JPH0720885B2 - Agent for preventing or treating herpes simplex virus infection - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention is directed to Herpes simplex vir.
us, HSV), which is a prophylactic or therapeutic agent for HSV infections, which comprises a human monoclonal antibody as an active ingredient.

(B) Conventional technology Using cell fusion technology, lymphocytes or B cells (antibody-producing cells) that produce specific antibodies but are destined to die eventually continue to proliferate permanently in an incubator. A method for establishing a hybridoma (fused cell) strain that permanently produces and secretes a specific antibody by fusing myeloma cells (myeloma cells) is known.

The monoclonal antibody produced by the hybridoma produced by such a method can be applied to a test reagent, a labeling reagent, affinity chromatography, etc. as a pure chemical reagent with high accuracy and reliability, as well as a therapeutic drug for various diseases and prevention. It can be expected to be applied as a medicine.

By the way, in order to obtain a monoclonal viral antibody, it is generally known that a viral antibody-producing hybridoma may be obtained by fusing a viral antibody-producing cell with a myeloma cell and cloning it. . And specifically, for example, Japanese Patent Publication No. 59-
In the 2276 publication, spleen cells (antibody-producing cells) of BALB / C mice immunized with influenza virus or rabies virus, and a hybridoma obtained by fusing myeloma cells of a mouse of the same species, by cloning it, Monoclonal antivirus. It has been disclosed that a hybridoma producing a mouse antibody was obtained. Further, JP-A-58-175489 discloses that a spleen cell of a mouse immunized with a herpes simplex virus and a myeloma cell of a mouse were fused to obtain a hybridoma producing an anti-herpes simplex virus / mouse antibody. Has been done.

(C) Problems to be Solved by the Invention As described above, a specific successful example of a hybridoma producing an antiviral antibody is antivirus. Only mouse-mouse hybridomas that produce mouse antibodies. However, for the prevention and treatment of human diseases, antiviral human antibody, which is a homologous protein, is more useful and safer, and for that purpose, mouse-human hybridoma cells are prepared using human antibody-producing cells. It is necessary to establish a human-human hybridoma. However, unlike the case of animals, in the case of humans, it is not possible to adopt a method in which humans are immunized with a large amount of virus in advance and effectively stimulated antibody-collecting cells are used for cell fusion. Since there are problems such as difficulty in collecting and preparing various antibody-producing cells, there are no reports of clear success cases.

(D) Means for Solving the Problems The present inventors obtained an anti-HSV human monoclonal antibody,
As a result of intensive research aimed at using this for the prevention and treatment of HSV infection, human antibody-producing cells sensitized with HSV or an HSV-derived protein or glycoprotein in the presence of mitogenes in vitro. Then, a mouse-human hybridoma producing an anti-HSV human monoclonal antibody could be obtained by the method of fusing the mouse with a myeloma cell of mouse. Then, this hybridoma and / or a cell line derived from it is cultured, and a human monoclonal antibody against HSV is collected from the culture, and it is confirmed that this has a neutralizing activity against HSV and an infection suppressing ability, and the present invention is completed. did.

That is, the present invention has the property that it reacts with the cell membrane and cytoplasm of cells infected with herpes simplex virus type 1 or type 2 and hardly reacts with non-infected cells. Is a prophylactic or therapeutic agent for herpes simplex virus infection, which comprises a human monoclonal antibody against Escherichia coli as an active ingredient.

Human antibody-producing cells refer to human lymphocytes (or B cells) that secrete antibodies or have the ability to secrete antibodies. It is contained in cells such as spleen, lymph nodes, peripheral blood, bone bones, tonsils, and adenoids. For the purposes of the present invention, lymphocytes of any source can be used, but preferred are those taken from the spleen or tonsils or adenoids.

As a mouse myeloma cell, it is advantageous to use an 8-azaguanine resistant strain, and as a known one, BA
LB / C mouse P3 × 63 Ag8 strain, P3-NS1 / 1-Ag4-1 strain, P3 × 6
3Ag8U1 strain, SP2 / OAg14 strain, P3 × 63Ag8.6.5.3 strain, MPC11-45.
6. There are TG1.7 strain, SP-1 strain, etc.

Prior to fusing human antibody-producing cells with mouse myeloma cells, it is preferable to sensitize human antibody-producing cells in vitro in the presence of mitogenes.

