KR100250714B1 - Therapeutic agent for treating infections of pseudomonas aeruginosa - Google Patents

Abstract

PURPOSE: An agent for treating Pseudomonas aeruginosa infectious disease containing human immunoglobulin specifically binding to cell envelop protein separated and purified from attenuated Pseudomonas aeruginosa strains as an active component is provided which has excellent treating effect on Pseudomonas aeruginosa infectious disease. CONSTITUTION: This agent for treating Pseudomonas aeruginosa infectious disease contains immunoglobulin separated and purified from blood plasma of a man in a normal state by CNBr-activated sepharose 4B resin column using cell envelop protein purely separated from attenuated new Pseudomonas aeruginosa strains as ligands. The attenuated Pseudomonas aeruginosa strains are CFCPA 10142, CFCPA 20215, CFCPA 30720 and CFCPA 60534 as strains of 1, 2, 3 and 6 type owing to Fisher Immunotype.

Description

Pseudomonas aeruginosa infection treatment

The present invention relates to a therapeutic agent for Pseudomonas aeruginosa infection. In particular, the present invention is directed to immunization isolated and purified from plasma of normal humans by CNBr-activated Sepharose 4B resin column using extracellular membrane proteins isolated from novel attenuated Pseudomonas aeruginosa strains as ligands. A therapeutic agent for Pseudomonas aeruginosa infections containing globulin.

Pseudomonas aeruginosa is 1.5 to 3.0 μm in length, 0.5 μm in width and has a single flagellar, motile, aerobic, aerobic Gram-negative bacterium, and is widely distributed in soil, water, sewage, and human intestines [Mol. Microbiol. 4, 1069-1075, 1990. Pseudomonas aeruginosa is a pathogenic bacterium that causes refractory infection in patients with systemic infection, chronic airway infection, and pancreatic cystic fibrosis. In particular, sepsis caused by Pseudomonas aeruginosa is a disease caused by the invasion of microorganisms or secretion of toxic constituents in the blood of patients whose resistance is reduced by surgery, burns and trauma. It is known to lead to death. In addition, Pseudomonas aeruginosa has recently been detected in the eye and urinary tract of contact lens users to attract more attention. Therefore, there is an urgent need for the development of a medicament capable of effectively preventing or treating a fatal damage to various organs due to toxicity caused by bacteria flowing into the blood through wound infection, pneumonia, urinary tract infection, and the like. However, Pseudomonas aeruginosa is not only resistant to most commercial antibiotics except for some antibiotics, but preventive vaccines and therapeutics have not been developed so far to provide a safe and good protective effect. There is a seriousness of the problem.

Representative methods for the generalization of Pseudomonas aeruginosa include seven classifications according to the Fisher Immunotype, O-antigen group classification method proposed by Terada, or International Antigenic Typing Scheme (IATS) classification. Method and the like. The most frequently detected Pseudomonas aeruginosas from patients with Pseudomonas aeruginosa infections at home and abroad are Fisher's immunotype / IATS serotype / O-group (Terada), which are 6/8 / C, 3/2 / B, 2/11 / E and 1/6. / G is the dominant type.

As a treatment for Pseudomonas aeruginosa infection developed to date, there is a method of neutralizing toxins produced by Pseudomonas aeruginosa by making antitoxins against P. aeruginosa. Antitoxin is a treatment used for patients with advanced sepsis, and has a disadvantage in that it cannot be used universally for general patients because of its high price. In addition, the biggest drawback is that the rate of cure that can be treated through the administration of this antitoxin is relatively low and the side effects are so severe that it is not suitable for use as a therapeutic agent.

In addition, as a treatment method for other Pseudomonas aeruginosa infections, there is a method of selecting and treating a wide range of antibiotics or highly selective chemotherapeutic agents for Pseudomonas aeruginosa, but P. aeruginosa is generally highly resistant and various types of treatment. Many victims of Pseudomonas aeruginosa were impossible.

Another method is a mouse monoclonal antibody against Pseudomonas aeruginosa using cell fusion technology [J. Inf. Dis. 152, 1290-1299, 1985] or human monoclonal antibody [FEMS Microbiol. Immunol. 64, 263-268, 1990], an antibody-producing cell line capable of producing and neutralizing Pseudomonas aeruginosa best among each antibody, and using this as a production cell line, attempts to develop low-cost therapeutics through mass production. However, this method is not able to treat all of the infections caused by dozens of Pseudomonas aeruginosas, which are known to be serologically and immunologically, by one type of monoclonal antibody, as well as the ability to neutralize them compared to polyclonal antibodies. It is very weak and can not be effectively cured. In order to overcome this drawback, cross-reactivity of each other is examined to administer several antibodies at the same time, blood of patients with Pseudomonas aeruginosa infection and the serological or immunological forms of Pseudomonas aeruginosa are identified here. Although a method of administering a suitable monoclonal antibody has been suggested, it is also time consuming, and thus it is not suitable for the treatment of struggling patients, and as described above, the neutralizing ability of one type of monoclonal antibody is very high. Low, the therapeutic effect is also unknown. Therefore, in recent years, a method of producing a therapeutic agent using a common antigen has been actively studied as one method of eliminating the disadvantages of the aforementioned Pseudomonas aeruginosa infection treatment.

