Drug to prevent rejection of the transplant, a monoclonal antibody to the cd3 antigen of t - lymphocytes person, hybridoma and a method of treating patients having a reaction of acute transplant rejection after kidney transplantation

 

(57) Abstract:

The invention relates to medicine and relates to drugs to prevent transplant rejection, monoclonal antibodies to the CD3 antigen of T-lymphocytes person, hybridoma producing these antibodies, and treatment of patients with reaction acute transplant rejection after kidney transplantation. The invention consists in that the proposed remedy is a solution of purified immunoglobulin IgG2a in physiological solution of phosphate-saline buffer, pH 7.2 to 7.4, containing active substances 1-10 mg/ml and 0.01-0.02% tween - 80. In the treatment of patients with reaction acute transplant rejection after kidney transplantation, the treatment of the proposed drug that is administered in the amount of 1-5 mg for 7-14 days. Monoclonal antibody to the CD3 antigen of T-lymphocytes human ICO-90, produced by the hybridoma strain of cultured animal cells Mus.musculus L., ICO-90, SCC(P)D, communicating with T peripheral blood lymphocytes and thymocytes person, and cell cultures of T lymphocytes of human rights and cell line Jurkat and does not bind to leukocytes, erythrocytes, myelocytes and monoblasts. The advantage of the invention is to reduce the cases of transplant rejection and allergization of an organism in patients after kidney transplantation. 4 c. and 7 C.p. f-crystals, 2 tab.

The invention relates to medicine and relates to drugs to prevent transplant rejection, monoclonal antibodies to SV-antigen T-lymphocytes person, hybridoma producing these antibodies and method of treatment of patients with reaction acute transplant rejection after kidney transplantation.

Background of the invention

The reaction of acute transplant rejection (ROOT) is a serious postoperative complication in patients who undergo transplantation of organs and tissues. The basis of this complication lies immune response of the host body against foreign antigens of the graft. Under the antigens in immunology understand the substance or structure that is able to induce an immune response against them.

We know that each person is characterized by its own set of antigens on the cells of various organs and tissues. This set is determined genetically and, accordingly, different antigens in different people (and animals) depends on the degree of genetic closeness between them. Grafted immune response to a foreign antigen, leading ultimately to the rejection of the graft.

It is known that the immune system of humans and animals consists of two interacting parts that carry out the humoral and cellular immunity. Humoral immunity is carried out by b-lymphocytes, which are formed and differentiated from stem cells hematopoietic system bone marrow and is associated with the production of b-cells antibodies to soluble antigens. Cellular immunity is T-cells, which are formed and differentiated from stem cells in the haematopoietic system in the thymus. While in the thymus, these cells called thymocytes, and after release into the bloodstream, they are called T-lymphocytes. For many immune responses requires the interaction of b - and T-cells.

The ROOT is carried out mainly by the mechanism of cell-mediated immunity. To suppress ROOT traditionally used drugs are immunosuppressants (azathioprine, cyclosporine, prednisone). These drugs are not specific inhibit the activity of the immune system and cause a number of related side effects (infection, tumor growth, and so on). In this regard, has been under constant search for drugs, more selectively podal of the promising areas of drug therapy, in General, is the use of so-called monoclonal antibodies (MAB) for the destruction or inactivation of the agents and agencies involved in the development of diseases.

MCA is produced by special technology of highly pure preparation of antibodies to a particular antigen. In General, antibodies are immunoglobulins that are produced by b-lymphocytes when ingested foreign antigen. With the help of antibodies, the body destroys or inactivates those antigens that caused the production of these antibodies.

Traditionally, the preparations of antibodies to a particular antigen was obtained by immunization of animals with this antigen and subsequent selection of antibodies from the blood of animals. However, since the animal organism is constantly exposed to the external environment and external antigens in his blood is constantly present a range of antibodies directed against different antigens. The highlight of this mixture of antibodies directed against a single antigen, technically difficult. Therefore, obtained in this way the preparations of antibodies to a particular antigen, as a rule, had impurities antibodies to other antigens and were used mainly for itiv one antigen, long been an obstacle to their wide clinical use.

In 1975 he developed a radically new technique that allows to obtain almost pure preparations of antibodies directed against a particular antigen (see Kohler et al., 1975). The basis of the method is to create a hybrid cell lines synthesizing antibody to only one specific antigen. As you know, the antibodies produced by b-lymphocytes, but lymphocytes in cell culture are not supported and in a few divisions perish. The idea of these authors was that b-lymphocytes were hybridisable with "immortal" culture of myeloma cells, supported indefinitely in cell culture. Thus, the resulting cell hybrid has the ability of b-lymphocytes to produce antibodies and the ability of myeloma cells be maintained indefinitely in culture.

