Compositions and method for immunosuppression

FIELD: medicine, immunology.

SUBSTANCE: invention relates to compositions and method for immunosuppression achievement. Claimed compositions contain two main agents: namely the first agent targeted to interleukin-15 receptor (IL-15R), and the second agent which inhibits costimulating signal transferred between T-cell and antigen-presenting cell (APC).

EFFECT: diagnosis and therapy of immune deceases, in particular autoimmune deceases with improved effectiveness.

45 cl, 3 dwg

 

The SCOPE of the INVENTION

The scope of the invention is immune suppression.

The premise of the INVENTIONS

An effective immune response begins when the antigen or mitogen initiates the activation of T cells. During activation of T cells there are numerous cellular changes, which include the expression of cytokines and receptors cytokines. One of the cytokines involved in the immune response, is interleukin-15 (IL-15), which is a growth factor for T cells, in vitro stimulates the proliferation and differentiation of b-cells, T-cells, natural killer (NK) cells and activated lymphocytes, killer cells (LAK). The proliferation of these cell types in vivo immune response.

Patients benefit as a result of suppressing the immune response in some cases, for example in the case of transplantation of organs or immune diseases, in particular autoimmune diseases. In other cases, for example when selected immune cells become malignant or autoaggressive, useful is their active destruction.

The INVENTION

This invention is based on the discovery of new ways of suppressing the immune response. In each case, the suppression is achieved by the introduction of the first agent whose target is a receptor, interleukin-15 (IL-15R), and the second agent, which is the first blocks of co-stimulating signal, which normally is transmitted from antigen-presenting cells (APCS) to T-cells. Thus, the essential characteristic of the invention are methods of treatment of the patient, which is useful immune suppression (for example, a patient who has received, or assigned to receive a transplant, or who has an immune disease, particularly autoimmune disease), by introducing one or more agents, the target of which is IL-15R, and one or more agents that block co-stimulating signal. The essential feature of the invention are therapeutic compositions that contain one or more of the above described first and second agents. Although such compositions must contain more than one agent, the methods according to the invention is not limited to the ways in which agents are necessarily imposed concurrently. The agents according to the invention and methods of their use are described below.

Many of the agents used in the context of this invention, possess features that have advantages in therapy. For example, agents that target is IL-15R, may represent a hybrid proteins that contain a mutant polypeptide of IL-15 (mIL-15). These agents apparently are not immunogenic, as part of a hybrid protein as the mutant IL-15 can about licatesi from IL-15 wild-type replaced only a few remnants. In addition, because the polypeptides mIL-15 can be contacted with IL-15α with the same affinity as IL-15 wild-type, they can effectively compete for the receptor. In addition, the agents according to the invention can activate components of the immune system of the host, such as complement fixation and phagocytosis, which ultimately mediate elimination, i.e. the depletion of the population of cells bearing the receptor, with which it communicates (for example, Poreba lysis or phagocytosis of target cells). Since the alpha subunit of the receptor for IL-15 (IL-15Rα) is activated or malignant immune cells, but not resting immune cells, the agents according to the invention can be used to make specific to hit the specified target cells that have been activated (e.g., activated antigen T cells) or that have become malignant. Another advantage of the invention is that when the agent whose target is IL-15R, injected with an agent that blocks of co-stimulating signal is induced tolerance. In the immunosuppression may be terminated without transplant rejection or secondary grafts may not be rejected without further immunosuppression.

Application of the compositions according to the invention, in order to supressive immune response, or p and obtaining drugs for the treatment of immune diseases, also is an essential element of the invention.

Other characteristics and advantages of the invention will be apparent from the following detailed description and from the claims. Although in practice or testing of the invention it is possible to use materials and methods similar or equivalent described herein, the preferred materials and methods are described below.

BRIEF DESCRIPTION of FIGURES

Figure 1 shows the nucleic acid sequence of IL-15 wild-type (SEQ ID NO:1) and the calculated amino acid sequence (SEQ ID NO:2).

Figure 2 presents the sequence of the nucleic acid mutant IL-15 (SEQ ID NO:3) and the calculated amino acid sequence (SEQ ID NO:4). Both codon, the codon wild-type, encoding glutamine at position 149, CAG, and codon wild-type, encoding glutamine at position 156, CAA, changed to GAC, which encodes aspartate (These provisions (149 and 156) are consistent with the provisions 101 and 108, respectively, in the Mature polypeptide IL-15, which lacks the signal sequence of 48 amino acids).

Figure 3 is a line graph depicting the percent survival of the graft after transplantation of cells of islets from donor mice DBA2 recipient B6AF1 mice. The recipients are either not exposed or subjected to processing to mIL-15/Ig, CTLA4/Ig, or a combination of mIL-15/Ig and CTLA4/Ig.

The DETAILED DESCRIPTION

An effective immune response begins when the antigen or mitogen initiates the activation of T cells. Fragments of the antigen are in Association with molecules of the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APCS). AIC also deliver co-stimulating signals, such as signals, mediated by CD40/CD154 and CD28/B7-1 or B7-2 ("co-stimulating signals to T cells" or "co-stimulating signals"). During activation of T cells there are numerous cellular changes, which include the expression of cytokines and receptors cytokines. One of the cytokines involved in immune response, is interleukin-15 (IL-15). IL-15 binds heterotrimeric receptor, consisting of β- and γ-subunits of other receptors of interleukins and unique subunit of the IL-15Rα. As described above, IL-15 is characterized as a growth factor for T cells, in vitro stimulates the proliferation and differentiation of b-cells, T-cells, natural killer cells (NK) and activated lymphocytes cells killer cells (LAK). In vivo proliferation of these types of cells enhances immune response.

This invention is based on the discovery of new ways of inhibiting the immune response. Inhibition can be achieved by the introduction of a combination of agents that target one of which is IL-15R. Means is I, the term "agent" encompasses essentially any type of molecule that can be used as a therapeutic agent. Proteins, such as antibodies, hybrid proteins and soluble ligands, any of which may be either identical to a protein of the wild type, or contains a mutation (i.e. a deletion, connection or replacement of one or more amino acid residues), and molecules of nucleic acids that encode them, are all "agents". The agents according to the invention it is possible to enter either systemically or locally, either through therapy, cell-based (i.e., the agent according to the invention it is possible to enter the patient through the introduction of cells, which expresses the agent of the patient). The cell can be a cell, injected into a patient for the sole purpose of expression of a therapeutic agent. The cell may be a cell of the cell, organ or tissue transplant, while transplantirovannam material is treated with an agent or transducer nucleic acid that encodes a therapeutic agent to, ex vivo or after transplantation. Thus transplantirovannam cell produces its own immunosuppressive factor(s). For example, a cell that produces insulin as a suitable phenotype can be modified to contain a gene's unstable past also produces the ith immunosuppressive factor(s) according to this invention. Specified and other routes of administration commonly used specialists in this area and are discussed next.

Agents that target is IL-15R

Agents that target is IL-15R, include agents that bind to IL-15R, and agents that bind and then destroy cells bearing IL-15R, such as activated T cells. Thus, agents that are suitable for achieving immune suppression, may contain two functional components: a guide to the target component, which directs the agent to the target cell bearing an IL-15R, and component, depleting the population of target cells (i.e. lytic), which leads to the elimination of cells bearing IL-15R. In one embodiment, the guide to the target component associated with IL-15R without effective transduction of the signal through this receptor. For example, the guide to the target component can include a mutant polypeptide of IL-15, and the depleting population of target cells, may contain the Fc region of immunoglobulin molecules, with the specified Fc-region derived from IgG, such as IgG1, IgG2, IgG3, human IgG4, or similar to mammalian IgG, or IgM, such as human IgM or similar mammalian IgM. In a preferred variant Fc region contains the hinge domain, CH2and CH3domains human IgG1 or IgG2a mouse. Although the invention is not limited to the prohibited agents, which operate according to any particular mechanism, it is assumed that the Fc region mediates managed complement or elimination by phagocytes cells bearing IL-15R.

The obtained mutant polypeptides of IL-15 that are associated with the receptor complex of IL-15 with affinity similar to the affinity of IL-15 wild-type, but cannot fully activate signal transduction. These mutant polypeptides effectively compete with the polypeptides of IL-15 wild-type and can inhibit one or more events that normally occur in response to the signaling of IL-15, such as cell proliferation. Listed here polypeptide IL-15 wild-type" is a polypeptide that is identical to IL-15 natural origin (for example, the polypeptide of IL-15 wild-type is shown in figure 1). By contrast, the mutant polypeptide of IL-15" is a polypeptide that has at least one mutation compared with IL-15 wild-type, and which inhibits at least one activity in vivo or in vitro, which usually stimulated by IL-15 wild-type.

Mutant polypeptide IL-15, which can be used according to this invention typically will block at least 40%, more preferably at least 70% and most preferably at least 90% of one or more activities of molecules of the I-15 wild-type. The ability of the mutant polypeptide of IL-15 blocking the activity of IL-15 wild-type, you can evaluate many tests, including the ones described here for the analysis of cell proliferation of BAF-BO3 (in which cells were transfusional design, coding IL-2Rβ). In addition, mutant polypeptides according to the invention can be characterized in accordance with a specific identity in the interest they show in relation to IL-15 wild-type. When determining percent identity between two polypeptides, the length of comparison sequences will generally be to be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 aminokislotami "identity"used in relation to polypeptide sequences or DNA sequences, refers to the identity between the subunits (amino acid residues of proteins or the nucleotides of DNA molecules) within two compared polypeptide sequences or DNA sequences. If the position of the subunit in the two molecules is occupied by the same Monomeric subunit (i.e. the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity of m is waiting for the two amino acid sequences or two nucleotide sequences is a direct function of the number of identical positions. Thus, a polypeptide that is 50% identical to a reference polypeptide, which has a length of 100 amino acids may be a polypeptide of 50 amino acids that is identical to part of the reference polypeptide has a length of 50 amino acids. Can also be a polypeptide with a length of 100 amino acids, which is 50% identical to a reference polypeptide throughout its length. Of course, the same criteria can answer many other polypeptides. Usually and most conveniently identity was measured using the computer program sequence analysis, such as a package of computer programs for sequence analysis Genetics Computer Group University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705), with the parameters used by default.