It is an object of the present invention to improve the disadvantages of conventional Pseudomonas aeruginosa infections, P. aeruginosa infections having excellent therapeutic effect containing human immunoglobulin as an active ingredient that specifically binds to extracellular membrane proteins isolated and purified from safe, attenuated P. aeruginosa strains. To provide a cure.

1 is a diagram showing the SDS-PAGE pattern of the extracellular membrane protein isolated and purified for use as a ligand from each attenuated Pseudomonas aeruginosa strain.

Figure 2 shows the SDS-PAGE pattern of human antibodies for treatment of Pseudomonas aeruginosa infection for each purification step.

The present inventors conducted extensive and intensive studies to develop a therapeutic agent that can be highly effective against various forms of Pseudomonas aeruginosa, and as a result, CNBr is used as a ligand for the extracellular membrane protein obtained from attenuated Pseudomonas aeruginosa strains. Coupling to activated Sepharose 4B resin and using it to isolate and purify immunoglobulin against small amounts of Pseudomonas aeruginosa present in the plasma of normal humans, and the immunoglobulin is not only severe Pseudomonas aeruginosa infection but also severe Pseudomonas aeruginosa. It was confirmed that the excellent therapeutic effect also for the infectious disease and completed the present invention.

The present invention relates to a therapeutic agent for Pseudomonas aeruginosa infection. In particular, the present invention relates to a therapeutic agent for Pseudomonas aeruginosa infections containing immunoglobulins that specifically bind to Pseudomonas aeruginosa outer membrane proteins. The immunoglobulin can be obtained by purely separating the extracellular membrane protein from the attenuated safe Pseudomonas aeruginosa strain, coupling it to a column with a ligand, and then separating and purifying it from the plasma of a normal person using this column.

The attenuated Pseudomonas aeruginosa strain used as a source of the extracellular membrane protein as a ligand for antibody binding in the present invention is described in patent application 93-10273 filed June 7, 1993 by the applicant as a novel strain. The attenuated Pseudomonas aeruginosa isolates were isolated from Pseudomonas aeruginosa infected patients, and these isolated microorganisms were classified into seven species according to Fisher's immunoforms. It is a safe strain obtained by purifying through the separation process and then selecting the most attenuated strain for each strain. The safe, attenuated Pseudomonas aeruginosa obtained in this way was CFCPA 10142 (Fischer Type 1), CFCPA 20215 (Fischer Type 2), CFCPA 30720 (Fischer Type 3), CFCPA 40057 (Fischer Type 4), CFCPA 50243 (Fischer Type 5), CFCPA Seven species of 60534 (Fischer 6) and CFCPA 70018 (Fischer 7). Each of these strains was deposited on May 12, 1993, at the Korea Microbiological Conservation Center, an international depository institution affiliated with the Korean spawn association. The accession numbers are as follows.

Strain name Accession number CFCPA 10142 KCCM 10029 CFCPA 20215 KCCM 10030 CFCPA 30720 KCCM 10031 CFCPA 40057 KCCM 10032 CFCPA 50243 KCCM 10033 CFCPA 60534 KCCM 10034 CFCPA 70018 KCCM 10035

Patent Application No. 93-10273, which describes these strains, is published as Publication No. 95-865 dated January 3, 1995, and is currently available for sale from the Korean spawn association.

The attenuated Pseudomonas aeruginosa was cultured in a fermenter maintained under optimal growth conditions to obtain a large number of cells, and the obtained cells were separated and treated according to the molecular weight size by ultrafiltration, ultracentrifuge, respectively. Extracellular membrane proteins are obtained by treatment in accordance with a series of procedures. In this process, tryptic soy broth is used as a medium for culturing attenuated Pseudomonas aeruginosa, or a special medium configured for cultivation of Pseudomonas aeruginosa is used. This special medium contains glucose (30 g / l), peptone (15 g / l), MgSO ₄ (0.5 g / l), CaCO ₃ (5 g / l), KH ₂ PO ₄ (1 g / l), FeSO ₄ (5 mg / L), CuSO ₄ (5 mg / L) and ZnSO ₄ (5 mg / L). This special medium is novel and specially designed by the present inventors to be suitable for the culture of attenuated Pseudomonas aeruginosa strains. When these attenuated Pseudomonas aeruginosa strains are cultured in a special medium, it is more preferable to use a special medium because more than 30% more dry cells can be obtained based on the weight of the dry cells than when cultured in tryptic soy broth medium.

