Method for the prevention and treatment of reaction of graft-versus-host stimulation antiidiotypic suppression of donor lymphocytes in experimental models


(57) Abstract:

The invention relates to medicine, in particular to the immunological treatment response graft-versus-host (GVHD). The proposed method is based on the stimulation antiidiotypic response in the body non-irradiated F1 mice(BALB/c x C57BL/6) recipient parent splenocytes (C57BL/6). This is achieved by immunization of recipient antibodies against surface antigens of lymphocytes of BALB/c - GVHD target. Antibodies obtained by immunization of an intermediate recipient of the third haplotype lymphocytes of BALB/c, and produce for the immunosorbent prepared from those cells, and to increase the immunogenicity immobilized on valdecaballeros. Immunization of the recipient, the conjugates of antibodies against target cells allows prevent and treat GVHD, reducing 5-7 times the severity of the disease. 3 Il.

The invention relates to medicine, in particular to the immunological treatment response graft-versus-host (GVHD).

The principle method of treatment - anti-idiotypic suppression of the synthesis of autoantibodies in patients with autoimmune diseases manifestations (patent 93032579) - formed the basis for the prevention and treatment of GVHD.

For lactationally antibodies.

Along with the obvious success of the treatment of GVHD can be noted main disadvantages:

1. The absence or lack of selectivity effects, what is the cause of serious complications, such as immunodeficiency, infectious complications.

2. Lack of effectiveness in some cases.

3. The need for continuous treatment.

To treat the disease graft-versus-host tested several ways immunological effects.

1. In experimental studies it was shown that blocking CD4 (5,9) and CD2 (1) antigens T cells contributes to the development of transplantation tolerance and can cause inhibition of GVHD.

2. Monoclonal antibodies against T cells prevent lethal outcome in allogeneic transplantation in the GVHD model: BALB/c (donor) - (C57BL/6 CBA/2)F1(the recipient). (6, 7)

3. Combining monoclonal anti-CD4 and-CD8 antibodies had a therapeutic effect on GVHD. (8)

4. It is shown that anti-B7 antibodies can cure T - cell-mediated disease of the immune system and can be used to suppress activation of T-cells, preventing graft rejection and the development of GVHD (1,2)

blocking T-lymphocytes, diseases caused by bone marrow transplantation or organs.

The main disadvantages of immunological methods of treatment-related bone marrow transplantation and organ:

1. treatment with the introduction of monoclonal antibodies are not always effective and in some cases the effect is temporary,

2. the lack of specificity in the inhibition of activated T-lymphocytes leads to disruption of the immune system.

3. there is a need for treatment with drugs throughout the duration of postoperative life.

The proposed method of treatment allows to overcome the problems associated with conducting passive immunotherapy, because it promotes the development of specific immunity. After a single cycle of immunization anti-BALB/c antibodies (antigens on target cells GVHD) produced tolerance to transplantat. The effect of therapy of GVHD provided by the technology of the immunization. Joining aldehydesilane antibodies against surface antigens of cells targets of GVHD increases their immunogenicity and promotes the synthesis antiidiotypic antibodies that selectively block the activated donor lymphocytes after immunization of the recipient. Immuniscience the next period of life of animals. Re-introduction of the same donor cells caused the disease, and the third death in the majority of untreated animals, whereas immunized animals did not show clinic reaction, graft versus host.

The opportunity the treatment and prevention of GVHD was studied on the model of non-irradiated hybrids F1mice inoculated with splenocytes one of the parent lines. As controls for specificity antiidiotypic effect used transplantation of syngeneic splenocytes, allotransplantation without treatment, allotransplantation immunization restored valdecaballeros, allotransplantation immunization with antibodies to the donor cells (anti-C57L/6), allotransplantation immunization with fixed cells of the donor (C57BL/6), allotransplantation immunization with fixed cells of the second parental line (BALB/c), allotransplantation passive injection antiidiotypic serum (1 ml/weekly). The development of GVHD investigated in dynamics, assessing its severity by the number of marked clinical symptoms: arthritis, splenomegaly, skin lesions, conjunctivitis, etc. on a weekly examination. For each mouse was calculated sum tazetta.

It was shown that one cycle of immunization prior to or after transplantation enough for successful treatment of GVHD, whereas passive immunization - transfusion antiidiotypic immunoglobulins was not effective. In the work investigated the cytotoxicity of serum in GVHD reaction cytotoxicity (MDGs).

Serum of mice with pronounced symptoms of GVHD, contained a high titer of cytotoxic anti-BALB/c or anti-DBA/2 autoantibodies. Their maximum accounted for 40-50 days, and throughout the studies in untreated mice titer was significantly higher (p < 0.05) than in the treated group. Comparison of dynamics of average cytotoxic test and clinical manifestations of GVHD showed that the reduction in the severity of the disease coincided with a decrease in the cytotoxic titer of autoantibodies in the group treated mice.

In the test neutralize the MDGs investigated the level antiidiotypic antibodies in sera of mice (BBF1) immunized direct (CBA/2)-anti-BALB/c antibody before or after allotransplantation.

