RU2385164C1 - APPLICATION OF Fc-FRAGMENTS OF IMMUNOGLOBULIN CLASS G AS ANTIGEN FOR TREATING RHEUMATOID ARTHRITIS, REMEDY AND METHOD OF TREATING - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention refers to medical technologies, immunology, pharmacology and particularly to drugs for treating the patients with rheumatoid arthritis (RA) and to methods of treating thereof to be used in rheumatology. Substance of the invention includes application of Fc-fragments of immunoglobulin class G as an antigen for treating RA, a remedy for treating RA, produced by mixing the antigen and an adjuvant, and the related method of treating RA. ^ EFFECT: improved effect to suppress autoimmune response. ^ 6 cl, 3 ex, 4 dwg

Description

The invention relates to medical technologies, immunology, pharmacology and, in particular, to medicines for the treatment of patients with rheumatoid arthritis (RA) and methods for their treatment, which can be used in rheumatology.

Nonsteroidal anti-inflammatory drugs, immunosuppressants, glucocorticoids are traditionally used to treat RA. The new directions in the treatment of RA is the use of monoclonal antibodies against inflammatory cytokines [1, 2, 3]. All these drugs and methods of treating RA are aimed at suppressing the realization of the pathogenic effect of autoantibodies or autoactive cells - autoimmune inflammation, but do not suppress the autoimmune process. Associated with this is their relatively low efficiency. A significant drawback of these drugs is also the presence of a large number of pronounced side effects and a large number of contraindications.

To date, clinical trials of a T-cell vaccine for the treatment of PA are being conducted [4]. A known method of producing a polyclonal T-cell vaccine for the treatment of PA [5], selected as a prototype of the method, which includes two stages: at the first stage, the patient’s mononuclear cells are cultured in the presence of an antigen - collagen proteins, after which the antigen-reactive blast T cells are separated from unreacted cells by gradient centrifugation on percol; at the second stage, the isolated T cells are increased by means of their nonspecific mitogenic stimulation, irradiated, then cryopreserved. The specificity of vaccinal T-lymphocytes is evaluated by their antigen-induced proliferative response. The immunotherapy regimen includes 4 weekly vaccinations with an interval of 1 month. However, vaccination was carried out against the background of basic therapy with well-known drugs (methotrexate, etc.), so the effectiveness of T-cell vaccination cannot be unambiguously assessed. In addition, despite the fact that this method has been known for a long time, it has not found wide application in clinical practice, which may also be a consequence of its low efficiency.

In immunochemistry, Fc fragments of immunoglobulin class G (IgG) of humans and animals are known, in particular, for use as an antigen for the production of anti-species antibodies in the construction of diagnostic enzyme-linked immunosorbent assays [6]. Methods for the isolation and purification of IgG Fc fragments are known from [7, 8]. To date, there have been no drugs or vaccines based on IgG Fc fragments, and therefore IgG Fc fragments have not been used to treat any diseases.

It is known to use non-specific human immunoglobulin preparations for autoimmune diseases, such as autoimmune thrombocytopenia, dermatomyositis [9]. Preparations of non-specific human immunoglobulins are closest in chemical nature to Fc fragments of human IgG. It is also known to use human immunoglobulin for intravenous administration in the treatment of rheumatoid arthritis, DAVID E. YOCUM, Use intravenous Immunoglobulin in therapy rheumatoid arthritis, in MARTIN L. LEE, VIBEKE STRAND, Intravenous immunoglobulin in clinical practice, 1997, pp.409-415. The specified tool is selected as a prototype. However, these drugs are not a vaccine and are not used for the treatment of RA due to their low efficacy in RA and variability of results [9].

An object of the present invention was to provide an agent (vaccine) and a method for treating RA based on it with a pronounced effect of suppressing an autoimmune reaction.