To obtain hybridoma producing antibodies to a specific antigen, mice subjected to immunization records antigen, receive cells of the spleen and hybridizing them with murine myeloma cells (typically myeloma P3X63.Ag8.653). After merging cells disperse one by one and subsequently select those colonies that produce antibodies to A-producer are the clone offspring of a single hybrid cell, the synthesized their antibodies are called monoclonal.

Using hybridoma technology to date, hundreds of μa to a variety of antigens of human and other organisms. These antibodies are widely used in experimental studies of etiology, pathogenesis and treatments of various diseases, for immunoassay for diagnosing and monitoring the course of disease, and to assess the hormonal and immune status of patients (see, for example, reviews Laurino et al., 1999 and Baryshnikov et al., 1996 and references in these).

T-lymphocytes of a human being are unique to them the CD3 antigen, which is a transmembrane glycoprotein with a molecular weight of about 20 kDa and is present on all Mature T-lymphocytes and thymocytes. The binding of the antibody with the antigen leads to a dramatic inhibition of cellular immunity, which may be due to the following reasons:

1) the binding of antibody with antigen leads to steric modifications associated with the CD3-antigen T-cell receptor antigens (TRA), resulting in blocking the activation of T lymphocytes by antigens;

2) the binding of an antibody to the antigen promotes opsonization by tsirkuliruyuschii occurs a short activation of the T-lymphocyte, in which of the cells are released cytokines, blocking proliferation and differentiation of progenitor cells T-lymphocytes. These cytokines include interleukin-2, -3, -6, -10, interferon gamma and tumor necrosis factor;

4) the binding of an antibody to the antigen initiates the process of apoptosis (programmed death) of T-lymphocytes.

In addition, suppose that the binding of an antibody to the CD3 antigen may cause the following:

1) the internalization of the complex CD3-TPA from the membrane and, consequently, a reduction in the ability of T-lymphocyte activation antigens;

2) increased expression in lymphocyte adhesion molecules, which leads to increased adhesion of lymphocytes to vascular endothelium;

3) cell-mediated cytolysis of T-lymphocytes.

As a result, within 1 hour after intravenous injection of antibodies to the CD3 antigen from the bloodstream disappear almost all of functionally active T-lymphocytes.

Already in the mid-1970s, it was known that the introduction of the patient of antibodies to surface antigens of T-lymphocytes allows to prevent or reduce the ROOT (Cosimi et al., 1976). In the late 1970s, by the method of hybridoma technology was the first monoclonal antibody to the antigen CD (Cosimi et al., 1981). The first drug based on monoclonal antibodies to CD3 T-lymphocytes person is drug Ortolon OCT company Ortho Pharmaceuticals (USA), who in 1986 was allowed by Management to control the quality of products and medicines of the USA for the prevention and treatment of reactions of acute transplant rejection in renal transplantation.

Monoclonal antibodies OKT are immunoglobulins of the IgG class, subclass IgG2, produced by hybridomas ADS CRL8001 cultivated in a suitable medium and received by the fusion of myeloma cells RH Ag8Ul and spleen cells of mice CAF1pre-immunized T cells, purified by the method of rosethorne (patent US 4361549).

In the patent US 4515893 the same company was patented hybridoma producing these antibodies, which react with more than 95% of T cells in human peripheral blood, but do not react with b-cells. In the patent discusses the possibility of therapeutic and diagnostic applications OCT.

Therapeutic drug ACT contains a mixture of pharmaceutically acceptable carrier with a therapeutically effective amount of Orthoclone OKT, effectively weakening or overwhelming exclusion is seven normal T-cells in human peripheral blood cells and cutaneous T-cell lymphoma, but is not associated with b-cells, null cells and macrophages. In the diagnostic process, for example, T-cell lymphoblastic leukemia mixing an effective amount of drug from peripheral blood lymphocytes obtained from a patient, and determine the percentage of lymphocytes that interact with antibodies. The patent also States that Ortolon OCT weakens or prevents the reaction of transplant rejection, however, specific data on the clinical efficacy of the drug is not given.