Optimal culture conditions in this cell culture step is a temperature of 37 ℃, aeration amount 1 v / v / m (volume / dose / min) 200 rpm by inoculating the strain in the amount of 5% (v / v) of the culture medium solution Incubate for 10 to 18 hours, preferably 12 to 14 hours at a stirring rate of. When the incubation is completed, cells are separated from the culture medium in a second step by a method such as centrifugation. In the third step, the isolated cells are treated with an organic solvent to inactivate microorganisms and remove lipid components in the extracellular membrane. The organic solvent is preferably acetone, ethanol, butanol and the like, most preferably acetone. Subsequently, in the fourth step, the extracellular membrane protein is extracted three to four times while preventing the cells from being destroyed. To do this, the microbial cells are mixed with a mixer or homogenizer in a solvent such as 10 mM phosphate buffer (10 mM PBS, pH 7.2), tris salt buffer (10 mM TrisCl, pH 7.5), preferably phosphate buffer. ), And the extraction process is preferably performed by stirring at 4 to 50 to 200 rpm. In addition, care must be taken not to destroy the cells in the fourth step, in order to prevent the incorporation of toxic proteins and nucleic acids in the cytoplasm into the desired extracellular membrane protein. Therefore, it is necessary to continuously check whether cells are broken by measuring lactate dehydrogenase, a cytoplasmic labeling enzyme, during the extraction process.

The extracellular membrane proteins of the attenuated Pseudomonas aeruginosa strains thus obtained as supernatants are subjected to primary and secondary ultrafiltration and fractionated so that only proteins having a molecular weight of 10 kDa to 100 kDa are obtained. At this time, it is very important that the molecular weight range of the extracellular membrane protein is 10 kDa to 100 kDa. Fractions with a molecular weight of less than 10 kDa are composed of pigments and low molecular weight proteins, and thus have no effect as antibody-inducing substances. Therefore, fractions having a molecular weight greater than 100 kDa have high molecular weight lipopolysaccharides. (LPS) is present and the antibody itself isolated and purified by the coupled lipopolysaccharide may be a secondary therapeutic agent for Pseudomonas aeruginosa infection, but the lipopolysaccharide coupled to the column will be freed. This can lead to toxicity.

The extracellular membrane protein fractions in the isolated molecular weight range 10 kDa to 100 kDa are again ultracentrifuged to remove trace lipopolysaccharides that may remain to obtain pure extracellular membrane proteins. In this way, extracellular membrane proteins purely isolated from each attenuated Pseudomonas aeruginosa were mixed at a ratio, followed by ultrafiltration using a molecular weight 10 kDa blocking membrane filter in Pellicon Cassette System (Millipore). By filtration, the process of concentration and buffer exchange is carried out simultaneously to dissolve in an appropriate volume of coupling buffer (Coupling buffer; 0.1 M NaHCO ₃ , 0.5 M NaCl, pH 8.3).

This protein was coupled as a ligand to a CNBr-activated Sepharose 4B resin from Pharmacia Biotech to prepare an affinity column, which was used to generate immunoglobulins from normal human plasma. Isolate and purify. The method for isolating and purifying immunoglobulins in detail is as follows.

The pre-purified plasma stock loaded on the column was treated with 2.77% (w / v) CaCl ₂ solution in normal FP plasma to 10% (v / v) of the final volume and left in a 37 ° C. constant temperature water bath for 2 hours. Was observed to remove the fibrinogen in the anticoagulant-treated FP plasma by centrifugation to collect only the supernatant, which was then filtered using sterile gauze and mixed with the same volume of phosphate buffer to prepare a final 0.02% (w chimerosal is added to prevent the growth of microorganisms.

When loading the plasma stock solution before purification, the flow rate is 400 ml per hour and the column is washed with 5 times, preferably 10 times, gel volume of phosphate buffer after loading. The effective antibody (immunoglobulin) bound to the affinity column is eluted using 0.2 M acetate buffer (pH 3.2), the elution of the effective antibody begins around pH 5.5, and the resulting eluate is titrated with 1 M carbonate buffer (pH 9.0). Neutralize to pH 7.0. The elution column of the eluted antibody was washed with 4 times the volume of the elution buffer and then again with 10 times the volume of phosphate buffer and finally washed with 5 times the volume of phosphate buffer containing chimerosal to 0.02%. Then stored at 4 ℃.

Protein A column chromatography is used to purify purified effective antibodies with high purity. Take 50 ml of Pharmacia Biotech's Protein A Sepharose CL-4B resin, add 10 times the volume of phosphate buffer, stir well, remove the fine resin particles suspended in the supernatant, and fill in an appropriate column. After washing the column packed at a flow rate of 150 ml per hour using a 10-fold volume of phosphate buffer, the purified effective antibody solution is loaded at a flow rate of 150 ml, preferably 80 ml per hour. The loaded column is washed well with 10-fold volume of phosphate buffer to remove non-specifically attached protein, and then eluted with 0.2 M acetate buffer (pH 3.2). Elution of the effective antibody begins around pH 5.5, and the eluate obtained is titrated with 1 M carbonate buffer (pH 9.0) to neutralize to pH 7.0, in which case the flow rate is maintained at 150 ml per hour. After eluting the effective antibody, the column was washed with 4 times gel volume of the above elution buffer, and then again with 10 times volume of phosphate buffer, and finally washed with 5 times gel volume of phosphate buffer containing 0.02% of chimerosal. Store at 4 ° C.

Ultrafiltration using a Millipore Prep / Scale TFF membrane (blocking molecular weight: 30 kDa, regenerated cellulose) was carried out to concentrate the effective antibody purified in high purity and to adjust the concentration and composition suitable for formulation. Conduct. The solution containing the high-purity effective antibody was ultra-filtered with a 10-fold volume of physiological saline solution while stirring slowly at room temperature using a magnetic stirrer, and the concentration of the antibody solution was concentrated to 1 mg / ml.

