JPS5872526A - Preparation of monoclonal hbs antibody - Google Patents

Abstract

PURPOSE:To obtain HBs antibody-producing cell having high antibody productivity from the transformed subculture cell, by heating an HBs antigen in the presence of a protein denaturating agent, and using the denaturated antigen as an immunogen. CONSTITUTION:An HBs antigen is recovered from the blood positive to human HBs antigen by conventional plasma fractionation method (e.g. ammonium sulfate precipitation method). The recovered and purified HBs antigen is heat- treated in the presence of a protein denaturating agent such as urea or guanidine (preferably 6-8mol) at 6.5-7.5pH and 60-120 deg.C. A solution of HBs antigen adjusted to a proper concentration and filtered to remove bacteria, is used as an immunogen and made to contact with human B lymphocyte cells, which is stimulated its antibody-producing activity by this process. The cells are sensitized with lymphotropic virus (e.g. EB virus) to effect the transformation to a productive cell. Cells producing HBs antibody is selected from the above productive cells, and concentrated and proliferated to produce the monoclonal HBs antibody continuously.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing monoclonal HBs antibodies, and more particularly to a method for producing monoclonal human antibodies using transformed subcultured cells.

HBsi antibodies have been shown to be effective in preventing the onset of hepatitis IB when injected into people contaminated with the hepatitis B virus, as well as in preventing post-transfusion hepatitis and HBs antigen positivity. B to the newborn child from the mother of
It has become increasingly clear that when there is a risk of BW hepatitis infection or any other type of BW hepatitis infection, it is possible to prevent infection by re-administering HBsBs antibodies, and this is a major contribution to medical science. Due to its high price, it is a drug for which there is a very high demand in the market for a preparation to be provided as soon as possible. In this way, experimental administration of HBs antibodies has been carried out in Japan for the purpose of preventing hepatitis infection. However, from the perspective of the manufacturer, it is difficult to find a way to continuously supply large quantities of HBs antibody preparations, and there is still very little commercially available HBs antibody preparation.

As a new technique for producing HBs antibodies, a technique for producing HBa antibodies using human cancerous lymphoid cell lines has recently been introduced and is beginning to attract attention. There are currently two types of technologies for these: cell fusion method and transformation method.
The former is a method in which B lymphocytes and bone marrow lung cells of an immunized donor are fused in vitro, and the latter is a method in which 33+7 lymphocytes of an immunized donor are fused with Epstein B cells.
This is a method in which the virus is infected with a lymphotropic virus such as arr virus (EB virus) and transformed into a form capable of propagation.

However, these methods are still in their infancy, and there are many unresolved issues such as the amount of antibody produced, the safety of the antigen being a virus, and the difficulty of purification due to contaminant antigens, and there are many difficulties in putting it into practical use. be.

Another object of the present invention is to provide a method for producing HBs antibody-producing cells with high antibody-producing ability by monoclonal and HBi antibody transformation methods. Another object of the present invention is to ensure the safety of the immunogen used as a virus and to provide a cell proliferation system free of contaminating antibody-producing cells.

The present invention uses HBs antibody, especially its aqueous solution, in the presence of whole urea or a protein denaturing agent such as guanidine, which increases the antigenicity of HBs antigen.
This method is based on the knowledge that HBs antibody production ability can be obtained by sensitizing lymphocytes.

That is, the present invention provides a method for producing a monoclonal human antibody tjR by contacting human IJ membrane cells with an immunogen to stimulate antibody production and transforming the cells into a proliferative form that can be cultured as successors. By using antigens heated in the presence of protein denaturing agents such as urea and guanidine as immunogens, human B lymphocytes can be
This is a method for producing monoclonal HBa antibodies in which the pH is adjusted to 3% to produce antibodies.

More specifically, the present invention involves sensitizing human B lymphocytes stimulated as described above with Q Epstein Barr Virus t to cause transformation into proliferative cells;
These proliferative cells are subcultured, and HBg is obtained from these cells.
Cells that produce antibodies are selected, purified, and reduced. ■ This selection
This is a method for producing a monoclonal human HBs antibody, which consists of growing concentrated cells in a growth medium to continuously produce HBs antibodies, and then collecting the HBs antibodies from the medium.

Original F! The HBs antigen used in AIC is generally recovered from human HBs antigen-positive plasma.

HBs antigen can be recovered from plasma using, for example, ammonium sulfate precipitation and Cohn's cold ethanol fractionation method [Cohn E.

