JPS6156220B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing immunosuppressive anti-human lymphocyte globulin (hereinafter referred to as AHLG). More specifically, as an immunogen, cultured Namalwa
The present invention relates to a method for producing immunosuppressive anti-human lymphocyte globulin derived from animals (excluding humans), which uses cell lines. Anti-human lymphocyte globulin has the effect of suppressing the immune response in vivo in patients who receive it. This globulin makes it possible to transplant skin, organs, etc. to patients to whom it is administered, by causing lymphocytes to lose their ability to carry out immune functions in vivo. Several methods for producing AHLG are already known. In particular, a method is known in which human lymphocytes are administered to animals as immunogens, blood from the animals is extracted, and antilymphocyte globulin (AHLG) is produced therefrom. However, fresh lymphocytes used as immunogens contain mixed immunogens such as red blood cells and platelets, which causes side effects in AHLG produced from these. To solve these problems, immunogen selection,
Development is underway. For example, a method using monkey-derived lymphocytes instead of human lymphocytes (British Patent No. 1303750), a method using a soluble extract of human lymphocytes (Japanese Patent Application Laid-Open No. 1983-2047), a method using human thymocytes (Japanese Patent Application Laid-open No. 1986-2047), Selection from five or more types of cultured human lymphoid cells (French Patent No. 2153193), and a method using a non-cellular supernatant of culture fluid of cultured lymphocytes (Japanese Patent Application Laid-open No. 1972-1902) −116222
No.) etc. are disclosed. However, although these methods have been able to significantly reduce the amount of mixed antibodies, they still only yield AHLG, which has side effects, and its use is still considerably restricted. That is, the above two methods using cultured lymphocytes are the methods with the most industrial advantages. In particular, AHLG is sterile and can be used in large quantities, and furthermore, through subculture, AHLG can be obtained with relatively low levels of mixed antibodies. However, even with this method, it is difficult to select lymphocytes, and normal lymphocytes cannot be cultured at this stage, so mutated cells must be used in some way. Therefore, AHLG produced using this immunogen will again contain other mixed antibodies, leading to new toxicity and this
The clinical use of AHLG is limited. In addition to the pursuit of immunogens, a method for removing contaminating antibodies from AHLG by fractional purification has also been disclosed. However, in these methods, it is difficult to obtain AHLG in a yield that can be used for therapeutic purposes, and furthermore, they are expensive and require complicated operations. With such prior art as a background, the inventors attempted to find a method for producing AHLG that is industrially suitable and free of toxicity and side effects by examining immunogens. Since the best method to date is to use cultured lymphocytes as immunogens, we selected cells that matched our purpose from among a large number of mutant lymphocytes. As a result, the present inventors discovered a mutant lymph cell that retained unprecedented safety and high lymphocyte antigenicity and completed the present invention. These are Namalwa cells, which are conventionally known as interferon-producing cells, and these cells have almost no ability to produce mixed antibodies.
Moreover, it has extremely low toxicity and high antilymphocyte activity.
It was found that AHLG is produced. The present invention provides an anti-human lymphocyte immunogen that is obtained by administering an immunogen obtained from cultured lymph cells to an animal (excluding humans) and collecting the γ-globulin of the antiserum generated from the plasma of the animal (excluding humans). Provided is a method for producing anti-human lymphocyte globulin, which is characterized in that Namalwa cell line cells are used as cultured lymphocytes in the production of lymphocyte globulin. The present invention will be explained in detail below. The Namalva cell line used in the present invention is a cell line selected from 20 types of lymphocyte lines derived from patients with Burkitt's lymphoma and leukemia, and this cell line is currently the cell line that produces the most interferon. known as. (Intern.J.Cancer
11 , 327 (1973)) The culture and propagation of this Namalva strain requires cell
RPMI1640 (Rosen Park Memorial Institute 1640) culture solution (released by Nissui Pharmaceutical Co., Ltd.)
The cells are suspended in a medium containing 8-15% fetal bovine serum. Passage is carried out once or twice a week, when the cell density is in the range of 1 to 5 x 10 ⁶ cells/ml. The culture conditions are preferably carried out at 37°C under a 5% carbon dioxide atmosphere, but are not particularly limited thereto. ^Cell proliferation reaches saturation in about 5 days in a 250 ml container and increases about 25 times. Sufficiently grown Namalva cell lines are 1000
The cells are centrifuged at ~1300 rpm x 10-20 min and collected as a precipitate. This was thoroughly washed with RPMI1640, and finally 2 to 5 × 10 ⁹ pieces/20
Prepare a cell suspension in mlRPMI1640. This preparation can be used for immunization as is, or the cells can be destroyed by ultrasonication, glycocol treatment, etc. Also, of course,
By repeatedly finding a fraction with higher antigenic specificity from this disrupted suspension, it can be used more efficiently. The immunogenic substance thus prepared is stored in a cold room below 10°C or lyophilized and used as necessary. Immunization of animals other than humans (preferably horses) is carried out using a single immunogen dose of 1 to 50 x 10 ⁹ cells/20 ml or its equivalent, either directly or in an equivalent amount of Freund's complete supplement. complete adjuvant)
Mixed with emulsion and applied at 2 week intervals for weeks 0 and 2.
Weekly and administered subcutaneously. 1-50×10 ⁹ cells are administered intravenously at 4 weeks. Thereafter, the titer of plasma collected from animals (excluding humans) is measured and booster immunizations are performed if necessary. Blood sampling will be done approximately 1 to 1 time per week after 4 weeks.
At step 15, blood sampling is performed by blood cell recirculation method. AHLG is recovered from plasma as follows. The obtained plasma is treated with an anticoagulant (such as heparin), and γ-globulin is recovered from the supernatant after centrifugation by a known method. If the supernatant contains a large amount of hemoagglutinin or anti-human protein antibodies, it is optionally treated with human red blood cells and human plasma to remove the respective antibodies. The simplest treatment is to remove the precipitate generated by the antigen-antibody reaction by centrifugation, but the most efficient method is to use the principle of affinity chromatography in which each antigen is adsorbed onto a carrier. It is preferable to use γ-globulin is recovered from animal (other than human) serum (hereinafter abbreviated as AHLS) from which unnecessary antibodies have been absorbed and removed, if necessary, by a known method. In other words, add 25 to 50% ammonium sulfate to serum that does not contain unnecessary antibodies.
to the saturation and recover AHLG. A more preferred method is to remove the fraction that precipitates at 25-30% saturation, and then collect the fraction that precipitates at 40-50% saturation. Another method is to contact AHLS with a weakly basic anion exchanger (e.g. DEAE-Cellulose, DEAE-crosslinked dextran, where DEAE represents a diethylaminoethyl group) to adsorb and separate components other than globulin. It's a method. The ion exchanger is not particularly limited, but equilibration conditions of pH 6 to 8 are preferred. Distilled water is convenient. AHLG remaining unadsorbed was 42% after filtration.
Obtained as a precipitate by adding ammonium sulfate to saturation. Other preferred methods include a method for producing intravenously injectable γ-globulin using a polyglycol copolymer (British Patent No. 1435816), a method for producing intravenously injectable γ-globulin using polyethylene glycol (British Patent No. 1372953), Purification of γ-globulin using acrinol (US Pat. No. 3,607,857); Process for producing γ-globulin using ethanol (US Pat. No. 2,543,215)
No. 2520076, No. 2437060, etc. are available. As additional treatments, known treatments with trypsin, plasmin, etc. to inactivate anti-complement activity, and known heat treatments at 60°C for 10 hours to inactivate pathogenic viruses such as hepatitis viruses, can be carried out. Be free. After centrifugation, AHLG is redissolved, filtered, and then dialyzed against distilled water or physiological saline for about 24 hours.
After dialysis, the aqueous solution is filtered for sterilization and then dispensed. Dispense 1 ampule from 4 to 15 ml or 50 mg to 50 mg of AHLG protein if freeze-dried.
Separate into 1000mg packets. The characteristics of the obtained AHLG preparation can be examined by the activity test described below. (1) Cytotoxicity test A lymphocyte cytotoxicity test is performed as a titer test for AHLG. Lymphocytes are prepared by the Ficoll method (Clinical Examination, 18 , 7, 1974) from helimph blood collected from a normal person with type 0 blood.
The obtained lymphocytes are adjusted to a suspension of approximately 1.5×10 ⁴ lymphocytes/ml with barbital buffer, and diluted 5 times with rabbit serum containing complement. Make a 2-fold dilution series of the sample with physiological saline. Each serial dilution of the specimen and each lymphocyte suspension
Transfer 0.1 ml each to a small test tube with an inner diameter of 5 to 6 mm and a length of 70 to 80 mm, keep at 37℃ for 90 minutes, centrifuge at approximately 1550 rpm for 6 minutes, and add 3 drops of 2% Trypan blue to the precipitate. Add and leave for 5 minutes. Judgment will be made immediately after staining. Judgment was made by counting the number of stained white blood cells under a microscope, and when it was 20% or more, it was considered positive, and when it was positive at a dilution of ×80 or more, the titer of AHLG was considered to meet the standard. The comparative experiments shown in Table 1 used Namalva strain according to the method of the present invention as well as human lymphocyte membrane components and human peripheral blood lymphocytes as immunogens, and the titer of the specimen obtained in each case was determined to be the maximum that was judged to be positive. The course after immunization was expressed as a dilution factor. As a result, obtained by the method of the present invention
AHLG has sufficient potency.