The extracellular membrane proteins of the seven attenuated Pseudomonas aeruginosa strains may be used in combination at a constant ratio, and generally at about the same ratio, using immunoglobulins obtained from normal plasma using an affinity column used as a ligand. However, considering the cross-reactivity of each other, preferably, the extracellular membrane proteins obtained from four strains of attenuated Pseudomonas aeruginosa CFCPA 10142, CFCPA 20215, CFCPA 30720 and CFCPA 60534 in a ratio of 1: 1: 1: 1 Immunoglobulins obtained from normal plasma using the affinity column used as a ligand can be used as an effective antibody for treating Pseudomonas aeruginosa infections, purified by high purity through protein A column chromatography and a tangential flow filtration system. Fischer serotypes 1, 2, 3, 4, 5, 6 and 7 can provide excellent therapeutic effects against infection by Pseudomonas aeruginosa.

An effective antibody for treating Pseudomonas aeruginosa infection according to the present invention is a lyophilized preparation or a liquid composition, preferably injectable, which further contains a pharmaceutically acceptable carrier, for example, a stabilizer, a preservative, an isotonic agent, etc., as necessary. It may be formulated in a form. Pharmaceutically acceptable carriers that can be used are preferably, for example, mannitol, lactose, saccharose and the like for lyophilized preparations, physiological saline, distilled water for injection, phosphate buffers and the like for liquid preparations.

Dosages vary widely depending on the age, weight, sex, health condition and depth of Pseudomonas aeruginosa infection, and the type of immunoglobulin formulated, but generally between 0.1 mg and 1,000 mg / kg of adult body weight per day, Preferably from 1 mg to 100 mg is administered intravenously.

The invention is further illustrated by the following examples, although the invention is not limited in any way by these examples.

Example 1 Isolation and Purification of Extracellular Membrane Proteins of Pseudomonas aeruginosa

(1) Cultivation of attenuated Pseudomonas aeruginosa

Four Pseudomonas aeruginosa strains isolated purely from clinical strains and attenuated [P. 150 liter incubator was used to obtain a large number of cells of aeruginosa CFCPA10142 (KCCM 10029), CFCPA20215 (KCCM 10030), CFCPA30720 (KCCM 10031) and CFCPA60534 (KCCM 10034). 100 L of the medium solution obtained by dissolving 30 g of Tryptic Soy Broth per 1 L of distilled water was adjusted to pH 7.2, and then sterilized. The culture temperature was 37 ℃, the aeration amount is 1v / v / m, the inoculum was used for 5% (v / v) conditions for the culture solution and after incubating for 12 to 14 hours after fixing the stirring speed at 200 rpm. The amount of cells obtained was slightly different depending on the type of Pseudomonas aeruginosa strains, but on average, at least 25 g of cells were obtained at approximately wet weight per liter of culture medium.

(2) Partial Purification of Extracellular Membrane Proteins from Attenuated Pseudomonas Aeruginosa

In order to partially purify the extracellular membrane protein from the four obtained Pseudomonas aeruginosa strains, the above-described method was used. As a representative example, the fischer type 3 strain CFCPA 30720 (KCCM 10031) was used. Purification was carried out using the same method as the purification method mentioned above.

After completion of incubation in a 150 liter fermenter, the cells were initially cultured using a Sartocon II system (Sartorius, Germany) equipped with a 0.1 μm membrane filter at room temperature. Concentrate to 5 liters of 1/20 of the solution, centrifuge the concentrate at 6,000 xg for 20 min at 4 ° C to remove supernatant, and then precipitate the cells at 4 ° C in 10 mM phosphate buffer (Na ₂ HPO ₄ per liter). 1.15 g, 0.2 g KCl, 0.2 g KH ₂ PO ₄ , 8.766 g NaCl, pH 7.2) and centrifuged again, followed by washing the cells with the same 10 mM phosphate buffer. Three times the volume of acetone was added to about 2,500 g of the obtained cells, and the mixture was left at 4 ° C. for 12 hours with stirring at regular time intervals. After removing acetone from the precipitated cells, double volume of fresh acetone was added again, stirred for 5 to 10 minutes, left at 4 ° C for 12 hours, and centrifuged at 8,000 xg for 20 minutes. Acetone in the upper layer was removed, and the same volume of acetone was added to the precipitated cells, stirred and left in the same manner as described above, centrifuged, and the supernatant was removed to obtain precipitated cells. The obtained Pseudomonas aeruginosa was suspended by adding 2,500 ml of the same phosphate buffer as above. Extracellular membrane proteins were extracted at a rate of 50 to 200 rpm for 10 minutes using a homogenizer (manufactured by EYELA Co., Ltd.) at 4 ° C. The obtained suspension was centrifuged at 8,000 to 10,000 × g for 30 minutes to obtain a supernatant, and then the precipitated cells were suspended by adding the same volume of phosphate buffer, followed by secondary extraction under the same conditions as the extraction method above, followed by the supernatant again. Got it.