In the case of humans, even normal people often have lymphocytes capable of producing antibodies to HSV, but due to the small number, it cannot be used as it is to obtain the desired hybridoma. . In contrast, in
According to the method of sensitizing human antibody-producing cells in vitro,
Sensitization can promote the differentiation and proliferation of cells, and can arbitrarily increase the number of antibody-producing cells of interest. Thus, by performing cell fusion using antibody-producing cells sensitized with HSV, a hybridoma that efficiently produces an anti-HSV monoclonal antibody can be obtained. HSV
Known as type 1 (for example, KOS strain, Hayashida strain) and type 2 (for example, YS-4 strain). For sensitization, not only these viruses but also proteins or glycoproteins derived from such viruses may be used.

Any mitogen may be used as long as it promotes differentiation and proliferation of lymphocytes, and examples thereof include pokewidmite-diene (PWM), protein A, and phytohemagglutinin (PHA) concanavalin A. Preferred is PWM, usually used in an amount of 2-200 μg / ml, preferably 20-100 μg / ml.

The method conditions for sensitization are not particularly limited, but the concentration of the antigen (HSV, or HSV-derived protein or glycoprotein) is 1 mg.
/ ml ~ 1μg / ml, concentration of lymphocytes (antibody producing cells) is 1x
10 ⁵ to 1 x 10 ⁷ cells / ml is suitable, and the culture temperature is 35 to 40 ° C.
The culturing time is 4 to 10 days, preferably 6 to 8 days. Any culture medium may be used as long as it contains serum of human, bovine, horse, etc., but especially a culture solution containing fetal calf serum (FCS) (eg RPMI1).
640) is preferred. The virus-sensitized human antibody-producing cells and mouse myeloma cells thus obtained are
Then, the cells are fused according to a known method. For example, antibody-producing cells and myeloma cells at 10: 1 to 1:10. Preferably, the mixture is mixed in a ratio of 1: 1 to 1: 3, and a suitable cell fusion solution, for example, about 35% polyethylene glycol (molecular weight 1,00
(About 0 to 6,000) and about 7.5% dimethylsulfoxide are added to RPMI1640, and the mixture is stirred at room temperature to 37 ° C for 1 to several minutes, and then gradually diluted with 10% FCS-added RPMI1640. Aminopterin-thymidine)
Adjust the cell concentration to 1-5 × 10 ⁵ cells / ml with selective culture medium. 0.2 ml each of this is dispensed into, for example, a 96-well plate and cultured in air containing 5% CO ₂ at 35 to 38 ° C. for 2 to 3 weeks. In the HAT medium, only the hybridoma survives, but the 8-azaguanine-resistant myeloma cells and the fused cells between the myeloma cells cannot survive (the unfused antibody-producing cells die within a few days).

After culturing, the antibody titer in the culture solution is checked, and only the hybridoma producing the target antibody is selected and isolated (cloning). The antibody titer in the culture solution can be checked by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),
It can be performed by a method such as a fluorescent antibody method of detecting a substance bound with an antibody to an antigen, a method of observing the activity of an antibody that inhibits the biological activity of a virus, or the like. The mouse-human hybridoma producing the antiviral human antibody of the present invention selected by cloning can be frozen and stored. By culturing a large amount of such a hybridoma cell line (cell line) and / or a cell line derived therefrom by an appropriate method, the human monoclonal antibody targeted by the present invention can be obtained from the culture supernatant. It is also possible to transplant this hybridoma into an animal to form a tumor and obtain a monoclonal antibody from the ascites or serum.

The present invention is a preventive or therapeutic agent for HSV infection, which comprises the monoclonal antibody thus obtained as an active ingredient. The form of the preventive or therapeutic agent is an aqueous solution or a freeze-dried powder thereof. This drug is 0.1mg / ml to 50mg / m
1, preferably 1-10 mg / ml of monoclonal antibody. The salt is 0.5 to 1.5% (w / v) sodium chloride, preferably 0.9% (w / v). Furthermore, from 0.001
0.1M, preferably 0.02M sodium phosphate (pH 7.0-pH
7.8) is added. As a protein stabilizer, 0.1 mg / ml-1
0 mg / ml human serum albumin and / or 0.1% to 5
% Mannitol and / or 0.01-1 mg / ml polyethylene glycol (molecular weight 1,000 to 10,000) is added.
The administration method is mainly intravenous injection or infusion, but intramuscular injection is also possible. Dosage is 0.01-10mg / kg body weight /
Day, preferably 0.1 to 1 mg / kg body weight / day.

