Interleukine-15 peptide antagonist
SUBSTANCE: invention relates to molecular pharmacology and specifically to a peptide which is part of an interleukine-15 (IL-15) sequence which can inhibit biological activity of the said molecule.
EFFECT: obtaining a peptide which inhibits T cell proliferation induced by IL-15, and apoptosis caused by tumour necrosis factor when bonding with the alpha subunit of the (IL-15R) receptor.
8 cl, 4 dwg, 5 ex
The technical field to which the invention relates
The present invention relates to the field of molecular pharmacology, in particular peptide of interleukin-15 (IL-15), which affects the binding of IL-15 with the alpha subunit of the receptor; therefore it may be useful for treatment of diseases associated with aberrant expression of IL-15 or IL-15Rα.
The cytokine known as IL-15, is a glycoprotein weight of 14-15 kDa, simultaneously belonging to two groups as a factor of activated T-cells (Grabstein, K.H. et al., Science 1994, 264, 965; Burton, J.D. et al., Proc. Natl. Acad. Sci. USA 1994, 91, 4935). the mRNA of IL-15 is widely expressed in various cells and tissues, however, to detect protein in these cells or in cell supernatant difficult due to the strong posttranscriptional control of its expression at the level of translation and intracellular transport (Bamford RN. et al., J. Immunol. 1998, 160: 4418-4426; Kurys G, et al., J. Biol. Chem. 2000, 275: 30653-30659). In addition, it was shown that IL-15 can exist in an active form in the form of a membrane protein (Musso et al., Blood 1999, Vol. 93, No. 10 (May 15),: pp 3531-3539), and recently it has been observed that it can function either as a ligand or receptor (Budalgian et al., JBC 2004, vol 279, No 40: pp 42192-42201)inducyruya through this path secretion of proinflammatory cytokines. High level expression of soluble protein was associated with patoh is a non-autoimmune and inflammatory diseases. IL-15 was detected in some diseases, including Crohn's disease (I. Kirman, 1996, Am. J. Gastroenterol. 91, 1789), psoriasis (Ruckert R. 2000, 165: 2240-2250), leukemia (Yamada Y. 1999, the Leukemia and Lymphoma, 35(1-2): 37-45) and rheumatoid arthritis (RA), (Mclnnes I.B. 1998, Immunology Today, 19, 75-79). The binding of ligand to T-cell receptor induces the expression of IL-15Rα and the expression of some antigens activation, such as CD69, CD25 and TNFRII. In addition, IL-15 is a chemoattractant for T lymphocytes of human blood (Wilkinson 1995, J. Exp. Med. 181, 1255-1259). All these data indicate that IL-15 is expressed by antigen presenting cells, may play an important role in the early activation of T-cells at the site of inflammation.
Mclnnes and others, have identified abnormalities in the expression of IL-15 in this disease, a high concentration of IL-15 in the synovial fluid and its expression in cells of the synovial membrane. They suggested that IL-15 is a precursor of TNFα in the cytokine cascade, representing a mechanism dependent on cell contact, in which T cells activated by IL-15, induce the synthesis of TNFα by macrophages. In addition, it is assumed that IL-15 as an important factor influencing the migration of T cells in synovial fluid (Mclnnes, 1997, NatMed, 3: 189-195).
Ziolkowska et al. reported that IL-15 induces the expression of IL-17 in the joints of patients suffering from rheumatoid arthritis, you already know that this cytokine stimulates the release of sin is visitame several inflammatory mediators, such as IL-6, IL-8, GM-CSF and prostaglandin E2that suggests the important role of IL-15 in the pathogenesis of rheumatoid arthritis (Ziolkowska y col 2000, J Immunol, 164: 2832-2838). The recruitment and activation of T cells can occur due to local synthesis of IL-15, and similar non-specific activation can result in infinite inflammation. All this suggests that the inhibition of IL-15 may have therapeutic potential for the treatment of this disease, and other autoimmune and inflammatory diseases.
