Thymus-specific protein

FIELD: medicine.

SUBSTANCE: thymus-specific protein T101 consisting of 84 amino acids is recovered from human thymus. The recovered full-length peptide T101 includes the signal peptide consisting of 33 amino acids and the peptide sequence T101 consisting of 51 amino acids and exhibiting immunomodulating activity. The full-length peptide T101, and also peptide fragments and derivatives are used as a part of a pharmaceutical composition for treating autoimmune and inflammatory diseases.

EFFECT: invention allows preparing polypeptide capable to stimulate lymphocyte proliferation of human peripheral blood, to inhibit the tumour growth and to modulate the immune system.

10 cl, 15 dwg, 1 tbl, 11 ex

 

2420-144113RU/031

The scope to which the invention relates

The present invention relates to a new thymus-specific protein.

Prior art

Listed below are the previous publication, which illustrate the prior art in the field to which the present invention is:

[1] Maurer, H.R., Eckert, K., Stange, R.: Einfluss der Therapie mit Thymoject auf die antitumorale Immunotoxizität der Leukozyten von Mamma-Tumorpatientinnen. Pers. Mitt. (1999).

[2] Mustacchi, G., Paves, L., Milani, S. et al.: High-dose folinic acid and fluouracil plus or minus thymostimulin for the treatment of metastatic colorectal cancer: results of a multicentered randomised trial. Anticancer Res. (1994) 14: 617-619.

[3] Schulof, R.S., Lloyd, M.J., Cleary, P.A., et al.: A randomized trial to evaluate the immunorestorative properties of synthetic thymosin-alpha1 in patients with lung cancer. J. Biol. Resp. Modif. (1985) 4: 147-158.

[4] Azizi, A., Brenner, H.J., Shoham, J.: Postoperative adjuvante Behandlung von Patienten mit malignem Melanom durch den Thymusfaktor Thymostimulin. Arzneim-Forsch/Drg. Res. (1984) 34(11): 1043-1046.

[5] Massimo, F., Gobbi, P., Moretti, G. Avanzini, P., Italian Lymphoma Study Group: Effects of Thymostimulin combination with Chemotherapy in patients with aggressive non-Hogkins lymphoma. Am. J. Clin. Oncol. (CCT) (1995) 18(1): 8-14.

[6] Peretti, P., Tonelli, F., Mazzei, T., Ficari, F., Italian study group on antimicrobal prophylaxis in abdominal surgery. J. Chemotherapy (1993) 5(1): 37-42.

[7] Gonelli, S., Petrioli, R., Cepollaro, C., Palmieri, R., Aquino, A., Gennari, C.: Thymostimulin in association with chemotherapy in breast cancer patients with bone metastases. Clin. Drug Invest (1995) 9(2): 79-87.

[8] Iaffaioli, R.V., Frasci, G., Tortora, G., Ciardiello, F., Nuzzo, F., Scala, S., Pacelli, R., Bianco, A.R.: Effect of thymic extract Thymostimulin on the incidence of infections and myelotoxicity during adjuvant chemotherapy for breast cancer. Thymus 1988) 12: 69-75. Kluwer Academic Publishers.

In the description of the invention these publications indicated above numbers in parentheses.

Background of invention

Currently identified many modifiers of biological responses (IBA). Examples of such modifiers are interleukins and cytokines. In General, in immunomodulation plays an important role in the thymus. We can say that the thymus is the Central organ of the immune system. It is believed that the thymus plays a key role in the development and functioning of the immune system and mechanisms of biological protection from cancer and chronically infected cells.

The tissue of the thymus is responsible for selective transformismo precursor cells in different T cells, that is, helper (CD4+) T-lymphocytes, which facilitates the differentiation of other lymphocytes, killer cells (NK-cells); cytotoxic cells and suppressor (cytotoxic) (CD8+) T-lymphocytes [1-3]. Lymphocytes after their release into the bloodstream, as well as in the intestinal and peripheral tissues are characterized by clearly defined antigens or markers of activation present on their surface. Their activity occur outside of the thymus.

There are very subtle interaction between the thymus and the active bone marrow. Thus there is a direct and positive correlation between hypofunction timesay declining production of colony-stimulating factors (CSF). Therefore, in case of insufficient production of CSF may be a useful therapeutic use timoszyk peptides.

Chronic hepatitis b (start), HIV infection, chronic hepatitis C (CHC) and malaria are chronic diseases, which currently affects about ten million people, without getting any treatment. The cellular component of the immune system responsible for immune “oversight”, aimed at the prevention of these chronic viral, fungal, yeast and parasitic infections as well as tumors and symptoms of aging. Timoshii extracts found clinical application in the treatment of various diseases, including some of the specified States. These extracts are administered orally or by injection as in pure form and in combination with other therapeutic agents. Timoshii extracts are used to treat severe and chronic allergies, including allergies, respiratory tract and skin, and also for the treatment of severe, acute and chronic infectious diseases. It was shown that these extracts reduce the risk of surgical infections and reduce the degree of damage caused by chemotherapy and radiation, and can also be used as an auxiliary means in the main course of therapy on the I treatment of tumor diseases. All of these States successfully treated with the use of extracts isolated from thymus cows.

In randomized trials of patients with malignant melanoma introduction timoszyk peptides led to the increase of the period of cessation of tumor growth, increase life expectancy and improve quality of life [4]. In another randomized study of patients with intermediate and vysokokachestvennoj non-Hodgkin lymphoma these patients in addition to standard chemotherapy were treated tempname peptides. Past the specified course of treatment patients are fairly well tolerated such treatment, and these patients showed a significantly higher rate of response than patients who did not enter timoshii peptides [5]. In another randomized trial, which involved patients undergoing surgery on the colon, it was found that in patients that in addition to cefotetan introduced timoshii peptides was observed a significant decrease in the level of development of abscesses in the abdomen and infections in the upper respiratory tract [6]. Randomized studies involving women with advanced breast cancer, have documented that women who in addition to chemotherapy was administered timoshii peptides were observed far the best part tolerability of chemotherapy and reduction of the level of development of secondary infections [7, 8].

In accordance with the above data it is evident that timoshii peptides can be used to enhance the function of bone marrow and to protect the patient from mielosupression caused by chemotherapy; to stimulate recovery of the bone marrow after irradiation and chemotherapy; to prevent the development of secondary infections due to immunosuppression caused by standard chemotherapy and surgery; to increase the rate of complete and partial response to anticancer therapy; and to improve the function of lymphocytes and biological defense mechanisms.

The immune system consists of many different cells including T-cells, B-cells, NK-cells, etc. These different cells vary in nature. Immature T-cells are formed in bone marrow, and then they migrate to the thymus. In the thymus there are various processes that lead to the production of Mature T-cells and the secretion of various peptides that regulate the immune response. T cells can be subdivided into several subgroups, such as T-helper cells, T-cytotoxic cells, T-memory cells and regulatory T cells. Cells of each such sub-groups play a role in immune response. Cells of these subgroups characterized by different antigens present on their membranes. So, for example,T-helper cells are the CD4 antigen on their membrane, and T-cytotoxic cells are CD8 antigen on their membrane.

CD4+T cells play a regulatory role and, obviously, associated with peripheral autotolerance. The assumption of the existence timoszyk regulatory T-cells was first suggested previously, based on the fact that autoimmune disease in mice begins to develop on the 3rd day after thymectomy. It was found that these disorders are caused by loss of peripheral CD4+-T-cells, constitutively expressing IL-2R alpha (CD25), which appear on the periphery of much later after birth. Physiologically generated CD4+-CD25+cells suppress autoimmune and inflammatory disorders a wide range, such as colitis.