Despite the high efficiency of suppression of the ROOT with the help of MCA ACT, approximately 6% of patients using Orthoclone OKT ineffective, and in 66% of patients observed in subsequent relapse ROOT. Patients who are not sensitive to other types of immunosuppressive drugs, the frequency of the lack of effect of Orthoclone OKT 20-25% (Ponticelli et al., 1986, Monaco et al., 1987).

Drugs similar to Orthoclone OKT have been developed by a number of other countries: IORT-3 (cube) and ICO-90 (Russia). In the works of Ivanov P. K. et al. (Ivanov, P. K., and others, 1998; Smith, P. K. et al., 1998) described their use for the treatment of reactions acute transplant rejection after kidney transplantation, as well as their biological activity.

In connection with islogan the post transplant rejection based on monoclonal antibodies ICO-90 and a pharmaceutically suitable carrier, ensuring the stability of the drug during prolonged storage, as well as high efficiency in the treatment of patients with reaction of acute transplant rejection after kidney transplantation with minimal allergization of an organism and the lack of subsequent relapses, when using the strain-producer, producing monoclonal antibodies broader spectrum and higher specificity compared to commercial preparations.

The invention

The invention includes a drug to prevent rejection of the transplant on the basis of monoclonal antibodies to the CD3 antigen of T-lymphocytes human ICO-90, is a purified immunoglobulin IgG2a subclass, containing from 1 to 10 mg of active substance in 1 ml of phosphate-saline buffer with a pH of 7.2-7.6 and 1 0.01-0.02% Tween-80.

A method of treating patients having a reaction of acute transplant rejection after kidney transplantation involves the use of this medicine.

In a variant of this method, the drug is administered daily in an amount of from 1 to 5 mg.

In a preferred embodiment of the method, the drug is administered in an amount of 5 mg is evno within 7-14 days.

Preferably injected the drug daily for 10 days.

Monoclonal antibody, on the basis of which is purchasing drug is a monoclonal antibody to the CD3 antigen of T-lymphocytes human ICO-90, produced by the hybrid strain of cultured animal cells Mus musculus L., ICO-90, SCC (P) D, communicating with 60.7 per cent of T-lymphocytes in peripheral blood and from 62.7% of thymocytes person, as well as with 97-98% of cells in cultures of T lymphocytes person (NK1, B12b, Hball) and with 80% of the cell line Jurkat.

In a variant of this invention, the specified monoclonal antibody is not associated with leukocytes (monocytes and granulocytes, erythrocytes and platelets in human peripheral blood and cell cultures of b-lymphocytes (JFP, JY, Raji, Namalva), pre-b-lymphocytes (Reh), erythroblasts (C), plasmic order has been revealed (HL60) and monoblasts (U937).

In another embodiment of this invention, the specified antibody is used for the diagnosis of diseases associated with decrease or increase of production D3+T-lymphocytes.

The specified monoclonal antibody is produced by a hybrid strain of cultured animal cells Mus musculus L., ICO-90, SCC (P) D.

In a variant of the invention the United States is our BALB/c, immunized T-lymphocytes human purified by reaction rosethorne with sheep erythrocytes.

The strain deposited in the special collection transplantable somatic cells of vertebrates of the all-Union collection of cell cultures.

Specific examples of the execution

Example 1. Getting hybridoma producing ICA ICO-90

Hybridoma obtained by fusion of murine myeloma cells and spleen cells of a mouse immunized with T-lymphocytes in human peripheral blood.

A) isolation of T-lymphocytes in human peripheral blood

T-lymphocytes isolated from a pool of mononuclear cells on the basis of the reaction of rosethorne with sheep erythrocytes treated with neuraminidase.

Blood from the cubital vein of healthy donors is collected in a solution of heparin (50 IU/ml). For whole blood, add 10% solution of gelatin to a final concentration of 1%, mixed and incubated for 45 min at 37oC. this precipitate erythrocytes. Plasma containing a suspension of cells, sucked off, and leukocytes precipitated by centrifugation and washed three times.

The leukocyte layer on the density gradient of ficoll-urografin (9 volumes of the preparation of cells at 3 odes washed with medium 199, if this is the first time centrifuged for 20 min at 200 g.

Mix equal volumes of a suspension of mononuclear cells (6106/ml), sheep red blood cells and fetal calf serum adsorbed with sheep erythrocytes. The mixture is incubated for 15 min at 37oC, centrifuged for 5 min at 150 g, and incubated overnight at 4oC. the Cells gently suspension, layered over a gradient ficoll-urografin and centrifuged in the same conditions. T-cells formed rosettes, are deposited on the bottom of the tube. The outlet of the sediment destroy hypotonic solution.