(E) The present invention will be described in detail below with reference to Examples and Examples.

Reference Example 1 (Hybridoma Producing Monoclonal Antibody Against HSV) (1) Preparation of HSV Antigen 4.4 × 10 ⁶ P was added to Vero cells (about 2 × 10 ⁸ cells) grown in a monolayer.
FU (plaque forming unit) / ml HSV (KOS strain) was inoculated. Contains 2% bovine serum after being adsorbed at 37 ℃ for 2 hours
The cells were cultured in MEM medium for 24 hours. The cells were washed with phosphate buffered saline and then sonicated to disrupt the cells.
This was centrifuged at 6000 ppm for 30 minutes to obtain a supernatant, which was 30%
It was layered on the sucrose solution and centrifuged at 30,000 rpm for 3 hours.
The pellet precipitated at the bottom of the centrifuge tube was used as the viral antigen.

(2) Lymphocyte sensitization with HSV Human tonsillar lymphocytes were cultured in culture medium A (RPMI1640 + 20% fetal bovine serum + 20 mM HEPES + 2 mM glutamine + 1 mM Na pyruvate +
0.02 mg / ml serine + 80 μg / ml gentamicin). The cell concentration was 17 × 10 ⁵ cells / ml. Each 1.2 ml of this cell suspension was placed in 12 wells of a culture plate (24 wells). They were divided into 4 groups with 3 wells each, the first group was non-added, the second group was HSV (KOS strain, partially purified standard) 16 ng protein / ml,
PWM of 20 μg / ml was added to the third group, and the same amount of HSV and PWM was added to the fourth group. The culture plate was placed at 37 ° C and 5% CO ₂ -air for 6 times.
Cultured for a day.

(3) Cell fusion with mouse myeloma cell P3 × 63Ag8U1 strain (abbreviated as P3U1).

P3U1 in culture medium B (RPMI1640 + 10% fetal bovine serum +
2 mM glutamine + 80 μg / ml gentamicin). The cell concentration at the time of use was 6 × 10 ⁵ cells / ml.
With 4 groups of sensitized lymphocytes (3 holes together) of (2) above
P3U1 was separately washed twice with serum-free RPMI1640. Lymphocytes from each group and 5 × 10 ⁶ P3U1 were combined in a test tube. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded. The cell pellet was well dispersed by tapping the test tube. Add 0.5 ml of polyethylene glycol solution (RPMI1640 5.75 ml + polyethylene glycol 1000 3.
5 ml + 0.75 ml of dimethyl sulfoxide (abbreviated as PEG solution) was added to gently suspend the cells. One minute later
0.5 ml + RPMI1640 was added, 1 minute later, 1 ml RPMI, and 2 minutes later, 4 ml of HAT culture medium (RPMI1640 + 20% fetal bovine serum + 80 μg / ml gentamicin + 95 μM hypoxanthine + 0.
4 µM aminopterin + 1.6 µM thymidine), and 2 minutes later, 4 ml of HAT culture medium was added. Finally, 2 with HAT medium
5 ml cell suspension was used. Culture plate (96 wells) 1
The pieces were sown and cultured at 37 ° C. in air containing 5% CO ₂ . Hybridomas were obtained by exchanging half of the culture medium with a new HT culture medium (HAT in which A was removed) every week.

(4) Measurement of human IgG and anti-HSV antibody It was measured by enzyme-linked immunosorbent assay (ELISA). Immobilize goat anti-human IgG antibody (10 μg / ml) to measure human IgG, or HSV (KOS strain) 1 μg protein / ml to measure anti-HSV antibody on Falcon Microtest III 96-well plate, respectively. did. 60 μl of hybridoma culture supernatant was added to this plate and left at room temperature for 1 hour. 0.05%
Phosphate buffered saline containing Tween-20 (Tween-PB
After washing 3 times with S), 60 μl of gear anti-human IgG antibody-alkaline phosphatase (2000-fold diluted solution) was added and reacted at room temperature for 1 hour. After further washing three times with Tween-PBS, 100 μl of a solution prepared by dissolving P-nitrophenylfoscuate in a pH 9.8 solution of 1M diethanolamine + 1 mM MgCl ₂ at a rate of 0.6 mg / ml was added. 405m after 30 to 60 minutes
The absorbance at μ was measured, and the value was calculated by comparison with the standard IgG solution or standard HSV-positive serum.