[Table] (2) Rosette formation inhibition potency The ability of AHLG to inhibit aggregation of immune cells is measured by
Rosette Test (The Journal of Immunology)
113 266 (1974)). Approximately 1.5x lymphocytes prepared by the Ficoll method
Suspend ¹⁰⁷ cells/ml in barbital buffer. Sheep red blood cells (SRBC) are approximately 1.0× ¹⁰⁸ cells/
Suspend in saline to ml. Mix 0.2ml of lymphocyte suspension and 0.25ml of SRBC in a small glass test tube.
(Mixing ratio of lymphocytes and SRBC is 1:8-10)
After adding 0.55 ml of Hank's solution and incubating at 37°C for 15 minutes, centrifugation is performed at 1500 rpm for 5 minutes. After allowing the precipitate to stand at 4° C. for 1 to 2 hours, 0.2 ml of Hank's solution is added and the test tube is shaken up and down or rotated in a circular motion to gently resuspend the lymphocytes. Lymphocytes with three or more SRBC aggregated around them are considered rosette-forming lymphocytes.
Calculate the rosette formation rate. To prevent rosette formation, sample the sample before adding SRBC.
It occurs by incubating lymphocytes with AHLG at 37°C. That is, 0.2 ml of lymphocyte suspension, 0.5 ml of diluted specimen, and 0.05 ml of 5-fold diluted guinea pig complement absorbed with human red blood cells and sheep red blood cells were mixed, incubated at 37°C for 90 minutes, and then 0.25 ml of SRBC suspension was added. Thereafter, the rosette formation rate is measured in the same manner as described above.
A rosette suppression rate of 25% or more is considered positive. The results of the comparative experiment shown in Table 2 below are based on the AHLG titer obtained in each case using the Namalva strain of the method of the present invention and human peripheral blood lymphocytes as immunogens, and the maximum dilution factor that was determined to be positive. The transition after immunization is shown. The results are shown in Table 2. As a result, there is no particular difference in the potency of AHLG of the present invention compared to others, and it has sufficient potency.