Despite numerous studies, the regulatory mechanism of action of CD4+-CD25+-T cells remains unclear. Some studies have shown that the regulation of in vivo depends on the production of cytokines-suppressors, such as IL-10 and GF-β and cell surface molecules such as CTLA-4. In vivo studies showed that the suppressor effect is not mediated by cytokines and cellular interactions.

In General it was reported that in vivo CD8+T-cells play a major role in the prevention of experimental autoimmune encephalomyelitis and promote the oral tolerance. Regulatory CD8+-CD28-T cells can be generated and propagated in vitro by conducting multiple rounds of stimulation of allogeneic APC, but it is not known whether this population in vivo. A new study has revealed a new population of regulatory CD8+cells also constitutively Express CD25 and which have properties very similar to the properties of CD4+-CD25+cells.

These cells produce IL-10, Foxp3 and CTLA-4 and inhibit CD25--T-cell response to stimulation with anti-CD3 antibody mediated intercellular interactions, efficacy, similar to the efficiency of CD4+-CD25+cells.

Obviously, these 2 subpopulations of CD4+-CD25+cells and CD8+-CD25+cells involved in the regulation of the immune response.

Description of the invention

The aim of the present invention is the selection in the thymus-specific protein from human thymus.

Using PCR libraries of human cDNA, isolated from 16 different tissues 20-year-old women were subjected to screening, and was identified unique cDNA of the human thymus. The peptide encoded by the indicated cDNA was identified T. Were subjected to screening banks of human, mouse and rat EST, but any similarities cDNA T with any of the known Geno is found. Similar results were obtained using banks proteins and full-length genomic DNA. The cDNA sequence and corresponding amino acid sequence are new sequences that have not been described previously.

The peptide encoded by the indicated cDNA has a length of 84 amino acids and includes a signal peptide consisting of 33 amino acids present at the N-end (see figure 1). Below are the cDNA sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) T:

atgatggcactcagaagccaggggctcatgttaccccaga

gctgcccacaactggctttcctcaccctaagtgccttggc

agcagtgtctttttcagctctgcatctctggcttagtggg

gagccagtccagagctctggaacaaaggacatgagatcca

aatccgattccaagcgagtgagtgacaagcagctaatttc

caaagctgtgtggtggacattttttcttccttcaaccctc

tgggagagaaaatga(SEQ ID NO:1)

MMLRSQGLMLPQSCPQLAFLTLSALAAVSFSALHLWLSG

EPVQSSGTKDMRSKSDSKRVSDKQLISKAVWWTFFLPSTL

WERK(SEQ ID NO:2)

The present invention also pertains to nucleic acid molecule SEQ ID NO:1 and peptide SEQ ID NO:2. The polypeptide SEQ ID NO:2 will hereinafter be called “full T101 peptide”.

Full-peptide T also includes a signal sequence of 33 amino acids. Thus, the present invention also relates to a peptide comprising the sequence of full-length peptide T that does not contain the specified signal peptide and having the following posledovatel the activity (SEQ ID NO:4):

LHLWLSGEPVQSSGTKDMRSKSDSKRVSDKQLISKAVWWT

FFLPSTLWERK(SEQ ID NO:4)

Peptide T that does not contain a signal sequence (SEQ ID NO:4), will hereinafter be referred to as “peptide C”.

The present invention also pertains to nucleic acid molecule containing a sequence encoding the peptide T. This sequence comprises the following sequence (SEQ ID NO:3):

catctctggcttagtggggagccagtccagagctctggaacaaag

gacatgagatccaaatccgattccaagcgagtgagtgacaagcag

ctaatttccaaagctgtgtggtggacattttttcttccttcaacc

ctctgggagagaaaatga(SEQ ID NO:3)

The present invention relates to modified nucleic acid molecules SEQ ID NO:1 or SEQ ID NO:3 and modified peptides SEQ ID NO:2 or SEQ ID NO:4, which added deleterows or substituted by one or more nucleotides or amino acid residues, respectively, where these modifications do not significantly affect the biological properties of the specified modified molecules compared to the unmodified molecule.

Used herein, the term “peptide” means a peptide, polypeptide or protein. This peptide can be obtained by synthesis by means of genetic engineering, by expression in a cell-the owner or any other appropriate methods.

The term “biological property”, referring to the peptide molecule means that the molecule has on me is greater least one in vitro or in vivo activity, which may have a full-sized peptide T or peptide T, including, but not limited to, biological activity, as described below in the “Examples”section. The term “biological property”, referring to a molecule of nucleic acid, refers to the ability of this molecule to encode a peptide having biological properties similar to the biological properties of the full-length peptide T or peptide T, including, in particular, (i) a nucleic acid molecule having a sequence that differs from the sequence of SEQ ID NO:1 or SEQ ID NO:3, but which due to the redundancy of the genetic code, encodes the full-sized peptide T or peptide T respectively; and (ii) a nucleic acid molecule encoding the amino acid sequence that differs from the sequence of the full-length peptide T or peptide T, but which has biological properties similar to the biological properties of the full-length peptide T or peptide T respectively.

The term “no significant effect on the biological properties of the modified molecules compared to the unmodified molecule” means that the modified molecule retains the biological activity, which by its qualities is similar to the activity of the unmodified molecule. In relation to the attachment of the modified peptide, the term refers to, the specified peptide retains one or more biological properties of the peptide SEQ ID NO:2 or SEQ ID NO:4, including, among others, its diagnostic and therapeutic effectiveness, described below, as well as in vitro and in vivo activity, described below in the “Examples”section. In order to determine whether this peptide biological activity, which is qualitatively similar to the activity of unmodified molecules, it is possible to conduct one or more tests, such as in vitro, in vivo or clinical experiment in which the modified peptide is compared with the corresponding unmodified peptide (namely with a full-sized peptide T or peptide T), which are subjected to a parallel analysis or experiment, wherein said modified peptide analyze in order to determine whether he has biological activity similar to the activity of the unmodified peptide, which was established in separately conducted experiment. Such experiments can be conducted, for example, as described below in the “Examples”section. In relation to the modified nucleic acid molecules, the term “no significant effect on the biological properties of the modified molecules compared to the unmodified molecule” means that the indicated molecule is capable of being in order to encode a modified peptide, have any of the above properties.

The modified peptide may be a peptide comprising a sequence of adjacent amino acids consisting of at least 8, 12, 15, 20, 25, 30, 35, 40 or at least 45 amino acid residues, at least 70%, preferably at least 80%, more preferably at least 90%, particularly at least 95% identical to the corresponding sequence consisting of at least 8, 12, 15, 20, 25, 30, 35, 40 or at least 45 amino acid residues present in the peptide T.

The present invention also relates to a peptide containing a partial sequence of contiguous amino acids derived from full-length peptide T and comprising at least 8 amino acid residues, where the specified continuous sequence is a sequence of contiguous amino acids present in the specified full-size peptide T. This peptide will hereinafter be called “incomplete peptide T”. In one of its variants the present invention relates to incomplete peptide T containing the following continuous sequence of 13 contiguous amino acid residues starting from the N-Terminus of the peptide T (amino acids 39-51):

WTFFLPSTLWERK(SEQ ID NO:5)

The present invention from OSISA also to a protein or polypeptide, containing the amino acid sequence of full-length peptide T, peptide T, modified peptide or partial peptide T (such peptide or polypeptide will hereinafter be called “T-containing protein”). T-containing protein may, for example, be a hybrid protein comprising a full-sized peptide T, peptide T, a modified peptide or partial peptide T; and it may be a conjugate of a protein or other peptide or polypeptide with a full-sized peptide T, peptide T, modified peptide or partial peptide T etc.

