 6 groups, each consisting of 5 mice. The mice were immunized against ovalbumin (OVA) antigen (Pierce, Rockford, Ill., USA) subcutaneously three times at 2-week intervals.

After immunization, sera were collected 2 weeks after each immunization, and OVA-specific IgG in the collected sera was measured by ELISA. The measurement results are shown in Table 1. As can be seen in Table 1, after the first immunization, the IgG antibody titers of groups 3 to 5 administered 20㎍ OVA with increasing doses of BCG-CWS from 25 to 100㎍ were similar to or slightly higher than that of group 2 administered 20㎍ OVA with alum as an adjuvant, and greatly increased as the immunization was repeated. After the third immunization, groups 3 to 5 administered OVA with BCG-CWS showed higher antibody titers than did group 2 administered with alum as an adjuvant, suggesting the BCG-CWS prepared according to the present invention greatly increased immunogenicity.

Table 1

2) Verification of efficacy as adjuvant against KLH antigen

As shown in Table 2 below, 6-week-old BALB/c mice were divided into 5 groups, each consisting of 5 mice. The mice were immunized against keyhole limpet hemocyanin (KLH) antigen (Pierce) subcutaneously three times at 2-week intervals.

Sera were collected 2 weeks after each immunization, and KLH-specific IgG in the collected sera was measured by ELISA. The measurement results are shown in Table 2. As can be seen in Table 2, after the first immunization, the IgG antibody titers of groups 3 to 5 administered KLH with increasing doses of BCG-CWS from 25 to 100㎍ were similar to or slightly lower than that of group 2 administered with alum as an adjuvant, but were generally greatly increased as the immunization was repeated, and after the third immunization, groups 3 to 5 all showed higher antibody titers than did group 2 administered with alum as an adjuvant.

Table 2

3) Verification of efficacy as adjuvant against BSA antigen

As shown in Table 3 below, 6-week-old BALB/c mice were divided into 3 groups, each consisting of 5 mice. The mice were immunized against bovine serum albumin (BSA) antigen (Pierce) subcutaneously three times at 2-week intervals.

Sera were collected 2 weeks after each immunization, and BSA-specific IgG in the collected serum was measured by ELISA. The measurement results are shown in Table 3. As can be seen in Table 3, the antibody titer of group 3 administered 100㎍ BCG-CWS together with 20㎍ BSA was higher than that of group 2 administered with alum as an adjuvant, suggesting that the BCG-CWS prepared according to the present invention greatly increased immunogenicity.

Table 3

The method for preparing mycobacterial CWS of the present invention can be used for the mass-production of mycobacterial CWS, which is useful for anticancer immunothrepy and adjuvant therapy.

Claims

A method for preparing mycobacterial CWS, the method comprising the steps of:

(A) adding a surfactant to killed mycobacterial cells and heat-treating the cells in the presence of the surfactant, thereby delipidating and deproteinating the cells; and

(B) washing the delipidized and deproteinized mycobacterial cells.
The method of Claim 1, wherein the surfactant is a nonionic surfactant and/or an anionic surfactant.
The method of Claim 1, wherein in the heat treatment in step (A), a nonionic surfactant and an anionic surfactant are sequentially used irrespective of a treatment order.
The method of any one of Claims 1 to 3, wherein a sonication process is additionally carried out before or after the heat treatment in step (A).
The method of any one of Claims 1 to 3, wherein the nonionic surfactant is either one of octyl phenol ethoxylate derivatives or nonyl phenoxylpolyethoxylethanol (NP-40).
The method of any one of Claims 1 to 3, wherein the anionic surfactant is either sodium dodecyl sulfate (SDS) or sodium lauryl ether sulfate (SLES).
The method of any one of Claims 1 to 3, wherein the washing step is performed by (1) washing the CWS with acetone; and then (2) re-washing the CWS with a 10-20% (v/v) aqueous solution of at least one alcohol selected from the group consisting of methanol, ethanol, propanol, butanol and benzyl alcohol.
The method of any one of Claims 1 to 3, wherein the CWS is prepared in the form of a suspension which is suspended in methanol, ethanol, propanol or butanol or benzyl alcohol.