Composition and method for control of activity of natural killer cells

FIELD: medicine.

SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.

EFFECT: invention provides for preparation of single type of antibodies, which controls activity of NK cells of various type, provides for amplification of their cytotoxicity, which may find application in therapy, for increase of activity or cytotoxicity of NK cells in individuals without preliminary detection of HLA type in individual.

7 cl, 13 dwg, 4 tbl, 7 ex

 

The technical FIELD

The present invention relates to antibodies, fragments of antibodies and their derivatives, which cross-react with two or more inhibitory receptors on the surface of natural killer cells (NK cells), and enhance the cytotoxicity of NK cells from individuals with mammals or in a biological sample. The invention relates also to methods of producing such antibodies, fragments, variants and derivatives, to pharmaceutical compositions containing them; and to the use of such molecules and compositions, in particular in therapy, to improve the activity or cytotoxicity of NK cells from individuals.

PRIOR art

Cells - natural killer, killer cells (natural killer cells, NK cells) is a subpopulation of lymphocytes that are involved in non-traditional (non-inherited) immunity. The NK cells can be obtained from various well-known in this field of ways - such as obtaining blood samples, ziefert, fees, etc.

Characteristics and biological properties of NK cells include the expression of surface antigens, including CD16, CD56 and/or CD57; the absence of the cell surface complex of T-cell receptor (TCR) alpha/beta or gamma/Delta; the ability to bind to cells that do not Express those antigens of the main complex tissue compatibility and antigen histocompatibility (MHC antigens/HLA), and kill these cells by activating specific cytolytic enzymes; the ability to kill tumor cells or other diseased cells expressing the activating NK cells receptor-ligand; the ability to release cytokines that stimulate or inhibit the immune response; and the ability to undergo multiple cycles of cell division and produce daughter cells with biological properties similar to properties of the parent cell.

In the context of the present invention, "active" cells NK mean biologically active NK cells, specifically the cells NK able to lyse target cells. For example, "active" NK cell ability to kill cells that Express the activating NK cells receptor-ligand and can't Express those antigens to the MHC/HLA (KIR-incompatible cells).

Based on the biological properties of NK cells in this area have been proposed a variety of therapeutic and vaccine strategies designed to modulate NK cells. However, the activity of NK cells is regulated by a complex mechanism that involves both stimulating and inhibiting signals. Therefore, for effective therapy, mediated by NK cells, may be required and stimulation of these cells, and neutralization of inhibitory signals.

There is a negative regulation of NK cells specification the definition for class 1 main complex tissue compatibility (major histocompatibility complex, MHC) inhibitory receptors (ärr and others, 1986; Öhlén and others, 1989). These specific receptors are associated with polymorphic determinants of class I molecules MHC or antigen histocompatibility (HLA)in other cells, and inhibit lysis by NK cells. A person group of alleles of HLA class I recognized some representatives of the family of receptors, called "killer lg-like receptors" (killer Ig-like receptors, KIR).

The KIR receptors are a large family of receptors available on certain subtypes of lymphocytes, including NK cells. The KIR nomenclature based on the number of extracellular domains (KIR2D or KIR3D) and on how long Lee (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS) cytoplasmic tail. In humans, the presence or absence of this KIR varies from one cell NK to another within the NK cell population in the possession of a particular individual. The human population has a relatively high level of polymorphism of KIR molecules, and certain KIR molecules are available for some individuals but not all. Some products KIR gene stimulate the activity of lymphocytes by binding with a suitable ligand. All KIR with a proven stimulant have a short cytoplasmic tail with a charged transmembrane portion which is associated with adaptarea molecule having immunostimulating Aitmatov (immunostimulatory motif, ITAM). Other products KIR gene are inherently any abscopal. All KIR with confirmed inhibitory activity have long cytoplasmic tail, and it turns out that they interact with different subtypes of HLA antigens, depending on the subtype of KIR. In the intracytoplasmic part of inhibitory KIR manifest one or more inhibitory motifs that use the phosphatase. Known inhibitory KIR receptors include representatives of the subfamilies KIR2DL and KIR3DL. The KIR receptors with two Ig domain (KIR2D), identify allotype HLA-C: KIR2DL2 (originally it meant R) or closely related gene product KIR2DL3 recognize an epitope common in allotype group 2 HLA-C (w1, 3, 7 and 8), whereas KIR2DL1 (R) recognizes an epitope common in allotype reciprocal group 1 HLA-C (Cw2, 4, 5 and 6). Recognition KIR2DL1 is determined by the presence of the Lys residue at position 80 of the alleles HLA-C. Recognition of KIR2DL2 and KIR2DL3 is determined by the presence of the Asn residue at position 80. Importantly, the vast majority of alleles of HLA-C is in position 80 or residue Asn, or Lys residue. One KIR with three Ig domains - KIR3DL1 (R) recognizes an epitope common alleles in the HLA-Bw4. Finally, glycosilated molecules with three Ig domains - KIR3DL2 (P140) recognizes HLA-A3 and HLA-A11.

