Methods and compositions for improving effectiveness of antibody for medical application with using compounds potentiating nk-cells

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns methods and compositions for improving effectiveness of antibody for medical application with using the compounds potentiating NK-cells. Substance of the invention covers a method of treating a disease and a pharmaceutical composition for treating a disease brought on or exacerbated partially by the cells which can be considered as targets and which can be eliminated by antibody for medical application, involving introduction to a patient of the first antibody blocking an inhibiting NK-cell receptor chosen of group: KIR2DL1, KIR2DL2, KIR2DL3 and NKG2A and then introductions of antibody for medical application.

EFFECT: advantage of the invention consists in higher clinical effectiveness.

40 cl, 2 ex, 1 tbl, 4 dwg

 

The technical field to which the invention relates.

The present invention relates, generally, to methods and compositions for improving the efficiency of antibodies for therapeutic purposes. More specifically, the invention relates to the use of antibodies for therapeutic purposes in combination with a compound that blocks the inhibitory receptor or stimulates an activating receptor of natural killer cells (NK-cells), and through this creates the possibility of potentiation of cytotoxicity of natural killer cells in mammals, in order to improve the effectiveness of treatment of people, in particular through increased ADCC mechanism.

The level of technology

Various therapeutic strategies for the human body based on the use of antibodies for therapeutic purposes (therapeutic antibodies). These include, for example, the use of antibodies for therapeutic purposes, designed to eliminate target cells, in particular of the affected cells, such as cells infected with a virus, tumor cells or other pathogenic cells. Such antibodies typically are monoclonal antibodies, species IgG, typically, Fc-parts of the human IgGI or IgG3. Such antibodies can be native or recombinant antibodies and are often "humanitarianism mouse antibodies [i.e. containing functional areas of different types, ipino, Fc-part of human origin or derived from Primate, non-human, and the variable region of an antibody or a hypervariable region antigenspecific sites of antibodies that define their complementarity (CDR, complementary determining region of murine origin]. On the other hand, monoclonal antibodies can be fully human, obtained by immunization of transgenic mouse, having a locus of human Ig, or obtained from a cDNA library from human cells.

Specific examples of such antibodies for therapeutic purposes is rituximab (rituximab, Mabthera®, Rituxan®)is a chimeric monoclonal antibody against CD20, derived from human γ1 and constant regions (so Fc-area human IgGI), United with murine variable regions, giving a specificity of CD20, In the last few years rituximab significantly changed the treatment strategy against malignant lymphoproliferative conditions, in particular non-Hodgkin's lymphomas (NHL). Other examples of humanized IgGI antibodies are alemtuzumab (alemtuzumab, campath, Campath-1H®), which is used in the treatment of b-cell malignant conditions, and trastuzumab (trastuzumab, Herceptin®), which is used in the treatment of breast cancer. In the technique disclosed other examples of antibodies developed for therapeutic purposes.

The mechanism of action of antibodies for therapeutic purposes is still a subject for discussion. Injection of antibodies leads to the disappearance of cells containing the antigen specifically recognized by the antibody. This loss may be mediated, at least three mechanisms: mediated by antibodies cellular cytotoxicity (ADCC, antibody-dependent cellular cytotoxicity, complement-zavisimym the lysis of cells and direct antitumor inhibition of tumor growth via signals from antigen-directed antibodies.

Although these antibodies represent a new and effective approach to the treatment of people, in particular to the treatment of tumors, they do not always show a stable performance. For example, although it is shown that rituximab, alone or in combination with chemotherapy, is effective in the treatment of NHL as low, medium, and high, from 30% to 50% of patients with NHL low-grade does not have a clinical response to rituximab. It is assumed that the level of expression of CD20 on lymphoma cells, the presence of high tumor mass at the time of treatment or low concentrations of rituximab in serum may explain the lack of efficacy of rituximab for some patients. However, the actual cause of treatment failure remain largely unknown.

In addition, the use of antibodies for therapeutic alamout limited side effects, caused by their introduction. For example, patients may manifest such side effects as fever, headache, nausea, hypotension, stercorosus (noisy) respiration, hives, infection, and other side effects, potentially limiting the number or the frequency with which you can enter antibodies.

Thus, it can be quite exciting improving the efficiency of antibodies for therapeutic purposes or the ability to achieve therapeutic efficacy with the use of reduced doses of antibodies, which are less likely to cause side effects. The present invention is directed to these and other needs.

The invention

The present invention discloses new approaches to improving the efficiency of antibodies for therapeutic purposes. Not limited to the following theory, believe that the unexpected results achieved with the application of the methods of the present invention are due to the ability of antibodies for therapeutic purposes, when they are injected, to strengthen the mechanism of ADCC in vivo. Of course, the present invention relates to new compositions and methods that overcome the difficulties associated with the effectiveness of antibodies for therapeutic purposes. The present invention shows that NK-cells of an individual (natural killer cells, natural killer cells, which appear the t cytotoxicity towards cells of its own body) can have poor therapeutic mediated mAb (monoclonal antibody, monoclonal antibodies ADCC due to insufficient activation of NK-cells, for example, by inhibition of inhibitory receptors of NK-cells. Preferably increased ADCC mechanism is achieved by introducing compounds that block inhibiting receptor or stimulates an activating receptor on natural cells killers, and through this promoterwise potentiation of cytotoxicity of natural killer cells in mammals. Preferably the compound is an antibody or its fragment.

These antibodies or other compounds can interact, for example, with any abscopal receptors of NK-cells - molecules inhibitory receptor killer (KIR, killer inhibitory receptor, or NKG2A/C) or with activating receptors on NK cells, such as NCR, such as NKp30, NKp44, or NKp46, and through this counteracted the inhibition of the cells and increases the activity of ADCC.

More specifically, the invention relates to the treatment of the subject, in which connection, preferably antibodies or fragments that blocks the inhibitory receptor or stimulates an activating receptor of NK cells, is administered to a subject in conjunction with antibodies for therapeutic purposes. This description demonstrates that the effectiveness of antibodies for therapeutic purposes can significantly enhance joint introduction, for example,joint injections such compounds, preferably antibodies or their fragments, which overcomes the inhibition of NK-cells, for example, by blocking inhibitory receptor or stimulate an activating receptor of NK cells.

The invention also relates to pharmaceutical compositions containing the antibodies for therapeutic purposes and connection, preferably antibodies or fragments thereof that blocks the inhibitory receptor or stimulates an activating receptor of NK cells. The invention also relates to kits containing antibodies for therapeutic purposes and connection, preferably antibodies or fragments thereof that blocks the inhibitory receptor or stimulates an activating receptor of NK cells.

The invention also relates to the use of compounds, preferably antibodies or their fragments, blocking inhibitory receptor or stimulates an activating receptor of NK-cells, to increase the effectiveness of treatment with antibodies for therapeutic purposes or to enhance ADCC in the subject, which is treated with antibodies for therapeutic purposes.

The invention also relates to the use of compounds, preferably antibodies or their fragments, blocking inhibitory receptor or stimulates an activating receptor of NK-cells, and antibodies for therapeutic purposes to obtain drugs for the treatment of diseases. Conques is Etnea, for treatment of the disease requires the removal of target cells, preferably of infected cells, such as cells infected with a virus, tumor cells or other pathogenic cells. Preferably the disease is a cancer, infection or immunopathology. Preferably a disease selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, and viral diseases. The disease also include graft rejection, including allograft rejection and disease graft-versus-host (GVHD, graft versus host disease).

The present invention also relates to a method for reducing the dosage of the antibodies for therapeutic purposes, for example antibodies that bind Fcy-receptor, preferably CD 16 (FcyRIIIa). For example, co-administration of antibodies for therapeutic purposes, and compounds that block the inhibitory receptor or stimulates an activating receptor of NK-cells, allows the use of a lower dose of antibodies for therapeutic purposes. Such antibodies can be used in the dose component 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% the dose recommended in the absence of a connection, or in a lower dose.

In addition, the invention relates to a method of determining a therapeutically effective low doses of antibodies for therapeutic purposes, for example antibodies that bind CD 16 including the properly

i) co-incubation of antibodies for therapeutic purposes in the first concentration of the target cells and NK-cells and in the absence of compounds that block inhibiting receptor or stimulates an activating receptor of NK-cells;

ii) co-incubation of antibodies for therapeutic purposes in the second, lower concentrations with target cells, NK-cells and in the presence of compounds that block inhibiting receptor or stimulates an activating receptor of NK-cells;

iii) determining whether the disappearance of target cells observed at stage (ii), as significant as in stage (i).

If you see that stage (ii) is as effective as stage (i), then the relative concentrations of compounds and antibodies for therapeutic purposes can be modified and observe the disappearance of the target cells in order to identify the different conditions that may be suitable for application to the patient, for example maximizing the disappearance of the target cells, a reduced dose of antibodies for therapeutic purposes or a reduced dose of the compounds, depending on the specific needs of the patient.

In the private aspect of the present invention relates to a method of treating disease in humans, in need thereof, including

a) the introduction of a specified subject compounds that block the inhibitory receptor or with Kulibayeva activating receptor of NK-cells; and

b) the introduction of a specified subject antibodies for therapeutic purposes, which may contact CD 16.

In one embodiment antibodies for therapeutic purposes, and the compound is administered to the subject simultaneously. In another embodiment the compound is administered to the subject within one week, within 4 days, within 3 days or the same day (e.g., within about 24 hours) after administration of antibodies for therapeutic purposes. In another embodiment the disease is a cancer, infection, or immunopathology.

In one embodiment, the method also includes the additional step, which assess the activity or the number of NK cells before or after the introduction of the connection. In another embodiment an additional step includes

i) obtaining NK-cells from the subject prior to the introduction;

ii) incubation of NK-cells in the presence of one or more target cells, recognizable by antibodies for therapeutic purposes, in the presence or in the absence of the compound;

iii) evaluation of the effect of the compound on the ability of NK cells to eliminate target cells; where revealing the fact that the compound enhances the ability of NK cells to eliminate target cells indicates that the compound is suitable for use in the method and that the method is suitable for application to the subject.

In another aspect, the present invention relates to pharmaceutical compositions containing the antibodies for therapeutic purposes is her for example, who may contact CD 16, the connection blocking inhibiting receptor or stimulates an activating receptor of NK-cells, and a pharmaceutically acceptable carrier. In another aspect, the present invention relates to a kit containing antibodies for therapeutic purposes, for example, which may contact CD 16, and one or more compounds that block inhibiting receptor or stimulates an activating receptor of NK-cells.

For any of the above methods, compositions or kits in one embodiment antibodies for therapeutic purposes contain the Fc-part of human IgG1 or IgG3. In another embodiment the compound is an antibody or its fragment. In yet another embodiment of antibodies for therapeutic purposes are monoclonal antibodies or fragments thereof. In another embodiment of antibodies for therapeutic purposes are not conjugated with radioactive or toxic group. In another embodiment, the compound inhibits the inhibitory receptors of NK cells. In another embodiment, the compound stimulates an activating receptor of NK cells. In another embodiment the compound is a human, humanitariannet or chimeric antibody or its fragment. In one embodiment antibodies for therapeutic purposes or compounds may represent fragments or derivatives of antibodies, t is the cue as, among other things, Fab fragment, Fab'2 fragment, and CDRs of ScFv.

In one embodiment antibodies for therapeutic purposes are human, humanized or chimeric antibodies, or fragments thereof. In another embodiment of antibodies for therapeutic purposes are rituximab or campath (Campath). In another embodiment the antibodies are rituximab, and these antibodies is administered in a dosage of less than 375 mg/m2in the week. In another embodiment the antibody is campath, and the antibody is administered at a dosage of less than 90 mg per week.

In one embodiment, the compound binds at least one human NKG2 receptors, KIR2DL or KIR3DL and inhibits the corresponding mediated NKG2, KIR2DL or KIR3DL, inhibition of cytotoxicity of NK-cells. In another embodiment of the connection blocks the inhibitory receptor NK-cells selected from the group consisting of KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, and NKG2E LILRB5. In another embodiment the connection is associated with a common determinant of human KIR2DL receptors and inhibits KIR2DL-mediated inhibition of cytotoxicity of NK cells. In another embodiment the connection is associated with a common determinant of human receptors KIR2DL1, KIR2DL2, and KIR2DL3 and inhibits mediated KIR2DL1, KIR2DL2, and KIR2DL3 inhibition of cytotoxicity of NK cells. In another embodiment of compounds which inhibits their binding allelic variant molecules HLA-C, containing a Lys residue at position 80, with the human receptor KIR2DL1, and linking allelic variant molecules HLA-C, containing the Asn residue at position 80, with the human receptor KIR2DL2 and KIR2DL3. In another embodiment, the compound binds to the same epitope as monoclonal antibody DF200 produced by hybridoma DF200. In another embodiment, the compound competes with the monoclonal antibody DF200 produced by hybridoma DF200, for binding to the receptor KIR on the surface of human NK cells. In another embodiment the compound is a monoclonal antibody DF200 produced by hybridoma DF200, or its fragment.

In one embodiment, the compound binds to a receptor selected from the group consisting of NKp30, NKp44, NKp46 and NKG2D. In another embodiment of the connection comes from, or competes with the monoclonal antibodies selected from the group consisting of AZ20, A76, Z25, Z231 and WAV.