As a result of scientific studies, they found the effect of suppressing the production of specific autoantibodies that trigger autoimmune inflammation in arthritis, and an improvement in the clinical manifestations of arthritis during immunization with IgG Fc fragments. The rationale for the use of IgG Fc fragments as an antigen in the treatment of RA was our studies on an experimental model of rat collagen-induced arthritis, which showed the dependence of the development of arthritis on the activity of lymphocytes producing rheumatoid factor (RF) in response to arthritis inducer. The high activity of lymphocytes producing RF was associated with resistance to arthritis [10]. Due to the fact that IgG Fc fragments are an antigen for lymphocytes producing RF, they can be used to stimulate the latter.

The technical result is achieved by obtaining funds (vaccines) for the treatment of rheumatoid arthritis (hereinafter - the means) based on antigen and adjuvant (adjuvants are substances that enhance the immune response). As an antigen in the composition of the agent (vaccine), Fc fragments of human IgG are used. For example, Fc fragments of human IgG with a purity of at least 95% are used, mixed in buffered saline solution with an adjuvant until a stable emulsion is obtained, and buffered saline solution is prepared from physiological saline and 0.15 M sodium phosphate buffer, pH 7.2-7, 4 in a ratio of 9: 1. The adjuvant may be Freund's incomplete adjuvant or ISA-51 Montanid.

The technical result is achieved in a method for the treatment of rheumatoid arthritis, including immunization with an agent (vaccine) obtained by combining an antigen and an adjuvant in a dose sufficient to elicit an immune response to the antigen, using IgG immunoglobulin Fc fragments as antigen.

The invention is illustrated by drawings:

- figure 1 shows the change in the tumor of the affected limbs (in points) in experimental and control animals (m ± σ). The arrow shows the introduction of the proposed funds (vaccines) to experimental rats and the introduction of NAF control rats;

- figure 2 shows the change in the number of affected limbs in experimental and control animals (m ± σ). The arrow shows the introduction of the proposed funds (vaccines) to experimental rats and the introduction of NAF control rats;

- figure 3 shows the kinetics of the rheumatoid factor in the peripheral blood of experimental and control rats before and after the introduction of funds (vaccines) based on Fc fragments of rat IgG obtained as described in example 1. The arrow shows the introduction of the proposed funds;

- figure 4 shows the kinetics of autoantibodies to type II collagen in the peripheral blood of experimental and control rats before and after the administration of an agent (vaccine) based on Fc fragments of rat IgG obtained as described in example 1. The arrow shows the introduction of the proposed agent.

The technical solution of the problem was carried out in two stages.

1. Preparation of funds (vaccines).

An agent (vaccine) was prepared by combining a solution of human IgG Fc fragments with an adjuvant. The method of connection depends on the properties of the used adjuvant. To obtain purified Fc fragments of human IgG, various methods can be used that provide the necessary degree of purity and immunogenicity of Fc fragments of human IgG.

2. The method of treatment of RA

It was carried out by immunization of patients with RA with a prepared agent (vaccine), and immunization is possible. Any known route of administration of the agent (vaccine), with the exception of intravenous, may be used. The choice of method of immunization depends on the immunogenic form of the agent (vaccine).

Example 1. The tool (vaccine) based on Fc fragments of IgG for rats

The agent (vaccine) was obtained by mixing a solution of Fc fragments of rat IgG with a concentration of 5.6 mg / ml (purity not less than 95% according to electrophoresis) in buffered saline with incomplete Freund's adjuvant (NAF) from Sigma to obtain a stable emulsion, for example 1: 1 (or in any ratio that allows you to get a stable emulsion). 1 dose of the drug contained 100-500 μg of Fc fragments of rat IgG. In this case, Freund's adjuvant composition, well known and commercially available, was used. The buffered saline solution consisted of 9 parts of physiological saline and 1 part of 0.15 M sodium phosphate buffer, pH 7.2-7.4.

Example 2. Means (vaccine) based on Fc fragments of IgG for humans

The agent (vaccine) was prepared from Fc fragments of human IgG dissolved in buffered saline mixed with adjuvant ISA-51 Montanide in a 1: 1 ratio (Syntex adjuvant based on a water-in-oil emulsion containing squalene, Plunorik L121, Tween 80) also commercially available from Syntex. Upon receipt of the agent, not only a standardized allogeneic preparation of human IgG Fc fragments was used, but also the patient’s IgG Fc fragments — an autoantigenic preparation.

