KR20110101547A - Preparation method for cell wall skeleton of mycobacterium - Google Patents

Abstract

The present invention relates to a method for producing antibacterial CWS through deproteinization, degreasing process from an antibacterial cell using a surfactant, and more specifically, (A) adding a surfactant to a sterilized antibacterial cell and heat treatment. Degreasing and deproteinization by the; And (B) the step of washing the degreasing and deproteinized antibacterial bacteria cells; relates to a method for producing an antibacterial CWS comprising a.
According to the present invention, the CWS of the antibacterial bacterium can be produced economically in large quantities by a simple process without using an enzyme, and the CWS of the antibacterial bacteria prepared by the present invention can be stably formed as a homogenized suspension without forming an aggregate. Because it can be stored, it can be used more effectively when used as an anticancer immunotherapy or adjuvant.

Description

Preparation method of antibacterial cell wall skeleton {Preparation method for cell wall skeleton of Mycobacterium}

The present invention relates to a method for producing an antimicrobial cell wall skeleton that can economically mass produce an antibacterial cell wall skeleton.

In general, antimicrobial cell wall components strongly stimulate local inflammatory reactions to induce granuloma formation and increase immune responses by increasing effective antigen presentation and inflammatory cytokine production. BCG, a representative antibacterial bacterium, is an attenuated live vaccine strain that has been proven to be safe since it has been inoculated to over 3 billion people worldwide since 1921. Currently, BCG has been studied not only as a tuberculosis vaccine but also as an anticancer immunotherapy for various malignancies including bladder cancer.

About 60% of the dry weight of the antibacterial cell wall is composed of complex lipid components, peptidoglycan, arabinogalactan, mycolic acid, lipoarabino-nannan, LAM), surface lipids and mycosides consisted of stacked structures with a thickness of about 20 nm (FIG. 1). The cell wall skeleton (CWS) among the antimicrobial cell wall components is an insoluble particle excluding free soluble components such as free proteins, nucleic acids and lipids in cells (J. Nat. Cancer Inst., 52, 95-101 (1974) ), Peptidoglycan, arabinogalactan and mycolic acid complexes.

Among the antibacterial bacteria, BCG's CWS is known as a potent adjuvant that stimulates innate immunity as immunoactive microparticles. In addition, by inducing CTL proliferation and NK cell activation, it also exerts anti-cancer immunity effect, and since 1970s, Yamamura et al. (Ann NY Acad Sci 277: 209-227) have studied the clinical usefulness of anticancer immunity drugs in lung cancer, Stomach cancer, intestinal cancer, breast cancer, tongue cancer, laryngeal cancer, acute myeloid leukemia, pancreatic cancer, ovarian cancer, etc. are being introduced as new immunotherapy in almost all cancer treatments.

Until now, BCG CWS anticancer immunotherapy has been used only as an adjuvant therapy in the immunocompromised state after surgery, chemotherapy or radiation therapy, but recently, the opinion that BCG CWS anticancer immunotherapy should be started from the early stage of cancer diagnosis. . This is because the extensive immune system damage caused by chemotherapy or radiation therapy results in severe immunosuppression, and the immune-boosting effects of BCG CWS cannot be expected. In fact, it is reported that BCG CWS monotherapy showed a much better anticancer immunotherapy than the combination of anticancer drugs and BCG CWS immunotherapy.

For immunotherapy with BCG CWS, dozens or more intravenous injections of 10 to 200 μg dry dry BCG CWS should be administered every week or month. Such injection therapy has the advantage of being able to inoculate a constant dose due to the high absorption rate of CWS, but it is very cumbersome and inconvenient and painful for patients who require repeated inoculations. In addition, due to the intensive concentration of immune cells on the inoculated skin, local skin reactions such as skin redness, sclerosis (stiffening of the skin), blisters and ulcers may occur, and in severe cases, local lymph nodes may also swell. In particular, the scar is left at the inoculation site and repeated injections are a great burden for patients.

Oral administration of BCG CWS can solve the above-mentioned intradermal injection problem and can be used more conveniently and widely in anticancer immunotherapy. However, in the oral administration method, the absorption rate through the intestinal membrane of CWS is very low compared to injection therapy, so the dry wt of CWS administered at once is up to 5 ~ 10mg unit (usually 0.05mg for intradermal inoculation). At least several hundred times more CWS than injectable therapy is needed because of the increase and the need for oral administration for 6 months to 1 year or more daily. However, practical application is difficult because there is no method for mass production of CWS yet.

Akira et al. Prepared BCG CWS through complex delipidation process using various organic solvents after deproteinase reaction by sequentially treating BCG cells with trypsin-chymotrypsin and pronase. A method was reported (US 6,593,096).