The present invention also relates to oligonucleotide consisting of at least 24 nucleotides and represent (i) an oligonucleotide that encodes an incomplete continuous sequence of contiguous amino acids of the peptide T comprising at least 8 amino acid residues, and which may include a continuous sequence of 24 contiguous nucleic acids present in SEQ ID NO:1; (ii) a nucleotide sequence that can gibridizatsiya with nucleotide sequence SEQ ID NO:1 under conditions of hybridization of high rigidity; (iii) an oligonucleotide having a sequence that is at least 24 contiguous nucleotides which is at least 70%, preferred the equipment at least 80%, more preferably at least 90%, particularly at least 95% identical to the corresponding continuous sequence of contiguous nucleotides present in SEQ ID NO:1.

The present invention also pertains to nucleic acid molecule, such as a vector to transfer or expression vector containing any of the above nucleic acid molecules.

In another aspect the present invention relates to the following additional partial peptides T:

SGEPVQSSGTKDMRSKSDSKRVS(SEQ ID NO:6)

DKQLISKAVWWTFFLPSTLWERK(SEQ ID NO:7)

PSTLWERK(SEQ ID NO:8)

AVWWTFFLPSTLW(SEQ ID NO:9)

KREWLTSPLFFTWWVA(SEQ ID NO:10)

WTFFL(SEQ ID NO:11)

Sequence of SEQ ID NO:6 is composed of amino acids 6-28 peptide T; the sequence of SEQ ID NO:7 is the amino acid 29-51 peptide T; the sequence of SEQ ID NO:8 is the amino acid 44-51 peptide T; the sequence of SEQ ID NO:9 is composed of amino acids 36-48 peptide T; the sequence of SEQ ID NO:10 is composed of amino acids 36-51 peptide T located in the reverse order; the sequence of SEQ ID NO:11 is composed of 39-43 amino acids of the peptide T.

The present invention relates to modified peptides derived from any of the peptides defined above, for example modified peptides in which one or more amino acid substitution is s other amino acids by conservative substitutions. Used herein, the term “conservative substitution” means the replacement of an amino acid of one class of amino acid of the same class, where the class of amino acids is determined by the General physico-chemical properties of the side chains of amino acids and a high frequency of substitutions in homologous proteins observed in nature. There are six General classes of side chains of amino acids, and such classes are: class I (Cys); class II (Ser, Thr, Pro, Ala, Gly); class III (Asn, Asp, Gln, Glu); class IV (His, Arg, Lys); class V (Ile, Leu, Val, Met) and class VI (Phe, Tyr, Trp). So, for example, replacement of Asp by another residue class III, such as Asn, Gln or Glu, is a conservative substitution.

In one embodiment of the invention in the amino acid sequence has only one replacement. In another embodiment of the invention in the amino acid sequence has two substitutions. In yet another variant of the invention in the amino acid sequence has three substitutions. The maximum number of substitutions must not exceed the number of amino acids that is at least 70%, preferably at least 80%, preferably at least 90%and most preferably at least 95% of all amino acids in the unmodified sequence. In one of the preferred variants of the invention replacements, which include up to 3, sometimes up to 6 amino acid residues, replaced by other residues, the two who are conservative substitutions.

In another embodiment, one or more amino acids may be replaced by D-amino acids, and preferably the corresponding D-amino acids.

In yet another embodiment, the present invention also include sequences located in the reverse order with respect to the above sequences.

For example, the present invention relates also to a full-sized peptides Et SEQ ID NO:2, or preferably a peptide Et SEQ ID NO:4, or to incomplete sequences T modified by one or more conservative substitutions.

The present invention also relates to a peptide comprising at least 10 or 15, or 20, or 25, or 30, or 35, or 40 amino acid residues, or polnorazmernomu the peptide T having the sequence:AA1,-AA2-...-AA51where

AA1selected from leucine, isoleucine, valine and methionine;

AA2selected from lysine, arginine and histidine;

AA3selected from leucine, isoleucine, valine and methionine;

AA4selected from tryptophan, phenylalanine and tyrosine;

AA5selected from leucine, isoleucine, valine and methionine;

AA6selected from serine, threonine, alanine, glycine and Proline;

AA7selected from serine, threonine, alanine, glycine and Proline;

AA8selected from g is alumina, glutamic acid, aspartic acid and asparagine;

AA9selected from serine, threonine, alanine, glycine and Proline;

AA10selected from leucine, isoleucine, valine and methionine;

AA11selected from glutamine, glutamic acid, aspartic acid and asparagine;

AA12selected from serine, threonine, alanine, glycine and Proline;

AA13selected from serine, threonine, alanine, glycine and Proline;

AA14selected from serine, threonine, alanine, glycine and Proline;

AA15selected from serine, threonine, alanine, glycine and Proline;

AA16selected from lysine, arginine and histidine;

AA17selected from glutamine, glutamic acid, aspartic acid and asparagine;

AA18selected from leucine, isoleucine, valine and methionine;

AA19selected from lysine, arginine and histidine;

AA20selected from serine, threonine, alanine, glycine and Proline;

AA2Iselected from lysine, arginine and histidine;

AA22selected from serine, threonine, alanine, glycine and Proline;

AA23selected from glutamine, glutamic acid, aspartic acid and asparagine;

AA24selected from serine, threonine, alanine, glycine and Proline;

AA25selected from lysine, arginine and histidine;

AA26selected from lysine, arginine and histidine;

AA27selected Slatina, isoleucine, valine and methionine;

AA28selected from serine, threonine, alanine, glycine and Proline;

AA29selected from glutamine, glutamic acid, aspartic acid and asparagine;

AA30selected from lysine, arginine and histidine;

AA31selected from glutamine, glutamic acid, aspartic acid and asparagine;

AA32selected from leucine, isoleucine, valine and methionine;

AA33selected from leucine, isoleucine, valine and methionine;

AA34selected from serine, threonine, alanine, glycine and Proline;

AA35selected from lysine, arginine and histidine;

AA36selected from serine, threonine, alanine, glycine and Proline;

AA37selected from leucine, isoleucine, valine and methionine;

AA38selected from tryptophan, phenylalanine and tyrosine;

AA39selected from tryptophan, phenylalanine and tyrosine;

AA40selected from serine, threonine, alanine, glycine and Proline;

AA41selected from tryptophan, phenylalanine and tyrosine;

AA42selected from tryptophan, phenylalanine and tyrosine;

AA43selected from leucine, isoleucine, valine and methionine;

AA44selected from serine, threonine, alanine, glycine and Proline;

AA45selected from serine, threonine, alanine, glycine and Proline;

AA46selected from serine, threonine, alanine, glycine and Proline;

AA47selected from leucine, isoleucine, valine and methionine;

AA48selected from tryptophan, phenylalanine and tyrosine;

AA49selected from glutamine, glutamic acid, aspartic acid and asparagine;

AA50selected from lysine, arginine and histidine; and

AA51selected from lysine, arginine and histidine.