Biological effects of IL-15 mediated by through its binding with the receptor cell membrane, consisting of three subunits α, β, and γ. IL-15Rα is subunit specific for this cytokine to which it binds with very high affinity Kd of 10-11and can be detected in the form of a membrane receptor, or a soluble form (Budagian, V. et al., JBC 2004, 279, 39: 40368-40375; Mortier et al., The Journal of Immunology, 2004, 173: 1681-1688).
With subunits β and γ also binds IL-2, a cytokine with high structural similarity to IL-15. Earlier it was reported that Asp56 in the molecule IL-15 plays an important role in binding to the β subunit of the receptor, and Gln156 plays an important role in binding to the γ subunit of the receptor.
Mutiny behave like molecules that are antagonists of IL-15 binding to the α subunit of the receptor and reducing the transmission signal is Ala through β and γ subunits. Antibodies that recognize these amino acids also act as antagonists of IL-15 (US 6177079, US 6168783, US 6013480, US 6001973, US 9706931, WO 9741232).
Ruchatz et al. (Ruchatz H. 1998, J. Immunol. 160: 5654-5660), created a soluble fragment of the α subunit of rat receptor (IL-15Rα) and demonstrated that the introduction of this fragment suppresses the development of collagen-induced arthritis (CIA) in mice DBA/1.
Genmab Company owns the patent on specific human antibodies to IL-15, WO 03017935, which describes 4 types of antibodies, including 2 species, 146B7 and 146H5, contact with IL-15 in the interaction region on the γ subunit of the receptor and inhibit IL-15-induced proliferation of cells in the cell line CTLL2 and PBMC (mononuclear peripheral blood cells)and antibodies 404A8 and 404E4 not inhibit proliferation. The use of antibody 146B7 (Amgen) called AMG714 for the treatment of rheumatoid arthritis is in phase II clinical trials.
Recently identified two sequences of IL-15 binding to the α subunit of the receptor with amino acids 44 through 52 and amino acids 64 to 68 (Bernard et al., JBC 2004, 279 (23), 24313-24322). They described mutiny, which can act either as agonists or as antagonists of IL-15.
So far not described any peptide antagonist of IL-15. The use of short peptide length (10 aa) as an antagonist of IL-15 has the advantage of selective block of the e binding of IL-15 with the α subunit of the receptor and mediating or reduce the effects of IL-15, manifested by the interaction of IL-15 receptor. For example, as described in the present invention, the peptide, named Sec.No.1 has a 10 amino acid regions of IL-15, which we identified as an area that interacts with the α subunit of the receptor (Figure 1). This peptide binds protein IL-15Rα-Fc in ELISA and in the analysis with Tentagel resin (Figure 2), inhibits IL-15-dependent proliferation of the cell line CTLL-2 (Figa and 3b) and protects against TNFα-induced apoptosis (Figure 4), this effect is mediated by the binding of IL-15 α chain of the receptor. This latter effect allows its use in diseases where the inhibition of apoptosis. Similarly, the binding of this peptide with soluble α-chain, as described in the present invention, may inhibit reverse signaling, mediated associated with membrane IL-15 (Budalgian et al., JBC 2004, vol 279, No 40: pp 42192-42201).
Detailed description of the invention
In particular, this invention relates to the identification of IL-15, which is capable of binding subunit of IL-15Rα. The peptide containing this region, called here Sec.No.1, chemically synthesized and are capable of binding IL-15Rα-Fc (Figure 2), inhibits IL-15-induced proliferation of CTLL2 and protects against TNFa-induced DMA fragmentation (Figure 4).
This invention also includes any homologous or mimetica VA is ianti specified peptide, which were obtained using recombinant or synthetic method, as well as any containing composition.
Similarly, the invention also includes the use of this peptide, alone or in combination with any other suitable molecule, for example, steroidal anti-inflammatory drugs (corticosteroids), drugs that modify the course of the disease (MTX) or other antagonists of cytokines used in the treatment of rheumatoid arthritis, and the use of this peptide to inhibit the binding of IL-15 with the α subunit of the receptor, both soluble and associated with the membrane forms.