Wataru et al. Improved the defatting process in the above, (A) crushed BCG microorganisms to collect cell walls, and then (B) sequentially treated with benzonase and pronase to deproteinize, and (C) surfactants under warming. 1D Triton X-100), (D) reported the preparation of BCG CWS by washing with organic solvent and drying (JP 2008214266 A). Wataru et al. Produced CWS up to 10 g units from 190 g of BCG cell wall pellets obtained by crushing 600 g of BCG bacillus by this method, but the production process is prolonged by the enzyme treatment time and proceeds through several steps. This is a low problem. In addition, in order to be used for adjuvant or chemoimmune therapy, effective dispersion of CWS is required.However, according to the above method, the final CWS is a very hard agglomerated mass. There is a problem that needs to be solved.

As such, in order to use CWS as a pharmaceutical raw material, a method capable of mass-producing high purity CWS is required, but it is difficult to achieve such a goal by the currently known technology. Therefore, the development of a manufacturing method that can produce a large amount of antibacterial CWS in a simpler process and lower cost is required.

US, 6,593,096 JP, 2008 214266, A

 J. Nat. Cancer Inst., 52, 95-101 (1974)

An object of the present invention is to provide a process that can economically mass-produce CWS of antibacterial bacteria in order to solve the problems of the prior art.

Still another object of the present invention is to provide a method for preparing CWS of acid bacteria, which can easily prepare a homogenized suspension from the prepared CWS of acid bacteria.

The present invention for achieving the above object relates to a method for producing the antibacterial bacteria CWS through deproteinization, degreasing process from the antibacterial cells using a surfactant, and more specifically, (A) sterile acid bacteria Adding a surfactant to the cells to heat treatment to degreasing and deproteinizing; And (B) the step of washing the degreasing and deproteinized antibacterial bacteria cells; relates to a method for producing an antibacterial CWS comprising a.

In the present invention, the antibacterial bacterium refers to bacteria belonging to the genus Mycobacterium , including Mycobacterium tuberculosis. In the embodiment of the present invention, only the preparation of CWS of Mycobacterium bovis BCG is an example of the antibacterial bacterium, but the acidophilic bacteria have the same CWS structure. Naturally, it can be applied to all of them.

Sterilized antibacterial cells used as starting materials in the present invention are known and can be easily prepared by those skilled in the art by known methods. For example, the antibacterial bacteria can be obtained by incubating in Sauton medium and centrifuging, and by sterilizing by heating (J. Nat. Cancer Inst., 52, 95-101 (1974)). ).

In the present invention, the step (A) is preferably used as a surfactant nonionic surfactant and / or anionic surfactant. Generally, nonionic surfactants are known to be more effective for degreasing and anionic surfactants for deproteinization. In the step (A), it is preferable to heat-treat using a nonionic surfactant and an anionic surfactant together, and more preferably, heat treatment using sequentially. At this time, it is more efficient to treat the anionic surfactant first and then treat the anionic surfactant, but the anionic surfactant may be treated first and then the nonionic surfactant may be treated. That is, the order of treatment of the surfactant is irrelevant.

In addition, in order to more efficiently remove lipids and proteins by the surfactant, it may be performed by adding an ultrasonic treatment process during the heat treatment of step (A). The ultrasonic treatment may be added at any stage before or after the heat treatment.

The nonionic surfactant is one of the Triton X-based octyl phenol ethoxylate compounds, including Triton X-100, or NP-40 (nonyl phenoxylpolyethoxylethanol), and the nonionic surfactant SDS (sodium dodecyl). sulfate) or sodium lauryl ether sulfate (SLES), but is not limited thereto. Naturally, the concentration of the nonionic and / or anionic surfactant should be appropriately selected depending on the type of the surfactant used, the treatment temperature, the amount of use, the pH, the ionic strength, and the type of the additive, but 0.5 to 5% ( Preference is given to using w / v) solutions. If the concentration of the surfactant is too low, it is natural that the degreasing and deproteinization effects are insufficient. Even if the concentration of the final surfactant is higher than the critical micelle concentration (CMC), the degreasing and deproteinization effects are not improved. On the contrary, the concentration of the residual surfactant may be increased. The surfactant solution is preferably used in an amount of 4 to 30 times (w / w) with respect to the acidophilic cells in the wet state. If the amount of the solution is too small, degreasing and deproteinization are not sufficiently made, and if the amount of the solution is too large, the production efficiency is lowered, so it is preferable to properly adjust in the above range. Degreasing and deproteinizing more completely during the treatment of the surfactant, especially when manufacturing high-purity CWS, or when the amount of the surfactant solution used is low due to problems in the equipment used. The number of treatments may be repeated.

