JPH04124142A - Production suppresser of anti-idiotype antibody - Google Patents

Abstract

PURPOSE:To obtain a production suppresser of anti-idiotype antibody induced by administration with a monoclonal antibody by containing an immunoglobulin as an active ingredient. CONSTITUTION:The objective production suppresser contains an immunoglobulin derived from the animal of species same as the origin of a monoclonal antibody, preferably a non-chemically modified gamma-globulin having a natural state and low anticomplementary value as an active ingredient. As the gamma-globuin, gamma-globulin for intramuscular injection is used and the gamma-globulin agglutinate causing anticomplementary action is preferably removed by using nonionic surfactant, although the gamma-globulin obtained by eliminating the agglutinate with an acid treatment is efficient. The immunoglobulin may be administered simultaneously with a monoclonal antibody or may be individually administrated with the monoclonal antibody. Thereby, side effect in administration of monoclonal antibody is conquered and the monoclonal antibody can be used for prevention or curing of a specific disease (e.g. virus infectious disease, hepatitis B or tumor).

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to new uses of immunoglobulins. Specifically, the present invention relates to an agent for suppressing the production of anti-idiotypic antibodies by administering a monoclonal antibody.

[Conventional technology]

High-titer globulin preparations generally refer to globulin preparations that have high antibody activity against specific antigens, for example,
Various viruses (cytomegalovirus, herpesvirus, HIV, influenza, etc.), virus-derived proteins (
Various bacteria (such as hard hepatitis B virus surface antigen and tetanus toxin)
Aeruginosa, streptococcus, staphylococcus, pneumococcus, etc.) or tumor-associated antigens (carcinoembryonic antigen, α-fetoprotein, etc.) It is being used for cancer treatment and is being considered for use in tumor treatment.

However, conventional high-titer globulin preparations are prepared using human-derived positive plasma as a raw material, and the titer is not sufficiently high, and it is difficult to obtain human-derived positive plasma. Problems such as the difficulty in obtaining products of uniform quality have also been pointed out.

On the other hand, techniques using monoclonal antibodies are being established as a method for preparing antibodies against specific antigens, and some are being considered for clinical application as globulin preparations.

[Problem to be solved by the invention]

However, since monoclonal antibodies are a homogeneous molecular population, it has been reported that when administered to conspecific animals, they induce the production of anti-idiotypic antibodies, which was not observed with plasma-derived globulin preparations (rmmuno
logy, 55.197-294.1985).

The produced anti-idiotypic antibody binds to the antigen-binding site of the administered monoclonal antibody, neutralizes the antibody activity, and weakens the ability to protect against infection.

That is, administration of a monoclonal antibody causes an immune response against the administered antibody, even if it is of the same species.

[Means to solve the problem]

Therefore, the present inventors conducted various studies in view of the above circumstances and found that the immune reaction against the monoclonal antibody could be avoided by administering the monoclonal antibody and immunoglobulin derived from the same type of plasma. That is,
The present invention was completed by discovering that the production of anti-idiotypic antibodies caused by administration of monoclonal antibodies can be suppressed by immunoglobulin.

(i) Plasma-derived immunoglobulin Plasma-derived immunoglobulin is derived from the same species as the monoclonal antibody (ii). The form of immunoglobulin is not particularly limited, and those prepared by known means can be used.

For example, non-chemically modified types (including PEG-treated types), chemically modified types (alkylated types, sulfosylated types), ion exchange resin-treated types (plasmin, pepsin, etc.), etc. are used. Examples include chemically modified γ-globulin.

The non-chemically modified γ-globulin of the present invention is: (1) Fab, F(ab'), which is natural and has not undergone any modification or change, and is therefore a fragment of γ-globulin.
2. Contains no Fc, etc. ■ No decrease in antibody titer or antibody spectrum at the same time ■ Anti-complement action (complement fixation) or 20 units (CH2O value).