B) Immunization of mice T-cells

Mice of BALB/c into the tail vein injected T cells in number 2107cells. The immunization is carried out 5 times with an interval in 2 weeks. Last immunization spend 5 days prior to the hybridization.

C) Obtaining cells of the spleen

The spleen of immunized mice extracted under sterile conditions and crushed in the Potter homogenizer. A suspension of cells filtered through a gauze filter and washed twice with medium 199.

G) Line myeloma cells

To obtain hybridoma use mouse myeloma P3X63.Ag8.653. Myeloma cells support among gibridizacija cells

The myeloma cells are washed twice with medium 199 and mixed with spleen cells at a ratio of 1:5-1:10. The mixture of cells washed with medium 199 and placed in a volume of 20 ml of medium 199 100-ml bottle, in which it is centrifuged for 3 min at 300 g and incubated for 20 min at 37oC. Then the medium is sucked off dry to cells add 3 ml of 50% polyethylene glycol. Over 75 into the vial, slowly pour in 20 ml of medium 199. Cells are washed 3 times, not shaken, and leave for 2-8 hours. After that, cells are poured into 0.2 ml in the wells of 96-cellular microplate. The next day the medium is changed on Wednesday GAT (environment DMEM containing of 13.6 μg/ml gipoksantina, 191 ng/ml aminopterin and 3.85 μg/ml thymidine). Visible colonies appear 10-14 days.

E) Selection of strains-producers

The selection of strains producing MCA, is carried out by indirect immunofluorescence analysis of samples of the culture fluid with cells used for immunization.

Cells in the amount of 500 thousand incubated with 20 μl of the supernatant diluted 1:10 in saline solution with 0.1% sodium azide. Incubation was performed at 4oC for 30 minutes then the cells washed twice with phosphate-saline buffer (FSB) with 2% fetal calf who's rabbit antibodies against mouse IgG, conjugated with FITZ or horseradish peroxidase. After that, cells are again washed twice with medium IFA and resuspension in saline with 1% formalin. In the resulting suspension analyze the fluorescence of cells on a flow cytometer.

To control the specificity of the staining as negative controls instead of the first antibody using the buffer, instead of the second antibody buffer or another labeled antibody specificity.

The strain selected to receive MCA, was named ICO-90.

F) Cloning and maintaining hybridoma

Producing hybridoma twice clone feeder cells from spleen and thymus of BALB/c mice. For preparation of feeder pieces of thymus and spleen crushed in a Potter homogenizer, a suspension of cells filtered through a gauze filter and cells are poured into the growth medium by 0.1 ml into wells of 96-cellular microplate. After 1-7 days these wells is used as a feeder. Cells producing clone is removed by gentle pipetting, and count in the camera Goriaev and diluted in growth medium to a final concentration of 50 cells in 10 ml of medium growth. Received a suspension of cells is poured into 0.1 ml in the wells of the feeder. 2 wells had 1 cell. Vidimus 75% of the holes.

Culture hybridoma supported in the female mice of BALB/c mice by serial passage of ascitic fluid.

Example 2. Getting the ICA ICO-90

ICA ICO-90 isolated from the ascitic fluid of BALB/c mice, which intraperitoneally inoculant cells producing hybridoma.

A) Obtaining ascitic fluid.

For 3-10 days prior to inoculation of cells hybridoma mice intraperitoneally injected with 0.5 ml of paraffin oil or 3% of peptone on vaseline oil for induction of growth hybridoma in ascitic form. 107cells hybridoma ICO-90 inoculant mice intraperitoneally and after 7-8 days, depending on the accumulation of ascites, collecting ascitic fluid. Ascitic fluid obtained from different mice are pooled and centrifuged for 20 min at 2.5 thousand g and 4oC. the Supernatant stored at -50oWith up to further processing (up to 3 months).

B) isolation and purification of the ICA ICO-90

ICA ICO-90 allocate by affinity chromatography sorbent protein a-sepharose (Pharmacia, Sweden). Ascitic fluid is thawed and centrifuged for 20 min at 2.5 thousand g and 4oC. the Supernatant deleteroute against the starting buffer (0.1 M sodium phosphate buffer, pH 8.0) overnight. Received the m (pH 8.0), and washed with the starting buffer until the lack of protein in the output solution. After contacting the sorbent immunoglobulins elute with 0.1 M sodium citrate buffer (pH 3.0). The obtained eluate concentrated and used as substrate for the further preparation of a medicinal product.