In another aspect, the present invention relates to a method of screening compounds for administration in combination with antibodies for therapeutic purposes, and specified the method includes

i) providing a test compound, inhibiting inhibitory receptor or stimulates an activating receptor of NK-cells;

ii) incubation of antibodies for therapeutic purposes with target cells specifically recognized by the antibodies for therapeutic purposes, the presence of NK-cells and in the presence or in the absence of the test compound; and

iii) evaluation of the activity of a compound on the ability of NK cells to eliminate target cells; where the discovery that the compound enhances the ability of NK cells to eliminate target cells indicates that the compound is suitable for use in the method.

In one embodiment the compound enhances the ability of antibodies for therapeutic purposes to destroy target cells in 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more. In another embodiment, the compound is chosen from the group consisting of antibodies, fragments of antibodies, monoclonal antibodies, fragment of a monoclonal antibody gumanitarnogo antibodies, chimeric antibodies and human antibodies. In another embodiment, the target cells are cancer cells, cells infected with a virus, or cells, supporting an autoimmune disorder. In another embodiment the antibodies for therapeutic purposes are rituximab or compat.

In another aspect, the present invention relates to a method of increasing the efficacy of treatment, including the introduction of the subject antibodies for therapeutic purposes, which may contact CD 16, and this method includes the introduction of a specified subject before, concurrently or after administration of the indicated antibodies for therapeutic purposes, a therapeutically effective amount of a compound that blocks the inhibitory receptor or stimulates advance the respective receptor NK-cells. In one embodiment, the compound increases the effectiveness of the treatment, enhancing ADCC in the specified entity.

Brief description of figures

Figure 1. Monoclonal antibody DF200 associated with a common determinant of the various human KIR2DL receptors.

Figure 2. Recovery lysis mAb (monoclonal antibodies) against KIR2DL on the target C1R Cw4 when the ratio of the effector/target 4/1. Monoclonal antibody DF200 inhibit KIR2DL-mediated inhibition of cytotoxicity KIR2DL1-positive NK cells (restored lysis) on Suu-positive target cells.

Figure 3. Mediated Rituxan enhancing ADCC KIR2DL1-positive NK clones on w4-positive cell lines EBV by blocking the interaction of KIR/HLA. The cytolysis NK clone containing KIR2DL1, tested against w4-positive EBV transformed (D20-positive) cell lines targeted at different ratios of effector/target (1 to 4) in the presence of 5 μg/ml of antibodies against CD20 (Rituxan) and 10 μg/ml of antibody EU (anti-KIR2DL1); one Rituxan; some EV or without any antibody. ADCC is greatly enhanced in the presence of antibodies against KIR2DL1 (EV).

Figure 4. Mediated Campton enhancing ADCC KIR2DL1-positive NK clones on w4-positive cell lines EBV by blocking the interaction of KIR/HLA. The cytolysis NK clone containing KIR2DL1, tested about the Yves w4-positive EBV transformed (CD20-positive) cell line target in the presence of Kampala and 100 μg/ml of antibody EU (anti-KIR2DL1); one Kanata; some EV or without any antibody. ADCC is greatly enhanced in the presence of antibodies against KIR2DL1 (EV).

Detailed description of the invention

The present invention relates to a method for improving the efficiency of antibodies for therapeutic purposes. More specifically, the invention discloses that the use of compounds, preferably antibodies or their fragments, potentiate NK-cells, preferably by blocking inhibitory receptor or activation of an activating receptor of NK-cells, can significantly improve the efficiency of antibodies for therapeutic purposes by co-injection of antibodies directed against NK-cell receptor, for example inhibition of the receptor.

Consequently, the invention relates to a method of treating disease in a subject in need of such treatment, including

a) the introduction of a specified subject compounds, preferably antibodies or their fragments, blocking inhibitory receptor or stimulates an activating receptor of NK-cells; and

b) the introduction of a specified subject antibodies for therapeutic purposes.

These antibodies for therapeutic applications can connect CD 16 NK-cells, preferably via Fc-plot.

Preferably these antibodies for therapeutic purposes contain the Fc-part of human IgG1 or IgG3, in particular monoclonal is ntotila or fragments thereof, also preferably humanized, human or chimeric antibodies, or fragments thereof, such as rituximab.

It is understood that the compounds, preferably antibodies or fragments thereof that blocks the inhibitory receptor NK-cells, you can enter the subject before, concurrently or after administration of antibodies for therapeutic applications. The method of introduction of different antibodies depends on their bioavailability and pharmacokinetics. Preferably antibodies for therapeutic purposes is administered within a week on the introduction of compounds, preferably antibodies or their fragments, blocking inhibitory receptors of NK-cells, preferably within a period of 5 or 2 days. Preferably antibodies for therapeutic purposes are administered before or simultaneously with the compounds, preferably antibodies or their fragments, blocking inhibitory receptors of NK cells.

In another aspect the invention relates to a method for enhancing ADCC for a subject receiving treatment with antibodies for therapeutic purposes, and this method includes the introduction of a specified subject before, concurrently or after administration of the indicated antibodies for therapeutic purposes is sufficient to enhance ADCC number of compounds, preferably antibodies or their fragments, blocking inhibitory receptors of NK cells. These antibodies for therapeutic purposes can masivatsa CD 16 NK-cells, preferably via Fc-plot. Preferably these antibodies for therapeutic purposes contain the Fc-part of human IgG1 or IgG3, in particular monoclonal antibodies, or fragments thereof, also preferably humanized, human or chimeric antibodies, or fragments thereof, such as rituximab.

In another aspect, the invention relates to a method of increasing the effectiveness of treatment of the subject antibodies for therapeutic purposes, and this method includes the introduction of a specified subject before, concurrently or after administration of the indicated antibodies for therapeutic purposes is sufficient to increase the effectiveness of these antibodies for therapeutic purposes, the number of compounds, preferably antibodies or their fragments, blocking inhibitory receptors of NK cells. These antibodies for therapeutic purposes may contact CD 16NK-cells, preferably via Fc-plot. Preferably these antibodies for therapeutic purposes contain the Fc-part of human IgG1 or IgG3, in particular monoclonal antibodies, or fragments thereof, also preferably humanized, human or chimeric antibodies, or fragments thereof, for example, rituximab.

Definition

Used in this description, the following terms shall have ascribed to them the values, unless otherwise indicated.

Used in this description, the term"NK"cells refers to a subpopulation of lymphocytes, which is involved in non-standard immunity. NK Cells can be identified on the basis of some characteristics and biological properties, such as the expression of specific surface antigens, including CD 16, CD56 and/or CD57, no alpha/beta or gamma/Delta TCR complex on the cell surface, the ability to bind to and kill cells that fail to Express "self" antigens to the MHC/HLA, by activating specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells expressing a ligand for the activating NK receptors, and the ability to release protein molecules called cytokines, which stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells using methods well known in the art.

The term "antibody", as used herein, refers to polyclonal and monoclonal antibodies. Depending on the type of the constant region in the heavy chain of an antibody refers to one of the five major classes: IgA, IgD, IgE, IgG and IgM. Some of them are also divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. an Example of structural units of immunoglobulins (antibodies) includes the tetramer. Each tetramer is composed of two and antichnyh pairs of polypeptide chains, each pair has one "light" (L, light, about 25 KD) and one "heavy" (H, heavy, about 50-70 KD) circuit. N-end of each chain defines a variable region at about 100-110 or more amino acids, which is primarily responsible for recognizing antigens. The term "variable region light chain (VL) and a variable region heavy chain (VH) refer to these light and heavy chains, respectively. Constant region heavy chains, corresponding to different classes of immunoglobulins are called "alpha", "Delta", "Epsilon", "gamma" and "mu", respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known. IgG and/or IgM are preferred classes of antibodies used in this invention, and IgG are especially preferred because they are the most common antibodies in the physiological situation, because they are easier to obtain in the laboratory, and because IgG is specifically recognized by Fc-gamma receptors. Preferably the antibodies of the present invention are monoclonal antibodies.

Especially preferred are humanized, chimeric, human or other suitable human antibody.

In the context of this invention the term "antibody or antibodies for therapeutic purposes" (therapetic antibody is specifically, for any antibodies that function as eliminating the patient's target cells. In particular, antibodies for therapeutic purposes, specifically bind to the antigens present on the surface of target cells, such as tumor-specific antigens that are present predominantly or exclusively in the tumor cells. Preferably antibodies for therapeutic purposes include human Fc-plots or capable of interacting with human Fc receptors. Antibodies for therapeutic purposes can mark cells by any means, such as ADCC or otherwise, and may be "naked", i.e. without conjugated particles, or may be conjugated with such compounds as radioactive labels or toxins.

The term "specifically bind" means that the antibodies can be contacted, preferably in competitive binding analysis with a binding partner, for instance an activating NK receptor, such as NKp30, NKp44, or NKp46, or human Fc-gamma receptor, when evaluating the use or recombinant forms of the proteins, epitopes, or native proteins present on the surface of isolated NK or relevant target cells. Competitive-binding assays, and other methods are also described below and is well known in the technique.

Antibody, "suitable for human-suitable) is any antibody, derivational antibody or antibody fragment that can be safely applied to humans in the case of, for example, methods of treatment described in this description. Antibodies suitable for a person, include all types of humanized, chimeric or fully human antibody or antibody, in which at least part of the antibody is derived from human or otherwise modified in order to achieve an immune reaction that is called when the use of native, non-human antibodies.

The term "immunogenic fragment" in this description refers to any polypeptide or peptide fragment that is able to detect an immune response, such as

i) an antibody that binds the specified fragment and/or linking to any form of molecules containing the specified portion, including membrane-bound receptor and going away mutants,

ii) stimulation of T-cell response involving T-cells in the interaction with the bimolecular complex, containing any MHC molecule and a peptide formed from the specified fragment

iii) binding of transfected media, such as bacteriophages or bacteria expressing the genes encoding the immunoglobulins mammals.

On the other hand, the term "immunogenic fragment" also refers to any structure capable of yablet immune response, described above, such as a peptide fragment, conjugated to protein carrier covalent bond, chimeric recombinant polypeptide construct that contains the specified peptide fragment in its amino acid sequence, and specifically includes cells transfected with a cDNA sequence which contains a section that encodes the specified fragment.

For the purposes of the present invention the determination of antibodies as humanitariannet" refers to an antibody in which the constant region and a frame area variable regions of one or more human immunoglobulin fused with the binding site, for example, a CDR of an immunoglobulin of the animal. Such humanized antibodies are creating in order to retain the binding specificity of antibodies, non-human, from which there are binding sites, but avoid the immune response against the antibody, which is not human.

"Chimeric antibody" is an antibody molecule in which

a) the constant region or part thereof altered, replaced or exchanged so that the binding site of the antigen (variable region) is linked to a constant region of a molecule of a different or altered class, effector function, or species, or an entirely different molecule which adds new properties x the measuring antibody for example, an enzyme, toxin, hormone, growth factor, lekarstvennym tool etc.; or

b) variable region or part thereof altered, replaced or exchanged with a variable region with a different or altered antigen specificity. In preferred embodiments of the present invention chimeric antibodies, however, retain the Fc-part of an immunoglobulin, preferably a human Fc-plot, what is possible due to interaction with a human Fc-receptors on the surface of target cells.

In the context of this invention, the terms "potentsiirovannye", "active" or "activated" NK-cells represent biologically active NK-cells, specifically NK cells are able to lyse target cells. For example, "active" NK cells are able to kill cells that Express ligands for activating NK receptors and unable to Express "self" antigens to the MHC/HLA (KIR-incompatible cells). Examples of suitable target cells for use in the retargeted killing-analyses (redirected killing assays) are cells R and C, but you can use any of the cells from several types of cells, which are well known in the art (see, for example, Sivori et al. (1997), J. Exp. Med., 186:1129-1136; Vitale et al. (1998), J. Exp. Med., 187:2065-2072; Pessino et al. (1998), J. Exp. Med., 188:953-960; Neri et al. (2001), Clin. Diag. Lab. Immun., 8:1131-1135). "Potentsiirovannye", "active" or "activated"cells can also be identified on any property or activity, known in the art as associated with NK activity, such as production of cytokines (such as IFN-γ and TNF-α) or increased levels of free intracellular calcium. For the purposes of the present invention, the terms "potentsiirovannye", "active" or "activated" NK-cells are definitely not for NK cells in vivo, which are not suppressed by stimulation of inhibitory receptor or in which such suppression is overcome, for example, through the stimulation of the activating receptor.

Used in this description, the term "NK-activating receptor" refers to any molecule on the surface of NK cells that, when stimulated, causes a measurable gain any properties or activities which are known in the art as associated with NK activity, such as production of cytokines (such as IFN-γ and TNF-α), increased levels of free intracellular calcium, the ability to bind to target cells in the retargeted killing-analysis, described, for example, elsewhere in this description, or the ability to stimulate the proliferation of NK-cells. The term "KIR-activating receptor" includes, but is not limited to, NKp30, NKp44, NKp46, NKG2D, IL-12R, IL-15R, IL-18R and IL-21R. The term "NK-activating receptor", as used herein, excludes the receptor for IL-2 (IL-2R). How to determine whether NK cell active or p is filerules or is not, described in more detail below and is well known to specialists in this field of technology.