The heat treatment of the surfactant is carried out by heating to a temperature of 70 ℃ or more to the boiling point. If the heat treatment temperature is too low, the degreasing and deproteinization reactions are not efficient, so the degreasing and deproteinization may be incomplete or the warming time may be long. Usually, when processing surfactant at the temperature of 70 degreeC or more-a boiling point, it is preferable to heat-process for 2 to 5 hours.

Since the surfactant remains in the CWS of the antibacterial bacteria prepared by degreasing and deproteinization by heat-treating the antibacterial bacteria under surfactant, step (B) is performed by the antibacterial bacteria obtained by degreasing and deproteinization. It is a step of washing the surfactant remaining in the CWS. In the washing step (1) after washing with acetone, (2) rewashing with an aqueous solution of 10-20% (v / v) of one or more alcohols selected from the group consisting of methanol, ethanol, propanol, butanol and benzyl alcohol. desirable. The washing solvent is preferably used 5-30 times (v / w) with respect to the wet weight of the CWS of the antibacterial acid obtained in step (A), it can be properly adjusted according to the wet degree of the CWS. The washing process may be repeated with the acetone or alcohol aqueous solution washing process, similar to the surfactant treatment process. It may also be subjected to a pre-washing process using the aqueous alcohol solution before the acetone wash.

In the present invention, after the surfactant treatment and washing, always bacteria or CWS are obtained by a conventional centrifugation process.

The antimicrobial CWS prepared by the present invention may be prepared as a solid after drying as in the embodiment, but once dried to form a solid, the hydrophobicity of the CWS may be entangled with each other in an aqueous solution or a hydrophilic solution to form agglomerates. As a result, there is considerable difficulty in preparing a homogeneous suspension. In order to solve this problem, the CWS of the antibacterial bacteria is preferably stored and used in a floating state in the alcohol or benzyl alcohol of C1 to C4 of methanol, ethanol, propanol and butanol without drying after the washing step. As a result of the preliminary experiments, the cell wall skeleton was suspended in one of the alcohols, and even when the concentration of CWS was 10% (dry weight of 100 mg / ml), it remained stable without forming aggregates at -20 ° C for at least 2 years.

When combined with adjuvant at the time of immunization using BCG CWS prepared according to an embodiment of the present invention, as compared with the administration of the antigen alone, as well as showing a similar or higher antibody value of alum currently used as an adjuvant It was confirmed that it can be usefully used for immunotherapy.

As described above, according to the present invention, the CWS of the antibacterial bacterium can be economically produced in large quantities by a simple process without using an enzyme.

In addition, the CWS of the antibacterial bacteria prepared according to the present invention can be stably stored as a homogenized suspension without forming aggregates, and thus can be used more efficiently when used as anticancer immunotherapy or adjuvant.

1 is a schematic diagram showing the cell wall structure of the antibacterial bacteria.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, these drawings and embodiments are only examples for easily explaining the contents and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.

< Example  1>

Manufacturing of BCG CWS

After sterilization of BCG cells cultured in Sauton synthetic medium, 2% Triton X-100 and 2% SDS were added sequentially, followed by delipidation and deproteination by warming and sonication. BCG CWS Was purified.

More specifically, Mycobacterium bovis BCG Pasteur-1173P2 was incubated in Sauton medium for 37 weeks, 6 weeks, then centrifuged (15,000 xg, 30 minutes) to collect the BCG cells and autoclaved at 121 ℃ for 15 minutes. Approximately 3,561 g (wet weight) of sterile BCG cells were added 15% of 2% (w / v) Triton X-100 and sonicated for 5 minutes using a VCX 750 ultrasonic processor (Sonics & Materials, Inc.). 35 kHz, 60 W). The suspension was centrifuged (15,000 × g, 30 minutes) in a 100 ° C. water bath and then centrifuged (15,000 × g, 30 minutes) to collect 1,769.3 g (wet weight) of degreasing BCG cells. Thereafter, 2% SDS corresponding to 15 times of the collected BCG wet weight was added, followed by ultrasonic crushing (35 kHz, 60W) at room temperature for 5 minutes in the same process as before, followed by centrifugation at 100 ° C for 3 hours. 881.5 g (wet weight) of BCG CWS pellets consisting of peptidoglycan, arabinogalactan and mycolic acid were obtained.

The BCG CWS pellet was washed sequentially with 15 times acetone and 20 times 10% 2-propanol. All the washing procedures were washed by adding washing solution and centrifugation (15,000 xg, 30 minutes) after shaking mixing to take BCG CWS pellets. If the absorbance of the final supernatant at 280 nm was greater than 0.05, it was further washed with 10 times 10% 2-propanol to sufficiently remove residual SDS.

2-propanol was added to the CWS pellet obtained by the above method, and stirred sufficiently to prepare a homogeneous CWS suspension. 1 ml of the CWS suspension was dispensed into 2 ml microtubes, and the CWS pellet obtained by microfuge for 10 minutes at 14,000 rpm was dried at 50 ° C. for 18 hours to measure dry weight, and then the total dry weight of the final purified CWS was calculated. As a result, 107.2 g of BCG CWS was obtained.