Example 3. A method of treating rat RA

Rats with collagen-induced arthritis in the stage of pronounced clinical manifestations of arthritis were immunized with the agent (vaccine) obtained according to Example 1. The agent was administered once intradermally and subcutaneously in a volume of 200 μl at a dose of 100-500 μg per rat.

To confirm the effectiveness of the proposed funds (vaccines) in RA, a model experiment was conducted. The effect of IgG Fc fragments on arthritis was studied using a model of rat collagen-induced arthritis, which is an adequate model of human RA [11]. Arthritis in white rats (m = 150-180 g) was caused by the administration of bovine type II collagen at a dose of 400 μg as a part of NAF intradermally. On the 14th day after immunization with bovine collagen, immediately when the first signs of arthritis appeared, the rats were immunized according to Example 3 with the preparation prepared in Example 1. Vaccinated animals made up the group of experimental animals (n = 7). The control group consisted of arthritic rats (n = 7), which were injected with Freund's incomplete adjuvant mixed with physiological saline in an equivalent volume and according to the same scheme as in the experimental group.

Within 5 weeks after the administration of the drug (vaccine), the clinical picture of arthritis was observed according to the indicators accepted in medical and research practice, reflecting the severity of arthritis, such as the severity of the tumor of the affected joints and the number of paw affected by arthritis. In addition, we studied the effect of the proposed tool on autoimmune reactions in arthritis, determined by the level of autoantibodies to type II collagen and the level of RF in the blood. The tumor of the affected limb was measured in points (1 point - slight swelling of several joints of the paw, 2 points - significant swelling of several joints of the paw, 3 points - swelling in the area of many joints of the paw, 4 points - a strong tumor of the entire paw, but the animal proceeds to the paw, 5 points - a strong swelling of the paw, the animal does not step on the paw, does not move).

In control animals, a paw tumor significantly increases within two weeks after the first appearance of arthritis (Fig. 1), during the first week after the first clinical manifestations, the number of affected limbs grows - new paws are involved (Fig. 2).

The development of the clinical picture (tumor enlargement of the affected joints, involvement of new joints) within 2 weeks after the first manifestations is typical for the rat KIA model [10]. In experimental animals that received the drug, neither an increase in the tumor of the affected extremities was observed (Fig. 1), nor the involvement of new paws in the pathological process (Fig. 2). In experimental animals, the tumor not only did not increase, but even subsided two weeks after the onset of clinical manifestations (Fig. 1) and was not observed for 3 weeks, there remained small thickenings in the area of the affected joints, which passed within the next three weeks. In control animals, as shown in figure 1, the paw tumor was observed for a long time, within 5 weeks after the onset of arthritis, and subsided only 6 weeks after the onset of arthritis with the formation of pronounced thickenings in the area of the affected joints.

Despite the fact that a high standard deviation from the average number of affected paws (figure 2) does not allow us to judge the significance of differences in the first two weeks after administration of the drug (vaccine), nevertheless, the observed limitation of the development of arthritis in vaccinated rats is further implemented in a significant difference in the number of affected paws between the animals that received the vaccine and the control. So, figure 2 shows that in animals that did not receive the drug (vaccine), the number of affected paws remains unchanged for 5 weeks, while in animals that received the drug, 3 weeks after injection, the state of all previously affected paws returns to normal . From this it follows that in rats immunized with the proposed agent, a vaccine prepared as described in Example 1, the development of the clinical picture of arthritis is suppressed, and a reduction in the symptoms of arthritis is observed.

To confirm that the proposed vaccine acts on an autoimmune reaction, titers of autoantibodies were determined: titer of autoantibodies to type II collagen and titer of rheumatoid factor. The titer of autoantibodies to type II collagen was determined in ELISA, RF in the agglutination reaction of erythrocytes loaded with rat IgG (Sigma).