Mutant polypeptide of IL-15 may be, at least 65%, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, 96%, 97%, 98% or 99%) identical to IL-15 wild-type. The mutation may be a change in the number or content of amino acid residues. For example, the mutant IL-15 may have more or fewer amino acid residues than IL-15 wild-type. Alternative or additionally, the mutant polypeptide can contain a substitution of one renesola amino acid residues, present in IL-15 wild-type. Mutant polypeptide of IL-15 may be different from IL-15 wild-type accession, deletion or substitution of one amino acid residue, for example by joining, deletion or substitution of the residue at position 156. Similarly, a mutant polypeptide can differ from the wild-type polypeptide accession, deletion or replacement of two amino acid residues, such as residues at positions 156 and 149. For example, the mutant polypeptide of IL-15 may be different from IL-15 wild-type substitution of glutamine to aspartate residues 156 and 149 (as shown in figure 2). Mutant polypeptides, suitable as guides to target agents like IL-15 wild-type, recognize and bind a component of the IL-15R. In one embodiment, the mutation of IL-15 is in the domain of carboxyl end of cytokine, which presumably binds to IL-2Rγ (a subunit of the receptor for IL-2). Alternative or additional mutant polypeptides of IL-15 can contain one or more mutations in the domain that binds IL-2Rβ (β-subunit of the receptor for IL-2).

In the case when the mutant polypeptide of IL-15 contains a substitution of one amino acid residue for another, such a substitution may, but not necessarily, be a conservative replacement, which includes replacement within the following groups: glycine, alanine; valine, isoleucine, leucine; and paracinema acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Instead of the use of or additionally to the use of the polypeptide of IL-15 (e.g., a mutant polypeptide of IL-15) the direction to the target IL-15R can be performed using antibodies that bind to a component of the IL-15R (for example, a subunit of IL-15Rα). The ways through which you can obtain antibodies, including humanized antibodies against a component of IL-15R, well known in this field. Preferably the antibodies must have the ability to activate complement or phagocytosis, such as subclasses of IgG3 and IgG1 person (preferably the latter) or the subclass IgG2a mouse.

As described above, agents suitable to achieve immune suppression, may contain two functional components: a guide to the target component, which directs the agent to the target cell bearing an IL-15R (such as just described mutant molecule IL-15), component, depleting the population of target cells, which, for example, analyzes, or otherwise leads to the elimination of cells bearing the IL-15R. Thus, the agent can be a chimeric polypeptide, which contains a mutant polypeptide of IL-15 and a heterologous polypeptide, such as the Fc region of antibodies of IgG subclasses and IgM. Fc-region may include mutati is, which inhibits complement fixation and binding of the Fc receptor or it can Deplete the population of target cells (i.e. capable of destroying cells by binding of complement or based on another mechanism, such as a dependent antibody lysis with the participation of complement).

Fc-region can be distinguished from a source of natural origin, obtained by recombinant means or to synthesize (like you can get any polypeptide described in this invention). For example, Fc-region, which is homologous C-terminal domain of IgG, can be obtained by cleavage of IgG dad's. Fc IgG has a molecular mass of approximately 50 kDa. The polypeptides according to the invention may include a complete Fc region or a smaller part, which retains the ability to lyse cells. In addition, full or fragmented Fc-region may be variants of the molecules of the wild type. Namely, they may contain mutations that may affect or not affect the function of the polypeptide.

Fc-region may be depleting the population of target cells" or "not depleting the population of target cells". In the case of Fc-region, not depleting the population of target cells, usually lacks the binding site high affinity Fc-receptor and the binding site C 1q. The binding site high affinity Fc receptor Fc mouse IgG the Leu residue at position 235 in the Fc of IgG. Thus, the binding site of the receptor Fc mouse can be destroyed by mutation or dellarovere Leu 235. For example, replacement of Leu 235 to Glu inhibits the ability of the Fc region to bind the high affinity Fc-receptor. The binding site C 1q mouse functionally to break through mutation or dellarovere residues Glu 318, Lys 320, and Lys 322 in IgG. For example, replacement of Glu 318, Lys 320, and Lys 322 on the Ala makes IgG1 Fc unable to stimulate dependent antibody lysis involving complement. In contrast, depleting the population of target cells Fc-region of IgG has a binding site high affinity Fc-receptor and the binding site C 1q. The binding site high affinity Fc receptor contains a Leu residue at position 235 IgG Fc, and the site linking C 1q, contains residues Glu 318, Lys 320, and Lys 322 IgG1. Depleting the population of target cells IgG Fc has in these sites the remains of wild-type or conservative amino acid substitutions. Depleting the population of target cells Fc IgG can do the cells with target-dependent cellular cytotoxicity antibody or caused by complement cytolysis (CDC). Corresponding mutations IgG man also known (see, for example, Morrison et al., The Immunologist 2: 119-124, 1994; and Brekke et al., The Immunologist 2: 125, 1994).

Agents that target is IL-15R, can also represent agents that contain mutant polypeptide of IL-15 and the polypeptide, which run the t as an antigenic tag, such as a sequence FLAG. Sequence FLAG recognized biotinylating, highly specific anti-FLAG antibodies as described here (see also Blanar et al., Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89: 8145, 1992).

Agents that inhibit co-stimulating signal

In order to inhibit the immune response, agents, targets which are described above cells bearing IL-15R, you can enter together with an agent which inhibits co-stimulating signal, which normally is transmitted to the APC to the T-cell. The agent that is administered together with another agent, it is possible, but not necessary, to enter at the same time. For example, in the context of this invention, the agent whose target is IL-15R, you can enter before or after the agent which inhibits co-stimulating signal. In order to inhibit co-stimulating signal, you can enter any agent that binds and blocks one or more molecules or T-cell or APC, known as they take part in the transfer of co-stimulating signal between these two cell types. For example, you can enter any agent that binds to a molecule B7 (including, but not limited to, B7-1 or B7-2) on AIC or CD28, which is expressed, for example, T-cells and binds B7. These agents include antibodies that bind to B7 molecules (e.g., anti-B7-antibody), CTLA4/Ig and antibodies that bind CD28. Alternative or additionally, you can enter any agent that binds CD40 on APC or CD40L (also called CD154), which is expressed, for example, T-cells and binds CD40. These agents include antibodies that bind CD40 or CD40L.

Methods of screening for agents that inhibit the immune response

In addition to testing the investigational agent (for example, a mutant polypeptide of IL-15) in the in vitro tests described in the examples below, you can use any of the following tests in vivo, to check what specific combination described herein agents most effectively induce immune suppression. For example, you can test one or more agents, the target of which is IL-15R in combination with one or more agents that either block co-stimulating signal, which normally is transmitted from the APC to the T-cells are TCR antagonists, inhibit calcineurin, inhibit cell proliferation, or which bind and inactivate the activated antigen T cells. These in vivo analyses represent only some of the most common methods with which the person skilled in the art can further test the effectiveness of the agents according to the invention. Selected these analyses are included here because they approach the Yat for many clinical conditions, treatable agents to target is IL-15R. For example, the assays are suitable for organ transplantation, immune diseases, in particular autoimmune diseases, and malignancies of the immune system (for example, malignant tumors that arise in the case when T-cells become malignant).

Principles of transplantation

To determine, does the agent according to the invention or combination of such agents, immune suppression, they are administered (either directly through based on gene therapy, either through therapy, cell-based) in the context of well-developed principles of transplantation.

The agents according to the invention or encoding molecules are nucleic acids can be entered systemically or locally by standard means any ordinary laboratory animal, such as rat, mouse, rabbit, Guinea pig or a dog, before the animal held allogeneic or xenogeneic skin grafts, organ transplantation or implantation of the cells. Strains of mice, such as C57B1-10, B10.BR and B10.AKM (Jackson Laboratory, Bar Harbor, ME)who have the same genetic background, but do not correspond to the locus H-2, are well suited for the evaluation of various organ transplants.

Heart transplantation

Method of performing heart transplants by anastomosans donor with a trunk vessel in the abdominal cavity of the host first published Ono et al. (J. Thorac. Cardiovasc. Surg. 57: 225, 1969; see also Corry et al., Transplantation 16: 343, 1973). With the help of specified surgical procedures impose an anastomosis between the aorta of the heart donor and the aorta the abdominal cavity of the host and put the anastomosis between the pulmonary artery of the heart donor and nearby Vena cava, using standard technology microvessels. After the heart is transplanted into place and heated to 37°C solution of lactate ringer, will restore normal sinus rhythm. The functioning of the transplanted heart can often be assessed by palpation of contractions of the ventricles through the abdominal wall. Rejection is defined as the stop of myocardial contractions. The agents according to the invention (for example, the combination of mutant IL-15/Fc and CTLA4/Ig) will be deemed to be effective in reducing organ rejection, if the owners, who received these agents, there is a longer period of engraftment of transplanted heart donor, than untreated hosts.

Skin grafts

The effectiveness of various combinations of agents according to the invention also can be assessed after skin grafting. To perform a skin graft on rodents animal donor anaesthetize and part of the tail, remove the skin in depth completely. Animal-recipient also anaesthetize and prepare the bed for transplantation removing the flap of the skin with shaved sides. Typically, the flap has a size of approximately 0.5×0.5 cm Skin donor shape corresponding to the shape of the bed for transplantation, put in place, cover with gauze and bintou. Grafts can be examined daily, starting from the sixth day after the operation, and the transplant is rejected in the case when more than half of the transplanted epithelium looks unsustainable. The agents according to the invention (for example, the combination of mutant IL-15/Fc and CTLA4/Ig) are effective in reducing rejection in transplantation of the skin in that case, if the owners, who received these agents, there is a longer period of healing of the skin of the donor, than untreated hosts.

Model allograft Islands

Allografts cells of the islets of DBA/2J can be transplanted rodents, such as mice B6 AF1 6-8 weeks of age, at which diabetes is caused by a single intraperitoneal injection streptozotocin (225 mg/kg; Sigma Chemical Co., St. Louis, MO). As a control syngeneic grafts of cells of the islets can be transplanted mice with diabetes. Transplantation of islet cells can be performed using the following published protocols (for example, see Gotoh et al., Transplantation 42: 387, 1986). Briefly, the pancreas donor perfusion in situ collagenase type IV (2 mg/ml; Worthington Biochemical Corp., Freehold, NJ). The village is e 40-minute period cleavage at 37° C islets secrete in a stepwise gradient Pichola. Then 300-400 islets transplanted under the renal capsule of each recipient. The functioning allograft can be monitored by serial measurements of blood glucose (Accu-Check IIITM; Boehringer, Mannheim, Germany). The primary function of the graft is determined in the form of glucose levels in the blood below 11.1 mmol/l on day 3 after transplantation, and graft rejection define as a rise in the concentration of glucose in the blood, more than 16.5 mmol/l (at least, in each of 2 consecutive days) after a period of initial functioning graft.

Models of autoimmune disease

Models of autoimmune disease provide other ways to evaluate combinations of agents according to the invention in vivo. These models are well known to specialists in this field, and you can use them to determine whether this combination of agents, which includes the agent whose target is IL-15R, therapeutically suitable for treating a specific autoimmune disease in the case of direct delivery as a result of genetic therapy or through therapy, cell-based.

