Promoting agent of proliferation of regulatory t-lymphocyte, and their promotion method

FIELD: biotechnologies.

SUBSTANCE: invention proposed N-end fragment of soluble suppressor of immune response with the length of 21 amino-acids, which has sequence of amino-acids as per Seq ID NO: 1, allowing to promote formation of regulatory T-lymphocytes, as well as a promotion method of formation of regulatory T-lymphocytes with N-end fragment of soluble suppressor of immune response with Seq ID NO: 1, at its introduction with the concentration of 0.1-50 microgram/ml.

EFFECT: invention can be used for propagation of regulatory T-lymphocytes obtained from a patient suffering autoimmune disease, for the purpose of further injection of the obtained T-lymphocytes into the body of the patient.

3 cl, 2 tbl, 3 ex


The invention relates to the field of biotechnology, biologics, medical devices, designed to impact on the immune system of the body, namely the activity of T-lymphocytes, namely a stimulator of proliferation of regulatory T-lymphocytes.

Minor subpopulation of T-lymphocytes called regulatory T lymphocytes (Treg), capable of using multiple mechanisms to inhibit the proliferation of normal T-lymphocytes, having an important role in suppressing the activity of T-cells directed against antigens of the organism. Phenotypic regulatory T-lymphocytes are distinguished by the presence of markers of CD4+CD25+FoxP3+ [Aune T.M. et al. // J. Immunol. - 1983. - v. 131. - p.2848-2852; Webb D.R. et al. // International Immunology. - 1990. -v.2. - p.765-774].

The high content of regulatory T-lymphocytes was observed in the lymphocytes of tumor and draining lymph nodes of tumor that is probably one of the reasons for the suppression of T-cell immune response of the body to the tumor antigens. In this regard, the increased activity and/or maintenance of Treg in the body may become the treatment of several autoimmune diseases, complications associated with transplantation of organs and tissues, including bone marrow transplantation, such as disease "graft versus host". In particular, in the present method of treatment of autoimmune and alloimmune disease is the second by a fence of a patient, in need of this treatment, leukocyte mass, the separation from her cell with the phenotype CD4+CD25+, further cultivation of these cells in vitro for up to 4 weeks with the purpose of reproduction and reverse the introduction of the received cells to the patient (WO 2005086781, 2004).

As agents stimulating the proliferation of regulatory T cells is suggested to use azacytidine, tumor necrosis factor-beta, retinoic acid, trichostatin a, anti-CD3, anti-CD28, oxidized ATP, interleukin-2 (WO 2009126877, 2008; WO 2009114097, 2008).

The disadvantage of these stimulants is that they are not strictly selective stimulators of proliferation of regulatory T-lymphocytes, causing the need to find a more specific activator of proliferation of such cells.

Closest to the claimed invention is the use as a stimulator of cytokines, particularly of interleukin-2, which is injected into the culture medium in a dose of 100 IU/ml (WO 2005086781, 2004). The disadvantage of this stimulant is the lack of specificity.

The problem faced by the authors, was expanding Arsenal of stimulators of proliferation of regulatory T cells on the proliferation of regulatory T-lymphocytes.

The technical result was achieved in the synthesis of peptide representing the N-terminal fragment of soluble su the spring immune response (SIRS 1-21), the amino acid sequence of which is presented in Annex 1, Seq ID NO: 1, which was having voting stimulating effect on the proliferation of regulatory T cells (Treg) in populations of lymphocytes in the thymus and spleen.

Protein SIRS (soluble immune response suppressor), the complete amino acid sequence of which is still unknown, is a polypeptide with a molecular weight of about 10-14 kDa, which is produced by CD8+ cells, activated mitogen, antigen or interferon (US 4771125, 1984) and has the ability to inhibit the production of antibodies and to inhibit the reaction of the delayed-type hypersensitivity in vivo [Aune T.M. et al. // J. Immunol. - 1983. - v. 131. - p.2848-2852; Webb D.R. et al. // International Immunology. - 1990. - v.2. - p.765-774]. The amino acid sequence of the N-terminal segment SIRS mouse, containing 21 amino acid residue (SIRS 1-21), set [Webb D.R. et al. // International Immunology. - 1990. - v.2. - R-774]. Information about the use of SIRS or its fragments as stimulators of proliferation of Treg cells in the reviewed literature was not found.