[Table] (3) Measurement of hemagglutinin titer The anti-erythrocyte antibody activity of AHLG is measured by the hemagglutinin titer. Red blood cell suspension is O, A,
Erythrocyte types B and AB were washed twice with 0.9% saline, and a 2% erythrocyte sediment layer was suspended in 0.9% saline to prepare. In a test tube (inner diameter 7 mm, length 80 mm), add 0.1 ml of this red blood cell suspension and 0.1 ml of a 0.9% saline dilution of the sample.
ml, shake to mix well, and centrifuge at 1000 rpm for 1 minute. To measure, shake the tube just enough to remove any blood clots at the bottom of the test tube, and read whether red blood cell clots have formed. The highest dilution that produces a clot is the hemagglutinin titer of the specimen. Comparative experiments used the Namalva strain of the invention as well as human lymphoid membrane components and human peripheral blood lymphocytes as immunogens and showed the course of the AHLG titers obtained in each case. The results are shown in Table 3. As a result, the AHLG of the present invention has virtually negligible anti-erythrocyte antibody activity.

[Table] (4) Platelet aggregation titer The antiplatelet antibody activity of AHLG can be assayed by an agglutination method. Platelet-rich plasma (PRP) is obtained by centrifuging type O human blood (heparin blood) at 1000 rpm for 10 minutes. This PRP contains approximately 300,000 platelets/ ^mm3 . Mix 0.05 ml of this PRP and an equal amount of AHLG at each dilution ratio on a slide glass with a hole at room temperature.
Cover with a cover glass and observe the presence of platelet aggregation under a microscope after 30 minutes. Aggregation of three or more platelets was regarded as aggregation, and the sample dilution factor at which this aggregation was observed was defined as the platelet aggregation titer. Comparative experiments were conducted using the Namalva strain of the method of the present invention as well as human lymphocyte membrane components and human peripheral blood lymphocytes as immunogens, and the AHLG obtained in each case.
The change in titer is shown. The results are shown in Table 4. As a result, obtained by the method of the present invention
AHLG has almost completely no antiplatelet antibody activity.