Autoimmune disease models obtained in animals include disease, rheumatism, such as rheumatoi the hydrated arthritis and systemic lupus erythematosus (SLE), diabetes type I and autoimmune diseases of the thyroid gland, bowel and Central nervous system. For example, models of SLE on animals include mice, MRL, BXSB mice and NZB mice and their hybrids F1. These animals could be crossed in order to explore specific aspects of the process of rheumatism; in the offspring line NZB severe form of lupus glomerulonephritis when crossed with NZW mice (Bielschowsky et al., Proc. Univ. Otago Med. Sch. 37: 9, 1959; see also Fundamental Immunology, Paul, Ed., Raven Press, New York, NY, 1989). Similarly, the shift to a fatal nephritis observed in progeny from crosses NBZ X SWR (Data et al., Nature 263: 412, 1976). Histological manifestations of kidney damage in mice SNF1well characterized (Eastcott et al., J Immunol. 131: 2232, 1983; see also Fundamental Immunology, above). Thus, the overall health of the animal, as well as histological type of renal tissue can be used to determine cupressinum whether effective immune response in an animal model of SLE with the introduction of agents to target is IL-15R, and that, for example, inhibit costimulation.

One of the lines of MRL mice who develop SLE, MRL-lpr/lpr, also develop a form of arthritis similar to rheumatoid arthritis in humans (Theofilopoulos et al., Adv. Immunol. 37: 269, 1985). Alternative, experimental arthritis can be induced in g is Uzunov by injection of collagen type II rats (2 mg/ml), mixed 1:1 with complete adjuvant's adjuvant (100 μl), the base of the tail. Arthritis develops 2-3 weeks after immunization. The ability of nucleic acid molecules encoding the agents according to the invention (for example, agents that target is IL-15R, and agents that inhibit costimulation or which bind and inactivate T cells activated by antigen), supressive immune response can be assessed by targeted delivery of nucleic acid molecules to T lymphocytes. One way targeted delivery to T-lymphocytes is as follows. Suspensions of spleen cells prepared in 2-3 days after the onset of arthritis and incubated with collagen (100 μg/ml) for 48 hours to induce proliferation of activated collagen T cells. At this time cells transducers vector coding genes of interest polypeptide agent. As a control, parallel cultures grown, but not transducer them, or transducers "empty" vector. The cells are then injected intraperitoneally (5×107cells/animal). The treatment efficiency estimate, watching for symptoms over the next 2 weeks, as described Chernajovsky et al. (Gene Therapy 2: 731-735, 1995). Less severe symptoms compared to the control suggests that the combined agents according to the but the invention and the nucleic acid molecule, they encode, function as immunosuppressants and therefore applicable for the treatment of immune diseases, in particular autoimmune diseases.

The ability of various combinations of agents to supressive immune response in case of diabetes type I can test on the line BB rats, which was obtained from a commercial colony of Wistar rats in the Bio-Breeding Laboratories in Ottawa. These rats spontaneously develop autoantibodies against islet cells and insulin, exactly as happens in the case of a type I diabetic person. Alternative as a model system, you can use NOD mice (diabetic mouse without obesity).

Autoimmune thyroid disease was modeled in chickens. Chicks line, which is observed obesity (OS), uniformly develops spontaneous autoimmune thyroiditis similar to Hashimoto's disease, (Cole et al., Science 160: 1357, 1968).

Approximately 15% of these birds produce autoantibodies to parietal cells of the stomach, just as in autoimmune thyroiditis person. Manifestation of the disease in chickens OS which can be observed in any treatment regimen that includes body size, fat, lipids in serum, sensitivity to cold and infertility.

Models of autoimmune disease in the Central nervous system (CNS) can also induce exp is rimentale. Inflammation of the CNS, which leads to paralysis can be induced with a single injection of the adjuvant in the tissue of the brain or spinal cord of many different laboratory animals, including rodents and primates. This model, called experimental allergic encephalomyelitis (EAE), mediated by T-cells. In this way it is possible to obtain experimentally induced myasthenia pregnant by a single injection of the acetylcholine receptor with adjuvant (Lennon et al., Ann. N.Y. Acad. Sci. 274: 283, 1976).

Autoimmune diseases of the intestine can be modeled in "knocked out" of mice by IL-2 or IL-10, or in mice that received enemas, containing bovine serum albumin.

Molecules of nucleic acids that encode the agents according to the invention

Polypeptide agents according to the invention, including agents that are hybrid proteins (for example, molecules of mutant IL-15/Fc and CTLA4/Ig)can be obtained not only by the expression of the nucleic acid molecule in a suitable eukaryotic or prokaryotic expressing the system in vitro and subsequent purification of the polypeptide agent, but you can also enter the patient using a suitable gene therapy expressing vector encoding a nucleic acid molecule. In addition, the nucleic acid can be introduced in CL is woven graft prior to transplantation of the graft. Thus, nucleic acid molecules encoding the above-described agents are within the scope of the invention. Just as the polypeptides according to the invention (for example, the polypeptides of mutant IL-15/Fc) can be described according to their identity with the wild-type polypeptides (e.g., the polypeptides of IL-15 wild-type), molecules encoding their nucleic acids are bound to have a certain identity with molecules that encode the corresponding wild-type polypeptides. For example, the nucleic acid molecule encoding a mutant polypeptide of IL-15 may be, at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably at least 95%, 96%, 97%, 98% or 99%) identical to the nucleic acid that encodes IL-15 wild-type. In the case of nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

Molecules of nucleic acids that encode the agents according to the invention may contain a sequence of natural origin, or sequences that differ from the sequences with the same origin, but due to the degeneracy of the genetic code, encode the same polypeptide. These molecules are nucleic acid can consist of RNA or DNA (e.g. genomic DNA, cDNA, or synthetic DNA, such, which is obtained by synthesis based on phosphoroamidite) or combinations or modifications of nucleotides within these types of nucleic acids. In addition, the nucleic acid molecule can be Dantewada or single-stranded (i.e. either sense or antisense strand).

Molecules of nucleic acids according to the invention referred to as "isolated", as they are separated from the 5'- or 3'-coding sequences with which they are directly adjacent in the genome of the organism of natural origin. Thus, the nucleic acid molecule is not limited to sequences that encode polypeptides; can also be enabled for some or all non-coding sequences that are "left" or "right" coding sequence. Experts in the field of molecular biology are familiar with standard procedures for isolation of nucleic acid molecules. Them, for example, can be obtained by treatment of genomic DNA with restriction endonucleases, or by performing polymerase chain reaction (PCR). In the case where the nucleic acid molecule represents the t of a ribonucleic acid (RNA), molecules can be obtained using in vitro transcription.

Isolated nucleic acid molecule according to the invention may contain fragments not identified as such in a natural form. Thus, the invention encompasses recombinant molecules, such as molecules, in which the sequence of the nucleic acid (e.g., a sequence encoding a mutant IL-15) is included in the vector (e.g. plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position different from its natural position in chromosome).

As described above, the agents according to the invention can be a hybrid proteins. In addition or instead of the above heterologous polypeptide molecule of nucleic acid encoding the agent according to the invention may contain sequences encoding "marker" or "reporter". Examples of marker or reporter genes include β-lactamase, chloramphenicolchloramphenicol (HUT), adenoidectomies (ADA), aminoglycosidetherapy (neor, G418r), dihydrotetrazolo (DHFR), hygromycin-B-phosphotransferase (HPH), timedancing (TK), lacZ (encoding β-galactosidase) and contingenciesliabilities (XGPRT). As in the case of many standard methods associated with the practical application of izaberete the Oia, the person skilled in the art will know of additional suitable reagents, such as additional sequences that can function as a marker or reporter.

Molecules of nucleic acids according to the invention can be obtained by introducing mutations in the agent according to the invention (for example, a molecule of IL-15 or a CTLA4 molecule, derived from any biological cells, such as mammal cells, or obtained by standard methods of cloning. Thus, nucleic acid according to the invention can be a nucleic acid mouse, rat, Guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog or cat. Preferably the nucleic acid molecule will be the molecules of the person.

The above-described nucleic acid molecule can be part of a vector which is able to control their expression, for example, in the cell, which was transducible vector. Accordingly, in addition to the polypeptide agents among the preferred options are expressing vectors, containing a nucleic acid molecule encoding the indicated agents, and cells transfetsirovannyh these vectors.

Suitable for use in this invention are vectors include vectors based on T7 for use in bacteria (see, the example Rosenberg et al., Gene 56: 125, 1987), expressing the vector pMSXND for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263: 3521, 1988), yeast expressing the system such as Pichia pastoris (for example, family expressing vectors PICZ from Invitrogen, Carlsbad, CA) and based on baculovirus vectors (for example, expressing the vector pBacPAK9 from Clontech, Palo Alto, CA) for use in insect cells. In such vectors, the insertion of nucleic acids that encode interest polypeptide can be operatively associated with a promoter that is selected, for example, based on the type of cells in which trying to get the expression. For example, the T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the promoter of cytomegalovirus or metallothionein can be used in mammalian cells. Also in the case of higher eukaryotes are widely used tissue-specific and specific for cell type promoters. These promoters are so named because of their ability to direct expression of the nucleic acid molecules in the body tissue or cell type. The person skilled in the art is familiar with numerous promoters and other regulatory elements that can be used to control the expression of nucleic acids.

In addition to sequences that contribute to the t of transcription of the integrated nucleic acid molecule, the vectors can contain the start replication and other genes encoding breeding marker. For example, the gene of resistance to neomycin (neor) confers resistance to G418, the cells in which it is expressed, and thus provides phenotypic selection of transfected cells. Other suitable genes of breeding markers that allow phenotypic selection of cells include various fluorescent proteins, for example protein with green fluorescence (GFP) and its variants. Specialists in this field will easily determine whether this regulatory element or breeding marker for use in a specific experimental situation.

Viral vectors that can be used in the invention include, for example, retroviral, adenoviral vectors, and vectors based on adeno-associated virus, herpes virus, monkey virus 40 (SV40), and human papilloma virus bull (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, New York).

Prokaryotic or eukaryotic cells that contain the nucleic acid molecule, which encodes the agent according to the invention, and Express the protein encoded by this nucleic acid molecule, in vitro, are an essential element of the invention. The cell according to the invention is transtitional CL is woven, i.e. the cell in which by means of recombinant DNA technology was introduced nucleic acid molecule, for example a nucleic acid molecule encoding a mutant polypeptide of IL-15. The progeny of such cells is also considered to be within the scope of this invention. The exact components of the expression system are not critical. For example, the mutant polypeptide of IL-15 can be obtained in a prokaryotic host, such as the bacterium E. coli, or in eukaryotic host, such as an insect cell (e.g., Sf21 cells, or mammalian cells (such as cells, COS cells, Cho cells, 293 cells, NIH 3T3, or HeLa cells). These cells are available from many sources, including the American type culture collection (Manassas, VA). When choosing expressing the system's importance is only that the components are compatible with each other. The person skilled in the art are able to determine this. In addition, if you need guidance in choosing expressing system, you can refer to Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

Eukaryotic cells that contain the nucleic acid molecule, which encodes the agent according to the invention, and in vivo Express the protein encoded by such a nucleic acid molecule, are also a significant feature of izopet the deposits.