Used in this description, the term "inhibiting" or "inhibitory NK receptor" refers to any molecule on the surface of NK cells that, when stimulated, causes a measurable weakening of any of the properties or activities which are known in the art as associated with NK activity, such as production of cytokines (such as IFN-γ and TNF-α), increased levels of free intracellular calcium or the ability to bind to target cells in the retargeted killing-analysis, described, for example, elsewhere in this description. Examples of such receptors are KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, and NKG2E LILRB5. How to determine whether NK cell is active or not is described in more detail below and is well known to specialists in this field of technology.

In the present invention, the term "blocking inhibitory receptor or stimulate an activating receptor of NK cells" refers to the ability of some compounds, preferably antibodies, their fragments or derivatives, preferably directly interact with at least one inhibiting or activating receptor of NK cells, such as KIR, NKG2A/C, NKp30, NKp44, NKp46, and others listed in this about what Isani, and/or to neutralize suppressing signals of the receptor (in the case of inhibitory receptors or to stimulate the receptor signals (in the case of activating receptors). In the case of inhibitory receptors, preferably the compound, preferably antibodies or fragments thereof, capable of blocking the interaction between HLA and receptor.

When the compound is an antibody, they can be polyclonal or preferably monoclonal. They may be produced by hybridomas or recombinant cells created for the expression of the desired variable and constant regions. The antibody can be a single-chain antibodies or other derivatives of antibodies that retain antigen-specificity and lower hinge section or fragment, such as Fab fragment, Fab'2 fragment, and CDRs of ScFv. They can be a multifunctional antibodies, recombinant antibodies, humanized antibodies, or their variants.

In the context of this invention, the term "common determinant" refers to a determinant or epitope, which is the part of several members of a group of related receptors, such as group human KIR2DL receptors. Determinant or epitope can present peptide fragment or conformational epitope, which is the part of members of the specified group. In a specific embodiment of the General determinant contains the epitope recognized by the monoclonal antibody DF200, NKVSF1 or EB6.

In the context of this invention the term "antibody that binds to a common determinant"refers to an antibody that binds to a specified determinant of the specificity and/or affinity, for example, which essentially does not bind with high affinity or specificity with other unrelated motifs or determinate or structures on the surface of human NK cells. In the more particular case of the binding of monoclonal antibodies according to this invention with the above determinant can be distinguished by the binding of these antibodies with other epitope or determinant.

Compounds, preferably antibodies, the ability to communicate with any abscopal NK receptors and prevent their stimulation, are thus "neutralizing" or "any abscopal" compounds, preferably antibodies, in the sense that they block at least partially inhibiting the signaling cascade mediated by any abscopal receptors of NK-cells, i.e. receptors KIR or NKG2A/C, so that such compounds, preferably antibodies, can be used for various subjects with high efficiency.

The term "recombinant"when used in relation to, for example, cells or nucleic acid, protein or vector, in which it shows, that the cell, nucleic acid, protein or vector modified by introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from cells modified in this way. For example, recombinant cells Express genes that are not found in native (not recombinant) form of the cell or Express native genes that are otherwise abnormally expressed, expressed insufficiently or not at all expressed.

In the context of the present invention by the subject or patient can be any mammal, preferably human.

Antibodies for therapeutic purposes

The present invention relates to the use of compounds that potentiate NK-cells, in combination with antibodies for therapeutic purposes. In the present invention can be any of a large variety of antibodies for therapeutic purposes. Essentially, you can use any of the antibodies for therapeutic purposes, whether they are naked or conjugated with radioactive labels, toxins or other groups, whether they are full-length or fragments, or whether they are true antibody or a modified derivative of the antibody. Preferably the methods they use to increase the effectiveness of treatment, which plays the role of the asset is ity of NK-cells without exception, the effectiveness of the injected antibodies for therapeutic purposes, and also preferably antibodies or fragments will include naturally, or will be modified to incorporate human Fc-area or another area, which enables detection of antibodies to human Fc receptors, such as Fc-gamma receptors.

Compounds of the present invention can be applied to enhance the ability of antibodies for therapeutic purposes to eliminate target cells expressing an antigen that is specifically recognized by the antibodies for therapeutic purposes. Accordingly, any disease or condition which is caused or exacerbated, at least partially cells, which can be targets for antibodies for therapeutic purposes, can be treated using the methods described in this description. Specific examples of target cells are tumor cells, cells infected with the virus, allogeneic cells, pathological immune cells (such as b-lymphocytes, T-lymphocytes, antigen-presenting cells and so on)involved in allergic reactions, autoimmune disease, allogeneic reactions and the like, or even healthy cells (e.g. endothelial cells in angiogenic therapeutic strategies). The most preferred target cells in the context of this izobreteniya tumor cells and cells infected with the virus. Antibodies for therapeutic purposes, for example, to mediate cytotoxic effect or lysis of the cells, in particular mediated by antibodies cell-mediated cytotoxicity (ADCC).

For ADCC requires leukocyte receptors for the Fc-region of IgG (FcγR), the function of which is communication-sensitive IgG antigens to cytotoxic cells containing FcγR, and the launching mechanism of cell activation. Therefore, antibodies for therapeutic purposes, capable of forming an immune complex. For example, the immune complex may represent a tumor-target coated with antibodies for therapeutic purposes. Specifically, antibodies may contact CD 16 preferably via their Fc-plots. Determining, contact Lee antibodies for therapeutic purposes with Fcγ-receptor, such as CD 16, can be accomplished in any suitable way, for example, determining the binding of the obtained recombinant polypeptide CD 16 or its fragment, optionally immobilized on a carrier, or, for example, determining the binding of antibodies for therapeutic purposes with cells known or expected that they Express CD 16.

Antibodies for therapeutic purposes can be polyclonal or preferably monoclonal. They may be produced by hybridomas or recombinant cells created for the expression of the desired variabeln the x and constant regions. The antibody can be a single-chain antibodies or other derivatives of antibodies that retain antigen-specificity and lower hinge area or its fragment. They can be multifunctional antibodies, recombinant antibodies, humanitarianism antibodies, fragments or variants. The specified fragment or derivative is chosen preferably from among the Fab fragment, Fab'2 fragment, and CDRs of ScFv. Preferably the fragment is an antigen-binding fragment. Antibodies for therapeutic purposes, including fragments of antibodies, can also include bespecifically antibodies, and another; one example of a suitable bespecifically antibody contains antigennegative site-specific CD 16, and antigennegative site specific for a tumor antigen. Other formats of antibodies containing fragments include recombinant derivatives bespecifically antibody combining sites linking two different antibodies in a single polypeptide chain, also called BiTE™ [Kufer P. et al., TRENDS in Biotechnology, 2004; 22(5):238-234; and Baeuerle et al., Current Opinion in Molecular Therapeutics, 2003; 5(4): 413-419, the work included in this description as the links].

Antibodies for therapeutic purposes, as a rule, are specific for surface antigens, such as membrane antigens. The most preferred antibodies of the La therapeutic purposes are antibodies, specific for tumor antigens (e.g., molecules specifically expressed by tumor cells, such as CD20, CD52, ErbB2 (or HER2/Neu), CD33, CD22, CD25, MUC-1, CEA, KDR, αVβ3, etc., in particular antigens lymphomas (e.g., CD20). Antibodies for therapeutic purposes contain preferably the Fc-part of human IgG1 or IgG3, or a Primate, non-human, preferably human IgG1.

In one embodiment the antibodies will include modifications in its Fc-plot, strengthening the interaction of antibodies with NK-cells during ADCC. Such modified antibodies for therapeutic purposes ("modified antibody"), as a rule, contain the modification preferably in the Fc section, altering the binding affinity of antibodies to one or more FcγR. Methods of modification of the antibodies with modified binding to one or more FcγR known in the art, see, for example, PCT publication no WO 2004/016750 (application for international patent PCT/US2003/025399), WO 99/158572, WO 99/151642, WO 98/123289, WO 89/107142, WO 88/107089 and U.S. patent No. 5843597 and 5642821 included in this description as a reference.

Antibodies for therapeutic purposes identified in this description, such as D2E7 [Cambridge Antibody Technology Group, pie (Cambridge, UK)/BASF (Ludwigshafen, Germany)], used for the treatment of rheumatoid arthritis, or infliximab (infliximab, Centocor, Inc., Malvem, PA; used to treat Crohn's disease and rheumatoi the aqueous arthritis), or antibodies disclosed in the application for international patent PCT/US2003/025399 (included in this description by reference), can be modified as indicated in the above and below the applications and use for the treatment of diseases, for which the typical use of such antibodies. In some embodiments the invention relates to altered antibodies with altered affinity or higher or lower affinity, with respect to an activating FcγR, e.g FcγRIII. Some preferred embodiments include modified antibodies with higher affinity in relation to FcγR. Preferably such modifications also have an altered Fc-mediated effector function.

Modifications that affect Fc-mediated effector function, well known in the art (see, for example, US 6194351 included in this description by reference). The amino acids that can be modified include Proline 329, Proline 331, lysine 322 and others. Proline 329 and/or 331, and lysine 322 can preferably be replaced by alanine, but also consider any other amino acid. Cm. International publication number WO 00/142072 and US 61945351 included in this description as a reference.

Thus, modification of the Fc-phase may consist of one or more amino acid changes located in the Fc-site antibodies. Such change is possible can lead to the production of antibodies with altered antibody-mediated effector function, with altered binding to other Fc receptors (e.g., activating Fc-receptors), with an altered ADCC activity, with altered activity Clq binding, with a modified complement-dependent cytotoxic activity, or any combination thereof.

In one embodiment, the antibodies specifically recognized by Fc-gamma receptor, such as FCGR3A [also called CD 16, FcγRIIIa, FCGR3, Fc receptor III immunoglobulin G; IGFR3, receptor for Fc fragment of IgG, low affinity IIIa (Low Affinity IIIa); see, e.g., OMIM 146740], FCGR2A (also known as CD32, CDw32, receptor for Fc fragment of IgG, low affinity IIA; FGG2, Fc-receptor II immunoglobulin G; see, e.g., OMIM 146740); FCGR2B (also known as CD32 receptor for Fc fragment of IgG, low affinity IIb; RS-gamma-KPV FCGR2B; see, e.g., OMIM 604590); FCG1RA [also known as CD64; receptor for Fc fragment of IgG, high affinity la (High Affinity Ia); IGFR1; see, e.g., OMIM 146740]; FCGR1 fragment of IgG (also called highly affine 1C, Fc-receptor IC immunoglobulin G, see, e.g., OMIM 601503); or FCGR1 (also known as CD64, receptor for Fc fragment of IgG, high affinity Ib; Fc-receptor IB immunoglobulin G; IGFRB; see, e.g., OMIM 601502).

Typical examples of antibodies for therapeutic purposes of this invention are rituximab, alemtuzumab and trastuzumab. Such antibodies can be used according to the clinical protocols approved for use for l is dei. Other specific examples of antibodies for therapeutic purposes are, for example, epratuzumab, basiliximab, daclizumab, cetuximab, labetuzumab, severums, taurima, palivizumab, infliximab, omalizumab, efalizumab, clenoliximab etc. is not necessary when the connection that stimulates an activating receptor of NK-cells, is a cytokine, antibodies for therapeutic use are different than rituximab or Herceptin, or, optionally, other than antibodies against CD20 or against HER2/neu. Other examples of preferred antibodies for therapeutic purposes according to the invention include anti-ferritine antibodies (publication of U.S. patent No. 2002/0106324), antibodies against P140 and anti sc5 pack (WO 02/50122) and antibodies against KIR (killer-inhibitory receptor) (KIR receptors are described in Camngton and Norman, The KIR Gene Cluster, May 3, 2003, available at http://www.ncbi.nlm.nih.gov/books), all of these works are included in this description as a reference. Other examples of antibodies for therapeutic purposes are listed in the table below, any of which (and others) can be used in the methods of the present invention. It should be borne in mind that, despite the fact that lists whether antibodies in the following table or not, or described elsewhere in this description, any antibodies that can eliminate target cells, preferably through ADCC can be pleased with what passed in the methods of the present invention, and that the following table 1 is not exhaustive nor in relation to enumerated antibodies, or in relation to targets or testimony for the listed antibodies.