In experiments with GVHD prophylaxis immunization hybrids BBF1conducted direct (CBA/2)-anti-BALB/c antibodies before transplantation. The study pools of sera from 20 to 50 mice in maiechka serum (dilutions 1:1-1:4) neutralized (CBA/2-anti-BALB/c) serum (dilutions 1:10-1:20) 62-75%. Thus, the cycle of direct immunization antibodies obtained through "intermediate" host and immobilized on aldehydesilane, leads to the induction of synthesis antiidiotypic antibodies. Immunization of recipients BBF1after transplant them parental splenocytes was on the same circuit, from which it follows that the serum of these mice should contain antiidiotypic antibodies. However, in the test neutralization of cytotoxicity was not detected differences in the level antiidiotypic antibodies in immunized and unimmunized mice. Most likely, antiidiotypic antibodies cannot be detected in this test, as they are in complex with direct antibodies and their presence indicates the difference in the level of direct autoantibodies: the level of cytotoxic anti-BALB/c mice was significantly lower (0-25% dead cells) from immunized mice compared with the control group (30 - 80%).

The results indicate the possibility idiotypical suppression of activated against alloantigens target cells T-lymphocytes by stimulation of the synthesis antiidiotypic anti-anti-(antigens on target cells) antibodies. The latter, as "images" antigens on target cells, blocking the receptor is x F1you how to treat and prevent.

This model of GVHD close to the events occurring in case of allogeneic transplantation in the clinic, along with donor cells in the recipient recovers his own blood.

Since GVHD models of autoimmune disease, is a promising treatment of diseases with autoimmune manifestations by induction of synthesis antiidiotypic antibodies autoantibodies, which pathogenetic factors.

The proposed method is as follows. Treatment is preceded by:

1. preparation of antigen-sorbents of the surface antigens of lymphocytes of the patient, which is the "target" reaction, graft versus host,

2. antibodies by immunization of animals with the same lymphocytes of the patient,

3. selection of antibodies from immune sera pre-cooked antigen-sorbent.

4. the chemical binding of the antibody with dialdehydes

5. immunization of recipient antibodies to antigens of cells-targets of GVHD associated with cellulose.

For immunization and preparation of antigen-sorbents using lymphocytes from the thoracic duct balsa to generate antibodies to membrane antigens patient immunization of rabbits (or mini-pigs) live cells of this patient. Another part is to prepare the corresponding immunosorbent assay. By treatment of cells with detergents get solubilization membrane antigens of cells and kongugiruut them with separate or oxidized valdecaballeros. Prepared from antigens of cells"targets" column is used for affinity selection of immune sera relevant antibodies. The selected antibodies are subjected to partial proteolysis to remove the FC fragment of immunoglobulin of the animal sterilized by ultrafiltration, and Fab fragments (or whole immunoglobulins) kongugiruut with dialdehydes.

After verifying the sterility of the drug is immobilized on cellulose antibody or Fab fragments) is used for the treatment of the patient. Immunization begin one week after bone marrow transplantation and hold for five weeks with a one week interval in a dose of 10-100 µg of protein (or 5-10 μg of Fab-fragments) per 1 kg of body weight per injection. If Fab fragments do not produce, ensures that immunological security.

The proposed method was tested in numerous series of experiments more than 800 mice.

1. In experiments with the treatment of disease of the graft CLASS="ptx2">

2. To prevent the cycle of immunization was performed before transplantation recipients - BBF1, the donor C57BL/6.

Unirradiated the F1 hybrids in the tail vein injected parental splenocytes C57BL/6 (donor), causing the disease graft-versus-host. Starting from the next day after transplantation to stimulate antiidiotypic response conducted immunization antibodies against the second parental line (anti-BALB/c), which is the target of transplant attack - therapeutic immunization. The effectiveness of preventive immunization were tested on the same model: non-irradiated F1 hybrids, were injected splenocytes (C57BL/6) after immunization with anti-BALB/c antibody. In both cases, the disease severity was reduced by 5-7 times and resistance to disease graft-versus-host (with repeated injections of donor cells) were maintained during the entire life of the animal. In experiments with GVHD prophylaxis immunization hybrids BBF1conducted direct (CBA/2)-aHTH-BALB/c antibodies before transplantation. The study pools of sera from 20 to 50 mice in macrotest neutralize the MDGs showed the presence of antiidiotypic (anti-anti-BALB/c) antibodies. Received antiidiotypic serum (dilutions 1: 1-1: 4) what ntially, obtained through an intermediate host and immobilized on aldehydesilane, leads to the induction of synthesis antiidiotypic antibodies. Immunization of recipients BBFi after transplantation parental splenocytes was on the same circuit, from which it follows that the serum of these mice should contain antiidiotypic antibodies, but not easily identified, as they are in combination with cytotoxic antibodies.

For each mouse was calculated the overall rate of disease severity (number of symptoms). Differences in mean values in the groups was assessed using a criterion Student.

Example 1.