In addition, eukaryotic cells according to the invention can be cells that are part of the cell transplant, transplant tissue or organ. Such grafts may contain either primary cells taken from the body of the donor, or cells that have been cultured, modified and/or have passed selection in vitro before transplantation into the body of the recipient, for example, lines of eukaryotic cells, including stem cells or precursor cells. Because after transplantation into the recipient organism may be proliferation of the cells, the progeny of such cells are also seen as part of the invention. Cell part cell transplant, transplant tissue or organ, can be transliterowany nucleic acid that encodes a mutant polypeptide of IL-15, and then transplanted into the body of the recipient, where the expression of a mutant polypeptide of IL-15. In addition, this cell can contain one or more additional structures of nucleic acids, allowing to apply the procedures of selection, for example, specific lines of differentiation of cells or types of cells prior to transplantation into the recipient's organism.

Expressed polypeptides can be cleaned from expressing system using standard biochemical methods and can be used as diagnostic tools or as therapeutic agents as described below (therapeutic compositions according to the invention can be understood as pharmaceuticals). Therefore, the essential feature of the invention is also a method of obtaining a therapeutic composition comprising a mutant polypeptide of IL-15, which connects subunit of IL-15R, and a polypeptide that binds a molecule B7. The method can be carried out, for example, by (a) purification of mutant polypeptide of IL-15 expressing system; (b) purification of the polypeptide which binds to B7, from expressing system; and (c) combining the polypeptide of IL-15 and a polypeptide which binds to B7. therapeutic composition can be prepared (as described here and by methods known to experts in this field) to treat patients with any described herein immune disease (e.g., any autoimmune disease).

Agents that target is IL-15R, suitable for diagnosing

Agents that target is IL-15R, can be used to determine whether the patient has the disease (e.g., immune disease, particularly autoimmune disease, which is treatable by a combination of agents described here. The diagnostic method can, for example, to perform, receiving a tissue sample from the patient, which assumes the presence of immune Zab the diseases, in particular autoimmune diseases or malignant tumors, which is found in the form of malignant immune cells, and subjecting the fabric to the impact of the labeled antigen polypeptide whose target is IL-15R. The sample can be any biological sample such as a blood sample, urine, serum or plasma. In addition, the sample may be a tissue sample (e.g., tissue biopsy), or effusion obtained from a joint (e.g., synovial fluid), from the abdominal cavity (for example, ascitic fluid), chest (eg, pleural fluid) or from the Central nervous system (e.g., cerebrospinal fluid). The sample may also consist of cultivated cells that the source was obtained from the patient (e.g., mononuclear cells from peripheral blood). The sample can be obtained from the body of a mammal, such as a patient man. If the sample contains cells that are associated with the agent, the impact of which they were subjected, then with high probability they will communicate with this agent (for example, an agent whose target is IL-15R) in vivo, and thus, the in vivo can inhibit their proliferation or destroy them. Symptoms patients-candidates for such testing and related tissues which need study the performance in the case of this particular set of symptoms, well-known specialist in this field.

Patients under treatment

Combinations of agents according to the invention can be used to treat patients who suffer from immune disease, particularly autoimmune disease, including, but not limited to, the following diseases: (1) a rheumatic disease such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren syndrome, scleroderma, mixed disease of connective tissue, dermatomyositis, polymyositis, Reiter syndrome or disease Becket; (2) diabetes type I or type II; (3) autoimmune thyroid disease such as Hashimoto's thyroiditis or graves ' disease; (4) an autoimmune disease of the Central nervous system, such as multiple sclerosis, myasthenia pregnant or encephalomyelitis; (5) various forms of pemphigus, such as pemphigus vulgaris, the pemphigus vegetans, pemphigus leaf syndrome Sideara-usher or Brazilian pemphigus, (6) diseases of the skin, such as psoriasis or neurodermitis, and (7) inflammatory bowel disease (e.g. ulcerative colitis or Crohn's disease). Combinations of agents according to the invention can also be used to treat acquired immunodeficiency syndrome (AIDS). Similarly, the ways in which administered these agents, you can use DL the treatment of the patient, who received a graft of synthetic or biological material, or a combination of both. Such grafts can be transplants of organs, tissues or cells, or synthetic grafts, seeding with cells, such as synthetic vascular grafts, seeding with cells of blood vessels. In addition, this method is useful for patients with vascular damage.

Since the invention involves the injection mold agent, depleting the population of target cells, the target of which is IL-15R, you can selectively kill self-reactive or "destructive transplant immune cells without massive destruction of normal T cells. Therefore, the essential feature of the invention is a method for killing cells that Express IL-15R in vivo, which include active or self-reactive or "destructive transplant immune cells or cancer cells. These methods can be accomplished by administration to a patient combinations of agents, which includes the agent whose target is IL-15R and which activates the complement system, analyzes cells through ADCC or otherwise kills cells expressing the receptor complex of IL-15 wild-type. This method can be used to treat patients with IL-15R+-leukemia, lymphoma other malignant diseases IL-15R +such as cancer of the large intestine.

Compositions for use and route of administration

Methods according to this invention and therapeutic composition used for their implementation, include "substantially purified" agents. For example, in the case where the agent is a polypeptide, a polypeptide at least 60% by weight (dry mass) consists of interest polypeptide such as a polypeptide that binds and destroys cells bearing IL-15R. Preferably the agents (e.g., polypeptides), at least 75%, more preferably at least 90% and most preferably at least 99% by weight consist of interest of the agent. Purity can be measured by any appropriate standard method, for example by chromatography on a column by polyacrylamide gel electrophoresis or HPLC analysis.

Although agents applicable in the methods according to this invention, can be obtained from sources of natural origin, it is also possible to synthesize or produce other way (for example, agents that bind and destroy cells bearing IL-15R, you can obtain the expression of the recombinant nucleic acid molecule). Polypeptides derived from eukaryotic organisms or synthesized in E. coli or on the ugogo the prokaryotes, or polypeptides that are synthesized chemically, will be largely exempt from components associated with them in natural conditions. In the case where the polypeptide is a Chimera, it can be encoded hybrid nucleic acid molecule containing a single sequence that encodes all or part of the agent (e.g., a sequence encoding a mutant polypeptide of IL-15, and the sequence encoding the Fc-region of IgG). The agents according to the invention (e.g., polypeptides) can be merged with the mark of hexaglycine to facilitate purification of the expressed bacterial protein, or hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells.

The methods that are necessary to obtain the agents according to the invention, are standard in this area, and the person skilled in the art can perform, without resorting to undue experimentation. For example, a mutation that consists of replacing one or more amino acid residues in IL-15, can be created using technology mutagenesis using PCR described here to create a mutant polypeptide of IL-15, in which the glutamine residue at positions 149 and 156 is replaced with aspartic acid residues. Mutations that consist of deletions or accessions am nekisnotnice residues (to the polypeptide of IL-15 or to any other described here is suitable polypeptides, for example, polypeptides that inhibit costimulation, or which bind activated T-cells), can also be standard recombinant methods. In therapeutic applications, the agents according to the invention can be entered with a physiologically acceptable carrier such as physiological salt solution. Therapeutic compositions according to the invention can also contain a carrier or excipient, many of which are known to the person skilled in the art. Fillers that can be used include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g. serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol and glycerol. The agents according to the invention can be prepared in the composition in various ways in accordance with the relevant route of administration. For example, for drinking or injection it is possible to prepare liquid solutions; gels or powders can be prepared for oral exposure, inhalation or topical application. Methods of obtaining such compositions are well known and can be found for example in "Remington''s Pharmaceutical Sciences".

Route of administration are well known to experienced pharmacologists and clinicians and include intraperitoneal, nutribase is Noah, subcutaneous and intravenous administration. Additional pathways include intracranial (e.g., vnutrisustavnogo or intraventricular), intraorbital, ophthalmic, vnutricapsulino, intraspinal, intraperitoneal, through the mucous membrane, local, subcutaneous and oral administration. It is assumed that the preferred will be intravenous or intra-arterial route for the introduction of agents, the target of which is the receptor for IL-15. Often you can also use the subcutaneous route, since the subcutaneous tissue provides a stable environment for polypeptides from which they can slowly be released.

In the case of cell-based therapies (gene therapy) cells/tissues/organs before transplantation to transliterate composition of nucleic acids by incubation, infusion or perfusion, so that therapeutic protein expressively and then released transplanted cells/tissues/organs in the body of the recipient. And cells/tissues/organs can be subjected to pre-treatment by perfusion or simple incubation with therapeutic protein prior to transplantation, in order to remove the associated with transplant immune cells adhering to the cells/tissues/organs of the donor (although this is only a side aspect, which may not buteimat any clinical significance). In the case of cell grafts of cells you can enter any manner implantation or by way of injection catheter through the wall of the blood vessel. In some cases, the cells can be activated by a release into the vascular system from which they are then distributed by the blood stream and/or migrate into the surrounding tissue (this is implemented by the transplantation of islet cells, when islet cells are released into the portal vein and then migrate to the liver tissue).

In medicine it is well known that the dose for any patient depends on many factors, including General health status, sex, weight, surface area and age of the patient, as well as a particular connection, you must enter the time and route of administration, and other drugs administered simultaneously. The dose of the polypeptide according to the invention will vary, but when administered intravenously may be given in doses of from 1 microgram to 10 mg/kg of body weight, or about 0.01 mg/l to 100 mg/l in blood volume. The dose, if necessary, you can enter one or several times a day, and the treatment may continue for long periods of time. Identifying the appropriate dose for a given application is not a problem for a qualified specialist in this field.