Table 1
Antibodies for therapeutic purposes
The specificity of the ABDCICommercial nameTypical indications
Against CD20rituximabMabThera®, Rituxan®NHLB
Against CD20of zevalinNHL
Against CD20bessacarNHL
Against CD52alemtuzumabcampath-1H®CLL, allograft
Against CD33SMART M195AML
Against CD33Tamil™ Acute myeloid leukemia
Against antigen HLA-DRSMART ID 10NHL
Against HLA-DRErmitage™NHLB
Against CD22epratuzumabLymphoCide™NHLB
Against HER2MDX-210Prostate cancer and other cancers
Against erbB2 (HER-2/neu)trastuzumabHerceptin®Metastatic breast cancer
Against CA 125OvaRaxOvarian cancer
Against MUCITriAbMetastatic breast cancer
Against MUCIBravaRexMetastatic cancer disease
Against antigen REMtaragin, TerexOvarian cancer, breast cancer
Against CD44belarusembHead and neck cancer
Against gp72Mab, idiotypical 105AD7Colorectal cancer
Against ArcamAgainst Ersam; NTIS-IL2Cancer

Against VEGFMab-VEGFMetastatic NSCLC, colorectal cancer
Against CD 18AMD FabAge-related macular degeneration
Against CD 18Against CD 18Myocardial infarction
Against VEGF receptorIMC-lclIColorect the local cancer
Against nuC242nuC242 DMI-Cancer colorectal, stomach and pancreas
Against EGFRMab425Cancer
Against EGFRABX-EGFCancer
Against EGFR (HER-1, erbB1)cetuximabCancer ENT and colorectal
Against MUC-1Terex®Breast cancer and epithelium
Against CEACEAVacColorectal cancer
Against CEAlabetuzumabCEA-Cide™Solid tumor
Against αVβ3vitaxinLeiomyosarcoma, colorectal cancer and other cancers
Against KDR (VEGFR2)Cancer (antiangiogenic)
Against the fused protein VRSpalivizumabsynagis®Viral diseases
Toonumax™Too
CMVseverumprotoverInfection with CMV
HBstobiramaOctaver™Hepatitis b
Against CD25basiliximabsimulect®Prevention/treatment of allograft rejection
Against CD25daclizumabZenapax®Prevention/treatment of allograft rejection
Against TNF-αinfliximabRemicade™Crohn's disease, reumatoide the arthritis
Against CD80IDEC-114Psoriasis
Against IgEF-26Allergic asthma and rhinitis
Against IgEomalizumabXolair™Asthma
Against IgERhu-mAb E25Allergic/asthma
Against integrin αL(CD11a, LFA-1)efalizumabxanelim™Psoriasis
Against integrin beta-2LDP-01Shock, allograft rejection

Against integrin αL(CD11a, LFA-1)against CD 11Psoriasis
Against CD4keliximab, siplizumab, MEDI-507GVHD, psoriasis
Against CD4ACTARejection of the allograft
Against CD3ACTSRejection of the allograft
Against CD3SMART-aCD3Autoimmune disease, allograft rejection, psoriasis
Against CD64Anemia
Against CD 147GvHD
Against integrin α4(α4β1-α4β7)natalizumabantegren®Multiple sclerosis, Crohn's disease
Against integrin P7Crohn's disease, ulcerative colitis
Alpha-4-beta-7LDP-02Ulcerative colitis/td>
Against HLA-DR10ancoliNHL
Against CD3nuvionT-cell malignancy
Against ganglioside GD2trigemMetastatic melanoma and small cell lung cancer
Against antigen SK-1Colorectal and pancreatic carcinoma
Against CD4*clenoliximab
Against IL-8ABX-IL8Psoriasis
Against VLA-4antegrenMS
Against CD40LantovaSLE, allograft rejection
Against CD40LMS,SLE
Against E-selectinCDP850Psoriasis
Against CD11/CD18Hu23F2GMS, kick
Against ICAM-3ICM3Psoriasis
Against CBLABX-CBLGVHD
Against CD 147
Against CD23IDEC-152Asthma, allergies
Against CD25simulectRejection of the allograft
Against T1-ACSACS-110Breast cancer
Against TTSTTS-CD2 Cancer of the pancreas, kidneys

Against TAG72AR54Breast cancer, ovarian cancer, lung
Against SAGivaRexCancer colorectal, pancreas, stomach
Against PSAProstaRexProstate cancer
Against HMFG1R1550Breast cancer, stomach
pemtumomabtaraginStomach cancer, ovarian
Against hCGP-16, P-21Multiple cancer disease
Against collagen types 1-VHU 177; HUIVI26; XL313Multiple cancer disease
Against CD46 Cmcell/J&JMultiple cancer disease
Against 17A-1edrecolomabpanorexColorectal cancer
Against NMAMNMultiple myeloma
Against CD38Against CD38Multiple myeloma
Against the IL15 receptorNimah-lymphomaLymphoma
Against IL6In-E8Lymphoma
Against TRAIL-R1TRM-1Multiple cancer disease
Against VEGF2Multiple cancer disease
Against BlySlymphostat
Against SCLC, CEA and DpanaceaLung cancer
Against CD52compatLeukemia, lymphoma
Against antigen Lewis YIGN311Cancer epithelium
Against cadherin VEE4G10Multiple cancer disease
Against CD56W, huN901DC1Cancer colorectal, lung
Against mertansine/mucincantuzumabCancer colorectal, lung, pancreas
Against AFPAFP-cideLiver cancer
Against CSApMu-9Colorectal cancer
Against the CD30 MDX-060Melanoma, Hodgkin's disease
Against PSMAMDX-070Prostate cancer

Against CD 15MDX-11Leukemia
Against TAG72MDX-020Colorectal cancer
Against biospecifics-
who CD19, CD3
MTLymphoma
Against antigen mesothelinSS1-PE38Brain cancer and ovarian cancer, mesothelioma
Against DNA and histonesKotarCancer colorectal, pancreatic, sarcoma, brain cancer and other cancers
Against integrin AVagainst A5 B1 Multiple cancer disease
Against RSGN17/19Melanoma
Against CD5genemonLeukemia, lymphoma

Compounds that regulate the activity of NK-cells

The activity of NK cells is regulated by a complex mechanism involving both stimulating and suppressing signals. Accordingly, effective mediated NK cell treatment can be achieved by stimulation of these cells or neutralization of inhibitory signals. It should be borne in mind that it is possible to use any compound which has the effect of blocking, inhibiting or otherwise negative regulation of inhibitory receptors of NK-cells, or activating, stimulating, or other action, promoting the activity or expression of the activating receptor of NK cells. These compounds include such compounds as cytokines and small molecules, polypeptides, and antibodies that can bind with the receptors of NK-cells and to inhibit or stimulate them. It should also be borne in mind that the mechanism by which receptors are blocked or stimulius is, is not a critical factor for the advantages of the invention. For example, the compounds can enhance the expression of the activating receptor or to inhibit the expression of the inhibitory receptor, the compounds can prevent the interaction between the ligand and inhibitory receptor or to enhance the interaction between the ligand and activates the receptor, or the connection can communicate directly with the receptor and to inhibit them (in the case of inhibitory receptors) or activate them (in the case of activating receptors). A critical parameter is the action that connection have on the ability of antibodies for therapeutic applications to resolve their target cells in vivo.

Any inhibitory receptor on the surface of NK cells may be a target for compounds of the present invention. NK Cells are negatively regulated by any abscopal specific receptors in the class I major histocompatibility complex (MHC) (Cage et al., 1986; Ohien et al., 1989; work included in this description as references). These specific receptors are associated with polymorphic determinants of molecules of class I major histocompatibility complex (MHC or HLA and inhibit the lysis of natural killer (NK) cells. People have a family of receptors called killer Ig-like receptors (KIR), recognizes groups of alleles to the ACCA I HLA.

There are several groups of KIR receptors, including KIR2DL, KIR2DS, KIR3DL and KIR3DS. KIR receptors containing two Ig domain (KIR2D), identify allotype HLA-C: KIR2DL2 (called first R) or closely related gene product KIR2DL3, recognizes an epitope common to allotype HLA-C group 2 (Cw1, 3, 7 and 8), while KIR2DL1 (R) recognizes an epitope common to allotype HLA-C reciprocal group 1 (Cw2, 4, 5 and 6). Recognition KIR2DL1 is dictated by the presence of the Lys residue at position 80 of the alleles HLA-C. Recognition of KIR2DL2 and KIR2DL3 is dictated by the presence of the Asn residue at position 80. Moreover, a significant majority of the alleles HLA-C has residue Asn or Lys at position 80. One KIR with three Ig domains KIR3DL1 (R) recognizes an epitope common to the alleles HLA-Bw4. Finally, glycosilated molecules with three Ig domains KIR3DL2 (P140) recognizes HLA-A3 and-Al 1.

Although KIR and other inhibiting receptor class I (floors are only et al., 1997; Valiante et al., 1997; Lanier, 1998; all work included in this description as a reference) can be co-expressed NK-cells, in any given individual NK repertoire there are cells that Express one KIR, and, thus, the corresponding NK-cells are blocked only cells expressing specific group of alleles of class I, Respectively, as described below, when inhibiting receptors are localized, the methods of the present invention will often include the introduction of compounds to the verge localize multiple inhibitory receptors, providing through this comprehensive action, which reaches the maximum range of NK-cells.

In some embodiments of the compound, preferably antibodies or their fragments, blocks the inhibitory receptor NK-cells, neutralizing the inhibitory signal, at least one inhibitory receptor selected from the group consisting of KIR2DL2, KIR2DL3, KIR2DL1, KIR3DL1, KIR3DL2, NKG2A and NKG2C. Preferably the connection, preferably antibodies or fragments thereof that blocks the inhibitory receptor NK-cells, is a compound, preferably antibodies or fragments thereof, neutralizing the inhibitory signal KIR2DL2, KIR2DL3 and/or KIR2DL1.

The invention also relates to the use of a combination of several compounds, preferably antibodies or their fragments, blocking various inhibiting receptors of NK-cells. Preferably the compounds, preferably antibodies or their fragments, blocking inhibiting receptors of NK-cells, specific inhibitory receptor, selected from among the KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, NKG2A and NKG2C, and is able to inhibit indirectly related KIR or NKG2A/C inhibition of cytotoxicity of NK cells. For example, compounds that block inhibiting receptors of NK-cells, can contain antibodies with the specificity of KIR2DL1, and other antibodies with specificity the awn to KIR2DL2 and/or KIR2DL3. Preferable combination of compounds that block inhibiting receptors of NK-cells, can inhibit mediated KIR2DL1, KIR2DL2, and KIR2DL3 inhibition of cytotoxicity of NK cells. In other embodiments will be a mixture of one or more compounds localizing one or more inhibitory receptors, as well as one or more connections, localizing one or more activating receptors.

For example, it was shown that monoclonal antibodies specific to KIR2DL1, block alleles KIR2DL1 Cw4 (or similar) (floors are only et al., 1993; the work included in this description by reference). As another example, it is reported that monoclonal antibodies against KIR2DL2/3 block alleles KIR2DL2/3 HLACw3 (or similar) (floors are only et al., 1993). It was shown that antibodies against NKG2A block the inhibitory interaction between NKG2A and HLA-E.

Optionally, the antibody can be selected from the group consisting of GL183 (KIR2DL2, L3, available from Immunotech, France and Becton Dickinson, USA); EB6 (KIR2DL1, available from Immunotech, France and Becton Dickinson, USA); AZ138 (KIR3DL1, floors are only accessible from et al., Univ. Genova, Italy); Q66 (KIR3DL2, available from Immunotech, France); Z270 (NKG2A, available from Immunotech, France); P25 (NKG2A/C, floors are only accessible from et al., Univ. Genova, Italy) and DX9, Z27 (KIR3DL1, available from Immunotech, France and Becton Dickinson, USA).

In a preferred aspect of the invention using monoclonal antibodies and their fragments and derivatives, where these antibodies, ragment or derivatives cross-interact with multiple KIR or NKG2A/C-receptors on the surface of NK-cells and neutralize the inhibiting signals.

In one embodiment of the invention using a monoclonal antibody that binds a common determinant of human KIRlDL-peuenTopoa and inhibiting the corresponding inhibitory cascade reactions. Preferably in the invention using a monoclonal antibody that binds KIR2DL1 and KIR2DL2/3 receptors on the surface of human NK cells and inhibiting mediated KIR2DL1 and KIR2DL2/3 inhibition of cytotoxicity of NK cells. Antibodies specifically inhibit the binding of molecules HLA-c with KIR2DL1 and KIR2DL2/3 receptors. Preferred antibodies contribute to the activity of NK-cells in vivo. As KIR2DL1 and KIR2DL2 (or KIR2DL3) is sufficient for grasping the majority of allotype HLA-C, respectively group 1 allotype HLA-C group 2 allotype HLA-C, such antibodies can be used to improve the efficiency of antibodies for therapeutic purposes, for most people, typically, for about 90% of people or more. In this embodiment according to the invention can use any of the antibodies described in the patent application, PCT No. PCT7FR04/01702, registered July 1, 2004, entitled "Compositions and methods for regulating the activity of NK-cells"that are included in this description by reference.

The specific purpose of the present invention are antibodies that block inhibitory receptors of NK-cells, representing a monoclonal antibody that binds the General will is menantu human KIR2DL-penenTOpoB and inhibiting KIR2DL-mediated inhibition of cytotoxicity of NK cells. The specific antibodies bind to the same epitope as monoclonal antibody DF200 or NKVSF1, produced by hybridomas DF200 and NKVSF1, respectively, and/or competing with a monoclonal antibody DF200 or NKVSF1, produced by hybridomas DF200 and NKVSF1, respectively, for the binding of KIR receptor on the surface of human NK cells. Examples of antibodies, functional tests and analyses to determine, arbitrate whether antibodies for binding to these antibodies, for discussion are described in the patent application PCT/FR04/01702.

In a particular embodiment, monoclonal antibodies are monoclonal antibody DF200 produced by hybridoma DF200. In another embodiment, monoclonal antibodies are EV, or antibody binds to the same epitope as monoclonal antibodies EV, or compete for binding with the monoclonal antibody EV. In other embodiments the antibodies are fragments or derivatives of antibodies DF200 or EV. Hybridoma producing DF200 deposited in culture collections CNCM, identification No. "DF200", registration number CNCM 1-3224, registered June 10, 2004, Collection Nationale de Cultures de Microorganismes, Institut Pasteur, 25, Rue du Docteur Roux, F-75724 Paris Cedex 15, France. Antibody NKVSF1 available from Serotec (Cergy Saint-Christophe, France), No. - MCA2243.