During the studies the authors found that the SIRS peptide 1-21 during culturing a population of cells containing T-lymphocytes that selectively stimulates the proliferation of regulatory T lymphocytes (Treg, CD4+CD25+FoxP3+ cells), practically no impact on the proliferation of total is opulatio cells, that may be associated with the inhibition of regulatory T-lymphocytes proliferation of normal lymphocytes.

Stimulation of the formation of regulatory T-lymphocytes is carried out by introducing the culture fluid stimulator SIRS 1-21 in a concentration of 0.1-10 µg/ml While the efficiency increases with the simultaneous introduction into the system of interleukin-1-beta at a dose of 100-300 PG/ml

The nature and advantages of the claimed group of inventions are illustrated by the following examples.

Example 1. Chemical synthesis of the N-terminal fragment of SIRS (SIRS1-21)

Synthesis of peptide SIRS1-21performed by a solid phase method on a synthesizer Vega Coupler 250 according to the method of in situ using Nα-Boc-protected derivatives of amino acids. In the work we used the following derivatives of amino acids Boc-Ser(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Pro-OH, Boc-Ile-OH, Boc-Asn(Trt)-OH, Boc-Gln-OH, Boc-Glu(OBzl)-OH, Boc-Met-OH, Boc-Asp(OcHex)-OH, Boc-Ala-OH, Boc-Lys(ClZ)-OH. The source of the aminoacyl-polymer served as Boc-Ser(Bzl)-PAM with specific capacity of 0.64 mmol/g.

Release temporary tert-butyloxycarbonyl protection held 50% triperoxonane acid (TFA) in methylene chloride (DCM). The condensation reaction was carried out in dimethylformamide (DMF). Neutralization in the first condensation was carried out by adding a threefold excess diisopropylethylamine (DIPEA) directly into the reaction mixture at the stage of joining AMI is kislotno residue; re-condensation was carried out after an additional washing peptidyl-polymer 10% solution of DIPEA in DMF. The joining of amino acid residues was performed by the method of activated esters derived from the corresponding derivatives of amino acids using aminobutiramida-carbodiimide, using a 5-fold excess of reagent in DMF. The mixture of amino acid derivatives were prepared immediately before introduction into the reaction vessel. The completeness of the condensation reaction was performed using ninhydrin and bromophenol tests.

After completion of the extension of the peptide chain enters the polymer was treated with 50% TFA twice for 1 minute, washed with DCM and diethyl ether was removed from the reactor and dried to constant weight.

Cleavage of the peptide from the resin and removal of side protective groups was performed using liquid hydrogen fluoride on Snl mechanism in the presence of scavengers.

Selected coarse product was subjected to purification by the method of prepreparation reversed-phase HPLC on a column (Waters Prep Nova-Pak HR C-18, C, 60Å, 19×300 mm (detection at 220 nm).

The fraction corresponding to the peak of the main product, after freeze drying were subjected to mass spectral and HPLC-analysis.

According to MALDI-TOF spectrometry molecular weight of the peptide (M+N)+- 2281. theoretical molecular weight in the calculation of the free peptide - 2280,38

The content of the basic substance, the SIRS peptide1-21amounted to more than 95% according to HPLC.

Example 2. The influence of the SIRS1-21on the proliferation of splenocytes

The SIRS peptide1-21bred in the medium RPMI-1640 with 1% fetal calf serum at a concentration of 1 mg/ml, were prepared serial dilutions and added into the wells of 96-Loup night - tablet 50 ál per well. Later in the wells contributed suspension of splenocytes in 100 μl of RPMI-1640 medium containing 10% fetal calf serum (TES), 3×105cells per well. A production test was carried out not less than 4 Parallels for each experimental point.

The Board was placed in CO2incubator and incubated at 37°C in conditions of absolute humidity. After 48 h from the beginning of incubation in cell culture was made3H-thymidine (Isotope, St. Petersburg) at a final concentration of 5 mccu/ml Through day cells were transferred to glass fiber filters using a FilterMate harvester 96 (Perkin-Elmer) and determined the intensity of the incorporation of thymidine on a tablet β-counter MicroBeta TriLux 1450 (Perkin-Elmer).

The results, expressed in number of H3-thymidine incorporated into acid-insoluble residue (imp/min) are presented in table 1.