EXAMPLES

Reagents

For the studies described here used the following reagents: recombinant IL-2 was obtained from Hoffman-La Roche (Nutley, NJ); rapamycin was obtained from Wyeth-Ayerst (Princeton, NJ); cyclosporine-A (CsA) was obtained from Sandoz (East Hanover, NJ); RPMI-1640 and fetal calf serum (FCS) was obtained from BioWittaker (Walkersville, MD), penicillin, streptomycin, G418 and streptavidin-RED670 were obtained from Gibco-BRL (Gaithersburg, MD); dexamethasone, PHA, lysozyme, Nonidet P-40, NaCl, HEPES and PMSF were obtained from Sigma (St. Louis, MO); fecal-hipac was obtained from Pharmacia Biotech (Uppsala, Sweden); recombinant IL-15 human and Antibodies against IL-15 person received from PeproTech (Rocky Hill, NJ); anti-FLAG Antibody and anti-FLAG affinity beads were obtained from International Biotechnologies, Inc. (Kodak, New Haven, CT); pRcCMV were obtained from InVitrogen Corporation (San Diego, CA); genistein was obtained from ICN Biomedicals (Irvine, CA); disuccinimidyl (DSS) was obtained from Pierce (Rockford, IL); the restriction enzyme was obtained from New England Biolabs (Beverly, MA); [3H]TdR was obtained from New England Nuclear (Boston, MA); and conjugated with a fluorescent dye antibody CD25-PE3, CD14-PE, CD16-PE, CD122-PE, CD4-FITZ, CD8-FITZ, IgG1-PE or IgG1-FITZ received from Beckton/Dickinson (San Jose, CA). FLAG-peptide was synthesized on equipment for the synthesis of peptides in the Harvard Medical School.

Getting a hybrid protein FLAG-HMK-IL-15

To investigate the expression pattern of the receptor for IL-15 human cells have constructed a plasmid that can be used for expression of the hybrid protein of IL-15. Plasmid encodes the polypeptide of IL-15 with N-end covalently associated with a sequence of FLAG-HMK of 18 amino acids (FLAG-HMK-IL-15). FLAG-posledovatelno and recognized biotinylating highly specific anti-FLAG-antibodies (Blanar et al., Science 256: 1014, 1992); LeClair et al., Proc. Natl. Acad. Sci. USA 89: 8145, 1992), whereas sequence HMK (the site of the kinase recognition of cardiac muscle, you can enter in the molecule, a radioactive label [32P] (Blanar et al., above, LeClair et al., above).

To construct the plasmid FLAG-HMK-IL-15 cDNA fragment size 322 BP that encodes a Mature protein of IL-15, amplified by PCR using synthetic oligonucleotides [sense 5'-GGAATTCAACTGGGTGAATGTAATA-3' (SEQ ID NO:5; the EcoRI site (underlined) plus the base 145-162); antisense 5'-CGGGATCCTCAAGAAGTGTTGATGAA-3' (SEQ ID NO:5; the BamHI site [underlined] plus Foundation 472-489)]. DNA template was obtained from PHA activated human PBMC. The PCR product was purified, digested EcoRI and BamHI and cloned in the plasmid pAR(DRI)59/60, split EcoRI-BamHI as described (Blanar et al., Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89: 8145, 1992). The main part of the plasmid pAR(DRI)59/60 frame contains the sequence coding sequences of peptides for recognition FLAG and HMK (Blanar et al, Science 256: 1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89: 8145, 1992).

Expression and purification of the hybrid protein FLAG-HMK-IL-15

The hybrid design of the FLAG-HMK-IL-15 related IL-15, expressed in strain BL-21 E. coli and affinity purified using beads coated with anti-FLAG, as described (Blanar et al., Science 256: 1014, 1992; LeClair et al. Proc. Natl. Acad. Sci. USA 89: 8145, 1992). The hybrid protein was suirable with affinity columns after thorough washing with 0.1 M glycine (pH 3.0). E. yet, containing FLAG-HMK-IL-15, were dialyzed against buffer containing 50 mm Tris (pH of 7.4) and 0.1 M NaCl, for 18 hours at 4°C, filtered through a membrane with pore diameter of 0.2 μm and stored at -20°C.

FLAG-HMK-IL-15 binds to a subunit of IL-15Rα

Purified hybrid protein FLAG-HMK-IL-15 was tested in order to determine whether it interacts with receptors of IL-15 on the cell surface. As described above, [32P]-FLAG-HMK-IL-15 was added to the PBMC cultures, which are activated by the mitogen PHA. In order to link the interacting proteins with each other, were constantly added chemical cross-linking agent disuccinimidyl (DSS). Cells were washed, literally, centrifuged and dissolved in the detergent proteins were separated in SDS-page. The autoradiograph separated in SDS-page proteins revealed a single band in the region of 75-80 kDa, corresponding to the total molecular mass of FLAG-HMK-IL-15 (15 kDa) and a subunit of the IL-15Rα person (60-65 kDa). The identity specified band the subunit of IL-15Rα confirmed by experiments on cross-linking performed in the presence of molar excess of hIL-15. Under these conditions, the authors could not detect radioactively labeled band with Mm 15 kDa. Thus, the conformation of the hybrid proteins [32P]-FLAG-HMK-IL-15 allows for site-specific binding subunit of IL-15Rα M.M. 60-65 kDa expressed on the surface of the mitogen-activated PBMC.

FLAG-HMK-IL-15 is a biologically active growth factor, which requires the expression of IL-2Rβ

In the next series of experiments the hybrid protein FLAG-HMK-IL-15 was tested in order to determine whether it can function as a biologically active growth factor. Activated PHA PBMC person proliferate in response to either FLAG-HMK-IL-15, or recombinant IL-2, which was determined through analysis of the inclusion of [3H]-TdR. The FLAG peptide in the absence of the sequence of IL-15 stimulates the proliferation of cells. As IL-2, a hybrid protein FLAG-HMK-IL-15 stimulates the proliferation of transfectants cells IL-2Rγ+BAF-BO3, which Express a subunit of IL-2Rβ. However, a hybrid protein FLAG-HMK-IL-15 stimulates the proliferation of the parental cells BAF-WO that were transliterowany vector, which lacks the sequence chain of IL-2Rβ. Thus, FLAG-HMK-IL-15 is a biologically active growth factor, which requires expression of the chains of the IL-2Rβ on target cells to stimulate cell proliferation.

Activated mitogen, but not resting PBMC Express subunit of IL-15Rα

A hybrid protein FLAG-HMK-IL-15, biotinylated anti-FLAG antibody and streptavidin-RED670 used to detect the expression of binding sites of IL-15 in human PBMC by C is colorimetrical analysis. Tested PBMC were either freshly isolated or activated by PHA. These cells were washed and incubated either in the environment or with FLAG-HMK-IL-15, after which biotinylating anti-FLAG-Antibody and streptavidin-RED670. Stained cells were analyzed by flow cytometry. PBMC, which were activated with PHA, expressed proteins IL-15Rαand resting PBMC is not expressed. In accordance with the above-described experiments on cross-stitching the binding of FLAG-HMK-IL-15 PHA activated PBMC blocked molar excess of rIL-15, testifying, thus, the specificity of the binding of FLAG-HMK-IL-15 binding sites of IL-15. Chain IL-15Rα Express as activated CD4+and cells CD8+. Induced upon activation chain IL-15Rα it was also detected in CD14 cells+(monocytes/macrophages) cells and CD16+(natural killer cells).

Subunit of the IL-2Rα and IL-2Rβ not required for binding of IL-15

FACS-analysis of PHA activated PBMC stained proteins FLAG-HMK-IL-15 and anti-CD25 MAB against subunit of the IL-2Rαreveals a population of cells expressing both IL-15Rαand IL-2Rα-subunit, as well as a population of cells that Express one of the two subunits, but does not Express both subunits. There are cells of the IL-2Rα+that do not bind FLAG-HMK-IL-15. Almost all of PBMC, which is were stimulated PHA within one day, Express or chain IL-15Rαor chain of IL-2Rβbut does not Express both proteins. In contrast, 3 days after PHA stimulation was noted a much larger population of cells IL-15Rα+IL-2Rβ+(double-positive) and a much smaller population of cells IL-15Rα+IL-2Rβ-(a positive one token). Interestingly, there are cells of the IL-2Rβ+which is not associated with the IL-15. Thus, the expression of the chains of the IL-2Rβ not sufficient for binding of IL-15.

Taken together our data indicate that IL-15 can be contacted with cells IL-15Rα+IL-2Rα-and IL-2Rβ-. A similar conclusion was made in the course of experiments in which they investigated the interaction of IL-15 cells IL-2Rα-that β-, transfitsirovannykh subunit of IL-15Rα (Anderson et al., J. Biol. Chem. 270: 29862, 1995; Giri et al., EMBO J. 14: 3654, 1995). In addition to the requirements of the expression of the subunit of the IL-15Rαrequires subunit of the IL-2Rβ and IL-2Rγ to make the cells sensitivity initiated IL-15 growth.

In conclusion, the above experiments showed that: (i) subunit of the IL-15Rα quickly expressed activated macrophages, T-cells and NK cells and (ii) induction subunit of the IL-15Rα is blocked by dexamethasone, but not CsA or rapamycin. In addition, the experiments confirmed that the subunits of the IL-15Rα necessary and sufficient for binding of IL-15 and that a hybrid protein FLAG-HMK-IL-15 is an extremely useful tool for the study of receptors IL-15.

Set phosphorylated at tyrosine proteins induced by IL-2, is the same as the set of proteins induced by IL-15

Subunit of IL-2Rβ is necessary for signal transduction as IL-2 and IL-15

Decreased viability of activated T-cells and thereby depletion of populations of activated T-cells gives way to reduce the production of lymphokines and mitogens, which contribute to accelerated development of atherosclerosis, allograft rejection, some leukemias and other immunologically mediated pathologies. In addition, blocking the path of signal transduction activated by IL-15 also provides a method of reducing the production of lymphokines and mitogens, which contribute to accelerated development of atherosclerosis, allograft rejection, some leukemias and other immunologically mediated pathologies. When T cells are activated, they proliferate and Express on their surface receptors of interleukins. In addition, activated T-cells release at least 3 lymphokine: gamma-interferon, factor of differentiation In cells II and IL-3. These lymphokines can cause a variety of undesired hair is a diversified events such as allograft rejection. In contrast, resting T-cells and T-cells in long-term memory does not Express the receptors for lymphokines. This difference in the expression of receptors provides a means for targeted impact on activated immune cells, without affecting the resting cells. Molecules designed to recognize any subunit of the IL-15R will recognize the activated monocytes/macrophages and activated T-cells, and can be used for selective inhibition or destruction of these cells. Derived IL-15 that are associated with the subunit of IL-15R, but have no activity of IL-15, because they either block the binding of and/or accumulation of the present IL-15, which is applicable in the method of the invention. Described below mutant molecule IL-15 is a working example of such a derivative.