Hybrids BBF1- (BALB/c C57BL/6)F1transplanted splenocytes of C57BL/6 mice. To stimulate antiidiotypic response conducted post-transplant therapeutic immunization anti-BALB/c antibodies. As a control of specificity receive therapeutic effect have examined the effect of GVHD direct synthesis of antibodies to the cells of both parental lines. To this end recipients after allotransplantation were immunized fixed cells of each of the parent lines of mice.

In Fig. 1 shows the dynamics of the manifestation of clinical symptoms. Kaa 5-7 times (p < 0.05) as compared with the control untreated group (curve 2). The severity of the disease treated group (curves 3,4) was on the same level with the group singando transplanted mice BBF1that have a low degree also developed GVHD (curve 1). Re-cycle post-transplant immunization is not further reduced the severity of disease (curve 4). When immunization recipients of fixed cells of any of the parent lines of donor splenocytes reduced the severity of GVHD (curve 6). After immunization with cells of the second parental line GVHD was slightly increased (curve 5). Untreated mice is most severe disease occurred in the period from 20 to 80 days, after which it was a "recovery" (curve 2). 4 months after transplantation clinical symptoms of GVHD in none of the groups was not found.

Example 2.

In the given experiment investigated the influence of repeated allotransplantation on the recipients of splenocytes C57BL/6, taken from the experiments described above. Half of each of the pre-treated and untreated groups of mice (respectively 6 mice) four months after recovery from GVHD (260 days after transplantation) was held again transplanta7cells/mouse). In Fig. 2 shows that mice previously treated by immunization with anti-BALB/c antibody groups (corresponding to curves 3, 4 in Fig. 1) don't get sick after re-transplantation (Fig. 2 group II, curve 2). All mice previously Neimoidians group (corresponding to curve 2 in Fig. 1) symptoms of severe GVHD (Fig. 2, group 1, curve 2).

After the third allotransplantation to the same group a few months after the second allotransplantation - all mice from the group of untreated mice showed symptoms of severe GVHD, which resulted in 4 of the 6 previously untreated mice died, whereas treated (6 mice) survived.

The content of donor cells into the mice through 270 days after transplantation ranged from 2 to 15% regardless of the research group. This indicates that the effect of re-transplant is associated with engraftment of donor cells and, apparently, depends on their immunological activity.

Example 3. The ability to prevent GVHD.

Hybrids BBF1- (BALB/c C57BL/6)F1transplanted parental spleen cells C57BL/6. Immunization anti-BALB/c antibody was performed before transplantation (preventive immunization). For comparison, the performance is completed, or separately. As controls were used transplantation of syngeneic splenocytes; allotransplantation without immunization, immunization with antibodies to donor antigens (aHTH-C57BL/6).

It is evident from Fig. 3 shows that untreated group of mice (curve 2) or in groups immunized with antibodies to donor cells (curve 6) or inert media (curve 7), the severity of GVHD was several times higher (p < 0.05) than in prophylactically or therapeutically immunized groups (curves 3, 4 and 5). The effectiveness of therapy is not dependent on the timing of immunization before or after transplantation.

The dose inoculated cells increased the severity of GVHD. "Recovery" of mice inoculated 6,5107cells/mouse, was incomplete. The effect of therapy was reduced from 5-7-fold at a dose of 3107cells/mouse up to 2-3 times the dose of 6,5107cells/mouse.

Captions to figures

Fig. 1. The results of immunotherapy GVHD.

7 groups of 12 mice (6 males and 6 females).

On the x-axis is days after transplantation.

On the y - axis the degree of severity of the disease.

1. Syngeneic transplantation (control 1).

2-7 - Allogeneic transplantation parental splenocytes C57BL/6. GVHD:


4. two cycles of post-transplant immunization anti-BALB/c antibodies,

5, 6 - post-transplant immunization fixed splenocytes parental strains of mice BBF1:

5. cells of BALB/c (control 3),

6. cells of C57BL/6 (control 4).

Fig. 2. Re allotransplantation "recovered" from GVHD groups of mice that were not previously immunized and immunized anti-BALB/c antibodies.

4 groups of 6 mice.

Group I. the severity of the disease mice without immunization

Group II. The severity of the disease mice previously immunized with anti-BALB/c antibody

1. re-transplantation of syngeneic (BBF1) splenocytes (control),

2. re-transplantation of parental splenocytes (C57BL/6).

Fig 3. The results of GVHD prophylaxis.

7 groups of 10 mice (5 males and 5 females).

On the x-axis is days after transplantation

On the y - axis the degree of severity of the disease

1. Syngeneic transplantation (control 1).

2-7-Allogeneic transplantation parental splenocytes C57BL/6. GVHD:

2. without additional immunization (control 2),

3. immunization with anti-BALB/c antibodies before transplantation,

4. emmalani

5. post-transplant immunization anti-BALB/c antibodies,

6. post-transplant immunization aHTH-C57BL/6. antibodies (control 3),

7. cycle post-transplant immunization inert carrier (control 4).

Method of prevention and treatment of disease graft-versus-host (GVHD) by immunotherapy, wherein the recipient subjected to immunization with getrootframe against surface antigens of lymphocytes are targets of GVHD, immobilized on an inert carrier, for receiving antiidiotypic suppression of activated T-lymphocytes of the donor.


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