It refers to something that has the following properties.

The non-chemically modified γ-globulin used in the present invention is
As long as it is in its natural state and has a low anti-complement value, it can be obtained by any method, or it can be manufactured using existing equipment and is already used as a medicine for intramuscular injection. The most efficient method is to use globulin and separate its aggregates with acidic treatment. However, if manufacturing complexity and yield reduction are not an issue, nonionic surfactants can be used to remove γ-globulin aggregates that cause anti-complement effects, and γ-globulin aggregates with low anti-complement value can be used. It is preferable to use globulin.

The preparation method may be performed by a known method, for example,
The method described in JP-A-53-47515, JP-A-57-32228, JP-A-63-183539, etc. may be followed.

It is also preferable to perform heat treatment. For example, as liquid heating, JP-A-61-191622, JP-A-63-1
No. 46832, and for dry heating, JP-A-61-787.
No. 30, No. 62-228024, No. 62-28393
No. 3, No. 62-289523, etc. may be followed.

The origin of plasma-derived immunoglobulin is not particularly limited. Specific examples include humans, mice, rats, etc., but humans are preferred.

(ii) Monoclonal antibody The monoclonal antibody is not particularly limited as long as it is derived from an animal of the same species as the immunoglobulin in (i).

The origin of a monoclonal antibody means the origin from the antibody-producing cell side. Therefore, when preparing monoclonal antibodies by the cell fusion method, either antibody-producing cells and proliferating cells are fused to prepare a hybridoma, and monoclonal antibodies are produced. The antibody-producing cells may be derived from the same species as the immunoglobulin (i).

In addition, in the case of the transformation method, antibody-producing cells are transformed with a virus to prepare transformants to produce monoclonal antibodies, or antibody-producing cells (i.e.
) immunoglobulin derived from the same species as the immunoglobulin.

These monoclonal antibodies are prepared by methods known per se.

This will be specifically explained using the cell fusion method.

As described above, the cell fusion method is performed by fusing antibody-producing cells and proliferative cells to prepare hybridomas.

Antibody-producing cells contain various viruses (cytomegalovirus,
Herpes virus, HIV, influenza) themselves, virus-derived proteins (hepatitis B virus surface antigen,
These cells produce antibodies against tetanus toxoid (tetanus toxin), various bacteria (pseudomonas aeruginosa, streptococcus, staphylococcus, pneumococcus, etc.), and tumor-related antigens (carcinoembryonic antigen, α-fetoprotein, etc.).

Antibody-producing cells are not particularly limited as long as they are derived from an animal of the same species as the immunoglobulin (i).

Specifically, those derived from humans, mice, rats, etc. are listed, and those derived from humans are preferably used.

Antibody-producing cells include tile cells, lymph node cells, and peripheral blood8
923 balls etc. are exemplified.

Examples of proliferative cells include myeloma cells.

As myeloma cells, those derived from mice, rats, humans, etc. are used, for example. For example, mouse myeloma P3
Examples include Ul, X63-Ag8-6.5°3, etc. Preferably, the antibody-producing cells and myeloid cells are derived from the same species.

Cell fusion can be achieved, for example, by C., Galfer (G.

Ga1fer) [Nature 266
, 550 (1977)] or a method similar thereto. At this time, the reaction is carried out using polyethylene glycol (average molecular weight t, ooo~4.000).

Monoclonal antibodies may be obtained by genetic engineering techniques. Furthermore, a chimerization technique may be used to create a chimeric antibody consisting of a C@ region derived from an animal of the same species as the plasma-derived immunoglobulin (i) and a V region derived from an animal other than that.

Furthermore, the monoclonal antibody may be fragmented by known means, such as Fab or Fab'.

The monoclonal antibodies obtained in this way are used for various viruses (cytomegalovirus, herpesvirus, HIV).
, influenza), virus-derived proteins (hepatitis B virus surface antigen, tetanus toxin), tumor-associated antigens (carcinoembryonic antigen, α-hue 1-
Examples include those against proteins (e.g. protein).