Mutant polypeptide of IL-15 whose target is IL-15R

Genetic structure of mutant IL-15

Protein IL-15 man carrying a double mutation (Q149D; Q156D), designed to purposefully influence the anticipated sites that are important for binding to the subunit of IL-2Rγ. Carried the mutation polar, but uncharged glutamine residues at positions 149 and 156 in acidic residues aspartic acid using mutagenesis using the HRC is. cDNA encoding the double mutant IL-15, amplified in PCR using synthetic sense oligonucleotide [5'-GGAATTCAACTGGGTGAATGTAATA-3' EcoRI site (underlined hexamer) plus the base 145-162] and synthetic antisense oligonucleotide (5'-CGGGATCCTC-AAGAAGTGTTGATGAACATGTCGACAATATGTACAAAACTGTCCAAAAAT-3'; BamHI site (underlined hexamer) plus the base 438-489; mutant base separately underlined]. The matrix was a plasmid containing the cDNA, which encodes the FLAG-HMK-IL-15 people. Amplificatory fragment was digested EcoRI/BamHI and cloned in the plasmid pAR(DRI)59/60, split EcoRI/BamHI as described (LeClair et al., Proc. Natl. Acad. Sci. USA 89: 8145, 1989). The presence of a mutation at residue 156 confirmed splitting SalI; mutation introduces a new restriction site SalI. In addition, mutations were confirmed by DNA sequencing according to standard method. Protein FLAG-HMK-IL-15 with a double mutation (Q149D; Q156D) was obtained, was purified and confirmed by sequencing as described above for protein FLAG-HMK-IL-15 wild-type.

Using the same strategy that got mutants, which contain a single amino acid substitution in either position 149 or position 156. As described above, these provisions (149 and 156) are consistent with the provisions 101 and 108, respectively, in the Mature polypeptide IL-15, which lacks the signal sequence of 48 amino acids.

In a similar manner specified is trategie used to enable any other amino acid instead of glutamine residues at positions 149 or 156, or to introduce amino acid substitutions at positions other than the position 149 and/or 156.

Cell proliferation of BAF-BO3 in the presence of proteins, related to IL-15

Double mutant polypeptide of IL-15 can inhibit the proliferation of BAF-BO3 dose-dependent manner: the addition of 30 µl (about 50 μg/ml) double mutant IL-15 inhibited the proliferation of more fully than adding 20 ál of double mutant IL-15 of the same concentration.

The proliferation of human PBMC stimulated by PHA

The ability of the double mutant polypeptide FLAG-HMK-IL-15 to link PHA activated human PBMC showed as follows. Activated PHA PBMC were washed and incubated only in the medium or in the presence of a double mutant of FLAG-HMK-IL-15. The cells are then incubated with biotinylated anti-FLAG antibody and stained with streptavidin, conjugated with RED670. Stained cells were analyzed by flow cytometry.

FACS-line analysis of leukemic cells, stained with FLAG-HMK-IL-15 wild-type

In a series of experiments similar to those described above have shown the ability of the polypeptide FLAG-HMK-IL-15 wild-type contact leukemia cells. Treated cells were obtained from lines leukemia cells MOLT-14, YT, HuT-102 and from cell lines that are currently created in Beth Isral Hospital (Boston, MA) and named 2A and 2B. Cultured cells were washed and incubated either in the environment or in an environment containing polypeptide FLAG-HMK-IL-15 wild-type. The cells are then incubated with biotinylated anti-FLAG antibody and stained with streptavidin, conjugated with RED670. Stained cells were analyzed by flow cytometry.

Genetic construction of the additional mutant chimeric polypeptides of IL-15

In addition chimeras FLAG-HMK-IL-15, in which the mutant IL-15 is equipped with antigenic tag, with any mutant IL-15 can bind a variety of other polypeptides. For example, the mutant IL-15 can bind to the Fc fragment of antibodies of the IgG subclass in accordance with the following method.

Genetic structure of mutant IL-15/Fc

cDNA Fcγ2a can be obtained from mRNA extracted from hybridoma, IgG2a secreting using the standard method using reverse transcriptase (MMLV-RT; Gibco-BRL, Grand Island, NY) and synthetic oligonucleotide oligo-dT (12-18) (Gibco BRL). cDNA mutant IL-15 can amplify on plasmid matrix in PCR using synthetic oligonucleotides that are specific for IL-15.

Construct 5'-oligonucleotide to incorporate unique restriction site NotI at a distance of 40 nucleotides from the 5'side of the start codon of translation, whereas the 3'oligonucleotide removes the termination codon and changes the use of the code is for C-terminal Ser residue with AGC on TCG, to ensure the creation of a unique BamHI site at the junction of the mutant IL-15/Fc. Synthetic oligonucleotides used for amplification of cDNA domain Fcγ2a, change the first codon of the hinge with Glu to Asp in order to create a unique BamHI site, covering the first codon of the hinge, and enter a unique XbaI site to the 3'side of the termination codon. Fc-fragment can be modified so that it will not exhaust the population of target cells, i.e. was not able to activate the complement system. To create the design of mutant IL-15, which does not Deplete the population of target cells (mIL-15/Fc), using site-specific mutagenesis using oligonucleotides to replace Glu 318, Lys 320, and Lys 322 motif that binds S 1q, Ala residues. Similarly, Leu 235 replaces Glu to inactivate the binding site FcγRI. Ligation components of the cytokine and Fc in the correct translational reading frame in the unique BamHI site gives an open reading frame length 1236 base pairs, coding for one polypeptide of 411 amino acids (including the signal peptide of IL-15 of the 18 amino acids), in which only 13 cysteine residues. It is estimated that the Mature secretory glycosilated IL-15/Fc has a total of eight intramolecular disulfide bond and three disulfide bonds between heavy chains and a molecular weight of approximately 85 kDa, not involved in the glycosylation.

Expression and purification of hybrid proteins mIL-15/Fc

Correct the genetic makeup of mIL-15/Fc can be confirmed by analysis of DNA sequences after cloning of the hybrid gene in the form of cassettes NotI-XbaI in eukaryotic expressing plasmid pRc/CMV (Invitrogen, San Diego, CA). The indicated plasmid carries the promoter/CMV enhancer, a polyadenylation signal growth hormone bull and the gene of resistance to neomycin for selection with G418. Plasmids carrying the hybrid gene mIL-15/Fc, transferout cells Chinese hamster ovary (CHO-K1, available for purchase from the American type culture collection) by electroporation (1.5 kV/3 μf/0.4 cm/PBS) and are breeding in serum-free medium Ultra-CHO (BioWhittaker Inc., Walkerville, MD)containing 1.5 mg/ml G418 (Geneticin, Gibco BRL). After sublimirovanny clones, which at a high level produce a hybrid protein, selected through a screening nadeshiko against IL-15 ELISA (PharMingen, San Diego, CA). Hybrid proteins mIL-15/Fc clear from nadeshiko cultures by affinity chromatography on protein a-sepharose, then cialiswhat against PBS and sterilized using a 0.22 μm filter. Purified proteins can be stored at -20°C until use.

Western blot analysis after SDS-page in reducing (DTT) and non (without DTT) conditions can be carried out using monoclonal or polyclonal anti-mIL-15 or anti-Fcγ primary antibodies, to assess the size and satiricheskuyu specificity of the hybrid proteins. The functional activity of the mutant IL-15/Fc can be estimated by the standard analysis of the proliferation of T-cells as described above.

The introduction of mIL-15 and CTLA4/Ig

The following work demonstrates that the viability of allograft strikingly increased in the case where the recipient of the allograft treated with an agent whose target is IL-15R, and an agent that blocks of co-stimulating signal. Untreated allografts islets implanted mice, which have caused the diabetes injection streptozotocin. All allografts from donors DBA/2J(H-2d) recipients B6AF1 (H-2b/d,k) constantly take root in the case when the mice were treated with CTLA4/Ig and mIL-15/Fc. Permanent engraftment was never observed in untreated mice or mice treated with a single protein. Completely mismatched for MHC allografts islets from Balb/c(2d)to C57B1/6(H2b)rejected with an average survival time of 15 days for untreated mice, 30 days in mice treated with one CTLA4/Ig and 30 days in mice treated with one mIL-15Fc. In contrast, treatment CTLA4/Ig and mIL-15/Fc led to the continued viability of the graft more than 70% of treated mice (see also figure 3). In addition, it was observed a stable condition that is arentnode to the allograft two recipients, not rejected the second allograft Islands Balb/c without immunosuppressive processing. Quantitative analysis of RT-PCR tissue graft and lymph nodes also showed a marked decrease in the expression of transcripts of T-cell receptors, as well as many genes lymphokines after the combined treatment. Histological analysis confirmed that the combined treatment protects the graft from leukocyte infiltration.

1. Therapeutic immunosuppressive composition comprising a first agent whose target is a receptor, interleukin-15 (IL-15R), and a second agent that inhibits co-stimulating signal transmitted between T-cell and antigen-presenting cell (APC).

2. therapeutic composition according to claim 1, used for supressive immune response in a patient.

3. therapeutic composition according to claim 1, used for obtaining a medicinal product for supressive immune response in a patient.

4. therapeutic composition according to claim 2 or 3, where the patient suffers from rheumatism, rheumatoid arthritis, type 1 diabetes, type II diabetes, autoimmune thyroid disease, autoimmune disease of the Central nervous system, various forms of pemphigus, psoriasis, inflammatory bowel disease, syndrome of acquired virus IMM is nondeficit (AIDS), the patient was transplanted biological organ, tissue or cell, or have vascular damage.

5. therapeutic composition according to claim 1, where the first agent includes a substantially purified mutant polypeptide IL-15, which connects subunit of IL-15R.

6. therapeutic composition according to claim 5, where the subunit is a subunit of IL-15Rα.

7. therapeutic composition according to claim 6, where the mutant polypeptide of IL-15 has a mutation at position 149 in SEQ ID NO:2.

8. therapeutic composition according to claim 6, where the mutant polypeptide of IL-15 has a mutation at position 156 in SEQ ID NO:2.

9. therapeutic composition of claim 8, where the mutant polypeptide of IL-15 also has a mutation at position 149 in SEQ ID N0: 2.

10. therapeutic composition of claim 8, where the mutation at position 156 in SEQ ID NO:2 is the replacement of glutamine to aspartate.

11. therapeutic composition according to claim 9, where the mutation at position 149 in SEQ ID NO:2 is the replacement of glutamine to aspartate.

12. therapeutic composition of claim 8, where the mutant polypeptide of IL-15 contains a substitution of glutamine to aspartate at positions 149 and 156 in SEQ ID NO:2.

13. therapeutic composition according to claim 5, where the first agent further contains a component that leads to the elimination of cells bearing the IL-15R.

14. therapeutic composition according to item 13, where the component that analyzes cells bearing IL-15R, CR is dstanley a Fc-region of IgG molecules.

15. therapeutic composition according to claim 1, where the first agent contains substantially purified anti-IL15R-antibody.

16. therapeutic composition according to claim 1, where the second agent contains substantially purified polypeptide that binds a molecule B7.

17. therapeutic composition according to item 16, where the molecule B7 is a B7-1.

18. therapeutic composition according to item 16, where the molecule B7 is a B7-2.

19. therapeutic composition according to item 16, where the polypeptide that binds B7, is a polypeptide containing the CTLA4/Ig.