The present invention relates to modified peptides derived from the full-length peptide T, peptide T or a partial peptide T, which includes the following subsequence (amino acids are numbered according to the numbering of the peptide T):

AA38-AA39-AA40-AA41-AA42where AA38and AA39represent amino acids of class VI, preferably tryptophan; AA40represents the amino acid class II, preferably threonine; and AA41and AA42represent amino acids of class VI, preferably phenylalanine;

AA38-AA39-AA40-AA41-AA42-AA43where AA38and AA39represent amino acids of class VI, preferably tryptophan; AA40represents the amino acid class II, preferably threonine; AA41and AA42are aminokisloty class VI, preferably henylalanine; and AA43represents the amino acid class V, preferably leucine;

Ala-Val-AA38-AA39-AA40-AA41-AA42where AA38and AA39represent amino acids of class VI, preferably tryptophan; AA40represents the amino acid class II, preferably threonine; and AA41and AA42represent amino acids of class VI, preferably phenylalanine;

Ala-Val-AA38-AA39-AA40-AA41-AA42-AA43where AA38and AA39represent amino acids of class VI, preferably tryptophan; AA40represents the amino acid class II, preferably threonine; AA41and AA42represent amino acids of class VI, preferably phenylalanine; and AA43represents the amino acid class V, preferably leucine.

A subsequence of 24 amino acids of SEQ ID NO:4 (amino acids 24-47):

SKRVSDKQLISKAVWWTFFLPSTL(SEQ ID NO:12),

has a close affinity with the following sequences originating from mammals other species, namely:

from dogs:SKQVSDKQLISKAVQRIFFFLQPS(SEQ ID NO:13); and

from rats:SKFMSDKQLISKAVQRIFFLSSTL(SEQ ID NO:14).

When comparing the sequences of SEQ ID NO:12-14 was identified following consensus sequence for mammalian what PEX species (shown in capital letters):

SKrvSDKQLISKAVwwtFFLpSTL(SEQ ID NO:12)

SKqvSDKQLISKAVQRIFFflqps(SEQ ID NO:13)

SKfmSDKQLISKAVQRIFFLsSTL(SEQ ID NO:14)

The present invention also relates to a peptide containing this consensus sequence. This peptide (referred to here as the “consensus peptide”) has one of the following formulas:

SKx1x2SDKQLISKAVx3x4x5FFLx6STL(SEQ ID NO:15);

SKx1x2SDKQLISKAVx3x4x5FFLx6(SEQ ID NO:16);

SKx1x2SDKQLISKAVQRIFF(SEQ ID NO:17); or

SKx1x2SDKQLISKAVQRIFFLx6STL(SEQ ID NO:18);

where

x1represents R, Q or F, or, alternatively, as a result of conservative substitutions, H, K, N, D, E, Y, or W;

x2is a V or M, or, alternatively, as a result of conservative substitutions, I or L;

x3represents W or Q, or, alternatively, as a result of conservative substitutions, F, Y, N, D, or E;

x4is a W or R, or, alternatively, as a result of conservative substitutions, F, Y, H, or K;

x5is a T or I, or, alternatively, as a result of conservative substitutions, S, P, A, G, L, V or M; and

x6represents P, L or S, or, alternatively, as a result of conservative substitutions, T, A, G, I, V or M.

This is e the invention relates also to a protein or polypeptide, which contains the amino acid sequence indicated a consensus peptide. The present invention also relates to a nucleotide sequence that encodes the above consensus peptide, or a protein or polypeptide containing the amino acid sequence indicated a consensus peptide.

T is soluble in aqueous solution. It's his property and biological properties, described below, as well as its tissue specificity and physical properties characteristic of the peptide hormone contribute to the fact that the peptide T, full-peptide T, a partial peptide T or any of their modifications can be used as therapeutic agents to induce a cellular immune response and to treat a variety of other clinical conditions such as HIV infection, hepatitis b, hepatitis C, cancer and malaria.

As will also be shown in the following examples, it was found that the peptide T and going away various modified peptides have the ability to modulate the immune system. This modulation is reflected in activation of the immune system (as indicated by, for example, an increase in the level of proliferation of peripheral blood lymphocytes (PBL)and in suppression of the immune system (as indicated by, for example, an increase in the level of production of I-10 and a decrease in the number of LPA). Thus, T has as immunoactivity and immunoinhibitory ability. Without pretending to any particular theory, and without limiting the scope of the present invention, it is possible to say that the mechanism of action T on the immune system may depend on its concentration.

The present invention relates also to methods of treatment, methods of diagnosis and to pharmaceutical compositions that use the specified peptide T, full-peptide T, a partial peptide T modified peptide T, or T-containing protein, or any nucleic acid molecule mentioned above. Peptide T can have the following diagnostic and therapeutic applications:

1. T can serve as a tool to diagnose the lack of immunocompetence after subcutaneous injection of toxins isolated from various organisms.

2. Determination of the level T in blood may be an indicator of increased or decreased function of the thymus and the indicator of the level of activity of the immune system.

3. Level T can serve as an indicator of autoimmune diseases.

4. T can serve as a stimulator of the immune system. For example, T can be used to treat the condition associated with lack of immunocompetence against bacteria, parasites and virusvaktsinu.

5. T can be used as diagnostic tools for the prevention of recurrent infections. For example, T should reduce the number of relapses of infections in the respiratory tract.

6. T can be used as a therapeutic agent for attenuating allergic and inflammatory reactions. For example, T can be used to reduce the incidence of asthma or alleviate its symptoms.

7. T can be used for the treatment of viral diseases, such as tuberculosis, herpes, acute and chronic hepatitis b, acute and chronic hepatitis C, chronic cholestatic hepatitis, cirrhosis, and stomatitis.

8. T can be used as a therapeutic agent for the treatment of diseases associated with immunodeficiency, such as AIDS and combined immunodeficiency.

9. T can be used as a therapeutic agent for the treatment of skin diseases such as atopic eczema and psoriasis.

10. T can be used for treatment of some other diseases. For example, T can be used as an inhibitor of the immune system for the treatment of autoimmune pathologies, such as inflammatory bowel diseases, severe myasthenia, multiple sclerosis, diabetes type I, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic autoimmune g is Politicheskaya anemia, colitis, Crohn's disease, etc.

11. T can be used as a stimulator of the immune system for the treatment of malignant diseases such as lung cancer, carcinoma of the larynx, carcinoma of the head and neck, carcinoma of the breast, Hodgkin's disease, non-Hodgkin's lymphoma, breast cancer, hepatocellular cancer, and melanoma.

12. T can be used to prevent infections after surgery and implantation. T can be used after transplantation for the inhibition of transplant rejection.

13. T can be used to enhance the immune response in elderly or younger people or people with compromised immune systems.

14. T can be used to treat skin infections after burns.

15. T can be used as a therapeutic agent for the treatment of infertility in men.

16. T can increase heart activity.

17. T can be used in a method of identifying cells of the thymus. T can also serve tempnam marker and indicator of the metabolic status of the thymus. For example, T can be used in ELISA-assays or other assays for measuring the metabolic condition of the thymus.

18. T can be used as a vehicle for the manifestation of a fluorescent dye in order to identify the value of specific subpopulations of leukocytes to highlight the various subpopulations of cells from various tissues.

19. T can serve as a General stimulator or inhibitor of various immune responses and may also directly or indirectly affect other organs such as the heart, lungs, etc.

20. T can be used to modulate the functions of the nervous system, such as memory and regeneration of the nervous system, as well as for the relief of pain and treatment of pathological conditions of the nervous system such as Parkinson's and Alzheimer's disease.