In another embodiment of the present invention the connection, and is used to increase the efficiency of antibodies for therapeutic purposes, stimulates an activating receptor of NK cells. You can use any activating receptors such as NKp30 (see, for example, PCT WO 01/36630 included in this description by reference), NKp44 (see, for example, Vitale et al. (1988), J. Exp. Med., 187:2065-2072, the work included in this description by reference), NKp46 (see, for example, Sivori et al. (1997), J. Exp. Med., 186:1129-1136; Pessino et al. (1988), J. Exp. Med., 188: 953-960; work included in this description as references), NKG2D (see, e.g., OMIM 602893), IL-12R, IL-15R, IL-18R, IL-21R, or activating KIR-receptors, such as receptor KIR2DS4 (Carrington and Norman, The KIR Gene Cluster, May 3, 2003, available at http://www.ncbi.nlm.nih.gov/books), or any other receptor that is present on the main part of NK-cells, activation of which leads to the activation or proliferation of cells, even preferably, if the cell is pre-ingibirovany inhibitory receptor, such as inhibitory KIR receptor. The connection may be any molecules, including polypeptides, small molecules and antibodies. Examples of compounds are any compounds, including natural, recombinant or synthetic ligands that interact with the activating receptors. For example, a compound that stimulates an activating receptor of NK-cells, may be a cytokine, such as IL-12 that interacts with the receptor of IL-12 (IL-12R), IL-15, which interacts with the receptor for IL-15 (IL-15R), IL-18, in imagestudio with the receptor of IL-18 (IL-18R), IL-21, which interacts with the receptor for IL-21 (IL-21R). Such compounds are known, for example, IL-12 (Research Diagnostics, NJ, DI-212), IL-15 (Research Diagnostics, NJ, RDI-215), IL-21 (Asano et al., FEBS Letter, 2002; 528: 70-76). Preferably the compound that stimulates an activating receptor of NK-cells, is a compound other than IL-2. Other examples of compounds that stimulate an activating receptor of NK cells include antibodies that bind the receptor NK-cells selected from the group consisting of NKp30, NKp44, NKp46, NKG2D, KIR2DS4, and other activating KIR receptors.

In some preferred embodiments, the activating receptor is a natural cytotoxicity receptor (NCR, natural cytotoxicity receptor)found on NK cells, preferably NCR selected from the group consisting of NKp30, NKp44, or NKp46, and the connection that stimulates an activating receptor that binds to the same epitope or competes for binding with any of the monoclonal antibodies selected from the group consisting of AZ20, A76, Z25, Z231 and BAB281.

Linking any connection with any of the described in this description of receptors of NK-cells can be detected using a number of standard ways. For example, you can use the analysis type colorimetric ELISA and immunoprecipitation and radioimmunoassay. You can use competitive analyses, for example, to compare the binding experience the constituent compounds and compounds, about which it is known that it binds to the receptor of NK-cells, in which the control (for example, WAV, specifically communicating with NKp46) and test compounds are mixed (or pre-adsorb) and applied to the sample that contain epitope-containing proteins, such as NKp46 if WAV. Protocols based on ELISA, radioimmunoassays, Western-blotting and the use of BIACORE, suitable for use in such simple competitive research and is well known in the technique.

The inhibition mediated by KIR or NKG2A/C inhibition of cytotoxicity of NK-cells or stimulation mediated NKp30, NKp44, NKp46, or NKG2D activation of NK-cells can be assessed by various assays or tests, such as binding, cytotoxicity, or other molecular or cellular assays.

In a specific embodiment, the inhibiting activity is illustrated by the ability of the compounds, preferably antibodies, to restore lysis KIR or NKG2A/C-positive NK clones, respectively, to the positive targets HLA-C or HLA-E. In another specific embodiment of the compound, preferably an antibody is defined as the linking molecule HLA-C receptors KIR2DL1 and KIR2DL3 (or closely related KIR2DL2), it is also preferred for its ability to alter the binding molecule HLA-C, selected from among Cw1, Cw3, Cw7 and Cw8 (or HLA-c, containing the Asn residue at position 80), KIR2DL2/3; and the binding molecules of the HLA-C, selected from among Cw2, Cw4, Cw5 and Cw6 (or molecule HLA-c, containing a Lys residue at position 80), KIR2DL1.

Inhibitory or potentiate the activity of the compounds of this invention, preferably antibodies, can be assessed by any number of ways, for example, through its effect on intracellular free calcium, as described, for example, Sivori et al. (1997), J. Exp. Med., 186:1129-1136, the work included in this description by reference. The activity of NK-cells can be assessed using assays based on cellular cytotoxicity, such as measuring the release of chrome, as it evaluates the ability of antibodies to stimulate NK cells to kill target cells, such as cells R, K, or the corresponding tumor cells, as disclosed in Sivori et al. (1997), J. Exp. Med., 186:1129-1136; Vitale et al. (1998), J. Exp. Med., 187:2065-2072; Pessino et al. (1998), J. Exp. Med., 188:953-960; Neri et al. (2001), Clin. Diag. Lab. Immun., 8:1131-1135;

Pende et al. (1999), J. Exp. Med., 190:1505-1516, all work included in this description as a reference. Suitable tests for cytotoxicity are also described in the examples section of the present description. In preferred embodiments, the antibodies cause at least a 10% increase in NK cytotoxicity, preferably at least 40% or 50% increase in NK cytotoxicity or predpochtitel is it at least 70% increase in NK cytotoxicity.

The activity of NK-cells can be assessed using analysis of cytokine release in which NK cells are incubated with antibodies that stimulate the production of cytokines by NK cells (for example, the production of IFN-γ and TNF-α). In an exemplary Protocol, the production of IFN-γ from RMS appreciate, staining the cell surface and intracytoplasmic, and exercising 4 days later analysis in culture by flow cytometry. Briefly, in the last 4 h of cultivation add brefeldin A (Sigma) at a final concentration of 5 µg/ml the cells are Then incubated with mAb against CD3 and anti CD56 before their permeability (increase in cell membrane permeability using IntraPrep™; Beckman Coulter) and staining with PE-anti-IFN-y or PE-IgGI (Pharmingen). The formation of GM-CSF and IFN-y from polyclonal activated NK cells measured in supernatant using ELISA (GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, MN; IFN-y: OpEIA set, Pharmingen).

In preferred embodiments evaluate the ability of antibodies to activate human NK-cells, where the ability to activate human NK cells at least as well as NK-cells, non-human, shows that the compounds suitable for use in the present invention. In particular, we can assess the ability of the compound to enhance the ability of antibodies Leche for the service of objectives to manage the elimination of suitable target cells NK-cells in vitro or in vivo.

The compounds of this invention, preferably the antibodies can detect partial inhibitory or stimulating activity, such as partially reduce KIR2DL-mediated inhibition of cytotoxicity of NK-cells, or partially activate NK cell stimulation NCR or other receptors at any level. The most preferred compounds are capable of inhibiting (or stimulating in the case of activating receptors), at least 20%, preferably at least 30%, 40% or 50% or more the activity of NK-cells, for example, when the dimension in the analysis of cellular cytotoxicity compared with cells in the absence of connection. Also preferred connection can improve the elimination of target cells on 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 1000% or more relative to the level of resolve in the absence of connection. On the other hand, preferred compounds of the present invention, preferably the antibody is able to induce lysis of the population suitable for a couple or HLA-compatible or autologous target cells, i.e. the population of cells that do not effectively lyse NK-cells in the absence of said antibody. Accordingly, the compounds of this invention can also be defined as contributing to the activity of NK-cells in vivo.

The invention also relates to embodiments, which which compounds stimulating activating receptors or preferably a blocking inhibitory receptors of NK cells represent fragments of these monoclonal antibodies with essentially the same antigen-specificity, including, without limitation, Fab fragments, Fab'2 fragments, CDR and ScFv. In addition, monoclonal antibodies can be humanitarianism, human or chimeric (e.g., bespecifically or functionalized antibodies). Although antibodies that stimulate an activating receptor, can also be phrases, they preferably are full-sized. Derivatives, for example, with modified sequences, or conjugated heterologous functional groups, or other compounds can be used in the case of any of the antibodies described in this description.

Antibodies according to the invention, the blocking inhibitory receptor or stimulates an activating receptor of NK-cells, can be obtained by various methods known in the art. Typically they are obtained by immunization of an animal, not a human immunogen containing polypeptide KIR, NKG2A/C, NCR (e.g., NKp30, NKp44, NKp46) or NKG2D, or immunogenic fragment of any of these polypeptides, and the set of spleen cells (for hybrid merge with the respective cell lines. Methods for obtaining monoclonal antibodies from various species are well known in the art (see, for example, Harlow et al., "Antibodies: A laboratory Manual", CSH Press, 1988; Coding, "Monoclonal Antibodies: Principles and Practice", Academic Press, 1986; included in this description as references). More specifically, these methods include the immunization of an animal, not a human, antigen, and subsequent extraction of spleen cells, which then merge with termed immortal cells, such as myeloma cells. Received hybridoma produce monoclonal antibodies and can be selected by limiting dilution to select individual clones. Antibodies can also be obtained by screening combinatorial libraries of antibodies, as disclosed, for example, in the work of Ward et al. (1989); included in this description by reference.

Preferred antibodies according to the invention, the blocking inhibitory receptor or stimulates an activating receptor of NK-cells, obtained by immunization with an immunogen containing the activating or inhibitory receptors of NK-cells, for example KIR2DL polypeptide, preferably human KIR2DL polypeptide. KIR2DL polypeptide can contain the full sequence of the human KIR2DL polypeptide or its fragment or derivative, typically immunogenic fragment, i.e. the part of the polypeptide containing the epitope, suppose the equipment T - or b-cell epitope. Such fragments typically contain at least 7 consecutive amino acid sequence of the Mature polypeptide, even more preferably at least 10 consecutive amino acids. They essentially derive from the extracellular domain of the receptor. In the preferred embodiment, the immunogen contains human KIR2DL, NCR or other polypeptide of the wild type in the lipid membrane is typically on the cell surface. In a particular embodiment, the immunogen contains intact NK-cells, in particular the intact human NK-cells, not necessarily processed or lysed.

Although antibodies for therapeutic applications can contain Fc-plots, modified in such a way that increases their ability to bind to receptors, such as CD 16, in some embodiments the antibodies potentiate NK-cells will contain Fc-areas that have changed in such a way that decreases their affinity to Fc-receptors, thereby decreasing the probability that NK-cell associated antibodies will be contacted and leiserowitz.

Antibodies that block KIR2DL-penenTopbi NK-cells, you can get ways, including:

i) immunizing an animal, not a human immunogen containing the KIR2DL polypeptide;

ii) obtaining monoclonal antibodies from the specified animal, where these monoclonal the specific antibodies bind the specified KIR2DL polypeptide;

iii) screening monoclonal antibodies from step (ii), cross-interact with at least two different serotypes of KIR2DL polypeptide; and

iv) selection of monoclonal antibodies from step (iii), inhibitory KIR2DL-mediated inhibition of NK-cells.

Implementation stages (iii) and (iv) can be changed. Optional method may also include an additional stage of obtaining fragments or derivatives of monoclonal antibodies, as described below.

In another embodiment, the method includes:

i) selecting from a library or repertoire of monoclonal antibodies, or their fragments, or derivatives, cross-interact with at least two different serotypes of KIR2DL polypeptide; and

ii) selection of antibodies from step (i), inhibitory KIR2DL-mediated inhibition of NK-cells.

It should be borne in mind that any of these methods can be used to select any of the antibodies or fragments of antibodies specific for any group (inhibiting or activating) receptors of NK-cells with a common one or more epitopes. For example, such methods can be used to generate antibodies that block KIR3DL and/or NKG2A/C receptors of NK-cells, or stimulate an activating receptor of NK-cells.

In the preferred embodiment of an animal, not a man used these with the family or with any described in this description of antibodies, is a mammal such as a rodent (e.g. mouse, rat, etc.), cow, pig, horse, rabbit, goat, sheep, etc.

Any of the antibodies described herein can be genetically modified or treated by the methods of genetic engineering so that they were suitable for a person, for example humanitarianism, chimeric or human antibodies. Methods of humanizing antibodies are well known in the art. As a rule, humanitariannet antibody of the present invention contains one or more amino acid residues built into it from the original antibody. Such mice or non-human amino acid residues are often referred to as "listed" (import) residues, which are typically taken from "made" variable regions. Humanization can be essentially to carry out following the method of winter (Winter) with employees (Jones et al. (1986), Nature, 321:522; Riechmann et al. (1988), Nature, 332:323; Verhoeyen et al. (1988), Science 239: 1534). In some cases, such "humanized" antibodies are chimeric antibodies (Cabilly et al., Pat. U.S. No. 4816567), in which, essentially, less than an intact human variable region is substituted by the corresponding sequence from the initial antibody. In practice, humanized antibodies according to this invention typically are human antibodies, to which x some CDR residues and, possibly some FR residues substituted by residues from analogous sites in the original antibody.