20. therapeutic composition according to item 16, where the polypeptide that binds B7, contains anti-B7-antibody.

21. therapeutic composition according to claim 1, where the second agent contains substantially purified polypeptide that binds CD28.

22. therapeutic composition according to item 21, where the polypeptide that binds CD28, includes anti-CD28-antibody.

23. therapeutic composition according to claim 1, where the second agent contains substantially purified polypeptide that binds to CD40L.

24. therapeutic composition according to item 23, where the polypeptide that binds to CD40L, is a polypeptide that includes an anti-CD40L-antibody.

25. therapeutic composition according to claim 1, where the second agent contains substantially purified polypeptide that binds CD40.

26. therapeutic composition according to claim 1, where the polypeptide that binds to CD40, is a polypeptide comprising an anti-CD40-antibody.

27. The method of suppressing the immune response in a patient, the method involves injecting the patient a therapeutic composition according to claim 1, containing the first agent whose target is IL-15R, and a second agent that inhibits co-stimulating signal transmitted between T-cell and antigen-presenting cell (APC).

28. The method according to item 27, where the patient has an immune disease, particularly autoimmune disease, or there is a risk of development of immune diseases, in particular autoimmune diseases.

29. The method according to p, where the autoimmune disease is a disease of rheumatism selected from the group consisting of systemic lupus erythematosus, Sjogren syndrome, scleroderma, mixed connective tissue disease, dermatomyositis, polymyositis, Reiter syndrome or disease Becket.

30. The method according to p, where the autoimmune disease is rheumatoid arthritis.

31. The method according to p, where the autoimmune disease is diabetes type I.

32. The method according to p, where the autoimmune disease is an autoimmune thyroid disease selected from the group consisting of Hashimoto thyroiditis or graves ' disease.

33. Pic is b on p, where the autoimmune disease is an autoimmune disease of the Central nervous system selected from the group consisting of multiple sclerosis, myasthenia pregnant and encephalomyelitis.

34. The method according to p, where autoimmune disease are various forms of pemphigus selected from the group consisting of pemphigus vulgaris, pemphigus vegetans, pemphigus leaf syndrome Sideara-usher and Brazilian pemphigus.

35. The method according to p, where the autoimmune disease is psoriasis.

36. The method according to p, where the autoimmune disease is inflammatory bowel disease.

37. The method according to p, where the patient has acquired immunodeficiency syndrome (AIDS).

38. The method according to p where the patient received a transplant biological organ, tissue or cell.

39. The method according to p, where the patient has damage to the blood vessels.

40. The method according to p, where the patient has diabetes type II.

41. Method of elimination of cells that expresses the receptor for IL-15, the method includes exposing the cells to a therapeutic composition according to claim 1, containing the first agent whose target is IL-15R, and a second agent that inhibits co-stimulating signal transmitted between the cell of the immune system and antigen-presenting cell.

42. The method according to paragraph 41, where the cell before the hat is a cell of the immune system.

43. The method according to paragraph 41, where the cell is a malignant cell.

44. Method of diagnosing a patient having a disease or condition that can be treated with therapeutic composition according to claim 1, the method comprises obtaining a tissue sample from the patient and the exposure of the sample with the labeled antigen is a polypeptide whose target is IL-15R, and the presence of binding of the polypeptide with the cell in the sample indicates that the cell can be associated in vivo with the agent whose target is IL-15R, and thereby to inhibit proliferation in response to IL-15 wild-type in vivo.

45. Method of preparation of therapeutic compositions containing the mutant polypeptide of IL-15, which connects subunit of IL-15R, and a polypeptide that binds a molecule B7, the method includes

a) purification of the mutant polypeptide of IL-15 expressing system and

b) purification of the polypeptide which binds to B7, from expressing system;

c) combining the polypeptide of IL-15 and a polypeptide which binds to B7.



 

Same patents:

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to derivative of triazaspiro[5.5]undecane of the formula (I): wherein R1 means compound of the formula (1): or (2): wherein G represents a bond, (C1-C4)-alkylene, (C2-C4)-alkenylene or -CO-; ring A represents: (1) C5-10-membered mono- or bicarbocyclic ring or (2) 5-10-membered mono- or bicyclic heterocycle comprising 1-2 nitrogen atoms and/or 1-2 oxygen atoms; substitute R6 means the following values: (1) (C1-C4)-alkyl, (2) halogen atom, (3) nitrile group, (4) trifluoromethyl group and others; R2 represents: (1) (C1-C4)-alkyl, (2) (C2-C4)alkynyl or (3) (C1-C4)-alkyl substituted with a substitute represented in claim 1 of the invention claim; each R3 and R4 represents independently: (1) hydrogen atom, (2) (C1-C4)-alkyl or (3) (C1-C4)-alkyl substituted with 1-2 substituted taken among: (a) Cyc 2 and (b) hydroxy-group (wherein Cyc 2 represents (1) C5-6-membered monocarbocyclic ring or (2) 5-6-membered monocyclic heterocycle comprising 1-2 nitrogen atoms and/or one oxygen atom), or R3 and R4 form in common group of the formula: wherein R26 represents (C1-C4)-alkyl or Cyc 2; R5 represents hydrogen atom or (C1-C4)-alkyl, its quaternary ammonium salt, its N-oxide or its nontoxic salt. Also, invention relates to pharmaceutical composition inhibiting HIV, regulator of chemokine/chemokine receptor and agent used in treatment and prophylaxis of some diseases, such as inflammatory diseases, asthma, atopic dermatitis, nettle rash, allergic diseases, nephritis, hepatitis, arthritis and other diseases that comprise as an active component above described compound of the formula (I) or its quaternary ammonium salt, its N-oxide or its nontoxic salt. Also, invention relates to (3R)-1-butyl-2,5-dioxo-3-((1R)-1-hydroxy-1-cyclohexylmethyl)-9-(4-(4-carboxyphenyloxy)phenylmethyl)-1,4,9-triazaspiro[5.5]undecane or its pharmaceutically acceptable salt and pharmaceutical composition based on thereof, and to (3R)-1-butyl-2,5-dioxo-3-((1R)-1-hydroxy-1-cyclohexylmethyl)-9-(4-(4-carboxyphenyloxy)phenylmethyl)-1,4,9-triazaspiro[5.5]undecane hydrochloride and pharmaceutical composition based on thereof.

EFFECT: valuable medicinal properties of derivative and composition.

16 cl, 32 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to new derivatives of triazaspiro[5,5]undecane of the formula (I):

wherein values of radicals R1-R5 are given in the invention claim, ort o their quaternary ammonium salts, N-oxides or nontoxic salts. Proposed compounds possess inhibitory and regulating activity with respect to chemokine/chemokine receptors and can be useful in prophylaxis and treatment of different inflammatory diseases, such as asthma, atopic dermatitis, nettle rash, allergic diseases, nephritis, hepatitis, arthritis or proliferative arthritis and other similar diseases. Also, invention relates to pharmaceutical compositions based on compounds of the formula (I).

EFFECT: improved control method, valuable medicinal properties of compounds.

9 cl, 5 sch, 36 tbl, 70 ex

FIELD: organic chemistry, antibiotics, pharmacy.

SUBSTANCE: invention relates to a new crystalline nonsolvated form of 40-O-(2-hydroxy)-ethylrapamycin that shows crystalline lattice with the following parameters: a = 14.37Å; b = 11.24Å; c = 18.31 Å, and volume value is 2805Å. Also, invention relates to a method for preparing this crystalline form that involves crystallization of 40-O-(2-hydroxy)ethylrapamycin from a solvent with mixture with aliphatic hydrocarbon of the formula CnH2n+2 wherein n = 5, 6 or 7. Also, invention relates to a pharmaceutical composition based on thereof and its using in preparing medicinal agents used in treatment or prophylaxis of organ or tissue transplant rejection, autoimmune, inflammatory states, asthma, proliferative disorders, tumor or hyperproliferative vascular diseases. Invention provides preparing the novel crystalline nonsolvated form of 40-O-(2-hydroxy)ethylrapamycin possessing immunosuppressive properties.

EFFECT: improved preparing method, improved and valuable medicinal properties of compound and composition.

7 cl, 2 ex

FIELD: transplantology.

SUBSTANCE: invention discloses pharmaceutical compositions containing substance effective as modulators of biological activity "induced by activation of lymphocytic immunomodulating molecule (AILIM)" (known also as "induced common stimulator (ICOS)"), in particular modulating transduction of AILIM-mediated signal.

EFFECT: achieved suppression, treatment, or prevention of rejection of transplant arising in case of transplantation of organ, a part thereof or tissue.

11 cl, 7 dwg

FIELD: organic chemistry, pharmacy, biochemistry.

SUBSTANCE: invention relates to new substituted 2H-pyrano[2,3-c] of the general formula (1) eliciting ability to inhibit activity of protein kinase. In the general formula (1) X represents oxygen atom or group NR3; R1 represents group -C(O)R4, optionally substituted and optionally condensed azaheterocycle; R2 represents optionally substituted hydroxyl group or optionally substituted amino-group; R3 represents hydrogen atom or inert substitute meaning optionally substituted low- or non-reactive radical including such as (C1-C7)-alkyl, (C2-C7)-alkenyl, (C2-C7)-alkynyl, (C1-C7)-alkoxy-group, (C7-C12)-aralkyl, heterocyclylalkyl, (C7-C12)-alkaryl, (C3-C10)-cycloalkyl, (C3-C10)-cycloalkenyl, phenyl, aryl, (C2-C12)-alkoxyalkyl, (C2-C10)-alkylsulfinyl, (C2-C10)-alkylsulfonyl, -(CH2)-O-(C1-C7-alkyl), -(CH2)m-N(C1-C7-alkyl)n, aryloxyalkyl, heterocyclyl wherein m and n have value from 1 to 7; R4 represents optionally substituted amino-group or hydrogenated optionally substituted azaheterocycle. Also, invention relates to combinatory and focused libraries consisting of compounds of the present invention and designated for the search of compound-hits and compound-leaders prepared by screening of these libraries for using in preparing medicinal agents.

EFFECT: valuable medicinal properties of compounds.

8 cl, 2 tbl, 6 ex

FIELD: organic chemistry, vitamins, medicine, pharmacy.

SUBSTANCE: invention relates to a new compound of the formula (I): wherein X means hydrogen atom or hydroxy group; R1 and R2 that can be similar or different mean hydrogen atom, (C1-C4)-alkyl; R3 means hydrogen atom, methyl group, fluorine or chlorine atom. Also, invention relates to its esters able to hydrolysis in vivo in combination with pharmaceutically acceptable acids. Also, invention relates to a pharmaceutical composition eliciting the inhibitory activity with respect to proliferation and promoting differentiation of cells and comprising the effective dose of compound of the formula (I) in common with pharmaceutically acceptable carriers and/or excipients. Also, invention relates to applying compound of the formula (I) for preparing a medicine used in treatment and prophylaxis of disease characterizing by abnormal differentiation of cells and/or proliferation of cells.