Final purified BCG CWS was stored suspended at -20 ℃ in 2-propanol at a dry weight of 50mg / ml concentration.

The protein content of BCG-CWS was measured by bicinchoninic acid (BCA) protein assay kit (Pierce) to compare, verify, and standardize the purity of BCG CWS prepared above with CWS prepared by the prior art. Protein content in 1 mg BCG CWS was analyzed for 5 preparation samples and the average content was 125.4 μg, accounting for 12.5% of dry BCG CWS, which was determined by Azuma et al. (J. Nat. Cancer Inst., 52, 95-101 (1974) Similar to 12.3%).

< Example  2>

Efficacy Assay of BCG CWS as Adjuvant

The efficacy of BCG-CWS prepared using the BCG CWS prepared in Example 1 as an adjuvant was assayed.

1) Efficacy assay as adjuvant for OVA antigen

Five 6-week-old BALB / c mice were divided into six groups, as shown in Table 2 below, to give an immune response to OVA (ovalbumin, Pierce, Rockford, Ill., USA) antigen through subcutaneous injection three times at two-week intervals.

Serum was taken at 2 week intervals after immunization and OVA-specific IgG was quantified by ELISA method and shown in Table 1. As can be seen in Table 1, the second to sixth groups administered Alum or BCG CWS with OVA showed higher IgG antibody titer compared to the OVA-only group, so that BCG CWS prepared by the present invention greatly increased immunogenicity. I could confirm that.

2) Efficacy assay as adjuvant against KLH antigen

Five 6-week-old BALB / c mice were divided into five groups, as shown in Table 2 below, to give an immune response to KLH (keyhole limpet hemocyanin, Pierce) antigen through subcutaneous injection three times at two-week intervals.

Serum was taken at two week intervals after immunization and KLH-specific IgG was quantified by ELISA method and shown in Table 2. As can be seen in Table 2, the IgG antibody titer at the first inoculation of the third to fifth groups in which BCG CWS was administered with KLH was similar to or slightly lower than the second group using alum as an adjuvant, but the inoculation was repeated. After the second immunization, all groups showed higher IgG antibody titers than the second group using alum as an adjuvant.

3) Efficacy assay as adjuvant against BSA antigen

Five 6-week-old BALB / c mice were divided into three groups, as shown in Table 3 below, to give an immune response to BSA (bovine serum albumin, Pierce) antigen through subcutaneous injection three times at two-week intervals.

Serum was taken at 2 week intervals after immunization to quantify BSA-specific IgG by ELISA method and are shown in Table 3. As can be seen from Table 3, the IgG antibody value of the third group administered BCG CWS with BSA is higher than the second group using alum as an adjuvant, confirming that BCG CWS prepared by the present invention greatly increases immunogenicity. Could.

Claims (8)

In the production method of the antibacterial cell wall skeleton,
(A) adding a surfactant to the sterilized acid bacteria bacteria and heat treatment to degreasing and deproteinization; And
(B) washing the degreasing and deproteinized antibacterial cells;
Method for producing an antimicrobial cell wall skeleton comprising a.
The method of claim 1,
The surfactant is a method of producing an antibacterial cell wall skeleton, characterized in that the nonionic surfactant and / or anionic surfactant.
The method of claim 1,
Step (A) is a method for producing an anti-bacterial cell wall skeleton, characterized in that the heat treatment using a nonionic surfactant and anionic surfactant sequentially irrespective of the order of treatment.
The method according to any one of claims 1 to 3,
Method of producing an antibacterial cell wall skeleton, characterized in that the addition of an ultrasonic treatment step before or after the heat treatment in step (A).
The method according to any one of claims 1 to 3,
The nonionic surfactant is A method for producing an antibacterial cell wall skeleton, characterized in that it is selected from one of octyl phenol ethoxylate compounds or NP-40 (nonyl phenoxylpolyethoxylethanol).
The method according to any one of claims 1 to 3,
The anionic surfactant is sodium dodecyl sulfate (SDS) or sodium lauryl ether sulfate (SLES) Method for producing an antibacterial cell wall skeleton, characterized in that.
The method according to any one of claims 1 to 3,
The washing comprises (1) acetone washing step; And (2) washing 10-20% (v / v) aqueous solution of one or more alcohols selected from the group consisting of methanol, ethanol, propanol, butanol and benzyl alcohol. Way.
The method according to any one of claims 1 to 3,
The cell wall skeleton is a method of producing a cell wall skeleton, characterized in that in the form of a suspended solid suspended in methanol, ethanol, propanol, butanol or benzyl alcohol.

Images