(ii) Usage and dosage of immunoglobulin The dosage of the immunoglobulin of the present invention may be about 1 to 100 times the dosage of the monoclonal antibody.

Regarding administration, the monoclonal antibody and immunoglobulin may be administered simultaneously or separately.

The preparation obtained according to the present invention can be administered as is in the case of a liquid preparation, or diluted with an appropriate solvent (e.g., distilled water for injection, physiological saline, glucose solution, etc.), or in the case of a dry preparation. is dissolved in a suitable solvent (for example, distilled water for injection) and used for prevention or treatment by intravenous administration, infusion, etc.

〔Effect of the invention〕

According to the present invention, side effects caused by administration of monoclonal antibodies (ie, those based on the production of anti-idiotypic antibodies) can be suppressed.

Therefore, by being able to overcome the side effects that have traditionally been a problem when administering monoclonal antibodies, it is possible to use monoclonal antibodies to prevent or treat specific diseases (infections caused by various viruses, hepatitis B, tetanus, tumors, etc.). I think this is now possible.

〔Example〕

EXAMPLES Examples and experimental examples are given to explain the present invention in more detail, but the present invention is not limited thereto.

Formulation Example 1 Fraction I [+H] obtained by Cohn's cold alcohol fractionation method
Dissolve 1 kg of the paste in 0.6% sodium chloride 101, adjust the pH to 3.8 with IN-hydrochloric acid, and incubate at 4°C for 6 hours.
The acid treatment was performed by stirring for 0 minutes.

Add 500g of polyethylene glycol with an average molecular weight of 4,000 to this solution, and while dissolving it, gradually increase the pE(5) with IN-sodium hydroxide.
, 0, and as soon as this value was reached, the precipitate was removed by centrifugation to obtain a clear supernatant. This supernatant has an average molecular weight of 4
,000 and added 700g of polyethylene glycol;
Adjust pH with IN-sodium hydroxide while stirring gently.
The temperature was adjusted to 8°0, and the immunoglobulin precipitated under these conditions was collected by centrifugation.

Highly purified γ-globulin can be prepared, for example, by adding 0.0% to physiological saline or 0.02M acetate buffer. It was redissolved in a solution containing 6% sodium chloride, 2% mannitol and 1% albumin, and subjected to sterilization filtration to obtain a clinically usable intravenous γ-globulin preparation.

Formulation Example 2 Cohn Fraction I obtained from human plasma by cold ethanol method [+11 kg and water IM? was added, and further 50 g of sorbitol was added per 100 d to adjust the pH to 5.5, followed by heat treatment at 60° C. for 10 hours.

After heat treatment, the pH was adjusted to 5.5, diluted 3 times with cold water for injection, and polyethylene glycol (average molecular weight 4.
000) was added to a final concentration of 6%, and centrifuged at 2°C.

The obtained supernatant was adjusted to pH 8 using IN=sodium hydroxide.
8, and then polyethylene glycol (average molecular weight 40
00) was added to a final concentration of 12%, and centrifugation was performed at 2°C to obtain an fgG fraction in the precipitate fraction. This precipitate was dissolved in water for injection, DEAE-Sephadex equilibrated with 0.4% saline was added to this solubility (2rrLl per 50d solution), and contact treatment was carried out at 0 to 4°C for about 1 hour. After the treatment, DEAE-Sephadex was removed by filtration to collect the filtrate ([gG solution)].

This [gG solution was adjusted to a 5% rgG solution with water for injection,
The pH of the solution was brought to about 5.5 with sodium acetate, and sorbitol was added to a final concentration of 5%. This aqueous solution (conductivity distance: about 1 mmha) was sterilized and filtered to obtain a liquid immunoglobulin preparation for intravenous injection.