The resulting ICA ICO-90 refer to the IgG2a subtype in the analysis method of immunodiffusion on Ouchterlony. The purity of the obtained ICA ICO-90 estimate by the method of pressure HPLC sorbent Superose 12 using as mobile phase 0.6 M potassium phosphate buffer (pH 6.8), 0.1 M potassium chloride. Peak ICA is not less than 95% of the total area of the chromatogram, and the area of individual peaks of impurities does not exceed 1% of the total area of the chromatogram. The authenticity of the ICA ICO-90 assessed by electrophoresis in polyacrylamide gel in the presence of dodecyl-sulfate sodium. MCA form 2 distinct bands corresponding to the molecular weight of 25 and 55 kDa. The specific activity of the ICA ICO-90 define a method of running cytofluorimetry in indirect reaction immunofluorescence assay with human lymphocytes (see below). On serial dilutions of antibodies determine the titer of antibodies, corresponding to the saturation point fluorescence. The titer of saturation of fluo-90

The eluate from the protein a-Separate deleteroute overnight against 0.01 M sodium phosphate buffer (pH 7.5) prepared in sterile physiological solution. Dialysate diluted to a protein concentration of 1-10 mg/ml and the resulting solution was added as a stabilizer tween-80 to a final concentration of 0.01-0.02%. The resulting preparation is sterilized by filtering through a filter with pore size 0,22 MK (Millipore, USA) and poured into sterile vials of 5 ml of the Finished preparation should be stored at +4oC.

Example 3. Characterization of the antigen detected by ICA ICO-90

The peripheral blood lymphocytes of donors allocate as described above, incubated with125I was literally and the resulting solution was labeled125I membrane proteins incubated with ICA ICO-90 immobilized on granulated sepharose. Then bound peroxidase proteins elute and separate by electrophoresis in polyacrylamide gel. Antibodies bind with the protein having a molecular weight of 25 kDa, which corresponds well-known from the literature, the molecular weight of the antigen CD3 (Kung et al., 1979).

Example 4. The activity of the obtained antibodies against T lymphocytes in vitro

It is known that the binding of the antibody to the CD3 antigen causes a temporary activation of the T-s T-lymphocytes person.

1106of human lymphocytes, isolated on a density gradient ficoll-urografin, incubated for 72 hours in 1 ml of RPMI-1640 medium containing 10% fetal calf serum in the presence of the obtained antibodies at various concentrations. The degree of lymphocyte proliferation appreciate the inclusion of 3H-thymidine. In intact lymphocytes (negative control) enable 3H-thymidine was 1714220 pulses/min. In lymphocytes incubated with the obtained antibodies at concentrations of 0.05-50 μg/ml, the inclusion of 3H-thymidine was increased in 15-20 times, and the effect of the antibody was weakly dependent on their concentration.

Example 5. Linking ICA ICO-90 with different types of human blood cells and cell cultures

Linking µa of ATAMA with the register cells in indirect immunofluorescence assay reaction, and the cells are incubated first with UA, and then labeled with a fluorescent label (fluoresceinisothiocyanate, FITZ) sheep antibodies against mouse IgG. Fluorescence of cells recorded on a flow cytometer.

A) Obtaining different fractions of blood cells

Donors receive venous blood in heparinised tubes. Before further processing the ora of gelatin, mix and incubated for 45 min at 37oC. Select adosados containing a suspension of cells. Leukocytes precipitated by centrifugation for 10 min at 150 g and room temperature and washed twice with medium 199 by resuspendable and centrifugation in the same conditions.

The resulting leukocyte resuspended in 6 ml of medium 199 and layered on 2 ml of density gradient ficoll-urografin; centrifuged for 40 min at 300 g at room temperature. Select the interphase containing a suspension of monocytes. Monocytes precipitated by centrifugation for 20 min at 150 g and room temperature and washed twice with medium 199 by resuspendable and centrifugation for 10 min at 150 g.

To obtain the fraction of T cells mixed with equal volumes of a suspension of mononuclear cells (6106ml), sheep red blood cells and fetal calf serum adsorbed with sheep erythrocytes. The mixture is incubated for 15 min at 37oC, centrifuged for 5 min at 150 g, and incubated overnight at 4oC. the Cells gently suspension, layered over a gradient ficoll-urografin and centrifuged in the same conditions. T-cells formed rosettes, are deposited on the bottom about who made the cell concentration with the camera Goriaev and if necessary, bring the concentration up to 1 million cells/ml of medium 199.