21. T can serve as a probe to identify specific cells of the immune system and their use for cell therapy.

For the above diagnostic and therapeutic purposes can also be used with full sized peptide T, a partial peptide T, T-containing protein or a modified peptide T.

Brief description of the graphical material

For a better understanding of the present invention and to facilitate its implementation will be described below, the results of the experiments conducted in accordance with the present invention, with reference to the accompanying graphic material, where:

Figure 1 shows the full-size cDNA sequence T and encoded it with full sized peptide T. “Full-size” peptide T consists of a signal peptide (shown in normal font) and peptide T (shows the IRNA font). Similarly shows the cDNA encoding the signal peptide.

Figure 2 presents a photograph of PCR-gel illustrating the peptide T. This gel contained 12 columns and was subjected to electrophoresis with two arrays of samples: one array was located in the upper part of the gel, and the other array was in the middle of the gel, as shown by the lines on both sides of the gel).

Figure 3 graphically illustrates the incorporation of BrdU in the human LPA, which were incubated with peptide T, compared with the control. Y axis shows the optical density at 450 nm, where the higher the OD value indicates a higher level of BrdU incorporation.

Figure 4 graphically illustrates the incorporation of BrdU in the thymocytes of mice Balb/C, which were incubated with peptide T, compared with the control. On the Y-axis delayed optical density at 450 nm, where the higher the OD value indicates a higher level of BrdU incorporation.

Figure 5 presents a fluorescent micrograph of lymphocytes after incubation with biotinylated peptide T.

Figure 6 presents a histogram showing the results of an experiment which was performed staining of mouse splenocytes peptide bT101 and/or CD25 using the CD25 antigen. On the Y-axis are given % of labeled cells.

Figure 7 presents a histogram showing the number of cells per microliter related to different populations of people the human leukocyte namely, monocytes (MONO), lymphocytes (LYM) and whole cells (WBC), after incubation of these cells either in physiological solution containing the peptide T (T)or in saline solution (saline). The number of monocytes was divided by 1000 to include all the results in one graph.

On Fig presents a histogram showing the number of cells per microliter related to human erythrocytes after incubation of these cells either in physiological solution containing the peptide T (T)or in saline solution (saline).

Figure 9 presents a histogram showing the number of cells belonging to different populations of blood cells of mice Balb/C: monocytes (MONO), lymphocytes (LYM), neutrophils (Neut), erythrocytes (RBC) and whole cells (WBC), after handling animals or peptide T, or saline (control).

Figure 10 presents a histogram showing the number of CD4 - and CD8-cells among lymphocytes in the spleen of mice Balb/C, after the injection, animals or C-containing physiological saline or physiological saline. On the Y-axis are given % of leukocytes.

Figure 11 presents a histogram showing the levels of IL-10 in mice which had been injected T in physiological solution, compared with the control mice that were injected with one dose of Phi is biologicheskogo solution through the day. Mice one experimental group were injected with the peptide T once a day (analysis No. 1), the mice of the second group were injected with the peptide once a day (analysis # 2), and mice of the third group were injected with the peptide daily twice a day (analysis No. 3).

On Fig presents a histogram showing the increase in tumor size in mice Balb/C mice that had induced the development of breast carcinoma that were treated either with saline containing peptide T or saline solution.

On Fig presents a graph illustrating the growth rate (in %) of the tumor shown in Fig.

On Fig presents the histogram, which shows the length of the colon in mice that received DSS in drinking water and are then subjected to processing. Were tested four groups of animals: one group of mice did not enter the DSS, and mice of this group were not subjected to the treatment (control); the second group of mice was injected DSS in drinking water and were injected with saline solution (DSS); the other two groups of mice were injected DSS in drinking water and were injected with physiological solution containing 52 and 13 micrograms/ml (DSS + BTL1 and DSS + BTL2 respectively).

On Fig presents the histogram, which shows the body weight of the mice described in Fig, which at the end of the experiment was introduced DS (end), compared to the body weight of mice at the beginning of the experiment (start).

EXAMPLES

Example 1

Amino acid and nucleotide sequence of the full-length peptide T presented in figure 1. Amino acid sequence was analyzed by the method of Phobius identifying signal peptides (described on the website www.phobius.cgb.ki.se) and used for the prediction of transmembrane topology and sequence of the signal peptide.

Method Phobius was predicted C-terminal sequence of the full-length peptide T, consisting of 33 amino acids and represents a hydrophobic transmembrane domain, a signal peptide. This signal peptide is shown in figure 1 in normal font.

In addition, using standard bioinformatics tools were found, which consists of 24 amino acid subsequence of SEQ ID NO:4,

SKRVSDKQLISKAVWWTFFLPSTL(SEQ ID NO:12)

has a close affinity with the following sequences originating from mammals other species, namely:

from dogs:SKQVSDKQLISKAVQRIFFFLQPS(SEQ ID NO:13) and

from rats:SKFMSDKQLISKAVQRIFFLSSTL(SEQ ID NO:14).

When comparing the sequences of SEQ ID NO:12-14 was identified following a consensus sequence of amino acid residues in these peptides (consensus amino acids are shown in uppercase letters):

<> SKrvSDKQLISKAVwwtFFLpSTL(SEQ ID NO:12)

SKqvSDKQLISKAVQRIFFflqps(SEQ ID NO:13)

SKfmSDKQLISKAVQRIFFLsSTL(SEQ ID NO:14)

Example 2

CDNA library isolated from 16 different fabrics used for screening of peptide T. These library purchased from Clontech, included libraries, isolated from leukocytes, testis, colon, prostate, small intestine, thymus, ovary, spleen, liver, kidney, brain, lung, pancreas, skeletal, placenta and heart.

The cDNA library was skanirovali by PCR using specific oligonucleotides for T. Sequences of the oligonucleotides were derived from the sequence T.

In PCR experiment used a 3 microliters of each cDNA libraries, 5 microlitres specific oligonucleotides (total), 20 microliters polymerase Taq Readymix and 17 microlitres DDW.

The PCR procedure was carried out as follows:

- 1 minute at 95°C;

- 30 cycles:

1 minute at 95°C,

1 minute at 52°C,

1 minute at 72°C.

After completion of the cycles, the samples kept at 72°C for 10 minutes.

Used the oligonucleotides had the following sequences:

(i) OLIGO No. 5 label: H10(180)E1P

5'-atggcactcagaagccaggg-3'(SEQ ID NO:19)

(ii) OLIGO No. 6 label: H10(180)E1C

5'-cactcgcttggaatcggatt-3'(SEQ ID NO:20)

The PCR results are presented in figure 2. Agarose gel showing the config in figure 2, contains 12 columns and was subjected to electrophoresis with two arrays of samples: the first array was located in the upper part of the gel, and the second array was in the middle of the gel, as shown by the lines on both sides of the gel). In the columns of the first array were the following samples: 1 and 12 MW markers (molecular masses are shown on the right side of the gel); 2 - growth hormone; 3 - leukocytes; 4 - testicles; 5 - colon; 6 - the prostate gland; 7 - the small intestine; 8 - thymus; 9 - ovary; 10 - spleen; 11 - the liver. In the columns of the second array were the following samples: 1 - MW marker; 2, kidney; 3 - the brain; 4 - light, 5 - pancreas; 6 - skeletal muscle; 7 - placenta; 8 - heart.

As you can see on the gel, it was found that the specific sequence T is only present in the tissue of the thymus and is absent in all other tested tissues. It was also found that growth hormone is used here as a positive control gave a positive result on T.