Using the same conditions as the extraction method used above, the extraction was repeated 3 to 4 times while maintaining the cells in a non-fragmented state. At this time, whether the cells were broken or not was determined by measuring a specific protein, ie, lactate dehydrogenase, a cytoplasmic marker. Each supernatant identified as having extracted the extracellular membrane protein without the incorporation of intracellular protein lactate dehydrogenase was collected, stirred, and finally recentrifuged at 12,000 xg for 30 minutes at 4 ° C to obtain a clear supernatant. Obtained. The protein solution thus obtained consisted of almost pure extracellular membrane protein only and was adjusted to maintain concentrations between 1 mg / ml and 2 mg / ml through protein quantification.

(3) Purification of Crude Extracellular Membrane Proteins

The following experiment was performed to purely separate crude extracellular membrane proteins adjusted to a concentration of 1 to 2 mg / ml to have only active ingredients. 10-liter phosphate buffer was added to 2-2.5 liter of crude extracellular membrane aqueous solution, diluted 4-5 times, and protein molecules with molecular weight of 100 kDa or more were first removed using a molecular weight 100 kDa blocking membrane filter in a Pelicon cassette system. In this way, proteins with molecular weights of less than 100 kDa exit the filtrate. Proteins with molecular weights greater than 100 kDa continue to circulate, separating molecules with molecular weights less than 100 kDa. An aqueous protein solution concentrated to 10-20% of the original volume was added to 20-25 liters of sterile phosphate buffer (1.15 g of Na ₂ HPO ₄ , 0.2 g of KCl, 0.2 g of KH ₂ PO ₄ , NaCl 8.766 per liter of 10 times the original volume). By continuing washing with g), 90% or more of protein having a molecular weight of 100 kDa or less was recovered. Proteins with a molecular weight of 100 kDa or less separated in the filtrate were removed using a molecular weight 10 kDa blocking membrane filter in the same system to remove proteins, pigments, and other components with molecular weights less than 10 kDa while simultaneously removing proteins with molecular weights between 10 kDa and 100 kDa. Concentrate to a concentration of mg / ml.

The extracellular membrane proteins of attenuated Pseudomonas aeruginosa strains were fractionated by primary and secondary ultrafiltration to obtain only proteins with molecular weights ranging from 10 kDa to 100 kDa. At this time, it is very important that the molecular weight range of the extracellular membrane protein is 10 kDa to 100 kDa. The fraction having a molecular weight of less than 10 kDa is a pigment and a low molecular weight protein, meaning that it is insignificant as an affinity ligand because it has no effect as an antibody-inducing substance. In the fraction of the range larger than 100 kDa, high molecular weight lipopolysaccharides are present. Antibodies isolated and purified by the coupled lipopolysaccharides may be a secondary therapeutic agent for Pseudomonas aeruginosa infection, but there is no problem. This is because if lipopolysaccharide coupled to the column is liberated, this may cause toxicity.

Finally, extracellular membrane protein fractions having molecular weights of 10 kDa to 100 kDa obtained above were ultracentrifuged to remove lipopolysaccharides or extracellular membrane related denatured fragments present in trace amounts. The ultracentrifugation was carried out at 180,000 xg to 200,000 xg for 3 hours at 4 ° C. to remove precipitates, and the resulting supernatant was then sterilized using a 0.2 μm filter to finally produce 4,000 to 5,000 mg of pure extracellular membrane protein. Obtained. In addition, as a result of confirming the purity of the protein solution by the 8 to 15% concentration gradient electrophoresis method, it was possible to confirm the presence of the extracellular membrane protein having a target molecular weight in the range of 10 kDa to 100 kDa (FIG. 1). In FIG. 1, lane 1 is a standard molecular weight marker, lane 2 is an extracellular membrane protein of CFCPA10142, lane 3 is an extracellular membrane protein of CFCPA20215, lane 4 is an extracellular membrane protein of CFCPA30720, lane 5 is an electric membrane protein of CFCPA60534 It shows the result of phoresis.

(4) Purification of extracellular membrane proteins from attenuated Pseudomonas aeruginosa with different immune forms

The remainder used in the present invention according to the same conditions and methods as the microbial culture and purification process for preparing the extracellular membrane protein composition for CFCPA 30720 Pseudomonas aeruginosa as described in Example 1 (1), (2), (3). Three attenuated Pseudomonas aeruginosa strains CFCPA 10142, CFCPA 20215 and CFCPA 60534 were treated. The final Pseudomonas aeruginosa extracellular membrane composition thus obtained showed the same band form as that obtained in CFCPA 30720 on SDS-PAGE at a concentration of 8 to 15%, and compared to the standard molecular weight marker, all proteins showed 10 It was confirmed that the protein is composed of more than kDa to less than 100 kDa (Fig. 1).

(5) Combination of extracellular membrane protein purified from each attenuated Pseudomonas aeruginosa

From the four attenuated Pseudomonas aeruginosa strains CFCPA 10142, CFCPA 20215, CFCPA 30720 and CFCPA 60534 by repeating the same conditions and methods as described in (1), (2), (3) and (4) of Example 1 For each strain, 4,000 to 5,000 mg of pure extracellular membrane proteins were obtained, and the obtained extracellular membrane proteins were mixed at a ratio of 1: 1: 1: 1 to prepare a total of 5,000 mg of pure extracellular membrane proteins to be used as ligands.