The peptide in this invention has a linear structure and is mainly characterized by its ability to be antagonistic to IL-15. On the other hand, in vitro effect produced by the peptide in the present invention, is demonstrated by analysis of cell proliferation CTLL-2 and analysis of inhibition of TNFa-induced apoptosis.
To identify the described peptide was used mapping technology on cellulose filter, which contains the full sequence of IL-15 in sequential peptides of 10 amino acids with 5 overlapping amino acids.
In the present invention the peptide Sec.No.1 was chemically synthesized using solid-phase method, cleansing is using HPLC, analyzed using mass spectrometry and finally assessed in relation to its impact on the activity of IL-15.
The results given in the present invention indicates that the identified and synthesized the region in the form of a peptide consisting of 10 amino acids (Sec.No.1)corresponds to the area of IL-15 that interacts with the α subunit of the receptor, and thus inhibits the binding of IL-15 with its receptor, inhibiting IL-15-induced proliferative activity of T cells.
Peptide (Sec.No.1)containing amino acids of IL-15 that interacts with IL-15Rα, simulates the effect of protection of IL-15 α-induced apoptosis, which is mediated by the binding of IL-15 to IL-15Rα (Bulfone-Paus et al., The FASEB Journal, 1999, September, Vol. 13).
The results obtained here suggest its use as a therapeutic agent in the treatment of the aforementioned diseases associated with high expression of IL-15, which justified the use of antagonists of IL-15 and those diseases in which the effect of protection from apoptosis, as well as diseases associated with high expression of soluble IL-15Rα. Similarly, antibodies recognizing the region contained in Sec.No.1 IL-15, will inhibit the binding of IL-15 to IL-15Rα and will be active antagonist of IL-15 by inhibiting the binding of molecules subunit is similar to the receptor, p is the cause of the peptide, associated with media molecule, or chemical conjugate MAP (multiantigenic peptide), can be used to generate antibodies to the antagonist of IL-15.
The purpose of the present invention also relates to the encoding DMA aforementioned peptide. As an alternative for expression of the peptide sequence may also be a vector containing the DMA sequence encoding the peptide of the present invention.
This peptide may be used in combination with other anti-inflammatory and immunosuppressive agents or other antagonists of cytokines used in rheumatoid arthritis, psoriasis, Crohn's disease, etc. Described peptide may be included in a therapeutic vaccine to provide humoral response against IL-15.
The present invention is the identification of the sequence of IL-15 (Sec.No.1)that interacts with the α subunit of the receptor. A similar sequence, synthesized in the form of a linear peptide of 10 amino acids, exhibits the property of antagonist IL-15 in terms of the induction of the proliferation of T-cells, and the effect of the agonist in protection against TNFa-induced apoptosis.
Brief description of drawings
Figure 1: mapping of the IL-15R on cellulose filter.
IL-15Rα-Fc recognizes a peptide of 8, corresponding Sec.No.1 and less than the degree of peptide 7.
Figure 2: Colorimetric analysis granules Tentagel s
The pellets, containing the peptide Sec.No.1, inkubiruemykh with IL-15Rα-Fc (R&D) at a concentration of 5 µg/ml was observed the appearance of a color.
2a) Incubation of resin containing (a) unbound peptide or b) a peptide Sec.No.1 contained resin, inkubiruemykh with 15Rα-Fc (R&D).
2b) Incubation of the resin containing the peptide Sec.No.1 with IL15Rα-Fc (R&D) (a) or peptide Sec.No.1, IL15Rα-Fc (R&D) is contained in excess IL-15 (b).
Figure 3: analysis of proliferation of CTLL-2 human IL-15 (R&D) with a specific activity of 108Ul/mg
3a), the analysis of CTLL-2 at various concentrations of IL-15α and fixed concentration of peptide 260 microns.
3b), the analysis of CTLL-2 at various concentrations of peptide Sec.No.1 and fixed concentration of IL-15α 300 PG/ml
Figure 4: analysis of apoptosis induction in cell line L929. Cells were incubated with α (100 ng/ml) alone or in combination with peptide Sec.No.1 (260 μm)
Examples of the types of exercise
The following examples are provided to illustrate embodiments of the present invention.