In another method of obtaining a "humanized" monoclonal antibodies as a mouse, used for immunization, use the XenoMouse® (Abgenix, Fremont, CA). XenoMouse is a mouse host, whose own genes of immunoglobulins replaced by functional genes of the human immunoglobulin. Thus, antibodies produced by such a mouse, or hybridoma derived from b-cells of this mouse are humanitarianism. XenoMouse described in U.S. patent No. 6162963 included in this description by reference. A similar method can be carried out using HuMAb-Mouse™ (Medarex).

Human antibodies can also be obtained according to various other methods, such as use for other immunization of transgenic animals, genetically engineered for the expression of a repertoire of human antibodies [Jakobovitz et al., Nature, (1993), 362:255], or the selection of the repertoire of antibodies using methods phage display (phage display). Such methods known to experts in the art and can be implemented on the basis of the monoclonal antibodies described in this application.

Antibodies of the present invention can also be derivateservlet to "chimeric" antibodies (immunoglo is ulinov), in which part of the heavy and/or light chain is identical with or homologous to corresponding sequences in the original antibody, while the remainder(s) part(s) chain(s) is identical to(s) or homologous(s) to corresponding sequences in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, as long as they do not show desired biological activity (Cabilly et al., see above; Morrison et al. (1984), Proc. Natl. Acad. Sci, 81:6851).

Also keep in mind that when the connection blocking inhibitory receptors of NK cells or stimulates an activating receptor of NK cells is an antibody, the antibody can be polyclonal or preferably monoclonal. Antibodies can be obtained by using a hybrid or methods cell engineering for the expression of the desired variable and constant regions. The antibody can be a single-chain antibody or other derived antibodies that retain antigen-specificity and lower hinge area or its variant. The antibody may be a polyfunctional antibody, recombinant antibody, humanitariannet antibody or fragment or derivative. These fragment or derivative is preferably chosen from among the Fab-fragm the same Fab'2 fragment, and CDRs of ScFv. Preferably the fragment is antigennegative fragment. Antibodies, including fragments of antibodies, also include bespecifically antibodies, but are not limited to these. One example is bespecifically antibody containing antigennegative site-specific activating receptor, and antigennegative site specific for a tumor antigen (see PCT publication no WO 01/71005 included in this description by reference).

Composition and introduction

The invention relates to compositions containing at least one compound, preferably an antibody or its fragment that blocks the inhibitory receptor or stimulates an activating receptor of NK-cells, and antibodies for therapeutic purposes, the use of specified composition to improve the efficiency of antibodies for therapeutic purposes, enhance ADCC in the subject, which is treated with antibodies for therapeutic purposes, or for the treatment of a subject with a disease, particularly a disease, which requires the elimination of target cells, preferably of infected cells, such as cells infected with a virus, tumor cells or other pathogenic cells. Preferably a disease selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, viral disease is. The disease also include graft rejection, including allograft rejection and disease graft-versus-host (GVHD).

Specifically, for the treatment of the disease requires the removal of the target cells are preferably infected cells, such as cells infected with a virus, tumor cells or other pathogenic cells. Preferably the disease is a cancer, infection or immune disease. Preferably a disease selected from the group consisting of cancer, autoimmune diseases, inflammatory diseases, viral diseases. The disease also include graft rejection, including allograft rejection and disease graft-versus-host (GVHD).

These diseases include neoplastic progression of hematopoietic cells. Optional these diseases are selected from the group consisting of lymphocytic leukemia, acute and chronic myeloid leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myelodysplastic syndrome, multiple myeloma and chronic lymphocytic leukemia. These diseases include cancers ENT, colorectal cancer, breast cancer, epithelial cancer. These diseases include infection with CMV and hepatitis C. These diseases in luchot Crohn's disease, rheumatoid arthritis, asthma, psoriasis, multiple sclerosis or diabetes. In particular, it is possible to treat any disease listed in the table above.

These antibodies for therapeutic purposes may contact CD 16 preferably via their Fc-plot. Preferably these antibodies for therapeutic purposes contain part of human IgG1 or IgG3, in particular monoclonal antibodies, or fragments thereof, also preferably a human, humanized or chimeric antibodies, or fragments thereof, such as rituximab.

The specified connection, preferably antibodies or fragments thereof that blocks the inhibitory receptor or stimulates an activating receptor of NK-cells, binds at least one human receptors KIR, NKG2A/C, NCR or NKG2D and or inhibits indirectly related KIR2DL, KIR3DL and/or NKG2A/C inhibition of cytotoxicity of NK-cells or stimulates indirectly related NCR or NKG2D activation of cytotoxicity of NK-cells. In one preferred embodiment of the use of human KIR2DL receptor, for example a receptor selected from the group consisting of human receptors KIR2DL1, KIR2DL2, KIR2DL3, or human receptor KIR3DL, for example a receptor selected from the group consisting of KIR3DL1 and KIR3DL2.

In one preferred embodiment the connection potentiate NK cell, the binding is seen with, at least one of the human KIR2DL receptors and inhibits indirectly related KIR2DL inhibition of cytotoxicity of NK cells. Preferably human KIR2DL receptor selected from the group consisting of human receptors KIR2DL1, KIR2DL2, KIR2DL3. In the preferred embodiment of the compound, preferably antibodies or fragments thereof binds a common determinant of human KIR2DL receptors and inhibits KIR2DL-mediated inhibition of cytotoxicity of NK cells. Preferably the specified connection, preferably the antibody binds a common determinant of human receptors KIR2DL1, KIR2DL2, KIR2DL3 and inhibits mediated KIR2DL1, KIR2DL2, KIR2DL3 inhibition of cytotoxicity of NK cells. In the private embodiment of the specified connection, preferably the antibody inhibits the binding of allelic molecules HLA-C, containing a Lys residue at position 80, with the human receptor KIR2DL1, and linking allelic molecules HLA-C, containing the Asn residue at position 80, with the human receptor KIR2DL2 and KIR2DL3. In another private embodiment the antibody binds to the same epitope as monoclonal antibody DF200 produced by hybridoma DF200. Optional antibodies compete with monoclonal antibody DF200 produced by hybridoma DF200, for the binding of KIR receptor on the surface of human NK cells. In one preferred the additional embodiment the antibodies are monoclonal antibodies DF200, produced by hybridoma DF200. In another embodiment, the antibody competes with or bind to the same epitope as monoclonal antibodies EV.

The composition of the present invention may contain a combination of several compounds, preferably antibodies or their fragments, blocking various inhibiting receptors of NK-cells and/or stimulating one or more activating receptors of NK-cells. Preferably the compounds, preferably antibodies or their fragments, blocking inhibiting receptors NK-cell-specific inhibitory receptor, selected from among the KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, NKG2A and NKG2C and is able to inhibit indirectly related KIR or NKG2A/C inhibition of cytotoxicity of NK cells. Preferably the combination of "neutralizing" of compounds able to inhibit mediated KIR2DL1, KIR2DL2 or KIR2DL3 inhibition of cytotoxicity of NK cells. Through a combination of connections maximum number of different inhibitory receptors will be blocked from the maximum possible number of patients. You can also use combinations of compounds that stimulate various activating compounds (or, as in the case of inhibitory receptors, contact with different epitopes in the same receptor), for example compounds that together lead to the activation of any combination of two or more h the SLA receptors selected from the group consisting of NKp30, NKp44, NKp46 and NKG2D. You can also use combinations containing one or more compounds that block the inhibitory receptor, and one or more compounds that stimulate an activating receptor. As described below, in the preferred embodiment it is possible to obtain from the patient a sample of NK-cells before the application of the methods of the present invention, and it is possible to evaluate the reactivity of NK-cells for various combinations of compounds, for example, in the presence of target cells and antibodies for therapeutic purposes.

The compositions of this invention may contain any pharmaceutically acceptable carrier or excipient, typically a buffer, an isotonic solution, aqueous suspension, optionally with the addition of stabilizers, preservatives, etc. Typical composition contains saline and optional protective or stabilizing substance, such as high molecular weight proteins (e.g. human serum albumin).

Also included are kits containing any combination of one or more species of antibodies for therapeutic applications and one or more compounds potentsiiruya NK-cells, and preferably the instructions on their application.

According to the methods and compositions of the present invention compounds, preferably antibodies or fragments thereof, to lock the bearing inhibitory receptor or stimulates an activating receptor of NK-cells, and antibodies for therapeutic purposes is administered in an "effective" or "therapeutically effective" amount.

An effective amount of antibodies for therapeutic purposes for the recipient can be from about 0.1 mg/kg to about 20 mg/kg, However, the effective amount of the antibody depends on the form of antibodies (total Ig or fragments), the affinity of mAb and pharmacokinetic parameters that should be determined for each specific type of antibody.

An effective amount of the compounds, preferably antibodies or their fragments, blocking inhibitory receptor or stimulates an activating receptor of NK-cells, enter the recipient, may be from about 0.1 mg/kg to about 20 mg/kg, However, the effective amount of the antibody depends on the form of antibodies (total Ig or fragments), the affinity of mAb and pharmacokinetic parameters that should be determined for each specific type of antibody.

In an important embodiment of the invention the use of compounds of the present invention may allow to achieve therapeutic efficacy with reduced doses of antibodies for therapeutic purposes. The application (for example, the dosage, the scheme of introduction) antibodies for therapeutic purposes may be limited by side effects, for example, in the case of rituximab, fever, headache, stertorously breathing, drop of blood on the compliance and others. Accordingly, although many patients the standard dose of antibodies for therapeutic purposes will be introduced in combination with the described in the description of compounds that potentiate NK-cells (i.e. the recommended dose in the absence of any other compounds), thereby increasing the efficacy of standard doses in patients requiring even greater therapeutic effectiveness; in other patients, for example, who suffer from side effects, the introduction of the compounds of the present invention allows to achieve therapeutic efficacy with reduced dose of antibodies for therapeutic purposes and thereby avoid side effects. In practice, the practitioner will be able to establish the ideal dose and scheme of administration of antibodies for therapeutic purposes and compounds potentiate NK-cells, for this patient, for example a therapeutic strategy that is most appropriate from the point of view of the special needs and the General condition of the patient. There are many references for guidance in establishing appropriate dosages for antibodies for therapeutic purposes, and for compounds that potentiate NK-cells, for example, Remington: The Science and Practice of Pharmacy, by Gennaro (2003), ISBN: 0781750253; Goodman and Gilmans, The Pharmacological Basis of Therapeutics, be Hardman, Limbird & Gilman (2001), ISBN: 0071354697; E.A. Rawlins, editor, "Bentley's Textbook of Pharmaceutics", London: Bailliere, Tindall and SOH (1977) and other publications.

One in which lewinii practicing doctor may gradually reduce the number of antibodies for therapeutic purposes, which provide in combination with the introduction of any compounds that potentiate NK-cells, from among the compounds of the present invention, in terms of either the dosage or frequency of administration and to monitor the effectiveness of antibodies for therapeutic purposes; for example, to control the activity of NK-cells, monitor the patient's target cells, to control the various clinical indications, or in any other way, and, taking into account the results of monitoring, to choose the relative concentration or methods of administration of antibodies for therapeutic purposes and/or compounds that potentiate NK-cells, to optimize treatment efficiency and limit side effects.

In another series of embodiments NK-cells will be obtained from the patient prior to the introduction of antibodies for therapeutic purposes and compounds that potentiate NK cells (and, in appropriate case, during treatment), and evaluate to determine the most appropriate connection or set of connections used for maximum efficiency. For example, you can set the identity of inhibitory and activating receptors present on NK cells, and the input connection, which is specifically localized on the most notable receptors. On the other hand, received NK-cells can be incubated with antibodies for therapeutic purposes and target cells and can assess the ability of one is about or multiple connections to increase the disappearance of the target cells. Then, any one or more of the compounds that are most effective in increasing disappearance in vitro, can be selected for use in the methods of the present invention.

Suitable doses of the compounds and/or antibodies for therapeutic purposes can usually be determined in vitro or in animal models, for example in vitro by incubation of antibodies for therapeutic purposes in various concentrations in the presence of target cells, NK-cells (preferably human NK-cells), optional other immune cells and various concentrations of one or more compounds that potentiate NK-cells, and assessing the degree or rate of disappearance of target cells in various conditions using standard tests (e.g., described in the examples section). On the other hand, you can give the animal models of various diseases that can be treated with antibodies (for example, animal models of NHL in the case of rituximab), different dosages of antibodies for therapeutic purposes, together with various dosages described in this description of the connections and it is possible to assess the effectiveness of the antibodies in the treatment of animals (for example, by definition, any suitable clinical, cellular, or molecular analysis or criteria).

The composition of the present invention can be injected directly to the subject typically intravenously is, intraperitoneally, intra-arterial, intramuscular, or transdermal means. It is shown that the composition of this invention can be used some monoclonal antibodies effective in clinical situations, such as Rituxan (rituximab) or Xolair (omalizumab), and similar schemes introduction (i.e. the compositions, and/or doses and/or protocols introduction).

Additionally, the compositions of this invention may also include or may be used in combination with other active ingredients or therapeutic programs, such as chemotherapy or other types of immunotherapy, or simultaneously, or sequentially.