EFFECT: valuable medicinal properties of compounds.

13 cl, 3 sch, 3 tbl, 6 ex

FIELD: medicine, transplantology.

SUBSTANCE: method involves applying (R)-ibuprofen methanesulfonamide and its nontoxic salts for preparing medicinal agents used for prophylaxis or treatment of ischemic, reprefusion and functional damages of transplanted organs. Invention provides prophylaxis such complications as delayed function of transplant arising in transplantation of organs.

EFFECT: valuable medicinal properties of medicinal agent.

5 cl, 5 tbl, 1 dwg, 2 ex

FIELD: medicine, infectious diseases.

SUBSTANCE: invention relates to a method for preparing capsule antigen of meliodosis pathogen eliciting anti-phagocytic activity. Method involves culturing the strain B. pseudomallei, inactivation, isolation of glycoprotein by gel-filtration and ion-exchange chromatography methods resulting to preparing glycoprotein with molecular mass 200 kDA consisting of 90% of carbohydrates, 10% of proteins and comprising 2.17% of nitrogen, 46.4% of carbon and 8.09% of hydrogen. Advantage of method involves preparing the purified glycoprotein eliciting anti-phagocytic activity.

EFFECT: improved method for preparing, valuable properties of antigen.

2 dwg, 5 ex

FIELD: medicine, therapy.

SUBSTANCE: invention relates to treatment of vasculitis and erythematosus lupus. Method involves administration of 15-deoxyspergualine or its analogous by two or more courses with break for 4 days to 5 weeks. Period between successive injections of preparation within the treatment course is up to 48 h and the treatment course is at least 5-7 days. Method provides high treatment effect and reducing the total dose of 15-deoxyspergualine or its analogous.

EFFECT: enhanced effectiveness of treatment.

25 cl, 2 dwg, 3 ex

Immunosupressor // 2253448

FIELD: drugs, medicine.

SUBSTANCE: invention relates to application of 1-methylindolyl-3-thioacetic acid tris-(2-hydroxyethyl)ammonia salt, which is known hypolipidemic agent, as immunosuppressor. Present invention ales it possible to produce pharmaceuticals for transplanted organ and tissue rejection prophylaxis or treatment of various immune-associated diseases.

EFFECT: new drug for transplant rejection prophylaxis or treatment of immune-associated diseases.

3 tbl, 3 ex

FIELD: medicine, infectious diseases, psychotherapy.

SUBSTANCE: method involves antiviral therapy, immune correction with thymus hormones and interferon inductors. Since the first day the relapse symptom method involves prescription of antiox+ (1 capsule per a day) and detox+ (1 capsule, 2 times per a day) for 30 days, profluzak (20 mg, 3 times per a day for 5 days) and then in the dose 20 mg, 1 time per a day for 20 days. Derinate is prescribed topically as installation into urethra in the dose 3-5 ml or with tampon into vagina and with simultaneous prescription of microenemas in the dose 10-40 ml for 10 days. Since 10-14 day in exacerbation period in the proliferative stage of an antiherpetic immune response derinate is prescribed by intramuscular injections in the dose 5 ml, 1 time in a day, 10 injections in total number. Then since 6-th day of exacerbation and intake of profluzak psychotherapy seances are carried out. The first seance of rational psychotherapy involves explanation to a patient in available form mechanism of the disease, the necessity of prolonged treatment and motivation for treatment is enhanced by suggestion. The second psychotherapy seance involves neurolinguistic programming wherein a patient colorful and detailed description of desirable function when he imagines achievement of the desire result, and positive emotional and vegetative symptoms are notes and the conditional-reflect association is formed by tactile contact. Under psychotherapist control a patient imagines "part of person" responsible for achievement of the desire result the patient attention is accented for the desire result and arisen physiological responses are fixed by using tactile contact. Also, new behavior methods are proposed to take for a patient that are directed for achievement of the desire result - avoiding sexual contacts during exacerbation of genital herpes in one of partner and during every month hormonal cycles, avoiding stress situations, and in case of each stress situation significant for patient profluzak has to be intake in a single dose 40 mg, using a condom in sexual contact in the exacerbation period. Patient analyzes the proposed new behavior methods that help avoiding relapses, provide good state of health, promotes to recovery process of genitals recovery and selects at least three the most rationally available for him behavior methods. In the case of the positive response that is controlled by physiological symptoms the result is fixed by tactile contact. The third seance involves the suggestive psychotherapy directed for fixing the attained result. The suggestive therapy seance is carried out once per a week for 6 months. Method provides declining the treatment time, to reduce relapse frequency of genital herpes and to recover the emotional state of patient.

EFFECT: improved treatment method.

2 cl, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves applying basic therapy until stroke type is unclear and differentiated therapy after having determined stroke type. Recombinant interleukine-2-roncoleukine as subcutaneous injections at a dose of 500 000 MU into external surface of intact arm at the fourth-fifth day after stroke beginning. Roncoleukine is administered as a course of 1-3 injections given with two-three days pause on the background of traditional stroke treatment.

EFFECT: enhanced effectiveness of treatment.

2 cl, 3 tbl

FIELD: medicine.

SUBSTANCE: method involves combining vaccine prepared from 107 cells of autologic tumor lysate with 60 mg of betaleukine and introducing it strictly in intracutaneous paravertebral mode in three points arranged 3 cm far from each other. The introduction takes place every 3 weeks so that the first two vaccinations are combined with 470 mg of betaleukine introduced into anterior abdominal wall. Vaccination treatment is continued on receiving positive retarded type hypersensitivity response to injection after every vaccination with tumor lysate without betaleukine.

EFFECT: enhanced effectiveness in inducing and supporting antitumor immune response.

FIELD: medicine, oncology.

SUBSTANCE: after removing malignant cerebral tumor one should conduct successive course of: cytokinotherapy consisting of 3 intramuscular injections of leukineferon at 48-h-long interval, adaptive immunotherapy with lymphokine-activated killer cells (LAKC) generated in the presence of recombinant interleukin-2 (IL-2). Moreover, LAKC should be introduced into the channel of removed tumor in combination with IL-2 as 2 procedures at 24-h-long interval. Then comes the course of adaptive immunotherapy with cytotoxic lymphocytes (CTL) generated due to cultivating patient's mononuclear blood cells together with dendritic cells loaded with a tumor antigen, in the presence of recombinant IL-2 to be introduced in combination with it into the channel of removed tumor as 2 procedures at 48-h-long interval. For obtaining dendritic cells in patients before operation it is necessary to isolate monocytes to cultivate with granulocytic-macrophageal colony-stimulating factor and interferon-α at maturating dendritic cells in the presence of monocytic conditioned medium and incubation of dendritic cells in the presence of tumor antigenic material for their loading with a tumor antigen. After immunotherapy with CTL on should conduct the course of vaccinotherapy with dendritic cells loaded with a tumor antigen in combination with subcutaneous injections of IL-2. The method enables to induce high specific anti-tumor immune response along with improved quality of life and prolonged duration of relapse-free period.

EFFECT: higher efficiency of immunotherapy.

2 cl, 2 ex

FIELD: medicine, gastroenterology, pharmacy.

SUBSTANCE: invention relates to agents used in treatment of ulcerous-erosion injures in gastroduodenal region. Method involves diluting 100 mcg of dry lyophilized powder of immunomodulating agent "Superlimf" in 3-5 ml of 0.9% isotonic solution and irrigation of ulcer or erosion with this solution 1 time per a day by the endoscopy method. The treatment course is 3-4 procedures with break for 4-5 days. Method provides alteration of cytokine pattern of tissues, induction of influx of mononuclear phagocytes to the injure focus that results to localization of inflammation and the complete epithelization of ulcers and erosions.

EFFECT: improved and effective method for treatment.

1 ex

FIELD: cardiovascular disorders.

SUBSTANCE: invention provides methods for modulating tissue antiogenesis using Raf and/or Ras protein, modified Raf and Ras proteins, nucleic acids encoding them. Antiogenesis is inhibited using inactive Raf or Ras proteins or nucleic acids encoding them, and antiogenesis is potentiated using active Raf or Ras proteins or nucleic acids encoding them. Use of gene transportation system to provide nucleic acids encoding Raf or Ras proteins or modified forms thereof.

EFFECT: enlarged choice of tissue antiogenesis control methods and means.

44 cl, 20 dwg

FIELD: medicine, immunology.

SUBSTANCE: method involves inhalation administration of combination of immunocorrecting agents wherein recombinant interleukin-2 is used or its combination with genetic engineering α2-interferon and with the complex immunoglobulin preparation in the daily doses for 5 days depending on age of patient. Before sanitation with an immunocorrecting agent the method involves assay of carrying type by anti-lysozyme activity of microorganisms (resident or transitory) and repeated examination after sanitation course is carried out. Carriers are subjected for additional sanitation with anti-bacterial preparations in change of carrying type from resident to transitory and with taking into account antibiotic-resistance property of the carrier strain. Method allows carrying out the effective sanitation and immune reablement of germ carriers due to recovery of the adequate natural resistance and complex of nonspecific factor of regional and systemic immunity.

EFFECT: improved and effective method for sanitation.

2 cl, 2 tbl, 5 ex

FIELD: medicine, phthisiology.

SUBSTANCE: method involves administration of roncoleukin by lymphotropic route: subcutaneously, in region of pretracheal cellular tissue in the equal dose 1/4-1/5 of the average daily therapeutic dose, once in one or two days, 3 injections for a course. Invention provides the local delivery of roncoleukin to the injure zone directly that promotes to making depot of the latter in the injure focus and to the rapid elimination of pathogen from body of patients. Invention can be used for correction of immune insufficiency in patients with pulmonary tuberculosis.

EFFECT: improved method for correction.

2 ex

The invention relates to medicine, more specifically to Oncology, and can find application in the treatment of colon cancer with liver metastases

The invention relates to medicine, namely to Pediatrics, neonatology and immunology, and can be used for the treatment of pneumonia in newborn infants

FIELD: medicine, phthisiology.

SUBSTANCE: method involves administration of roncoleukin by lymphotropic route: subcutaneously, in region of pretracheal cellular tissue in the equal dose 1/4-1/5 of the average daily therapeutic dose, once in one or two days, 3 injections for a course. Invention provides the local delivery of roncoleukin to the injure zone directly that promotes to making depot of the latter in the injure focus and to the rapid elimination of pathogen from body of patients. Invention can be used for correction of immune insufficiency in patients with pulmonary tuberculosis.

EFFECT: improved method for correction.

2 ex

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