Example 1: A. identification of the region that communicates with IL-15Rα. Synthesis on cellulose substrate 10-dimensional peptides corresponding to the amino acid sequence of IL-15
To identify areas IL-15 is involved in the binding of IL-15Rα, the method was applied for the synthesis of peptide fragment, the wound is described by Frank et. al. Receipt paper Whatman 540 was carried out using tarifitsirovana first anchor component, Fmoc-β-Ala-OH using N,N'-diisopropylcarbodiimide (DIG) and N-Mei (NMI) in dry N,N-dimethylformamide (DMF). The sequence of the fragment on the cellulose membrane was determined by fixing Fmoc-β-Ala-OH in the pre-marked positions, in accordance with the required number of 10-dimensional peptides (22 peptide, overlapping 5 residues, 114 amino acid sequence of IL-15). In addition, unbound peptide of 10 amino acids was synthesized in the fragment 23, as well as in fragment 24 was fixed only Fmoc-β-Ala-OH, both as a control. To build all of these molecules used standard chemical method Fmoc-/tBu. After the final cycle of the synthesis, the N-Terminus and the side chains of all peptides were deprived of protection.
Binding of anti-IL-15 Abs peptides synthesized on cellulose substrate
A sheet of cellulose was soaked in ethanol to prevent possible hydrophobic interactions on it between peptides. The ethanol was replaced by saline Tris-buffer (TBS) (150 mm NaCl, 10 mm Tris, pH 7,6) using sequential washing. Nonspecific binding was blocked with incubation of the membrane over night in 10 ml of blocking buffer IBS (5% poroshkovanie milk in IBS). The sheet was subsequently incubated for 3 casos receptor IL-15, dissolved in 10 ml of buffer sample T-TBS (5% dry milk and 0.5% Tween-20 in IBS). Used a serum dilution of 1:50. IL-15α-receptor was prepared at a concentration of 5 µg/ml in the same buffer solution. A sheet of cellulose was washed four times with buffer T-TBS. Then for 1 hour in the analysis of IL-15α-receptor was added anti-IgG alkaline phosphatase conjugate (Fc specific) (Sigma), dissolved in buffer sample T-TBS (anti-human IgG dilution 1:25000). A sheet of cellulose was again washed four times with T-TBS, and was performed by detection of the peptide bond with incubation of the membrane with 0.5 mg/ml 5-bromo,4-chloro,3-indolylmethane (BCIP) (Sigma) in the main buffer (100 mm NaCl, 2 mm MgCl2, 100 mm Tris, pH 8.9). Positive fragments were stained in blue/purple color. Staining was stopped by washing with PBS. At the end of the sheet of cellulose has been updated for conducting other analyses described previously (Frank, R. (1992)Tetrahedron48, 9217). We observed the recognition of two peptides, 8 and 7. As a control experiment, the membrane was incubated with humanized monoclonal antibody containing the Fc region of human lgG1. In this case, we did not observe any recognition on the membrane of any peptide.
Colorimetric analysis of granules, demonstrating the binding of the peptide to IL-15Rα. Synthesis of peptide Sec.No.1 resin NH2-7 Tentagel-s Resin NH2-Tentagel-S (0.24 mmol/g) was washed several times dichlo is methane (DCM) and methanol. Then it was incubated in 30% solution triperoxonane acid (TFA) for 10 minutes; promelas DCM several times and inquires in DCM with 5% content of diisopropylethylamine (DIEA) for 1 minute. This procedure caused the activation of NH2groups for synthesis. Then the resin was washed with DCM and were incubated in dimethylformamide (DMF) for 5 minutes for the synthesis of the peptide. Conventional methods of synthesis of Fmoc/tBu. The reaction with the formation of the communication was accompanied by a ninhydrin test. After the analysis of peptide Sec.No.1 side chains of the amino acids were not protected and left in a state of fixing the C-ends to the resin.