In a specific preferred example, the method of the invention also includes one or more injections of two or more compounds that block the inhibitory receptor or stimulates an activating receptor of NK cells. Thus, these two or more compounds can be used in combination. This may serve to cause an even greater increase in ADCC and effectiveness of antibodies for therapeutic purposes, and/or may serve to reduce the dosage of a compound that blocks the inhibitory receptor or stimulates an activating receptor of NK cells. For example, it is known that compounds such as IL-1, toxic in high doses is H. Consequently, the invention relates preferably to a method of treating disease in a subject in need of such treatment, including:

a) the introduction of a specified subject, at least two compounds, preferably antibodies or their fragments, blocking inhibitory receptor or stimulates an activating receptor of NK-cells, and

b) the introduction of a specified subject antibodies for therapeutic purposes.

For example, it is preferable scheme, when these two compounds are:

i) a first compound selected from the group consisting of antibodies, stimulating NCR - or MC-receptor or activating KIR receptor, and antibodies that block inhibitory NCR - or NKG2D-receptor, and

ii) a second compound selected from the group consisting of IL-12, IL-15, IL-18 and IL-21. Therefore, the invention also relates to a method of treating disease in a subject in need of such treatment, comprising (a) introducing a specified subject compounds of the present invention, preferably antibodies or their fragments, blocking inhibitory receptor or stimulates an activating receptor of NK-cells, b) the introduction of a specified subject antibodies for therapeutic purposes, and (C) the introduction of a specified subject IL-2. IL-2 is available from Research Diagnostics, NJ, RDI-202, or Chiron Corp. (Emerville, CA).

The cytokine can be entered according to any suitable scheme is e introduction and you can enter up, simultaneously and/or after administration of compounds that block the inhibitory receptor or stimulates an activating receptor of NK-cells, and before, simultaneously and/or after administration of antibodies for therapeutic applications. In a typical example, the cytokine is administered daily for 5-10 days, with the cytokine(s) is first injected on the same day that the first injection of compounds that block the inhibitory receptor or stimulates an activating receptor of NK cells. This method preferably includes one or two injections of a cytokine(s) per day subcutaneous method.

The dosage of the cytokine is selected depending on the condition of the patient being treated. In preferred examples, you can use a relatively low dose of the cytokine. For example, the effective dose is typically less than 1 million units of the cytokine(s) per square meter per day when cytokinemia pharmaceutical composition is used for daily subcutaneous injection. In a preferred example, IL-2 is injected subcutaneously daily doses of less than 1 million units/m2within 5-10 days. Other details of the application of cytokines are described in published International patent application number PCT/EP/0314716 and the application for U.S. patent No. 60/435344, entitled "Pharmaceutical compositions affect the proliferation of NK-cells, and the method of their application is", included in this description as a reference.

It should also be borne in mind that antibodies for therapeutic purposes and compounds potentiate NK-cells, you can enter together, for example together to be injected, or you can enter simultaneously in different compositions, or you can enter independently, for example compounds injected for hours, days, weeks before or after the introduction of the connection.

Other aspects and advantages of the invention are disclosed in the following experimental section, which should be seen as explanatory and not limiting the scope of this application.

Examples

Example 1. Antibodies pan KIR2DL

Cleaning PBL and getting polyclonally or riveting NK-cell lines.

The lymphocytes of the peripheral blood (PBL, peripheral blood tymphocytes) obtained from healthy donors by centrifugation in a density gradient with picollo Hypaque and removal is attached to the plastic cells. To get more NK cells, PBLs incubated with mAb against CD3, anti CD4 and anti-HLA-DR (30 min at 4°C)and then with goat artemisinine magnetic granules (Dynal) (30 min at 4°C), and the ways immunomagnetic breeding, known in the art (Pende et al., 1999). CD3 negative, CD4 negative and DR minus cells grown on the underlayer irradiated feeder cells in the presence of 100 u/ml interleukin 2 (Proleukin, Chiron Corporation) and 1.5 ng/ml phytohemagglutinin the and A (Gibco BRL) and get a polyclonal population of NK-cells. NK-Cell clone by the method of serial dilution and clones NK cells characterized by flow cytometry for the expression of receptors on the cell surface.

In this study use the following clones:

- CPU, CN5 and CN505 representing KIR2DL1-positive clones, stainable antibody EV or HA-141;

- CN12 and CN502, representing KIR2DL3-positive clones, stainable antibody GL-183.

Analysis by flow cytometry

mAb is used to produce in the laboratory JT3A (IgG2a, anti CD3), EV and GL-183 (IgGI, against KIR2DL1 and KIR2DL3, respectively), HA-141 IgM against KIR2DL1 (same specificity when compared with EV against CD4 (NR)against DR (D1.12, IgG2a). Instead JT3A, NR and DR1.12 you can use commercially available mAb of the same specificity, e.g., from Beckman Coulter Inc., Fuller-ton, CA. HA-141 commercially available, but you can use EV to oversee the restoration of the lysis, as described in floors are only et al., 1993.

Flow cytometry

Cells stained with appropriate antibodies (30 min at 4°C), then PE or FITC conjugated polyclonal artemisinine antibodies (Southern Biotechnology Associates Inc.). Samples analyzed cytofluorometric analysis on the device FACSAN (Becton Dickinson, Mountain View, CA).

Cytotoxic experiments

The cytolytic activity of NK clones appreciate the standard analysis on the release SG to 4 hours, in which the effector NK cells have Cw3 or Cw4-positive cell lines are known for their sensitivity to lysis by NK cells. All targets used in the amount of 5000 cells per well in titrations the microplate, and the ratio of effector and target is indicated on the figures (usually 4 of effector to target cells). Cytolytic analysis is performed with the supernatant of these monoclonal antibodies at a dilution1/2or without it. The procedure is essentially the same, what floors are only described in et al., 1993.

Getting new mAb

Get mAb immunization 5-week-old Balb From mice with activation of polyclonal or monoclonal NK-cell lines, as described in floors are only et al., 1990; the work included in this description by reference. After the merger of different cells mAb initially selected based on their ability to provide cross-react with EV and GL 183-positive NK cell lines and clones. Then positive monoclonal antibodies are screened for their ability to restore lysis EV-positive or CL183-positive NK clones Cw4 - or Cw3-positive targets, respectively.

DF200 - new monoclonal antibodies against the General determinants of KIR2DL human NK receptors

Discovered that one of the monoclonal antibodies DF200 mAb interacts with various members of the KIR family, that is the number of KIR2DL1, KIR2DL2/3. As for the staining of NK-cells DF200 mAb, as KIR2DL1+and KIR2DL2/3+ brightly stained (figure 1).

NK clones expressing one or the other (or even both) of the HLA class I-specific inhibiting receptors, are used as effector cells against target cells expressing one or more alleles of HLA-C. As expected, KIR2DL1+NK clones show (displayed) weak cytolytic activity against target cells expressing HLA-Cw4, a KIR2DL3+ NK clones show weakly active (or not active) against Cw3-positive targets. However, in the presence of DF200 mAb (used to "disguise" their KIR2DL receptors) NK clones become unable to recognize their HLA-C ligands and show a strong cytolytic activity on Cw3 or w4 target.

For example, cell line CIR (cell line CW4+ EBV, ATCC no CRL 1993) was not killed KIR2DL1+ NK clones (CN5/CN505), but inhibition can effectively return to the previous level when using DF200 mAb or normal against KIR2DL1. On the other hand, NK clones expressing the phenotype KIR2DL2/3+ KIR2DL1 (CN12), effectively killing CIR, and such activity is not affected DF200 mAb (figure 2). Similar results can be obtained with KIR2DL2 or KIR2DL3-positive NK clones on Cw3-positive targets.

The Biacore analysis of interactions DF200 mAb/KIR 2DL1 and DF200 mAb/KIR 2DL3

Materials and methods

Production and purification recombinant the x protein.

Recombinant proteins KIR2DL1 and KIR2DL3 get in E. coli. The cDNA molecule encoding the complete extracellular domain of KIR2DL1 and KIR2DL3, amplified by PCR from the vector pCDM8 clone 71.11 (Biassoni et al., 1993; the work included in this description by reference) and vector RSVS(gpt)183 clone 6 (Wagman et al., 1995; the work included in this description by reference), respectively, using the following primers:

- sense 5'-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3';

- antisense 5'-CCCAAGCTTGGGTTATGTGACAGAAACAAGCAGTGG-3'.

Their clone in expressing vector pML1 in frame with the sequence encoding the signal biotinidase (Saulquin et al., 2003; the work included in this description by reference).

The expression of the proteins is carried out in a bacterial strain BL21(DE3) (Invitrogen). Transfetsirovannyh bacteria grown until OD600≈0,6 at 37°C in medium with addition of ampicillin (100 μg/ml) and induction of 1 mm IPTG.

Proteins extracted from Taurus inclusions (inclusion bodies) under denaturing conditions (8 M urea). Refolding (new folding of the tertiary structure) of recombinant proteins is carried out in buffer 20 mm Tris, pH of 7.8, 150 mm NaCl, containing L-arginine (400 mm, Sigma) and β-mercaptoethanol (1 mm)at room temperature, reducing the concentration of urea by dialysis selectedimage(4, 3, 2, 1, 0,5 and 0 M urea, respectively). During the stages of dialysis with 0.5 and 0 M urea add vosstanovleniya oxidized glutathione (5 mm and 0.5 mm, accordingly Sigma). Finally, proteins extensive cialiswhat against buffer 10 mm Tris, pH 7.5, 150 mm NaCl. Soluble, again folded proteins are concentrated and then purified on a column of superdex method 200 replacement chromatography (Pharmacia; system ACT).

The Biacore Analysis.

Measurement method of surface plasma resonance is carried out on the Biacore instrument (Biacore optical biosensor). In all experiments with Biacore as a working buffer is the buffer HBS with the addition of 0.05% surfactant P20.

Immobilization of proteins.

Recombinant proteins KIR 2DL1 and KIR 2DL3 immobilized covalently bound to the carboxyl groups in the dextran layer on the sensor chip CM (Biacore). The surface of sensor chip activate EDC/NHS (hydrochloride of N-ethyl-N'-(3-dimethylamino-propyl)carbodiimide hydrochloride and N-hydroxysuccinimide, Biacore). Proteins injected in binding buffer (coupling buffer, 10 mm acetate, pH 4.5). Decontamination of the remaining activated groups carried out with 100 mm ethanolamine, pH 8 (Biacore).

Measurement of affinity.

For kinetic measurements on immobilized samples applied different concentrations of soluble antibodies (10-7up to 4×10-10M). The measurements were carried out at a constant flow rate of 20 µl/min For each cycle of the surface of sensor chip regenerate injection of 5 μl of 10 mm NaOH, pH 11.

<> Results of the analysis using the program BIAlogue Kinetics Evaluation (BIAevaluation 3.1).

Results

The BIAcore analysis of binding of mAb DF200 with immobilized KIR2DL1 and KIR2DL3

KD(10-9M)
KIR2DL110,9+/-3,8
KIR2DL32,0+/-1,9

KD: dissociation constant

Soluble analyte (40 μl at various concentrations) is injected with a flow rate of 20 μl/min in HBS buffer layers of dextran with reflectivity 500 or 540 units (RU, reflectance units) and 1000 or 700 EN for KIR2DL1 and KIR2DL3, respectively. Data represent 6 independent experiments.

Example 2. Enhancing ADCC through the use of a combination of Rituxan and mAb against KIR

Obtaining human NK clones.

Mononuclear blood cells deprived of T cells with negative D3-immunomagnetic selection (Miltenyi), sow under conditions of serial dilutions, activate phytohemagglutinin (PHA) (Biochrom KG, Berlin, Germany) and cultured with interleukin (IL)-2 (Chiron BV, Amsterdam, the Netherlands) and irradiated feed the cells. The efficiency of cloning is equivalent to all donors and ranges from 1 to 5 to 1 to 10 seeded NK-cells. Cloned NK cells subjected SK is Inigo on alloreactivity standard method of determining cytotoxicity on release of the 51Cr against transformed by Epstein-Barr lines B-lymphoblastoid cells known HLA type with a ratio of effector to target is 10:1. Clones that detect ≥30% lysis, evaluate how alloreactive. Typically, clones or find <5% >40% lysis.

Enhancing ADCC clone KIR2DLI-positive NK-cell-mediated Rituxan

The cytolytic activity of NK-clone appreciate the standard analysis release51SG 4 hours, in which the effector NK cells have Cw4-or Cw3-EBV positive cell lines (D20-positive), known for their sensitivity to lysis by NK cells. All targets used in the amount of 5000 cells per well in the microplate for titration, and the ratio of effector (clone NK-cells) and targets indicated in figure 3. In some experiments to a mixture of effector and target type chimeric antibodies for therapeutic purposes rituximab (Rituxan. Idec) in an amount of 5 μg/ml In some experiments to a mixture of effector and target type antibodies EV (against KIR2DLI) at 10 μg/ml.

This experiment shows that one Rituxan, essentially no mediates ADCC KIR2DLI-positive NK clones on w4-positive targets. ADCC KIR2DLI-positive NK clone is greatly enhanced in the presence of antibodies against KIR2DLI.

Example 3. Enhancing ADCC clone IR2DL1-position is positive NK cells, mediated Campton.

In an experiment similar to that described in example 2, autologous Cw4+ PHA blast cells incubated in the presence of NK cells with the addition of alemtuzumab (campath, Berlex), antibodies EV (100 μg/ml) or Kampala and EV. The results, shown in figure 4, show that the presence of EV significantly enhances the ability of NK cells to eliminate autologous cells: approximately 4% of the target cells literoitca in the presence of one Kanata, while over 30% of the cells undergo lysis in the presence of Kampala with adding EV.