B) cell Culture

Use culture T-lymphocytes human NK1, B12b, Hball, Jurkat, culture of human lymphocytes JFP, JY, Raji, Namalva, culture pre-b-lymphocytes human Reh, culture erythroblasts person C, culture plasmic order has been revealed human HL-60 and culture monoblasts human U937 supported in the Russian Oncological Scientific Center. N. N. Blokhin.

In) Cytofluorimetrically analysis

Antibodies at a dilution of 1:1000 is mixed in a glass tube with lymphocytes of peripheral blood in a ratio of 0.2 ml of the antibody solution: 0.5 ml cell suspension concentration of 1 million/ml of the resulting mixture was incubated for 30 min at room temperature and washed twice with medium IFA (0.01 M phosphate-saline buffer containing 2% fetal calf serum) by centrifugation for 10 min at 150 g and a temperature of 2-8oWith and resuspendable.

To 1 ml of the resulting suspension of cells add 0.1 ml of the diluted solution labeled FITZ of sheep antibodies against mouse IgG (this solution was prepared in accordance with the individual manufacturer's instructions included with each batch of labeled antibodies). PV for 10 min at 150 g and a temperature of 2-8oC.

The precipitate resuspended in 1 ml of fixing solution (0.9% sodium chloride solution with 1% formalin and stored at 4oTo analyze on a flow cytometer.

Analysis on a flow cytometer Becton Dickenson FacScan carried out in accordance with the instruction manual of the device. Register a histogram of fluorescence of the cells. As a negative control using monoclonal antibodies IR-109, directed against the immunoglobulins of the rat.

G) Results

The percentage of cells binding the obtained monoclonal antibodies listed in table 1.

Thus, ICA ICO-90 contact 60,7% of T-lymphocytes in peripheral blood and 62,7% of thymocytes person and not associated with other types of blood cells. ICA ICO-90 are also linked to the cultures of T cells, but not with the cultures of other blood cells and bone marrow.

Example 6. Comparison of the affinity of the ICA ICO-90 and mA Ortolon OCT-3 to D3 antigens T cells

The affinity of the ICA ICO-90 and Ortolan OCT-3 to D3 antigens of T cells is determined in an indirect immunofluorescence assay reaction with T-lymphocytes in human peripheral blood. On serial dilutions of antibodies determine the titer of antibodies, soutetsu sample and then conjugated with fluoresceinisothiocyanate (FITZ) polyclonal sheep antibodies against mouse IgG. Fluorescence of cells was detected by flow cytometer Becton Dickenson FacScan.

A method of obtaining the fraction of T-lymphocytes in human peripheral blood as described in example 5. Cytofluorimetrically analysis is performed as described in example 5, except that use serial dilution µa(1:10, 1:100, 1:500, 1:1000, 1:2000, 1:5000, 1:10000, 1:20000).

The results obtained are presented in table 2.

Thus, the saturation binding ICA ICO-90 T-lymphocytes in human peripheral blood is observed when breeding µa 1:1000, and mA Ortolon OCT-3 at a dilution of 1:10 000. a 10-fold difference saturating concentrations of these MCA indicates a higher affinity (affinity) mA Ortolon OCT-3 to the CD3-antigen T cells compared with ICA ICO-90. From literature it is known that the MCA, with the average level of affinity for the CD3 antigen, can repeatedly contact molecules CD3/TCR complex during T-cell activation inducyruya so-called activation-destruction of cells (see Orchansky P. L. et al. J. Immunol., 1994; 153:615, Hurdato J. et al. J. Immunol., 1997; 158(6): 2600, Radvanyi L. et al. J. Immunol., 1993; 150: 5704).

Prima is Aliza and kidney transplantation Moscow regional research clinical Institute (MONIKI). M. F. Vladimirsky was hospitalized b-Naya K., 20 years, with a clinical diagnosis of "chronic renal failure". The last two years she was on constant dialysis, waiting for a suitable transplant for kidney transplantation.

The reason for hospitalization was finding a suitable transplant. On the third day after the operation showed signs of acute transplant rejection, diuresis was absent, the plasma creatinine increased. There is a risk of graft loss.

For health reasons, with the consent of the patient and upon prior approval of the Directors of the Research Institute of experimental diagnostics and tumor biotherapy Russian Cancer Research Center (research Institute Edito research center). N. N. Blokhin Russian Academy of medical Sciences and MONICA K. was conducted treatment drug on the basis of the ICA ICO-90. Use the following mode of administration: daily 5 mg drug intravenously for 10 days.