Analysis on the granules
The resin beads with a fixed peptide were washed several times with saline solution (PBS). Non-specific interactions were blocked by incubation with BSA (1%) in PBS for 1 hour in real time. Then they were incubated with the merged protein IL-15Rα-Fc (R&D 147-IR) at a concentration of 5 μg/ml in BSA (1%/PBS) for 16 hours at 4°C. the pellets were washed three times in PBS for 5 minutes with stirring, and then were incubated with anti-Fc IgG conjugate human phosphatase, diluted 1:25000 in BSA (1%/PBS) for three hours in real time. They were thoroughly washed with saline (TBS/Tween-20,1%) and were incubated with BCIP (0.45 mg/ml) substrate solution (100 mm Tris, pH 8.9; 100 mm NaCl; 2 mm MgCl2/sub> for about 30 minutes. They were washed four times in PBS to stop the reaction. Intense blue staining was only observed during incubation of the resin containing the peptide Sec.No.1 with protein IL-15Rα-Fc, and was not observed during the incubation of resin containing unbound peptide from IL-15Rα-Fc. In this case, the chromogenic substrate is not praecipitium and not painted. Similarly, we did not observe staining in the presence of excess human IL-15α.
The peptides were synthesized according to the Fmoc/tBu, applying the resin Fmoc-AM-MBHA at a concentration of 0.54 mmol/g and protocols of synthesis with mechanical stirring. After processing TFA peptide was liofilizovane and tested using HPLC-MS
Example 2: the Impact of these peptides on the proliferation of cell line CTLL2
Cell line CTLL-2-dependent cytokines, proliferate in the presence of IL-15α. IL-15 binds molecules that impair receptor-mediated signaling, inhibit the proliferation of this cell line.
To assess the neutralizing ability of the peptide according to the invention were prepared serial dilution in 96-well plates (Costar, USA) in a volume of RPMI medium (Gibco) 25 ál, backed up with 10% fetal bovine serum (Gibco). Pre-washed cells CTLL-2 were added to 5x103cells on the cell and were incubated for 30 is in. Then in each cell were added to 300 PG of IL-15A. The plate was incubated for 72 hours in medium with 5% CO2and 37°C. the Results are shown in figure 3b. We found that a peptide called Sec.No.1, inhibits IL-15-induced proliferation with IC50130 microns. To determine proliferation was applied staining of mitochondria MTT (Cosman et al. 1984, Nature, 312: 768-771). We also evaluated the antagonistic effect of this peptide at a dose of 260 μm at various concentrations of IL-15 (figure 3a). The inhibitory effect of the peptide was dependent on the doses of IL-15A.
Example 3: induction of apoptosis in L929 cells
Analysis of DNA Fragmentation allows to determine the number of DMA, which was subjected to decay, after treatment of cells TNF-alpha. Cell DNA radioactively marked by growing cells in the presence of 3H-thymidine to radioactive 3H-thymidine was incorporated into DNA. After 24 hours, cells were treated with Tripsin/EDTA, washed and seeded at a concentration of 5000 cells per well 96-well plate. Then the labeled cells were incubated with TNFa (100 ng/ml), IL-15 (100 ng/ml), peptides (260 μm) or with a combination of TNFa with different peptides.
During this incubation, added agent (e.g., TNFa induces cell death by apoptosis and, consequently, fragmentation of DNA, whereas DNA of untreated cells remains intact). Cells were collected after 24 hours: all the time what I gather, cells were washed from the wells of 96-hole plates, double-distilled water: cells and organelles were destroyed and released DNA. Fragments of cells and DNA was entered through the filter membrane (fiberglass). Through the filter can pass only particles less than 1.5 μm. Thus, intact DNA (chunk lengths in the millimeter or even centimeter range) will not be able to pass through the filter, but will be collected on the filter membrane. The DNA that was broken/shattered into fragments of approximately 5000 bp or less, will have a sufficiently small size to pass through the pores of the filter and will not be collected on the filter. The membrane filter was dried in scintillation counter has been counting radioactivity (which corresponds to the number of intact DNA). By comparing the counted radioactivity (units per minute = cpm) of cells that were not treated cpm, and cells that were treated with the agent, was calculated the percentage of DNA fragmentation. As a result, we found that the peptide Sec.No.1 protected from TNF-induced apoptosis.