Example 2 Preparation of Affinity Columns for Antibody Preparation

(1) Pretreatment of Ligand for Antibody Binding

The procedure of concentration and buffer exchange was carried out by ultrafiltration of 10 mM phosphate buffer containing a total of 5,000 mg pure extracellular membrane protein obtained in Example 1 (5) using a molecular weight 10 kDa blocking membrane filter in a Pelicon cassette system. Simultaneous runs were made to dissolve in the final 800 ml coupling buffer (0.1 M NaHCO ₃ , 0.5 M NaCl, pH 8.3).

(2) pretreatment of column resin and coupling of ligand

145 g of Pharmacia Biotech's CNBr-activated Sepharose 4B resin was sufficiently suspended in 1,000 ml of 1 mM HCl, and then washed with 9,000 ml of 1 mM HCl using a sintered glass filter to remove fine particles or preservatives. It was.

CNBr washed above in 800 ml of coupling buffer (0.1 M NaHCO ₃ , 0.5 M NaCl, pH 8.3) containing 5,000 mg of pure extracellular membrane protein prepared by the method as described in (1) of Example 2 Remove the activated Sepharose 4B resin well, shake it for 12 hours at 4 ° C without using a magnetic stirrer, gently wash it with a coupling buffer using a sintered glass filter, and then remove 1,000 ml of blocking solution (1 M). It was suspended in ethanolamine, pH 8.0) and shaken at 4 ° C. for at least 3 hours to prevent CNBr reactive substrates of the remaining resin from remaining unreacted. The gel was washed lightly with phosphate buffer using a sintered glass filter and then loosened in 1,000 ml of phosphate buffer and then degassed sufficiently using a vacuum pump until no bubbles were observed.

The gels coupled in this manner are washed three times in alternating times using two volumes of 0.1 M acetate buffer (pH 4.0, 0.5 M NaCl) and the same volume of coupling buffer to prevent electrostatic reaction on the gel without reaction. Remaining extracellular protein ligands were removed.

Filled resin is packed into an appropriate column, washed three times with alternating volumes of 0.1 M acetate buffer (pH 4.0, 0.5 M NaCl) and equal volume of coupling buffer, followed by a 10-fold volume Rinse well with phosphate buffer. As a result of quantitative comparison of the amount of protein before and after coupling, the amount of ligand coupled through the above process was 18.5 mg per g of resin on a dry weight basis, and a total of 2,682 mg of purified pure extracellular membrane protein ligand was separated from Sepharose CL. Coupling was performed on a -4B resin, and the coupling efficiency was 54%.

Example 3 High Purity Purification of Effective Antibody (Immune Globulin) for Treatment of Pseudomonas Aeruginosa Infection from Normal Plasma

(1) pretreatment of normal plasma

12 units (220-230 mL / unit) of normal FP plasma purchased from the Red Cross blood source were collected, 2.77% CaCl ₂ solution was treated to 10% of the final volume, and left in a 37 ° C constant temperature bath for 2 hours to form a fibrin mass. When observed, fibrinogen in anticoagulant-treated FP plasma was removed by centrifugation for 40 minutes under 10,000 × g, 4 ° C. conditions. Approximately 2 L of plasma obtained through the above treatment from 12 units of plasma bags was filtered using 4 to 5 layers of sterile gauze, and then mixed with the same volume of phosphate buffer to obtain a total of 4 L of pre-purified plasma stock solution. It was prepared and 0.8 g of chimerosal was added to the final 0.02% to prevent the growth of microorganisms.

(2) Purification of Effective Antibody Using Affinity Column Chromatography

4 L of the pre-purified plasma stock solution of Example 3 (1) was loaded into the affinity column prepared in Example 2 (2). At this time, the flow rate was 400 ml per hour, and the column was washed with phosphate buffer of 10 times gel volume, preferably 5 times after loading, preferably no chimerosal. Elution of the effective antibody bound to the affinity column was performed using 0.2 M acetate buffer (pH 3.2), and elution of the effective antibody began around pH 5.5, at which concentration 20 mM was used to prevent denaturation of the effective antibody in the eluate. Phosphate buffer (pH 7.2) was prepared in advance so that the eluate was eluted into the solution. The obtained eluate was neutralized to pH 7.0 by titration with 1 M carbonate buffer (pH 9.0), and the final volume was 1,200 ml, and a total of 576 mg of effective antibody was obtained as a result of protein quantification (Preparation No. 1, Table 1). After elution of the effective antibody, the affinity column was washed with 4 times gel volume of the above elution buffer, and then again with 10 times volume of phosphate buffer, and finally with 5 times gel volume of phosphate buffer containing 0.02% of chimerosal. Store at ℃.