Already by the 6th hour from the start of injection of the antibodies, the number of T-lymphocytes (determined by indirect immunofluorescence analysis as the number of cells bearing the antigen CD3) in the peripheral blood decreased p is Cherno, that decrease, and then increase the number of T-lymphocytes was performed at the expense and T-helpers (CD4+) and T-suppressor (CD8+).

Proof that with the introduction of the ICA ICO-90 in the first hours is precisely the death of T-lymphocytes, and not just the disappearance of the antigen, is a mass appearance in the peripheral blood of kariolou with hypodiploid DNA content, which is typical for the development of apoptosis (programmed cell death).

3 days after the start of therapy in the treatment of K. appeared diuresis, creatine index has fallen, restored the function of the transplanted kidney, and after 4 weeks the patient was discharged home in good condition. Currently, her state of health is satisfactory. During the observation period (3.5 years) recurrence reaction, transplant rejection occurred. With the same technical result of the treatment of two patients.

Specified drug similar to other commercial preparations can be used for the diagnosis of diseases associated with decrease or increase in the number D3+-lymphocytes, for example, T-cell chronic lymphocytic leukemia and roar of purified monoclonal antibodies to the CD3 antigen of T-lymphocytes human ICO-90, which remains active during long-term storage and does not relapse reactions acute transplant rejection after kidney transplantation. Themselves antibodies ICD-90, produced by the hybrid strain of cultured animal cells Mus musculus L., ICO-90, SCC (P) D, broad spectrum of activity and high selectivity with respect to the various cultures of T-cells.

Thus, the invention and provided their options well suited to achieve the goals and outcomes described in the beginning. In the described techniques and tools can be modified without affecting the nature and scope of this invention. It is assumed that each element or step described in each of the following claims, should be attributed to all equivalent elements or steps to achieve the same results with the same or equivalent means. It is planned to expand the scope of the present invention in any form, which can be used its principles.

A list of the attached literature

1. Kohler g, Milstein C. Continuous culture of fused cells secreting antibody of predefined specificity". Nature, 1975; 256: 495-497.

2. Laurino, J. P., Shi Q., Ge J. "Monoclonal antibodies, antigens and mole and clinic. Moscow, 1996.

4. A. B. Cosimi et al. "Randomized clinical trial of ATG in cadaver renal allograft recipients: importance of T-cell monitoring". "Surgery", 1976; 40: 155-163.

5. Cosimi, A. B., Burton R. C., Colvin, R. B. et al. "Treatment of acute renal allograft rejection with OKT3 monoclonal antibody". "Transplantation", 1981; 32: 535-539.

6. US patent N A, 30.11.82.

7. US patent N A, 07.05.85.

8. US patent N A, 31.03.87.

9. Ponticelli C, Rivolta E., Tarantino A. et al. "Clinical experience with OKT3 in renal transplantation". "Transplant Proc", 1986; 18: 942-948 (In Russian).

10. Monaco, A., Goldstein G., Barnes L. "Use of OKT3 monoclonal antibody to reverse acute renal allograft rejection unresponsive to treatment with conventional immunosuppressive regimens". "Transplant Proc", 1987; 19: 28-31.

11. Ivanov P. K. , Baryshnikov, A. Y., polosukhina E. R. and other "Establishment and characterization of monoclonal antibodies to prevent reaction of acute transplant rejection". Proc. Dokl. V Russian national Congress "Man and medicine", Moscow, Russia, 21-25 April 1998, S. 489.

12. Smith P. K., Polosukhina E. R., T. Zabotina N. et al. "Study of biological activity of anti-CDS monoclonal antibodies". Proc. 2ndWorld Meet. API/APV Pharmaceutics, Biopharmaceutics and Pharm. Technology, Paris, 25-28 May, 1998, pp. 1189-1190.

13. Kung, P. C., Goldstein G., E. L. Reinherz, Schlossman S. F. "Monoclonal antibodies defining dictinctive T-cell surface antigens". Science, 1979; 206: 347.

14. Kaneoka h , Perez-Rojas G., Sasaki T. et al. "Human T-lymphocyte proliferation induced by a pan-T monoclonal antibody (anti-Leu-4): heterogenicity of response is a function of monoculytes". "J. I the monoclonal antibody to the CD3 antigen of T-lymphocytes human ICO-90, characterized in that it is a solution of purified immunoglobulin IgG2a in phosphate-buffered saline with a pH of 7.2 and 7.6, containing 1-10 mg of the active substance in 1 ml and 0.01-0.02% tween-80.