Example 4: obtaining monoclonal antibodies
Monoclonal antibodies were obtained as described Georges Kohler and Cesar Milstein (Nature, 256:495-497, 1975). For induced monoclonal antibodies that bind and inhibit the activity of IL-15 was used in the n peptide Sec.No.1, conjugatively with KLH, or chemical conjugate containing 4 molecules of this peptide.
Mice were subcutaneously immunized with conjugated peptide, which was made for the introduction using emulsification with an auxiliary tool Freund's in the range from 10 to 100 mg, with subsequent weekly subcutaneous immunization with peptide in incomplete auxiliary tool Freund's. The immune response to IL-15 was tested using ELISA. Mice with sufficient titers of anti-IL-15 antibodies received intravenous stimulant therapy for 3 days before the killing and removal of the spleen. To generate hybridomas producing monoclonal antibodies to IL-15, we used the above Protocol, published in Nature, 256:495-497, 1975. Resulting from hybridoma were subjected to screening concerning the production of specific antibodies to IL-15 or peptide by ELISA and by identifying inhibitory activity of IL-15 effect in the analysis of CTLL-2. Positive clones were inoculated into the peritoneal cavity of syngeneic mice for the development of ascites, and the resulting monoclonal antibodies were purified by ammonium sulfate precipitation and affinity chromatography based upon binding of antibody to protein A fromStaphylococcus aureus.
Example 5: Evaluation of peptide Sec.No.1 in the creation of neutralizing antibodies in monkeys Macacus computer.
Under the scheme the e immunization of monkeys was assessed three groups, immunized with the peptide conjugated to a protein carrier, chemically-conjugated protein in the form of a tetramer (MAP); and placebo. Proteins were injected at concentrations ranging from 100 μg to 200 μg per vaccine, in the auxiliary tool Freund's. The second immunization was performed one month, and the third immunization was performed two months later. Two weeks after the second and third immunization were taken blood samples to assess the level of antibodies anti-IL15 in the serum of monkeys. The neutralizing capacity of antibodies present in the serum of monkeys was determined by analysis of CTLL-2 in the presence of 300 PG of IL-15.
The advantages of the proposed solution
Peptide Sec No.1 selectively inhibits the binding of IL-15 and IL-15Ra. Peptide Sec No.1 is antagonism to the effect of IL-15-induced proliferation of T-cells (cells CTLL-2) and, in addition, it is agonist protective against apoptotic effect of IL-15 in cells that are sensitive to α-induced apoptosis.
1. Peptide antagonist activity of IL-15, which represents an amino acid sequence described as Sec. No. No. 1.
2. The peptide according to claim 1, characterized in that it binds to cellular subunit of IL-15Rα or its soluble fraction.
3. The peptide according to claim 1, characterized in that it inhibits IL-15Rα-dependent biological active is th IL-15.
4. The peptide according to one of claims 1 to 3, characterized in that it is obtained by genetic manipulation or chemical synthesis.
5. The peptide according to one of claims 1 to 3, characterized in that it is an active component of the pharmaceutical composition and is able to inhibit dependent cell receptor IL-15Rα biological activity of IL-15.
6. Chain nucleic acid, characterized in that it encodes the peptide according to claim 1, and its product is capable of binding cellular IL15Rα or the soluble fraction and inhibits the biological activity of IL-15.
7. Chain nucleic acid according to claim 6, characterized in that as part of the expression vector, it is able to inhibit IL-15Rα-dependent biological activity of IL-15.
8. Vaccine composition comprising the peptide of claim 1 conjugated with protein carrier and is able to cause inhibition-dependent cellular IL-15Rα biological activity of IL-15.