In all groups, cells were plated on one 96-well plate, and the number of wells in which 96 hybridomas had grown, the number of wells with hybridomas producing human IgG, and anti-HSV antibody were produced. The number of bored holes is shown in Table 1. Table 1 shows an example in which lymphocytes were isolated from three tonsils, and in all cases, the most anti-HSV antibody-producing hybridomas grew when HSV and PWM were added.

(5) Cloning of hybridoma producing anti-HSV antibody Cloning was performed by the limited dilution method. Remove the cells from the anti-HSV antibody-positive holes, count the number of cells, and use culture medium B 1
Cells were seeded at 10 cells / hole or 10 cells / hole. After 2 weeks, the cells were sufficiently grown, and thus the presence or absence of anti-HSV antibody in this supernatant was measured by ELISA to select anti-HSV antibody-producing hybridomas.

(6) Preparation of anti-HSV monoclonal antibody One of the obtained hybridomas, D34, was added to serum-free medium ITEM (R
The cells were cultured in 2 volumes of PMI1640 + 1 volume of Dulpette MEM + 1 volume of F12 + insulin 8.5 µg / ml + transferrin 2 µg / ml + ethanolamine 20 µm + selenite 2.5 × 10 ^-8 M. Obtained 480 ml of the culture broth and passed through ultrafiltration (Amicon PM3
0) to 14 ml. Add this to 0.02M sodium phosphate (pH 7.
8) DE52 column (2 cm x 14 c) which was deposited and equilibrated with the same buffer
m). Human monoclonal antibody was recovered in the non-adsorbed fraction (21 ml). When measured by the enzyme-antibody method, 1.9 μg / ml of purified monoclonal antibody was found in the culture supernatant and 2 in the
It contained 8 μg / ml human IgG. When subjected to sodium dodecyl sulfate-polyacrylamide (5% gel) electrophoresis, a single band was formed at a position of a molecular weight of about 160,000.

Reference Example 2 Properties of anti-HSV monoclonal antibody (1) The specificity of the monoclonal antibody was examined by the fluorescent antibody method. Vero cells infected with HSV1 type KOS strain were fixed on a slide glass with acetone, and the hybridoma culture supernatant containing the monoclonal antibody was allowed to react at room temperature for 1 hour, washed, and then labeled with fluorescein isothiocyanate. The obtained goat anti-human IgG (10-fold diluted solution) was reacted at room temperature for 1 hour. The slide thus prepared was observed with a fluorescence microscope. As a result, hybridoma D34,
It was found that all of the monoclonal antibodies produced by 7-4,5-11, H2 and H3 react with the cell membrane and cytoplasm of virus-infected cells, but not with non-infected cells.
Furthermore, it was found that it also reacts with cells infected with HSV type 1 Hayashida strain and HSV type 2 YS-4 strain.

(2) The MCA, D34, H2, and H3 subclasses and L chains produced by the cloned hybridomas were examined. As a result of looking at the subclass by the octaroni method using sheep anti-human IgG1, anti-human IgG2, anti-human IgG3 and anti-human IgG4,
All three MCA's produced a sedimentation line only with the anti-IgG1 antibody.
Furthermore, alkaline phosphatase-goat anti-human K
Of D34 by K-chain or anti-human in-chain ELISA
It was found that the chains, H2 and H3, had an incoming chain.

(3) Enzyme-linked immunosorbent assay (ELISA was used to examine the reactivity of D34, H2, and H3 against herpes virus. As shown in Table 2, both D34, H2, and H3 were tested for HSV type 1 and type 2 viruses. Reacts to all, non-infected host HEL (human enb
ryonic lung) cells did not react. Other herpes viruses, VZV, CMV and Epstein-Barr virus (E
BV) did not react. Therefore, these three monoclonal antibodies were found to react specifically with HSV type 1 and type 2.