Jo, Strong L, K., Hewes, W. L., Mal7ord, D. J., Ashworth, J. N., Melin, M., and Tiller, H. L., (Cobn E.,
J., StrongL., E.

Hughes W, L, Mulford D-J.
, ,Aghworth J, N., ,Melin M.
and Jaylor H, L*): Journal of American Chemical Engineering (Jo
Urnal of American Chemica
The plasma protein fractionation method is carried out by a well-known plasma protein fractionation method such as that described in 1946]. For example, α and β-globulin fractions can be obtained as fractions ① and IV-1 by Kohn's fractionation method, and as a fraction that precipitates at 35% saturation by ammonium sulfate precipitation. If the aqueous solution is cloudy, clarification can be performed to facilitate subsequent operations. Clarification is carried out, for example, by centrifugation, heating at about 60°C even in neutral conditions, and filtration. Heat treated in the presence of porcelain. The amount of protein denaturant used is 2 to 8 mol, preferably 6 to 8 mol. i, I: carried out in the presence of a heating medium, such medium comprising water and an aqueous solution of physiologically acceptable salts; the solution to be heated is preferably neutral, the pH of which is between 5 and 5; 9, preferably 6.5
~7.5.

It is recommended to use a buffer solution tube to maintain a predetermined pH, and inorganic salts such as phosphorus salts, carbonates, Tris hydrochloride, buffer solutions, and organic salt buffer solutions such as acetates and glycine hydrochloride are recommended. Used as a medium.

The heating temperature is usually in the range of 60°C to 120°C.The heating time varies depending on the heating temperature, but in short, it is a sufficient time to improve the antigenicity expressed by the antigen titer and destroy the infectivity. It is preferable to heat for about 3 minutes to 24 hours. For example, ij, 60'C=lθ~'24 hours, 120℃-3~
0 After heat treatment, filtration and clarification as necessary, and further water,
Alternatively, denaturing agents, its salts, produced low-molecular substances, etc. are removed by dialysis against an isotonic solution such as physiological saline. Concentration can also be performed by a known method such as vacuum dialysis. The f(Bs antigen solution prepared to an appropriate concentration can be used as an immunogen by sterilizing and filtering.

Human lymphoid cell lines, which stimulate antibody production with an immunogen, and B cells, which are responsible for antibody production in vivo, are used. Stimulation may be achieved by administering an immunogen into a living body and then collecting B lymphocytes from humans; however, a more preferred embodiment is to collect B lymphocytes outside the living body, or This method involves culturing and then stimulating with an immunogen.

The separation of B cells is preferably carried out by the specific gravity centrifugation method of Ficoll-Conray (J, Cl1n, Lab, Inv@s
t.

21, 5uppi, 77-89 (1968))
In vitro stimulation with 0 immunogens performed by, for example, purified HBs
Appropriate amount of antigen and B lymphocytes at t-30~40℃ for 10~
Leave in contact for 50 hours. This is generally carried out in a medium, and RPMI 1640 or the like is preferably used as the medium. When carried out in vivo, it can be carried out according to the usual vaccine administration method.

Lymphotropic viruses such as E-pst are used for transformation.
Ein Barr virus (EB virus) is used to transform normal cells into proliferative cells.C Nature 2'69 420-422 (
1977)) known as a virus. Transformation is carried out, for example, as follows. Using oB95-8-derived EB virus (obtained as a supernatant from mycoplasma-free Bs5-8 medium), an amount of virus capable of cell transformation is prepared in advance. For the culture of OB lymphocytes, drop an appropriate amount onto the culture medium of virus t-B9 lymphocytes and contact the cells preferably at 37°C for 5 to 20 days, for example, using RPMI 1640 at 37°C under 5% Co
+I O~20- Carry out in tallow calf serum. Furthermore, this culture may be carried out in a culture medium to which glutamine has been added. Cells that produce antibodies may be cultured using, for example, the PHA method (
Japanese Unexamined Patent Publication No. 53-29920), Radioimmunoassay (App
l, Microbiol, 25.567 (197
3)) Tracked by etc.

Selection of HBs antibody-producing cells is carried out by a method known per se, such as affinity chromatography. Specifically, this is carried out by suspending a cane, for example, a cultured cell, and then contacting it with immobilized HBa antigen and absorbing the antibody-producing cells.