Example 3

Peptide T tested for its ability to activate the proliferation of mouse splenocytes or thymocytes were used two mouse strain: CD1 and Balb/C), and human peripheral blood lymphocytes (PBL). The activity of proliferation was measured by incorporation in these cells with 5-bromo-2'-dose irradiation on neurogenesis (BrdU).

Method

Mouse SP is entity or thymocytes were isolated from the spleen or thymus of mice Balb/C. These splenocytes were sown in 96-well tablets at a concentration of 107cells/well. Used medium RPMI 1640 + 10% FCS and Pen/Str. These cells were treated with various concentrations of peptide T (or 0.1 or 0.01 μg/well) or saline (control), and after 48 hours the cells were labeled BrdU for 6 hours and then tested for the incorporation of BrdU into their kernel.

Human LPK allocated on the gradient ficoll from blood taken from volunteers, and then were sown in 96-well tablets using medium RPMI 1640 + 10% FCS + Pen/Str.

The obtained cells were treated with various concentrations of peptide or saline (as described above), and after 48 hours the cells were labeled BrdU for 6 hours and then tested for the incorporation of BrdU into their kernel.

Results

As shown in figure 3 and 4, respectively, in human LPK and thymocytes of Balb/c T found the ability to increase the levels of BrdU incorporation, which means that T able to induce an increase in the rate of cell proliferation.

Example 4

In the following experiment were designed and synthesized several deletion mutants of the peptide T. These mutants were tested for their ability to stimulate the proliferation of human FIC. The right column indicates the fold increase of proliferation rate in sravnenie what with the proliferation of untreated cells.

TABLE 1
Stimulation of proliferation of human LPK
PeptideAmino acid sequenceThe magnification of
Wild-typeLHLWLSGEPVQSSGTKDMRSKSDSKRV
SDKQLISKAVWWTFFLPSTLWERK (SEQ ID NO:3)
2
Mutant No. 1WTFFLPSTLWERK (SEQ ID NO:5)2,5-3
Mutant No. 2SGEPVQSSGTKDMRSKSDSKRVS (SEQ ID NO:6)1,4-1,6
Mutant # 3DKQLISKAVWWTFFLPSTLWERK (SEQ ID NO:7)2,2-2,8
Mutant No. 4PSTLWERK (SEQ ID NO:8)1,1-1,3
Mutant No. 5AVWWTFFLPSTLW (SEQ ID NO:9)2-2,2
Mutant No. 6KREWLTSPLFFTWWVA (SEQ ID NO:10)2-2,4

Based on the above results, the following conclusions were made:

. Mutant No. 1 consists of 13 N-terminal amino acids of peptide T and is very active. It is obvious that this sequence (or part thereof) plays an important role in the measured activity, stimulating proliferation.

2. Mutant No. 2 consists of 23 amino acids located above the specimen No. 1, and has a relatively low activity. Obviously, this part of the peptide T plays a less important role in the specified biological activity.

3. Mutant No. 3 consists of 23 N-terminal amino acids of peptide T and compared with specimen No. 2 is very active. Thus, it can be noted that some parts of the peptide T play a more important role in stimulating activity in comparison with other parts.

4. Mutant No. 4 consists of 8 N-terminal amino acids of peptide T and has almost no activity. This indicates that the activity of this peptide, play a decisive role five C-terminal amino acid of the mutant No. 1.

5. Mutant No. 5 consists of 13 amino acids, which partially overlap with amino acids of mutant No. 1. It has activity similar to the activity of the peptide T, but its activity is less than the activity of mutant No. 1.

6. Mutant No. 6 consists of the amino acid sequence of mutant No. 5, located in the reverse order. Unexpectedly, it was found that its activity is similar to activity of mutant No. 5.

So about what atom, obviously, to carry out activities that stimulate proliferation, do not need all of the amino acid sequence of the peptide T.

Example 5

Was synthesized biotinylated peptide T and determined the level of its binding with human LPK under a fluorescent microscope.

Method

Biotinylated peptide T in various concentrations was added to 106cells/tube (human LPK). Was also used in the control containing no peptide T. In the next stage was added fluorescent antibody against Biotin at concentrations of saturation, and the cells 3 times washed with PBS. Then these cells were observed under fluorescent microscope.

Results

The results presented in figure 5, showed that the antibody against Biotin was associated with cells treated with biotinylated T. It was found that increasing the concentration of the biotinylated peptide T led to an increase in fluorescence intensity (FACS results not shown). If biotinylated peptide T was first associated with the antibody, and then this conjugate was incubated with the cells, the fluorescence is not detected. Control cells did not show fluorescence (in this figure the results are not presented).

In most cases, cells labeled biotinyl the private peptide T, formed units.

This experiment clearly showed that the peptide T may contact human FIC. Because the antibody is not able to penetrate into cells, and cells, unlabeled T, were not labeled with the antibody against Biotin, it can be concluded that were only labeled cells, the outer surface of which is contacted T.

Similar results were obtained using mouse splenocytes.

Example 6

In order to determine which subpopulation of lymphocytes is associated with the peptide T was carried out FACS analysis using mouse spleen cells or thymus. In this analysis, we used several antibodies: (1) an antibody against mouse CD3, (2) antibody against mouse CD4, (3) antibody against mouse CD8 and (4) the antibody against murine CD25.

Method

106the splenocytes were labeled biotinylated peptide T, PV-streptavidin and FITZ-conjugated antibody against mouse CD25. For each antibody as a control was used antibody of the same isotype.

Results

The results of the FACS analysis are presented on Fig.6 (on the Y-axis gives the percent of labeled cells). Based on these results, can be made the following conclusions:

(1) Adding the biotinylated peptide T leads to large fluorescent cells, then the and as in the control experiment, these cells are not detected (results not shown).

(2) All CD25+cells were also labeled biotinylated peptide.

(3) Part CD3+-, CD4+- and CD8+cells were labeled biotinylated peptide (results not shown).

Peptide T binds to CD25+-cells with CD4+-cells and CD8+-cells. CD25+cells also are probably the CD4+cells and CD8+-cells. Thus, the peptide T associated with cells that regulate the immune response.

Example 7

This experiment was conducted to determine how the peptides T affect the immune response in mice, and to determine whether the peptide T affect any of the components of the immune system, namely at the level of cells of different types and the correlation between different subpopulations.

Method

50 micrograms/kg of peptide T or saline were injected with (twice a day for 8 days) 7-8-week-old female Balb/C mice (10 mice per group). Then there was the CDC blood test and determined the percentage of different subpopulations of leukocytes (WBC) (monocytes (MONO), lymphocytes (LYM) and total leukocyte count (WBC)). As control was used platelets and erythrocytes.

Results

As shown in Fig.7, 7-8-week-old female Balb/C mice were seen in approximately 30%increase in the total number of leukocytes after in the eccii peptide T compared with the total number of leukocytes in mice which was injected with saline. When testing subpopulations of leukocytes in the experimental group were seen in approximately 30%increase in the number of lymphocytes and monocytes compared with a control group that was injected with saline.

As a control also determined the levels of platelets and red blood cells (RBC). It was found that the difference between groups, which were injected with saline and peptide T, was less than 5%, as shown in Fig (results for platelets are not given).

Analysis of CD4+- and CD8+-populations did not reveal any significant differences between the group of mice treated T, and the group of mice treated with saline (results not given).