Table 1
In Janie SIRS peptide 1-21on the proliferation of mouse splenocytes in vitro
SIRS1-21µg/mlThe intensity of the proliferation of splenocytes (imp/min)
0 (control)8305±1254

Analysis of the results indicate a weak inhibition of the proliferation of splenocytes in the presence of 0.1 μg/ml of the investigated peptide. Lower and higher concentrations SIRS1-21during the analyzed period of time (1 day) effects on the proliferation of splenocytes did not.

Example 3. Assessing the impact of SIRS peptide1-21on the proliferation of regulatory T-cells in vitro.

During the experiment came from the fact that cytofluorimetric regulatory T cells can be identified so CD4+ CD25+ lymphocytes, also expressing the transcription factor FoxP3.

The thymocytes or splenocytes mouse were incubated with peptide SIRS1-21in the presence of 100-300 PG/ml recombinant IL-1β or without it for 3 days. To determine the percentage of regulatory T-cells in suspense and thymocytes and splenocytes cells were stained with monoclonal antibodies to surface markers CD4, CD8 and CD25, and intracellular marker FoxP3. Used antibodies to mouse eBioscience: anti-Rohr-FITC (clone FJK-16s), anti-CD4-PE (clone GK1.5), anti-CD25-PE-Cy5 (clone RS), anti-CD8a-PE-Cy5 (clone 53-6 .7). For staining with antibodies to FoxP3 used a set of Foxp3 / reduced Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions. Analysis of the expression of relevant markers was performed in a three-color mode on cytofluorimetry EPICS XL (Beckman-Coulter). The results obtained are presented in table 2.

Table 2
The influence of the SIRS1-25on the CD4+CD25+FoxP3+cells in cultured thymocytes and splenocytes mouse
SIRS1-21µg/mlThe CD4+CD25+FoxP3+ cells (%)
The thymocytesSplenocytes
Without IL-1βWith IL-1β3 PG/mlWithout IL-1βWith IL-1β3 PG/ml
100200300 100200300
16,50,630,770,850,797,37of 5.45,565,1
500,410,900,990,95 7,395,585,61the ceiling of 5.60

Table 2 shows that the best results are achieved by incubation of the cells in the presence of 16.5 ág/ml SIRS1-21. Thus, there is a 1.5 to 2-fold increase in the content of CD4+CD25+FoxP3+ in suspensions of thymocytes and 2-3-fold increase in the content of the cells of this phenotype in suspensions of splenocytes.

For thymocytes effect SIRS1-21on the CD4+CD25+FoxP3+cells is more pronounced on the background of IL-1β, which is itself on the content of the data cells in the population of thymocytes virtually no effect. Thus, SIRS1-21stimulates the proliferation of Treg in populations of thymocytes and splenocytes, including the presence in the environment of the cultivation of IL-1β.

From the above data it follows that the SIRS peptide1-21when culturing a population of cells containing T-lymphocytes that selectively stimulates the proliferation of regulatory T lymphocytes (Treg, CD4+CD25+FoxP3+cells), practically no impact on the proliferation of the total population of cells.

This property makes this peptide promising for breeding ex vivo regulatory T-lymphocytes obtained from a patient suffering from autoimmune disease associated with chronic inflammation, for example, a patient with rheumatoid arthritis, psoriasis and psoriaticescom arthritis, diabetes 1-g is of type autoimmune Hashimoto (Hashimoto's disease), ulcerative colitis, Crohn's disease, granulomatosis vasculitis, sarcoidosis, scleroderma, multiple sclerosis, systemic sclerosis, glomerulonephritis autoimmune nature and other similar ailments, graft rejection, diseases, graft versus host, with subsequent introduction of the obtained T-regulatory T-lymphocytes of this patient for treatment of the above diseases and pathological conditions.

1. The N-terminal fragment of soluble suppressor of the immune response length of 21 amino acid having the amino acid sequence according to Seq ID NO: 1, as a stimulant formation of regulatory T-lymphocytes.

2. Method of promoting the formation of regulatory T-lymphocytes by culturing cells containing T-lymphocytes, in the presence of the stimulator, wherein the stimulator into the culture medium injected N-terminal fragment of soluble suppressor of the immune response according to claim 1 in a concentration of 0.1-50 μg/ml

3. The method according to claim 2, characterized in that in the environment of the cultivation of additionally injected interleukin-1-beta at a dose of 100-300 PG/ml


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