Regarding the immobilized HBa antigen, as the HBs antigen, the one used as the immunogen is preferably used,
Examples of immobilization carriers include agarose and cellulose. After absorption, the carrier is preferably washed with a culture solution, physiological saline, etc., and then the carrier is decomposed with an enzyme such as dextranase to recover all attached cells. After washing the cells, for example by centrifugation, #! It is tightened. This can be seen, for example, in J. Immunol, I
O, 313 (1973). Other methods for recovering specific antibody-producing cells include cell fusion and transformation techniques [for example,
Separation by immunofluorescence (Rev, Set, In5
tru, 43.404 (1972) Method for forming rosettes with red blood cells bound to antigens (Nature, 43.404 (1972)
269.420 (1977)) etc. Thus, from the selected and concentrated HBs antibody-producing cells, tiJRPMI 1640+lO~2
Permanently cultured cells are obtained by culturing the cells in a medium containing 0.0% tallow calf serum or glutamine 'kfA at 37°C, 51 CO, and a hazardous atmosphere. Culture is usually carried out for 5 to 15 days, and the culture is repeated again using a new medium.

Antibodies are thus produced in the medium.

Recovery of HBa antibodies is performed, for example, by a method for recovering HBs antibodies using an immobilized HBs vaccine (4).
1111 Showa 53-44620n. However, the method is not limited to K, and other known r-globulin recovery methods can also be used. For example, ammonium sulfate fractionation method,
Examples include DEAE-exchanger chromatography method and polyethylene glycol fractionation method. In addition, as an additional process,
Of course, the purified Jfll HB a antibody may be subjected to any known treatment (acid treatment, #substance treatment, polyethylene glycol treatment) in order to prepare an r-globulin preparation for intravenous injection. Thereafter, appropriate stabilizers and excipients are removed at %, followed by sterilization filtration if necessary, freeze-drying, and a formulation can be prepared according to a conventional method.

When using the method for producing a monoclonal HBtz antibody according to the present invention, it is possible to obtain cells that can be subcultured and have a better antibody-producing ability than when a normal HBss antigen is used as an immunogen. Furthermore, when using the production method of the present invention, there is little contamination with contaminant proteins and the recovery efficiency is extremely high. Furthermore, since the infectivity of the immunogen used in the present invention is reliably inactivated by heat treatment, safety is sufficiently maintained for manufacturers when practicing the invention.

The invention is further illustrated in the following examples.

Example 1 B 95-8 jllmtRPMI-1640K20
*Culture in a culture medium (hereinafter simply referred to as culture medium) to which beef tallow calf serum (hereinafter abbreviated as FC8) was added, and the culture supernatant on the 7th day was separated and TD. 1 O/l of EB virus source was obtained.

Human tonsil cells removed after surgery] Lymphoid cells
-7 Ficoll-Conray specific gravity centrifugation, and the immunogen of the present invention was added to 1 MI (1 to 2 x 10' cells/l). The immunogen was prepared by the following method.

HBm & cost 81: ('IES method) total pooled plasma 1
Fraction IV-1 obtained by fractionating Ooy using Cohn's ethanol fractionation method and a phosphate buffer solution of pH 7.6 at 1100°C.
After heating for 10 minutes, the resulting precipitate was removed, and urea was added to 180 mL of the supernatant with a tofu antigen titer of 16]C to a concentration of 6 molar, followed by heating at 85°C for 1 hour. After heating for 1 hour, the antigen titer was 32x. The solution is dialyzed under reduced pressure against physiological saline to remove phosphates and urea, and then sterilized and filtered to obtain a solution to be used as an immunogen.90 This solution is used as an ldf immunogen to culture the lymphoid cells described above. In addition to the culture medium (RPMI 164G + 10 to 2 oqb tallow calf blood), the cells were cultured at 37°C for 48 hours under 5SCO□, and then the above-mentioned 0°2d EB virus supernatant was added per 1 m of culture solution for infection. This was cultured statically for 3 weeks in a t-culture medium, and the antibody production was tracked using the tPHA method.In order to recover HBa antibody-producing cells from the proliferated cells, the immunogen Immobilized HBs antigen was obtained by covalent bonding to agarose activated with cyanogen bromide, which was adsorbed onto HBa antibody producing cells by t-contacting the cells with a sufficient amount of the antigen. The immobilized HBs antigen was added to a suspension of cultured cells.
7 and 1IIi were added.

After sufficient contact, the cells were washed with culture medium to remove unadsorbed cells.