The experiment was repeated for 3-4-week-old female Balb/C mice (7 mice per group). These mice were injected with 72 micrograms/kg of the indicated peptide or saline twice a day. The results are shown in Fig.9.

In this experiment, in contrast to the above experiment, the peptide T induced some degree of suppression of the immune system observed at the level of cells of different types. The degree of this suppression was approximately 30%.

Example 8

The level of the different lymphocyte subpopulations in the spleen was assessed at 3-4-week-old female Balb/C mice to the m once a day were injected with peptide T (72 micrograms/kg of body weight). The results of the systematic figure 10.

As shown in figure 10, T reduced levels of CD4+- and CD8+-cells in the spleen of mice. In cells of the thymus any differences in the levels of CD4+- and CD8+cells were observed (results not shown). In the lymph nodes of the mice were observed 35-50%reduction in levels of CD4+cells, and the levels of CD8+cells did not change (results not shown).

Example 9

IL-10 is an interleukin, which can suppress the immune system (TH2 interleukin). The level of IL-10 was evaluated in three groups, each of which consisted of 3 mice: (1) mice of one group were injected with the peptide T twice a day (analysis No. 3); (2) mice of the second group were injected with the same concentration T once a day (analysis No. 2); (3) mice of the third group were injected with a single dose of the indicated peptide in a day (analysis No. 1). All injectable doses were similar (72 micrograms/kg of body weight). As control was used mice that were injected with saline (control). The results are presented in figure 11.

As mentioned above, mice of all groups after the introduction of peptide T the level of IL-10 was increased. The best scheme injections involved the introduction of a single dose every other day.

These results confirmed the efficiency of peptide T, ispolzuemogo is as a suppressor of the immune system to treat autoimmune diseases.

Example 10

Adult Balb/C mice with the introduction of lower concentrations T there was a slight increase in the levels of immune cells. This experiment was performed in order to determine whether this increase levels of cells effective in immunotherapy of malignant tumors.

Method

8-week old mice Balb/C were injected with the cells of breast carcinoma EMT6/CTX and after the tumor size reached 4 mm2the mice twice a day for 8 days were injected with peptide T (50 micrograms/kg). Tumor size was measured every other day and at the end of the experiment.

Results

The results of measurement of tumor size on day 14, were systematized in Fig. In mice treated with peptide T, tumor size, increased 4.3 times compared to the size of the original tumor prior to injection. In mice treated with physiological solution, the size of the tumor increased 6.7 times. Thus, T contributed to a significant reduction of tumor growth.

The growth rate of the tumor shown in Fig. As you can see, T able to significantly reduce the rate of tumor growth.

Pathological studies of these tumors showed that some T-treated mice showed a significant increase in the number of limfotsitov and neutrophils in the tumor compared with cont Olam (results given).

These results showed that T can be used as a therapeutic agent for the treatment of malignant tumors.

Example 11

To assess the feasibility of using T for the treatment of autoimmune diseases used a mice model of colitis/Crohn's disease.

8-week old female Balb/c mice were injected DSS (dextran sulfate) in drinking water (5%) for 14 days. In this period, the mice were injected with saline or saline containing T, as described below. Then monitored the content of blood in the faeces, body mass and composition of feces. At the end of the experiment, mice were killed, removed the colon and measured its length.

The mice were divided into 4 groups, 3 mice per group:

Group # 1 - control mice that were given normal drinking water (no DSS) and which is not subjected to processing.

Group # 2 - DSS-mice who were given drinking water containing 5% DSS, and were injected with saline.

Group # 3 - DSS+BTL1-mice who were given drinking water containing 5% DSS, and that once in every 3 days were injected with T (at a dose of 52 micrograms T per 1 kg of body weight).

Group # 4 - DSS+BTL2-mice who were given drinking water containing 5% DSS, and that once in every 3 days were injected with T (at a dose of 13 micrograms T per 1 kg of body weight).

Results the system is matsurowanu on Fig and 15. Obviously, the peptide T has a strong activity aimed at relieving symptoms of the disease in these animals, compared with mice that were injected DSS and only saline.

These results showed that the peptide T can serve as an immunosuppressant for the treatment of autoimmune diseases such as colitis/Crohn's disease.

1. The selected polypeptide is capable of stimulating the proliferation of peripheral blood lymphocytes (PBL) of a person, to inhibit tumor growth and/or to modulate the immune system to treat autoimmune diseases and/or inflammatory diseases, where the polypeptide is a peptide T with the amino acid sequence SEQ ID NO:4.

2. The selected polypeptide which is a fragment of the selected peptide T according to claim 1, where the fragment has the same type of activity and the polypeptide consists of amino acids 29-51 specified peptide T and has the amino acid sequence represented in SEQ ID NO:7.

3. The selected polypeptide representing fragments is selected peptide T according to claim 1, where the fragment has the same type of activity and the polypeptide consists of amino acids 39-51 specified peptide T and has the amino acid sequence represented in SEQ ID NO:5.

4. The selected polypeptide which is a fragment of the selected peptide T according to claim 1, where the fragment has the same type of activity and the polypeptide consists of amino acids 44-51 specified peptide T 101 and has the amino acid sequence represented in SEQ ID NO:8.

5. The selected polypeptide which is a fragment of the selected peptide T according to claim 1, where the fragment has the same type of activity and the polypeptide consists of amino acids 36-48 specified peptide T and has the amino acid sequence represented in SEQ ID NO:9.

6. The selected polypeptide which is a fragment of the selected peptide T according to claim 1, where the fragment has the same type of activity and the polypeptide consists of amino acids 24-47 specified peptide T and has the amino acid sequence represented in SEQ ID NO:12.

7. The selected polypeptide which is a derivative of the selected polypeptide according to any one of claims 1 to 6 and has the same type of activity in which from 1 to 3 amino acid residues replaced by another amino acid residue by conservative substitutions.

8. The selected polypeptide which is a derivative selected the second polypeptide according to any one of claims 1 to 7 and has the same type of activity, in which one or more amino acids replaced by the corresponding D-amino acid.

9. Pharmaceutical composition for stimulating the proliferation of peripheral blood lymphocytes (PBL) of a person, inhibiting tumor growth and/or modulating the immune system to treat autoimmune diseases and/or inflammatory diseases containing the selected polypeptide according to any one of claims 1 to 8 as an active agent.

10. The use of the polypeptide according to any one of claims 1 to 8 as an active agent pharmaceutical compositions for stimulating proliferation of peripheral blood lymphocytes (PBL) of a person, inhibiting tumor growth and/or modulating the immune system to treat autoimmune diseases and/or inflammatory diseases.



 

Same patents:

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic and protein engineering and can be used in biomedical industry. A genetic make up is proposed, which codes a peptide in which two domains bonding the growth hormone (GH) receptor are bonded into a tandem by a "semirigid" or "rigid" linker, which consists of at least 1-4 copies of the A(EAAAK)A amino acid sequence.

EFFECT: as a result of expression of the nucleotide sequence coding the said tandem, GH-linker-GH polypeptides are obtained, which exhibit growth hormone receptor agonist properties, which determine the possibility of their use in medicinal agents for treating diseases related to the need to administer the growth hormone.

10 cl, 31 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention concerns biotechnology, particularly obtaining cytokine proteins, and can be applied in cell technology. Polypeptide binding Zalpha11 ligand receptor is obtained. Nucleotide sequence coding new polypeptide is introduced into host cell as part of expression vector, and polypeptide is produced.

EFFECT: possibility of efficient adjustment of proliferation and/or development of hemopoietic cells in vitro and in vivo.