Experimental animals in response to the introduction of funds (vaccines) there was a gradual decrease in the titer of the Russian Federation (figure 3) 5 weeks after the introduction of the proposed funds (which corresponds to 7 weeks after immunization with BC) RF in the blood of vaccinated animals was not determined (figure 3). While in animals that did not receive the drug (control), the titer of the Russian Federation gradually increased and at 7 weeks after immunization with CD was 1: 1024 ± 451 (figure 3).

The titer of autoantibodies to type II collagen in experimental animals decreased and remained low in the range of 1: 100 ± 58 to 1: 267 ± 189 in response to the administration of the proposed agent, and the average period from 3 to 7 weeks after immunization with CD was 1: 160 ± 88 (Fig. 4). In control rats, the level of autoantibodies to type II collagen during the observation period varied significantly from 1: 100 ± 100 to 1: 3200 ± 1600, which is typical for the kinetics of autoantibodies in arthritis, and on average for the observation period from 3 to 7 weeks after immunization BC was 1: 1750 ± 680. Thus, in response to the introduction of the proposed agent, both the RF level and the level of autoantibodies to type II collagen are significantly reduced, while in control animals the titers of these autoantibodies increased during the entire observation period.

A decrease in the level of autoantibodies to type II collagen, the rheumatoid factor in response to the administration of the agent, confirmed the suppression of the autoimmune reaction in RA as a result of immunization with an agent containing IgG Fc fragments and an adjuvant.

It is generally recognized that the experimental model of collagen-induced arthritis in rats is the most adequate model for the development of RA in humans [11]. However, to conduct effective treatment of RA in humans with an agent based on Fc fragments of human IgG, the vaccination schedule (dose, number of repeated administrations, and also the adjuvants used) can be individual for each patient due to the severity of the disease, stage, and the body's ability to express an immune response to Fc fragments of IgG in parallel with drug therapy, etc. In human experiments, the used incomplete Freund's adjuvant is replaced by the commercial adjuvant Montanide (Syntex, France), working on the principle of Freund's incomplete adjuvant, but with minimal side effects (primarily inflammatory).

Thus, from the results of experimental studies, we can conclude about the effectiveness of the proposed means and method of treating RA based on it for humans and animals.

Information sources

1. Puppo F, Murdaca G, Ghio M, Indiveri F. Emerging biologic drugs for the treatment of rheumatoid arthritis // Autoimmun Rev. - 2005. Vol. 4. - P.537-541.

2. Till A.R., Gary T. Jennings, Marcela Hemandez, Paula Grest2, Markus Beck, Yu Zoul, Manfred Kopf and Martin F. Bachmaim Vaccination against IL-17 suppresses autoimmune arthritis and encephalomyelitis // J. Immunol. - 2006. - Vol. 36. - P.2857-2867.

3. Baslund B, Bendtzen K. Biopharmaceuticals in the treatment of rheumatoid arthritis // Ugeskr Laeger. - 2008 .-- Vol. 170. - P.2108-2110.

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6. Nikolaenko I.V., Galkin A.Yu., Raevskaya G.E. and others. - Obtaining monoclonal antibodies to the Fc fragment of human IGG and the use of enzyme-linked immunosorbents based on them. // Clinical laboratory diagnostics. - 2005. - No. 11. - S.8-11.

7. Andrew SM, Titus JA. Fragmentation of immunoglobulin G. // Curr Protoc Cell Biol. 2003. Chapter 16. Unit 16.4.

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Claims (6)

1. The use of Fc fragments of immunoglobulin class G as an antigen for the treatment of rheumatoid arthritis. 2. An agent for treating rheumatoid arthritis obtained by combining an antigen according to claim 1 and an adjuvant. 3. The tool according to claim 2, characterized in that the Fc fragments of class G immunoglobulin with a purity of at least 95% in physiological saline buffered with 0.15 M sodium phosphate buffer pH 7.2-7.4 are mixed with an adjuvant to obtain persistent emulsion. 4. The tool according to claim 2, characterized in that as an adjuvant use incomplete Freund's adjuvant. 5. The tool according to claim 2, characterized in that as an adjuvant use Montanide ISA-51. 6. A method of treating rheumatoid arthritis, comprising immunizing with the agent obtained according to claim 2, in a dose sufficient to elicit an immune response to an antigen.

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