The adsorbent was separated, suspended in the culture medium again, 1 mg/d of dakitranase was added, and the mixture was incubated at 23°C for 1 hour.
Time treatment. In this case, only the cells are separated by centrifugation,
The flBg antibody subculture produced cells are obtained. This cell'i
The cells were suspended in a medium with an initial concentration of i 0.5 to 1×lθ cells/d and cultured for 15 days. The culture solution was aliquoted every 3rd to 4th day, and the amount of HBm antibody purified from the culture solution was performed.

Purification was carried out by contacting 10 tf of the HBs antibody-containing culture solution with PHAil: 20,000 to the immobilized HBs antigen, adsorbing the HBI antibody, washing with Linlong buffer (PBS) of pH 7.2, and adding 3M KSCN solution. through H
Bs+ antibody is eluted, and after elution, KSCN is removed by dialysis against PBS under reduced pressure, and sterilization filtration is performed to show a PHA titer of 1:800X10', resulting in HBs% heteroantibody concentration r that can be injected into humans. -Glopurin solution 24dt- was obtained.

Example 2 Lymphocytes were isolated from human leukocytes using the Schiffercourt-Conley density centrifugation method.
PMI 164 G + 15% tallow baby blood fft+
The cells used in Example 1 and treated with daanidine instead of the friendly immunogen were used in a cell culture medium containing 2 mM glutamine (cell density of 1 to 2 x lO' cells/d) and cultured. Add 1 d of solution to 1 d of solution,
After culturing at 7°C for 48 hours, the EB virus supernatant prepared in Example 1 was added to each m of the culture solution for infection. After infection, the cells were statically cultured for 3 weeks under 5 sco.

To select HBm antibody-producing cells, cellulose and immunogen were covalently bonded using the method used in Example 1 to obtain immobilized HBs antigen for adsorption of HBI antibody-producing cells. Thereafter, HB Hatake antibody subcultured cells were obtained by the same procedure as in Example 1. This is 0.5
The cells were cultured in a medium for several days to form an initial dense layer of ~1X10' cells/d, and the HBs antibody was purified from this culture solution. Is refining carried out? The procedure was carried out in the same manner as in IJl to obtain 20 ml of a concentrated r-globulin solution of IBs-specific antibody (which can be injected into humans) with an antibody titer of 1.0OOXIO'.

Procedural amendment (spontaneous) December 23, 1980 Director General of the Patent Office 1. Indication of the case Relationship to Patent Application No. 170511 of 1988
Patent applicant 7, subject of amendment (1) Correct the application as per the attached correction application.

(2) The title of the invention in the specification, ``Method for producing monoclonal HBa antibodies'', is entirely corrected.

(3) Claims in the specification shall be corrected as per the attached sheet 0 (4) Specification page 2, line 6, page 2, line 8, page 4, line 1
In line 6, page 5, line 1, page 5, line 12, and page 12, line 6, the words "monoclonal" are corrected to "monoclonal."

(5) On page 4, line m1 of the specification, the word "monoclor" is corrected to "monoclonal."

(91 2, Claims (1) Manufacture of monoclonal human antibodies by contacting human B lymphoid cells with an immunogen, stimulating antibody production, and transforming the cells into a proliferative type that can be cultured as successors. HBs antigen heated in the presence of a protein denaturing agent such as whole urea or guanidine can be used as an immunogen to stimulate antibody production by B lymphocytes. Take it as a percentage! Z! Method for producing Ronaner B dust antibody.

(2) Human B lymphocytes stimulated to produce antibodies (i)
Epatein Barr, Virus K sensitization and transformation to cause proliferative cells, select and concentrate cells that produce HBa antibodies from these proliferative cells, ■ use the selected and concentrated cells as a growth medium. Grow with H
Ba antibodies were continuously produced and then HBs were extracted from the culture medium.
A method for producing a monoclonal HBa antibody according to claim (1), which comprises recovering the antibody.

Claims (2)

[Claims] (1) HBa anti-IfAt- Production of monoclonal HBs antibodies characterized by the ability to stimulate antibody production by B lymphocytes by heating them in the presence of protein denaturing agents such as urea and guanidine as whole immunogens. Method. (2) Q to B9 cells (stimulated antibody production)
Epstein Barr Virus F sensitization to cause transformation to produce proliferative cells, ■ Select and concentrate cells that produce HBs antibodies from these proliferative cells, ■ Use the selected and reduced cells as a growth medium. Grow with H
Bss antibodies are produced continuously and then HBs are extracted from the culture medium.
Claim No. (1) arising from the collection of a-antibodies
A method for producing a monoclonal human antibody as described in Section 1.