(3) High Purity Purification of Effective Antibodies Using Protein A Column Chromatography

Protein A column chromatography was used to purify the effective antibody purified by the method of Example 3 (2) with high purity. 50 ml of Pharmacia Biotech's Protein A Sepharose CL-4B resin was added, 10-fold volume phosphate buffer was added, mixed well, and allowed to stand to remove fine resin particles suspended in the supernatant, and then filled in an appropriate column. The packed column was washed with a 10-fold volume of 500 ml of phosphate buffer at a flow rate of 150 ml per hour, and then 150 ml, preferably 80 ml, of the effective antibody solution purified by the method of Example 3 (2). Loaded at a flow rate of. The loaded column was washed well with a 10-fold volume of 500 ml of phosphate buffer to remove non-specifically attached protein, followed by eluting the effective antibody using 0.2 M acetate buffer (pH 3.2). Elution of the effective antibody began around pH 5.5, in which a double concentration of 20 mM phosphate buffer (pH 7.2) was prepared in advance to prevent denaturation of the effective antibody in the eluate, and the eluate was eluted into the solution. . The eluate obtained was titrated with 1 M carbonate buffer (pH 9.0) and neutralized to pH 7.0, in which case 150 mL per hour flow rate was maintained. As a result of purification using a Protein A column, the final volume of the eluate was 720 ml. As a result of protein quantification, an effective antibody purified with a high purity of 351 mg was recovered (Product No. 1, Table 1). The eluted column of the effective antibody was washed with 4 times gel volume of the above elution buffer, and then again washed with 10 times volume of phosphate buffer and finally washed with 5 times gel volume of phosphate buffer containing 0.02% of chimerosal. Keep at. As shown in Table 1, the same purification process was performed three times, and the final amount of purified human antibody was 353 mg on average, indicating a very stable antibody recovery for each batch of preparation. In addition, the SDS-PAGE results of the 8 to 15% concentration gradient carried out using the samples obtained for each purification process are shown in Figure 2 (lane 1, standard molecular weight marker; lane 2, total plasma protein; lane 3, affinity column Eluted protein; lane 4, the final purified human antibody protein eluted from the Protein A column), the human antibody that specifically binds to the final purified Pseudomonas aeruginosa extracellular membrane protein is at least 95% pure (Figure 2, lane 4) ), It was confirmed that the purification was very pure.

Yield for each step of purification of human antibodies for treatment of Pseudomonas aeruginosa infection Manufacturing number Volume of plasma used (ml) Affinity Column Eluent: Concentration (elution volume) Total amount of eluted antibody (mg) Protein A column eluate: concentration (elution volume) Total amount of final purified human antibody (mg) One 2,000 ml 480 μg / mL (1,200 mL) 576 mg 488 μg / mL (720 mL) 351 mg 2 2,000 ml 554 μg / mL (1,000 mL) 554 mg 483 μg / mL (720 mL) 348 mg 3 2,000 ml 586 μg / mL (1,050 mL) 615 mg 507 μg / mL (710 mL) 361 mg

(4) Concentration of Effective Antibodies Using a TFF (tangential flow filtration) system

Millipore's Prep / Scale TFF membrane (blocking molecular weight 30 kDa, regenerated cellulose) in order to concentrate the effective antibody purified in high purity in Example 3 (2) and (3) to adjust the concentration and composition suitable for formulation Ultrafiltration was performed. 720 ml of a solution containing the high-purity effective antibody obtained in Example 3 (3) (Manufacturing No. 1, Table 1) at a room temperature was slowly stirred with a magnetic stirrer at a volume of 10 times 7200 ml for injection physiology Ultrafiltration of saline was concentrated to a final 340 mL of the effective antibody solution at a concentration of 1 mg / mL.

Example 4 Therapeutic Effect of Purified Effective Antibody on Pseudomonas Aeruginosa Infectious Disease

The therapeutic effect of the effective antibody obtained in Example 3 against Pseudomonas aeruginosa infection was confirmed by the following method. In the following, 20 mice were used in each group.

Fisher's immunotypes 1, 2, 3, 4, 5, 6 and 7 2-5 hours after intraperitoneal injection of 0.5 mL intraperitoneally with 0.2 mg per 5 weeks old male ICR mice in pure human purified antibodies. The pathogenic Pseudomonas aeruginosa was injected intraperitoneally with 3.6 to 7.3 × 10 ⁷ cells corresponding to 5 to 10 times LD ₅₀ , respectively, to cause Pseudomonas aeruginosa infection and to test the therapeutic effect of the purified antibody. The control group was intraperitoneally injected with 0.5 ml of physiological saline for injection instead of the effective antibody. As a control strain of the control group, 4.6 × 10 ⁷ cells were administered using Fischer immunotype 1, and survival rate was observed in the same manner.

As a result, the control group administered with only saline for injection showed a mortality rate of 50% or more within 48 hours and 100% in 72 hours, whereas in all test groups to which an effective antibody was administered, the survival rate was at least 85% even after 72 hours. It was. Therefore, an effective antibody against Pseudomonas aeruginosa purified from normal human plasma has proven to be effective for the treatment of infection by pathogenic Pseudomonas aeruginosa of all Fischer immunotypes. Therefore, an effective antibody composition against Pseudomonas aeruginosa purified from normal plasma according to the present invention can be used as an effective therapeutic agent for infection by Pseudomonas aeruginosa strains of the entire Fisher immunotype. The results are summarized in Table 2.