(4) Immunoprecipitation analysis of anti-HSV monoclonal antibody An immunoprecipitation analysis was performed in order to determine what is the constituent component of virus particles with which the human monoclonal antibody reacts. HSV type 1 (Fukuda strain) or type 2 (UW268) in Vero cells
Strain) and wasotope-labeled with H-glucosamine or S-methionine. Labeled cells with 0.01M
Tris.HCl-0.15M NaCl-1% sodium deoxycholate-1% Triton × 100-0.1% sodium dodecylsulfate (SDS) -1 mM Phenylmethylsulfonyl fluoride (pH 7.4) (dissolving solution). A monoclonal antibody was added to this to form an antigen-antibody complex, and the complex was adsorbed and purified with protein A-Sepharose 4B. Add this to 0.125M Tris HCl-1% S
DS-3% 2-Mercaptoethanol 15% Glycerin (pH
It was treated with 8.2) for 3 minutes at 100 ° C., and the supernatant was subjected to SDS-polyacrylamide gel electrophoresis. After running, the gel was dried and exposed to X-ray film at -70 ° C. The results are shown in Fig. 1 and Table 3, and show that hybridomas D34 and 5-11.
It was found that the monoclonal antibodies produced by S. cerevisiae were antibodies to HSV type 1 or type 2 glycoprotein B based on the degree of overlap of the X-ray film bands. From similar experiments, it was found that 7-4, H-2 and H-3 were also antibodies against glycoprotein B.

Example 1 Virus Neutralizing Activity of Anti-HSV Monoclonal Antibody The virus neutralizing activity (titer) of the monoclonal antibody was determined as follows. 96 μl of 25 μl of serially diluted monoclonal antibody solution with 25 μl of virus solution containing 150 PFU) and 25 μl of fresh guinea pig serum (7.5% w / w) as complement
Mixed in well microplate. After reacting at 37 ° C for 60 minutes, the cells were transferred to a Vero cell monolayer culture in a 96-well macro plate for infection. After culturing in a carbon dioxide incubator at 37 ℃ for 2 days, 10
The cells were fixed with% formalin and stained with 0.15% crystal violet, and plaques in each well were observed. The virus neutralization titer was expressed as the highest dilution of the monoclonal antibody solution (1 mg / ml) that reduced the number of plaques by 80%.
As shown in Table 4, the monoclonal antibody D34 binds to HSV but does not neutralize either HSV type 1 or type 2.

The other two monoclonal antibodies, H2 and H3, neutralized both type 1 and type 2 in the absence of complement. Table 5 shows the neutralizing spectrum (range) of the monoclonal antibody H2. H2 possessed high neutralization titers against all of the HSV type 1 and type 2 strains used.

Example 2 Protective effect of various monoclonal antibodies against HSV infection A serially diluted antibody solution (1 ml) was added to H2, H3 and HIgG (human Ig).
BALBc mice (5) for G) and 10 for D34 were intraperitoneally administered as a group. 30 minutes later HSV1 type (Miyama
GC ⁺ strain), 2.5 × 10 ⁵ PFU was intraperitoneally administered, and the survival rate was determined 15 days after the sensation. The results are shown in Fig. 2. The amount of antibody required to protect mice from infection death by 50% was 0.20 for each H2.
mg / kg, H3 was 0.35 mg / kg, D34 and HIgG were 40,380 mg / kg or more, respectively. Therefore, H2 has the ability to protect 1900 times more effectively than HIgG.

Example 3 The monoclonal antibody H2 was purified from the hybridoma culture supernatant by DEAE Sephacel column chromatography and Toyopearl HW-65. The H2 aqueous solution was thoroughly dialyzed against a 0.14 M sodium chloride aqueous solution containing 0.02 M sodium phosphate (pH 7.4). After adjusting the protein concentration to 5 mg / ml, human serum albumin was added to a final concentration of 1 mg / ml. This protein mixture was freeze-dried to prepare a human monoclonal antibody preparation.

[Brief description of drawings]

FIG. 1 is an illustration of an X-ray film showing the results of immunoprecipitation analysis of anti-HSV monoclonal antibody. Figure 2 shows
It is a figure which shows the HSV infection protection effect (survival rate) of a monoclonal antibody.

Front Page Continuation (72) Inventor Yoichi Matsumoto 4-3-2 Asahigaoka, Hino-shi, Tokyo Inside Teijin Biomedical Research Institute (56) Reference JP-A-58-175489 (JP, A) JP-A-59- 42397 (JP, A) JP 58-128323 (JP, A) JP 58-216125 (JP, A) Infection and Immunity, vol. 29, no. 2 (1980) p. 724−732

Claims (1)

[Claims] 1. An IgG type herpes simplex virus type 1 or type 2 sugar having the property of reacting with the cell membrane and cytoplasm of cells infected with herpes simplex virus type 1 or type 2 and hardly reacting with non-infected cells. A preventive or therapeutic agent for herpes simplex virus infection, which comprises a human monoclonal antibody that reacts with protein B and has neutralizing activity as an active ingredient.