Links

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Karre K., H.G. Ljunggren, Piontek g, Kiessling R. Selective rejection ofH-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature, 1986,319:675-678.

Larder L.L. NK cell receptors. Annu. Rev. ImmunoL, 1998,16:359-393.

Floors are only A., Bottino C, Pende d, G. Tripodi, G. Tambussi, Viale A., Orengo, A., M. Barbaresi, Merii A., Ciceone E., et al. Identification of four subsets of human CD3-CD16+ natural killer (NK) cells by the expression ofclonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition. J. Exp. Med. 1990,172:1589-1598.

Floors are only A., Vitale M., C. Bottino, A. M. Orengo, Morelli L., Augugliaro, R., M. Barbaresi, Ciceone E., and floors are only L. P58 molecules as putative receptors for major histocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specificities. J Exp Med. 1993,178:597-604.

Floors are only A., floors are only L. HLA class I specific inibitory receptors. Curr. Opin. ImmunoL 1997, 9:694-701.

Ohien C., Kling, G., Hoglund P., Hansson M., Scangos, G., Bieberich, C., Jay G., Karre K. Prevention ofallogeneic bone marrow graft rejection by H-2 transgene in donor mice. Science, 1989,246:666-668.

Pende D., S. Parolini, Pessino, A., Sivori, S., Augugliaro, R., Morelli L., Marcenaro, E., Accame L., A. Malaspina, Biassoni R., et al. Identification and molecular characterization ofNKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J Exp. Med. 1999,190:1505-1516.

Ruggeri L.; Capanni m, Urbani E., Perruccio K, Shiomchik W. D., Tosti A., Posati, S., Rogaia D., Frassoni f, Aversa F., et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science, 2002,295:2097-2100.

X. Saulquin, Gastinel L.N., Vivier E. Crystal structure of the human natural killer cell activating receptor KJR2DS2 (CD158J). J. Exp. Med. 2003,197 (7):933-938.

Valiante N.M., Lienert K., Shilling H.G., Smits B.J., Parham P. Killer cell receptors: keeping pace with MHC class I evolution. Immunol. Rev. 1997,155:155-164.

Wagtmann n, Biassoni R, Cantoni C., Verdiani s, Malnati M.S., Vitale M., Bottino, C., floors are only L., floors are only A., Long, E.O. Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra - and intracellular domains. Immunity, 1995,2 (5):439-449.

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All publications and patent applications cited in this specification, are included as reference as if each individual publication or patent application is specifically and individually included in the description by reference.

Although the foregoing invention is described with some detail for explanations and examples for purposes of clarity of understanding, those skilled in the art it will be obvious that in the light of the indications of this invention can be certain is that substitutions and modifications without departing from the spirits or scope of the attached claims.

Any combination of the above-described elements in all possible variations covered by the invention, this description is not specified or the context there are clear contradictions.

Terms with a definite and an indefinite article (original and similar to the ratio used in the context of describing the invention, should be considered as used for both singular and plural, unless otherwise indicated or the context there are clear contradictions.

It is expected that listing of the intervals of the values in this description serves only as a method of reducing the treatment individually to each value within a specified interval, if this description is not specified, and each separate value is included in the description as if it were quoted separately. Unless otherwise stated, all exact values listed in this description represent the corresponding approximate values (for example, all the exact values given as examples in connection with a specific factor or dimension can be seen as reflecting well as the corresponding approximate measurement, modified, where appropriate, the adverb "about").

All methods described in this specification can be implemented in any suitable poradek is, unless otherwise stated, or the context there are clear contradictions.

The use of any and all examples, or expression for examples (e.g., "such as")provided herein, is intended only for the best explanation of the invention and is not meant to limit the scope of the invention unless otherwise stated. There are no expressions in the description of the invention, which should be considered as indicating that a particular item is essential for the practical implementation of the invention, unless it is.

Citation and inclusion in the description of patent document is for convenience only and do not reflect any views about the suitability, patentability and/or security of such patent documents.

Description in this application any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing", related to the element or elements is intended to maintain such aspect or embodiment, which "consists of", "consists essentially of" or "essentially includes this a single element or elements, unless otherwise specified or the context there are clear contradictions (for example, the composition described in this invention, containing a specific element, should p is establet also, as a composition, is described as consisting of a particular item, unless otherwise stated or the context there are clear contradictions).

The invention includes all modifications and equivalents of the subject matter listed in the aspects or the claims presented herein, to the maximum extent permitted by relevant regulations.

1. A method of treating diseases caused or exacerbated by, at least partially cells that can be targeted and which can be eliminated by antibody for therapeutic purposes, the subject is a human, in need thereof, including
a) the introduction of a specified subject of the first antibody that blocks the inhibitory receptor NK-cells, and specified inhibitory receptor selected from the group consisting of KIR2DL1, KIR2DL2, and KIR2DL3 NKG2A; and
b) the introduction of a specified subject antibodies for therapeutic purposes, which can communicate with the CD 16 through the Fc-part of the indicated antibodies for therapeutic purposes.

2. The method according to claim 1, where the specified antibody for therapeutic purposes contain the Fc-part of human IgG1 or IgG3.

3. The method according to claims 1 or 2, where the aforementioned first antibody is an antibody or includes antigennegative fragment.

4. The method according to claim 1, where the specified antibody for therapeutic purposes is monoclonal and is the antibodies or includes antigennegative fragment.

5. The method according to claim 1, where the specified antibody for therapeutic purposes conjugative with radioactive or toxic group.

6. The method according to claim 1, where the specified first antibody is a human, humanitariannet, or chimeric antibody, or includes antigennegative fragment.

7. The method according to claim 1, where the specified antibody for therapeutic purposes is a human, humanitariannet, or chimeric antibody, or includes antigennegative fragment.

8. The method according to claim 1, where the specified antibody for therapeutic purposes is a rituximab or compat.

9. The method of claim 8, where the aforementioned antibody is a rituximab, and this antibody is administered at a dosage of less than 375 mg/m2a week.

10. The method of claim 8, where the aforementioned antibody is campath, and this antibody is administered at a dosage of less than 90 mg per week.

11. The method according to claim 1, where the specified first antibody binds to a common determinant of human receptors KIR2DL1, KIR2DL2, and KIR2DL3 and inhibits mediated KIR2DL1, KIR2DL2, and KIR2DL3 inhibition of cytotoxicity of NK-cells.

12. The method according to claim 11, where the aforementioned first antibody inhibits the binding of allelic molecules HLA-C, containing a Lys residue at position 80, with the human receptor KIR2DL1 and linking allelic molecules HLA-C, containing the Asn residue at position 80, chelovecheskim.finalnye KIR2DL2 and KIR2DL3.

13. The method according to claim 1, where the specified first antibody binds to the same epitope as monoclonal antibody DF200 produced by hybridoma DF200, or monoclonal antibody EV.

14. The method according to claim 1, where the specified first antibody competes with a monoclonal antibody DF200 produced by hybridoma DF200 or a monoclonal antibody EB 6 for binding to the receptor KIR on the surface of human NK cells.

15. The method according to claim 1, where the specified first antibody is a monoclonal antibody DF200 produced by hybridoma DF200, or its fragment, or derivative, or monoclonal antibody EV or its fragment or derivative.

16. The method according to claim 1, where the specified antibody for therapeutic purposes, and specified the first antibody is administered to a specified subject at once.

17. The method according to claim 1, where the specified first antibody is administered to a specified entity within one week on the introduction of the indicated antibodies for therapeutic purposes.

18. The method according to claim 1, where the disease is a cancer, infection or immune disease.

19. Pharmaceutical composition for treating diseases caused or exacerbated by, at least partially cells that can be targeted and which can be eliminated by antibody for therapeutic purposes, containing
(a) antibody for therapeutic purposes, which morespecific CD 16 through the Fc-part of the indicated antibodies for therapeutic purposes, (b) a first antibody that blocks the inhibitory receptor NK-cells, and specified inhibitory receptor selected from the group consisting of KIR2DL1, KIR2DL2, and KIR2DL3 NKG2A, and (C) a pharmaceutically acceptable carrier.

20. The composition according to claim 19, where the specified antibody for therapeutic purposes contains the Fc-part of human IgG1 or IgG3 or Fc-part of the IgG1 or IgG3 human Primate, non-human.

21. The composition according to PP or 20, where the aforementioned first antibody is an antibody or includes antigennegative fragment.

22. The composition according to PP or 20, where the aforementioned first antibody is a monoclonal antibody or includes antigennegative fragment.

23. The composition according to claim 19, where the aforementioned first antibody is a human, humanitariannet or chimeric antibody or includes antigennegative fragment.

24. The composition according to claim 19, where the specified antibody for therapeutic purposes is a monoclonal antibody or includes antigennegative fragment.

25. The composition according to paragraph 24, where the specified antibody for therapeutic purposes is a human, humanitariannet or chimeric antibody or includes antigennegative fragment.

26. The composition according to paragraph 24, where the specified antibody conjugative with radioactive or toxic group.

27. Composition pop, where the specified antibody for therapeutic purposes is a rituximab or compat.

28. The composition according to claim 19, where the aforementioned first antibody binds to a common determinant of human receptors KIR2DL1, KIR2DL2, and KIR2DL3 and inhibits mediated KIR2DL1, KIR2DL2, and KIR2DL3 inhibition of cytotoxicity of NK-cells.

29. The composition according to p, where the aforementioned first antibody inhibits the binding of allelic molecules HLA-C, containing a Lys residue at position 80, with the human receptor KIR2DL1 and linking allelic molecules HLA-C, containing the Asn residue at position 80, with the human receptor KIR2DL2 and KIR2DL3.

30. The composition according to claim 19, where the aforementioned first antibody binds to the same epitope as monoclonal antibody DF200 produced by hybridoma DF200, monoclonal antibody NKVSF1 or monoclonal antibody EV.

31. The composition according to claim 19, where the aforementioned first antibody competes with a monoclonal antibody DF200 produced by hybridoma DF200, a monoclonal antibody NKVSF1, or a monoclonal antibody EV for binding to the receptor KIR on the surface of human NK cells.

32. The composition according to claim 19, where the aforementioned first antibody is a monoclonal antibody DF200 produced by hybridoma DF200 or a fragment or a derivative thereof, a monoclonal antibody NKVSF1 or its fragment, or derivative, or monoclonal antibody is B6 or its fragment or derivative.

33. The method of selection of the first antibody for administration in combination with the antibody for therapeutic purposes, including
i) providing a test of the first antibody that blocks the inhibitory receptor NK-cells, and specified inhibitory receptor selected from the group consisting of KIR2DL1, KIR2DL2, and KIR2DL3 NKG2A;
ii) incubation of the indicated antibodies for therapeutic purposes with target cells specifically recognized the indicated antibody for therapeutic purposes, in the presence of NK cells in the presence or in the absence of this experience of the first antibody; and
iii) assessment of the actions specified first antibodies on the ability of these NK cells to eliminate these target cells;
where is the discovery that the first antibody increases the ability of these NK cells to eliminate these target cells, indicates that the first antibody is suitable for use in the method according to any one of claims 1 to 18.

34. The method according to p, where the aforementioned first antibody increases the ability of the indicated antibodies for therapeutic purposes to destroy these target cells by 30%.

35. The method according to p, where the aforementioned first antibody increases the ability of the indicated antibodies for therapeutic purposes to destroy these target cells by 50%.

36. The method according to p, where the aforementioned first antibody selected from the group consisting of antibodies, fragments of antibodies which, a monoclonal antibody, a fragment of a monoclonal antibody gumanitarnogo antibodies, chimeric antibodies and human antibodies.

37. The method according to p where these target cells are cancer cells, cells infected with the virus, or cells, supporting an autoimmune disorder.

38. The method according to p, where the specified antibody for therapeutic purposes is a rituximab or compat.

39. The way to increase the effectiveness of treatment of a disease caused or exacerbated by, at least partially cells that can be targeted and which can be eliminated by antibody for therapeutic purposes, and said treatment includes an introduction to the subject antibodies for therapeutic purposes, which can communicate with the CD 16 through the Fc-part of the indicated antibodies for therapeutic purposes, including the introduction of a specified subject before, concurrently or after administration of the indicated antibodies for therapeutic purposes therapeutically effective amount of a first antibody that blocks the inhibitory receptor NK-cells, and specified inhibitory receptor selected from the group consisting of KIR2DL1, KIR2DL2, KIR2DL3 and NKG2A.

40. The method according to § 39, where the aforementioned first antibody increases the efficiency of the specified treatment due to the enhancement of ADCC in the specified subject.



 

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SUBSTANCE: there is offered a method to assess of enzyme's ability to the level of phosphorylation of polypeptide that implies a reaction of the analysed enzyme and a substratum presented with a biotin-conjugated fragment of 516 to 777 residues of a human insulin 1 receptor substratum (hIRS-1-p30), binding of the reaction product and immobilised streptavidin and detection of the level of phosphorylation by antibodies specific to the phosphorylated polypeptide residues.

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3 tbl, 2 ex

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FIELD: medicine.

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1 tbl, 1 ex

FIELD: medicine.

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