Effectiveness test of human antibody for treatment of Pseudomonas aeruginosa infection by passive immunity Attack strain Dose (cfu / mouse) Survival Mice / Whole Mice Survival rate (%) 0 days 1 day 2 days 3 days 5 days F1 4.6 x 10 ⁷ 20/20 19/20 19/20 18/20 18/20 90 F2 5.5 x 10 ⁷ 20/20 20/20 20/20 19/20 19/20 95 F3 4.1 x 10 ⁷ 20/20 20/20 20/20 20/20 20/20 100 F4 7.0 x 10 ⁷ 20/20 19/20 18/20 18/20 18/20 90 F5 3.6 x 10 ⁷ 20/20 18/20 18/20 18/20 18/20 90 F6 5.2 x 10 ⁷ 20/20 20/20 19/20 19/20 19/20 95 F7 7.3 x 10 ⁷ 20/20 18/20 17/20 17/20 17/20 85

Example 5 Formulation of Purified Effective Antibodies

The combined composition of the extracellular membrane protein obtained by culturing and extracting the four attenuated Pseudomonas aeruginosa strains CFCPA10142, CFCPA 20215, CFCPA 30720 and CFCPA 60534, as in Example 1, was prepared in the same manner as in Examples 2 and 3. 1 to 100 mg of effective antibody per kg of body weight can be administered to an effective antibody for treating Pseudomonas aeruginosa infection isolated and purified from normal plasma using affinity column and protein A column chromatography prepared by coupling to Paros CL-4B resin. Formulated with various pharmaceutically acceptable carriers. 0.2 mg of the formulated effective antibody was intraperitoneally administered to 20 mice in each test group, followed by intraperitoneal administration of the attack strain after 2 hours, and the difference in the effectiveness of human antibodies according to the type of carrier was observed. The attack strain used at this time was GN11189 and was injected intraperitoneally by 1.0 × 10 ⁷ cells corresponding to 5 × LD ₅₀ , causing Pseudomonas aeruginosa infection and comparing the effects by viability. The results of this test are shown in Table 3 below. As a result, in the case of liquid formulations, an effective antibody formulation using physiological saline or phosphate buffer as a carrier showed a similarly high efficacy, and in the case of lyophilized formulations, liquids were used when mannitol, saccharose or lactose was used as a carrier. It showed high efficacy similar to the formulation.

Effectiveness test results of effective antibody formulations containing different pharmaceutical carriers Article Used carrier Survival rate (%) effectiveness Liquid phase Saline solution 95 +++ Phosphate buffer 95 +++ Tris salt buffer 80 + Freeze drying Mannitol 95 +++ Saccharose 90 ++ Lactose 95 +++ Human albumin 85 +

(+++; Excellent, ++; Normal, +; Poor)

Example 6: Validity of Formulated Effective Antibody Compositions

The phosphate buffer, which was observed to be highly effective according to the test results of Example 5, was used to confirm the effectiveness of the formulated effective antibody as a prophylactic and therapeutic agent for secondary skin infections in laceration, burns, trauma and the like. Known image test method [J. Infect. Dis. 131, 688-691, 1975], induces burns in 5-week-old male ICR mice and shows a survival rate of 95% or more after 15 hours, and then a liquid formulation containing 1.0 mg of human antibody for treating Pseudomonas aeruginosa infection per ml of phosphate buffer. Was administered intraperitoneally at 500 μl per test group mice. Two hours after intraperitoneal administration, Fisher's immune type 6 pathogenic Pseudomonas aeruginosa GN11189 was injected intraperitoneally into 1.0 x 10 ⁷ cells corresponding to 5 x LD ₅₀ to cause Pseudomonas aeruginosa infection and its effect was administered intraperitoneally with physiological saline Judging was compared with survival in one treatment antibody untreated group. As a result, the group of mice intraperitoneally administered with the effective antibody-phosphate buffer liquid formulation showed a superior survival rate compared to the non-treated group, and thus the effective antibody-containing formulation for treating Pseudomonas aeruginosa infection according to the present invention was excellent in treating P. aeruginosa infection. It can be seen that the effect. The results of this test are shown in Table 4 below.

Effectiveness Test Results of Human Effective Antibody Preparation for Pseudomonas Aeruginosa Infection in Burn Model Test group Antibody Dose (mg / mouse) Attack Group GN11189 Dose (cfu / mouse) Survival Mice / Whole Mice Survival rate (%) 0 days 1 day 2 days 3 days 5 days Control 0 1.0 x 10 ⁷ 20/20 3/20 1/20 1/20 1/20 5 Antibody Treatment Group 0.5 1.0 x 10 ⁷ 20/20 19/20 19/20 19/20 19/20 95

Human immunoglobulins that specifically bind to extracellular membrane proteins isolated and purified from safe, attenuated Pseudomonas aeruginosa strains exhibit excellent therapeutic effects against not only various Pseudomonas aeruginosa infections, but also severe infections. As an agent for treating Pseudomonas aeruginosa infectious disease, it is excellent in medical efficacy.

Claims (1)

A therapeutic agent for Pseudomonas aeruginosa infections comprising human immunoglobulins that specifically bind to extracellular membrane proteins isolated and purified from safe, attenuated Pseudomonas aeruginosa strains as active ingredients.

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