14 cl, 1 dwg, 55 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns angiogenesis-preventing immunotherapy. Invention substance includes immunogenic compositions for treatment of disorders associated with angiogenesis intensification, containing oligonucleotides, coding polypeptides VEGFR2, introduced as a part of plasmid or viral vectors, as well as polypeptides VEGFR2, oligonucleotides, coding autologous VEGF with damaged function of receptor activation, polypeptides VEGF and their combinations. Immunogenic compositions can be used for treatment of malignant neoplasms and metastasises, at benign neoplasm and chronic inflammatory and autoimmune diseases. Advantage of invention lies in humoral and cellular immunity induction by means of specified compositions.

EFFECT: development of effective method angiogenesis preventive immunotherapy.

19 cl, 11 ex, 7 tbl

FIELD: biotechnology, molecular biology, proteins.

SUBSTANCE: invention relates to a method for preparing cytokines of class II and can be used in medicine. Prepared proteins zcyto20, zcyto22, zcyto24 and zcyto25 are the most relative with interferon-α at amino acid sequence level. Receptor of cytokines of class II represents a receptor for this family of proteins. Proteins can be prepared by recombinant way using a cell-host transformed with expression vector that comprises nucleic acids corresponding to proteins. Base on proteins xcyto20, xcyto21, zcyto22, zcyto24 and zcyto25 antiviral pharmaceutical composition and specific antibodies are prepared. Invention provides preparing the novel cytokine that stimulates cells of differentiation hemopoietic line and possesses the expressed antiviral activity.

EFFECT: valuable biological and medicinal properties of polypeptide, improved preparing method.

24 cl, 21 tbl, 32 ex

FIELD: gene engineering, in particular method for treatment of viral infections.

SUBSTANCE: protein ZCYTO21 has amino acid sequence which is nearly similar to amino acid sequence of interferon-α. Protein and antibodies thereto have antiviral activity and are useful in treatment of hepatitis B and C as well as other diseases.

EFFECT: new protein with antiviral activity.

71 cl, 1 dwg, 6 tbl, 7 ex

FIELD: biotechnology, medicine.

SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.

EFFECT: valuable properties of new cytokine.

18 cl, 5 tbl, 1 dwg, 55 ex

The invention relates to the field of biotechnology, specifically to obtain protein/factor inhibiting osteoclastogenesis (OCIF), and can be used for treatment and immunological diagnosis of diseases involving bone resorption

The invention relates to the field of genetic engineering and can be used in the biomedical industry

The invention relates to a protein characterized by the following properties: (a) molecular weight during electrophoresis in polyacrylamide gel in the presence of LTOs (SDS-PAGE), which constitutes approximately 60 KD in terms of recovery, approximately 60 KD and 120 KD in non conditions; (b) high affinity for the cation-exchange column and a column of heparin; (b) biological activity of inhibiting the differentiation and/or maturation of osteoclasts, and this activity is reduced by heating at 70oC for 10 min or 56oC for 30 min, the activity is lost when heated at 90oC for 10 min; (g) internal amino acid sequences presented in SEQ ID NO: 1, 2, and 3, and (d) with optional N-terminal amino acid sequence represented in SEQ ID NO: 7; a method for production of such proteins by culturing human fibroblasts; protein purification by a combination of ion-exchange column chromatography, affinity column chromatography and column chromatography with reversed phase

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.

EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.

8 cl, 10 dwg, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention relates to a novel fluorescent protein from Entacmaea quadricolor and its functional mutants. The invention discloses nucleic acids which code the said protein, a vector which contains the said nucleic acids, a transgenic cell which carries the vector and a method of obtaining the said fluorescent proteins from transgenic cells. The composition of the said proteins and nucleic acids can be used in various applications and methods, particularly for labelling biomolecules, cells or cell organelles. The disclosed protein and nucleotide sequences can be used for testing activity of promoters under various conditions.

EFFECT: obtaining proteins with primarily red or far-red fluorescence.

7 cl, 7 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology. In particular, the invention relates to an Escherichia coli BL21 (pVEGF-A165) strain and can be used to produce a vascular endothelial growth factor - GST-VEGF-A165 protein. A novel Escherichia coli BL21 (pVEGF-A165) cell strain is obtained, which is transformed by the pGEX-VEGF-A165 plasmid. This strain produces a recombinant GST-VEGF-A165 protein.

EFFECT: invention enables to obtain a Escherichia coli BL21 (pVEGF-A165) strain which is stably transformed by plasmid which codes VEGF, and which secrete this factor in extracellular space when cultured in vitro.

3 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: recombinant virus containing human gene p53, its application, manufacture method and pharmaceutical composition are offered.

EFFECT: invention can be used for gene therapy in human malignant neoplasm treatment and prevention.

8 cl, 14 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.

EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.

3 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: method is based on assessment of ACTN3 genotype, where genotype 577RR is positively related to sprinter or power abilities; genotype 577XX is negatively related to sprinter or power abilities; genotype 577XX is positively related to stamina; genotype 577RX is positively related to sprinter or power abilities; and genotype 577RX is negatively related to stamina in women.

EFFECT: method makes it possible to select type of sports or competitions for an individual, for instance speed-power type of sports or type of sports that requires stamina, and to develop more optimal training regime for a single sportsman.

1 dwg, 6 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: gene of human vascular endothelial growth factor (VEGF) optimised for expression in mammal cells is presented.

EFFECT: due to introduction of VEGFopt gene into pC4W-VEGFopt plasmid, the invention allows improving the yield of an end product in 1,5 times in comparison with the technology of pC4W-hVEGF165 plasmid containing a natural gene.

3 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention concerns technology of genetically engineered constructs to be applied in cell-based and gene therapy. Gene of human hepatocyte growth factor optimised for expression in mammal cells is presented.

EFFECT: invention allows doubling the production of hepatocyte growth factor in comparison with a human natural gene.

3 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: there is offered application of group of survival-improving polypeptide cone cells originated from rod cells and designated as RDCF, and also coding molecules of nucleic acid to prepare medicines, particularly pharmaceutical compositions used to treat retinal dystrophy. Methods for preparing RDCF by recombinant DNA technologies, and required aids, as well as preparation of antibodies distinguishing said polypeptides are described.

EFFECT: improved clinical effectivenesses.

12 cl, 19 dwg, 1 ex

FIELD: microbiology.

SUBSTANCE: invention represents plasmid vector for transfer of DNA, which comprises sequence coding various fragments of oncoprotein p185neu, which are able to induce immune response in respect to tumors, which hyperexpress p185neu. Invention is also related to pharmaceutical composition on the basis of vector for prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

EFFECT: invention makes it possible to increase efficiency of prophylactics or treatment of patients with risk of development of p185neu-positive tumors, or patients with primary tumors, metastases or relapses of p185neu-positive tumors.

10 cl, 14 dwg, 2 tbl, 2 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to genetic engineering and pertains to a novel plasmid pL-ASP-08 and a novel Escherichia coli XL 1-blue/pL-ASP-08 bacteria strain containing the said plasmid. The recombinant plasmid DNA pL-ASP-08 has molecular weight of 2.9 MDa (4471 base pairs) and is designed for L-asparaginase synthesis.

EFFECT: invention enables to obtain highly pure genetically engineered L-asparaginase for preparing medicinal agents used in treating lymphoblastic leucosis, lympho- and reticulosarcoma in combined chemotherapy.

2 cl, 1 dwg, 2 ex

Up!