2. A method of treating patients having a reaction of acute transplant rejection after kidney transplantation, including the introduction of patient drug monoclonal antibody to the CD3 antigen of T-lymphocytes human ICO-90, characterized in that the monoclonal antibodies used drug under item 1.

3. The method according to p. 2, wherein the drug is administered daily in an amount of 1 to 5 mg.

4. The method according to any of paragraphs. 2 and 3, characterized in that the drug is administered preferably in an amount of 5 mg daily.

5. The method according to any of paragraphs. 2-4, characterized in that the introduction of a medicinal product shall exercise daily for 7-14 days.

6. The method according to any of paragraphs. 2-5, characterized in that the introduction of the drug carried out preferably within 10 days.

7. Monoclonal antibody to the CD3 antigen of T-lymphocytes person ICA ICO-90, produced by the hybrid strain of cultured animal cells Mus musculus L. , ICO-90,OK line Jurkat.

8. Monoclonal antibody under item 7, characterized in that it is not associated with leukocytes (monocytes and granulocytes, erythrocytes and platelets in human peripheral blood and cell cultures of b-lymphocytes (JFP, JY, Raji, Namalva), pre-b-lymphocytes (Reh), erythroblasts (C), plasmic order has been revealed (HL60) and monoblasts (U937).

9. Monoclonal antibody according to any one of paragraphs. 7-8, characterized in that it is used for the diagnosis of diseases associated with decrease or increase of production of CD3+T-lymphocytes.

10. The hybrid strain of cultured animal cells Mus musculus L. , ICO-90, SCC (P) D used to obtain monoclonal antibodies to the CD3 antigen of T-lymphocytes man in PP. 7-9.

11. The hybrid strain of cultured cells under item 10, characterized in that it is obtained by fusion of murine myeloma cells of strain RH. Ag8.653 and spleen cells of mice BALB/C immunized with T-lymphocytes human purified by reaction rosethorne with sheep erythrocytes.

 

Same patents:

The invention relates to immunology and biotechnology and can be used to test rapamycin

The invention relates to immunology

The invention relates to the field of medicine and relates to a composition for inhibition of angiogenesis, monoclonal antibodies, polypeptide and method of inhibiting tumor growth

The invention relates to biotechnology, in particular to recombinant IL4-antibodies used for treating disorders associated with the activity IL4

The invention relates to a monoclonal antibody having the ability to inhibit homing hematopoietic stem cells and to identify surface antigen stromal cells, having the ability to maintain homing hematopoietic stem cells, as well as to hybridoma producing monoclonal antibody

The invention relates to the complementarity determining regions (CDR, hypervariable regions) and variable regions (V regions) of murine monoclonal antibodies to human interleukin-8 (IL-8), human/mouse chimeric antibody to human IL-8, and reconstructed human antibodies, and region, defining a complementary variable region, a human light chain (L-chain) and variable regions of the heavy chain (H-chain) of the person replaced the CDR of mouse monoclonal antibodies to human IL-8

The invention relates to the field of medicine and biotechnology, namely to new proteins, which factors in the growth and development of megakaryocytes (MGDFs; mostly labeled Mp1-ligands), the biological activity of which is to stimulate the growth of megakaryocytes and their differentiation or maturation, which ultimately leads to the formation of platelets

The invention relates to biotechnology and concerns gumanitarnogo immunoglobulin specific for the protein L-selectin person

The invention relates to immunology and can be used to produce monoclonal antibodies having immunostimulatory action

The invention relates to propertytaxsession systems that require cleavage product a predecessor to the new polypeptide capable of restoring dichloroindophenol and oxidized glutathione to DNA that encodes this polypetide, farmkompanijam comprising the polypeptide, monoclonal antibodies against the indicated polypeptide

The invention relates to the field of Virology, immunology and biotechnology, namely, hybridoma technology, and represents a new hybrid strain of cultured animal cells Mus musculus L. producing cell cultures and ascitic fluids monoclonal antibody (MAB) to the virus vesicular disease swine (WBS) strain T-75, which can be used for scientific research and preparation of diagnostics, prevention and treatment VBS

The invention relates to hybrid technology and can be used in veterinary medicine and medicine in the diagnosis of brucellosis

The invention relates to hybridoma technology and can be used in the diagnosis of hepatitis b

The invention relates to medicine and organic chemistry and relates to the problem of creating new drugs, inhibiting the proliferation of lymphocytes
Up!