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to application of peptide of formula (I): ((X)1(Y)m)n where peptide contains 3 to 200 amino acids and where 1, m and n represent integers within 0 to 10; X and Y which can be identical or different, represent cationic amino acids chosen from arginine and lysine in preparing drugs for treating fungal infection.
EFFECT: ensured applicability of peptide for preparing a drug for treating fungal infection.
26 cl, 53 dwg, 2 tbl, 19 ex
SUBSTANCE: invention discloses novel synthetic RGD-like peptides capable of dose-dependant inhibition of thrombocyte aggregation.
EFFECT: obtaining novel compounds capable of dose-dependant inhibition of thrombocyte aggregation.
2 tbl, 1 ex
SUBSTANCE: invention relates to biotechnology and is a peptide which induces killer T cells ex vivo and which has an amino acid sequence as shown in one SEQ ID NOS: from 1 to 3. The disclosed peptide is used in an ex vivo agent for inducing anti-tumour immunity, in an ex vivo agent for inducing antigen-presenting cells, in an ex vivo agent which induces tumour-reactive T cells, as well as in an ex vivo pharmaceutical agent when treating or preventing tumours. The invention also relates to an antibody against the said peptide.
EFFECT: disclosed agents enable identification of glypican-3-derivative peptide, which can bond with HLA-A2, and activation of human killer T cells in order to provide an immunotherapy agent which may be effective in approximately 40% Japanese patients suffering from certain types of malignant tumours, accompanied by high level of GPC3 expression.
7 cl, 4 dwg, 1 tbl, 2 ex
SUBSTANCE: invention relates to contrast agents which contain a peptide vector linked with uPAR, marked with a visualising group.
EFFECT: obtaining a contrast agent for detecting urokinase plasminogen activator receptor.
6 cl, 6 ex
SUBSTANCE: invention relates to novel peptide compounds and their use in diagnostic optical visualisation techniques. More specifically, the present invention pertains to use of such peptide compounds as targeted vectors which are related to receptors associated with angiogenesis. The compounds are labelled using at least one cyanine reporter dye.
EFFECT: obtaining compounds which can be used as contrast agents in optical visualisation when diagnosing diseases related to angiogenesis.
9 cl, 5 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to bioengineering and specifically to obtaining biologically active substances of peptide nature, which have growth factor activity towards fibroblast proliferation and can be used in medicine. An oligopeptide of formula A-X1-X2-X3-X4-X5-B is obtained through in silico construction, where A is F; X1 is E, or Q, or S; X2 is N, or Q, or A, or G; X3 is K, or R, or T; X4 is K, or E, or is absent, X5 is K, or L, or is absent and B is OMe - methyl.
EFFECT: invention enables obtaining an oligopeptide with transformation growth factor (TGF-β) and oncostatin M (OSM) towards fibroblast proliferation, and expansion of the range of effective therapeutic agents with wound-healing effect, which take part in closing wounds during inflammation and cicatrisation.
4 dwg, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to bioengineering and specifically to obtaining biologically active substances of peptide nature, which have growth factor activity towards collagen synthesis stimulation and can be used in medicine. An oligopeptide of general formula A-X1-X2-X3-X4-X5-B (I) is obtained through in silico construction, where A is Ac - acetyl; X1 is G or A or is absent; X2 is P or I, or L, or V, or A; X3 is G; X4 is P or I, or L, or V, or A; X5 is G or A, or is absent and B is OMe - methyl.
EFFECT: invention enables obtaining an oligopeptide with acidic (aFGF) and transformation (TGF-β) growth factor activity towards stimulation of collagen biosynthesis, and expansion of the range of effective therapeutic agents with wound-healing effect, which speed up regeneration of damaged tissue and cicatrisation.
4 dwg, 2 ex
SUBSTANCE: invention is related medicine and concerns applications of antibodies specifically recognising any prevailing variants of beta-amyloid peptide, Aβ40 and Aβ42, in preparation of a drug applied for prevention and-or treatment of Alzheimer's disease.
EFFECT: invention provides prevention of progression or reduction of symptoms, and/or decrease in amyloid deposition in an individual when administering an immunostimulating dose of peptide or specific antibody.