[Table] (5) Local irritation test The local irritation of AHLG is tested by macroscopic and histological examination. Test weight 2.5-3.0
10 g of AHLG freeze-dried specimen prepared from blood collected 4 weeks after immunization from 3 Kg male rabbits per group.
Dissolve in 2 ml of physiological saline and administer 1 ml. For administration, the hair on the back of a rabbit is shaved, and the test sample is injected into the center of the left and right dorsal sacrospinous muscles, respectively. On the 2nd and 7th day of administration, the rabbits were killed by bloodletting, and a scalpel was placed in the center of the injection site to observe muscle changes.
Perform a visual inspection according to the criteria shown in (1).
Furthermore, the muscle tissue of the area is removed, fixed with Bouin's solution, HE stained, and histologically examined according to the criteria in (2). (1) Visual inspection criteria

[Table] (2) Criteria for histological examination The disorders were classified as follows, taking into account the nature and intensity of changes such as hemorrhage, cell infiltration, edema, degeneration, necrosis, and fibrosis in muscle tissue.

[Table] Table 5 shows the average score of the findings on the 2nd and 7th days when the Namalva strain, human lymphocyte membrane components, and human peripheral blood lymphocytes were used as immunogens in a comparative experiment.
It was shown to. As a result, AHLG obtained using the Namalva strain as an immunogen did not exhibit any irritation.

[Table] (6) Skin graft experiment Using 15 chimpanzees, the AHLG of the present invention was
100 mg/Kg/day was administered intravenously continuously for 10 days to examine the effect on skin grafts. The drug was administered in advance 24 hours before transplantation, and then once a day.
Surface observation after 12 days showed a survival rate of 100%, compared to 0% in the control. This suggests the effectiveness of the pharmacological effect of this drug as an immunosuppressant. (7) Acute toxicity test An acute toxicity test of the AHLG of the present application was conducted using 1 group of 5 rats weighing approximately 200 g. After dissolving each compound in physiological saline, 500mg/Kg, 1000mg/
Kg, 1500mg/Kg, 2000mg/Kg were administered intraperitoneally, 5
Although observation was conducted for several days, no deaths were observed. Thus, from the Namalva strain according to the present invention.
The method for producing AHLG makes it possible to obtain standardized antiserum on an industrial scale, which was previously impossible, because the Namalva strain is extremely easy to expand and has an extremely high proliferation rate. The AHLG obtained by this method has high lymphocyte activation and immunosuppressive ability, and is virtually free of the side effects of anti-erythrocyte and anti-platelet activities that have limited the use of anti-lymphocyte globulin in humans so far. Furthermore, the present invention provides an extremely safe method for producing pharmaceuticals, since there is almost no toxicity or local irritation due to cultured cells. Example Namalva cells were subcutaneously administered to horses in an amount of 20×10 ⁹ cells at week 0 and week 2, and at week 4, 20×10 ^{9 cells} were administered subcutaneously to horses.
Administer the cell amount intravenously. At the 5th week, plasma of No. 5 was collected by blood cell recirculation. Add heparin sodium injection (manufactured by Midori Juji Co., Ltd.) to the obtained plasma.
50,000 units (5 ml) were added, centrifuged at 1,000 rpm for 10 minutes, and the supernatant was collected. Add ammonium sulfate to this supernatant.
Add to 30% saturation, remove precipitate, then 40%
Fractions that precipitated upon saturation were collected. The obtained precipitate was dissolved in distilled water (1) and dialyzed against a sufficient amount of physiological saline. This dialysate is 0.22μ
Sterilization filtration was performed using a filter with a diameter of . This filtrate was freeze-dried to recover about 20 g of AHLG. About this AHLG, cytotoxicity test,
A rosette test, red blood cell aggregation test, platelet aggregation test, local irritation test, skin transplantation test, and acute toxicity test were conducted, all of which yielded good results. Furthermore, the presence or absence of serum antibodies was investigated using the Ouchterlony method and multilayer sedimentation method, but no serum antibodies were detected.

Claims (1)

[Scope of Claims] 1. An anti-human anti-human drug comprising administering an immunogen obtained from cultured lymphocytes to an animal (excluding humans) and collecting antiserum γ-globulin produced from the plasma of the animal (excluding humans). - A method for producing anti-human lymphocyte globulin, which comprises using Namalwa cell line cells as cultured lymphocytes in the production of lymphocyte globulin. 2. The method according to claim 1, wherein the recovery of antiserum γ-globulin includes the step of removing hemoagglutinin or anti-human protein antibodies. 3. The method according to claim 1, wherein the immunogen is cultured Namalva cells themselves. 4. The method according to claim 1, wherein the immunogen is a disrupted product of cultured Namalva cells.