This preparation substantially contains only IgG monomers, has an anti-complement value of about to-t5cHso, and passes the main criteria for intravenous immunoglobulin.

Experimental Example Effect of suppressing anti-idiotype antibody production by combined administration of monoclonal antibody and immunoglobulin (purpose) By repeatedly administering a monoclonal antibody, anti-idiotype antibodies against the monoclonal antibody are induced. Anti-idiotypic antibodies form immune complexes with monoclonal antibodies and not only reduce the efficacy of the administered antibody, but also cause various side effects due to the immune complexes.

The effect of mouse immunoglobulin (MGG) on the induction of anti-idiotype antibodies is investigated in a mouse model system. The antigen is hepatitis B virus surface antigen (HBs), and the monoclonal antibody is mouse anti-HBs monoclonal antibody (ID6).
Use.

(Method) Mouse: BALB/c, 6-8 weeks old, male antigen Komotsuklonal anti-HBs, 1D6-a (hereinafter abbreviated as ID6. ■Medori Juji) Polyclonal mouse IgG (below, abbreviated as MGG) ): MabTrap G (Ph
armacia).

Adjuvant: Ajuvant Bordetella
Pertussis.

BP (Kaken Pharmaceutical), Alu Get-s: Alum adjuvant, Alu (Serva) ID as antigen
Sensitization with 6 or a mixture of MGG and ID6 (see Table 1): Mice (1 group, 4-5 mice) ↓ BP, 3 X 10'10.5 i.p. injection ↓ 4
After hr, 0.54 (Alu+antigen) 1 intraperitoneal injection (1p
) ↓ IP administration of the above antigen three times every week ↓ Whole blood collection from the lower limb artery one week after the final immunization: Mix 1 Alu and 0.5 ml' of antigen (5 mg/J) for 1 hour. After standing, add 1.5 ml of physiological saline.

Preparation of ELrSA Coating of 96-well plate: 20 μg/
Add 1D6 (0.05m7) and leave it overnight in a cold room, wash, and block with 0.1-1.5% skim milk. Add 50μ of anti-idiotype antibody EL [SA: 1.06 to a coated plate. ! Add serum and 10 μl biotinylated HBs, 9.3 mg/mj' (genetically recombinant HBs, Y523-2), and incubate at 37°C.
Time - 6 times, washing → 50μl avine-peroxidase (Vector, BA3000゜2000 times diluted) 37°C 11 hours -〇-phenylenediamine + H2
0□→Colorimetric determination (A4Q2) (Results) Anti-idiotype antibodies in sensitized mouse serum (Figure 1, Table 1
Reference): The inhibition of the binding of biotinylated HBs to ID6 plates by anti-idiotype antibodies was measured. In group 1 without antigen ID6, no induction of anti-idiotype antibodies was observed. Increase in antigen (10-500 ag/ani
mal), the inhibitory ability increased. 8 for 500μg
It showed 7% inhibition.

There was no significant difference in the mean value of group 5 sensitized with ID610 μg + MGG 490 μg, because the inhibitory ability tended to be higher than that in group 2, which received ID6 alone, or the variation was large. Group 6 sensitized with 100 μg of 1D6 + 400 μg of MGG showed clearly lower inhibitory ability than Group 3 with ID6 alone. There was a significant difference between the two with a risk rate of less than 5%.

This result indicates that when the monoclonal antibody is administered in an amount sufficient to induce anti-idiotype antibodies, the induction of anti-idiotype antibodies is suppressed by MGG spikes.

Table 1 Anti-idiotype antibody width in serum of sensitized mice: No difference due to upbringing ”: <0.05%, significant difference Significant difference test is 5tu
dent' was performed by method.

[Brief explanation of drawings]

FIG. 1 is a graph showing the results of an experimental example. (3 others)

Claims (1)

[Claims] An agent that suppresses the production of anti-idiotypic antibodies by administering a monoclonal antibody, which contains immunoglobulin as an active ingredient.