7 cl, 3 ex, 2 dwg
SUBSTANCE: small peptides of formula X1-X2-X3-X4-X5-X6-X7-R1, containing 7-12 amino acid residues are proposed.
EFFECT: said peptides are MC4 receptor agonists and are therefore useful in treating obesity and related diseases.
31 cl, 2 tbl, 82 ex
SUBSTANCE: present invention concerns a compound representing a selective agonist of a melanocortin-4 receptor of formula: Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2 (SEQ ID NO:50) and its pharmaceutically acceptable salts, pharmaceutical compositions and methods for application thereof in preparation of drugs.
EFFECT: higher effectiveness of compound application.
20 cl, 8 dwg, 4 tbl, 4 ex
SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.
EFFECT: applicable in medicine for preparing a vaccine.
52 cl, 32 dwg, 7 tbl, 29 ex
SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.
EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.
14 cl, 1 tbl, 6 ex
SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.
EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.
35 cl, 13 dwg, 36 tbl, 14 ex
SUBSTANCE: there are disclosed polypeptide variants containing Fc-areas IgG, having amino acid modifications providing changed effector functions Fc in specified polypeptides. There is disclosed composition for antibody targeting on antigen, containing the specified polypeptide. There is described method for preparing the specified polypeptide. Also, there are disclosed the methods for treating V-cell tumour or a malignant disease characterised by V-cell expression of CD20, treating chronic lymphocytic leukosis, relieving the symptoms of the V-cell controlled autoimmune disease, treating a angiogenesis-associated disorder, treating HER2-expressing cancer, treating LFA-1-mediated involvement, treating IgE-mediated involvement wherein specified methods imply introduction to the patient of the therapeutically effective amount of said polypeptide.
EFFECT: higher clinical effectiveness.
63 cl, 6 ex, 13 dwg, 10 tbl
SUBSTANCE: present invention relates to biotechnology. Proposed is an antibody against interferon-α/β-binding protein I or against its mutein, obtained through conservative substitution. Proteins, against which the antibody is directed, are extracted from urine and link interferon-α/β-binding protein I with Kd constant from 3.6x10-9 to 1.6x10-10 M.
EFFECT: use of the invention allows for detecting presence of interferon-α/β-binding protein I in different samples.
5 cl, 10 dwg, 6 tbl, 17 ex
FIELD: chemistry, biochemistry.
SUBSTANCE: claimed invention relates to field of biotechnology. Claimed is antibody against interferon-α/β-connecting protein II or against its mutein, obtained by conservative substitutions. Here protein, against which antibody is directed, is separated from urine and connects interferon-α/β-connecting protein II with constant Kd equal 2.12x10-9 M.
EFFECT: obtaining possibility to detect interferon-α/β-connecting protein II presence in different samples.
5 cl, 10 dwg, 6 tbl, 17 ex
FIELD: chemistry; medicine.
SUBSTANCE: claimed are polypeptide and respective polynucleotide zcytor17lig and molecules of antibody against human zcytor17. Human zcytor17lig is novel cytokine. Claimed invention also relates to methods of protein obtaining, its application for stimulation of immune reaction in mammal. Described is method of obtaining antibodies to said protein and respective antibodies.
EFFECT: polypeptides can be used in realisation of methods stimulation of immune system, proliferation and development of hemopoietic cells in vitro and in vivo.
17 cl, 3 dwg, 21 tbl, 47 ex
SUBSTANCE: full-size human monoclonal antibodies against MCP-1 antigene are obtained. Antibodies are used for MCP-1 level definition in patient sample, as well as in tumour and inflammatory disease treatment. Invention allows obtainment of monoclonal antibodies highly specific to MCP-1 antigene.
EFFECT: efficient antibody use for treatment of the said diseases and in diagnostic purposes.
16 cl, 18 dwg, 17 tbl, 11 ex
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: biotechnology, immunology.
SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.
EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.
26 cl, 16 dwg, 1 tbl, 8 ex
SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.
EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.
17 cl, 1 dwg, 7 tbl, 7 ex