Although the inhibitory KIR and other inhibitory receptors, class I (floors are only and others, 1997; Valiante, etc., a; Lanier, 1998) may jointly ex is to reservats NK cells, in the set of NK cells each given individual, there are cells expressing single KIR, and therefore, the NK cells are blocked only cells expressing specific group of alleles of the I class.

It was found that the population of NK cells or clones that do not meet KIR, i.e. the population of NK cells that Express KIR incompatible with the HLA molecules of the host, are the most likely mediators of anti-leukemic action of transplant observed in allogeneic transplantation (Ruggeri and others, 2002). One way to reproduce this effect in a given individual could be the use of reagents that block the interaction of KIR/HLA.

It was shown that specific to KIR2DL1 monoclonal antibodies block the interaction between KIR2DL1 with Cw4 allele (or similar) (floors are only and others, 1993). Were also described monoclonal antibodies to KIR2DL2/3, blocking the interaction of KIR2DL2/3 alleles HLACw3 (or similar) (floors are only and others, 1993). However, the use of such reagents in clinical situations would require the development of two therapeutic monoclonal antibodies (monoclonal antibodies, mAb) for the treatment of all patients, regardless of, whether expressed the patient alleles HLA-C, 1st class or 2nd class. Moreover, before deciding what type of therapeutic antibodies using the ü, would need to pre-determine what type HLA expressed each patient. This would lead to a strong rise in the cost of treatment.

Watzl, etc. (// Tissue Antigens. 2000. T. S) received cross-reacting antibodies that recognize multiple isotypes KIR, but these antibodies had no enhancing effect on the activity of NK cells. Spaggiara G.M. and others (// Blood. 2002. T. S-4107) performed experiments using multiple monoclonal antibodies to different KIR. It was shown that one of these types of antibodies, namely NKVSF1, recognizes a common epitope in CD158a (KIR2DL1), CD158b (KIR2DL2) and R (KIR2DS4). It was not suggested that NKVSF1 may enhance the activity of NK cells, and was not intended that these antibodies can be used as a therapeutic tool. Therefore, the practical and effective approaches to the modulation of the activity of the cells still were not available in this area, and it still needs specific to the HLA alleles influence using specific reagents.

The INVENTION

The present invention now provides new antibodies, compositions and methods that overcome the existing difficulties in the activation of NK cells and provide the best features and benefits. In one cited as an example aspect of the invention provides adinin the second type of antibody, amplifying the activation of human NK cells in virtually all people. More specifically, the invention provides a new specific antibodies cross-reacting with a diverse group of inhibitory KIR and neutralizing their inhibitory signals, which leads to increased cytotoxicity of the NK cells that Express such inhibitory KIR receptors. This ability to cross-reactions with many products KIR genes allows efficient use of the antibodies of the present invention for increasing the activity of NK cells, the majority of human individuals without difficulty and costs associated with the preliminary determination of an individual's HLA type.

In the first aspect, the invention provides antibodies, antibody fragments and derivatives of any of them, with the indicated antibody, fragment or derivative cross-reacts with at least two inhibitory KIR receptors on the surface of NK cells, neutralizes the inhibitory signals to NK cells and enhances the activity of NK cells. More preferably, the antibody binds to a common determinant of KIR2DL receptors person. More specifically, the antibodies according to the present invention are associated at least with receptors KIR2DL1, KIR2DL2, and KKIR2DL3. For the purposes of the present invention, the term "KIR2DL2/3" refers to any receptor KIR2DL2 and KIR2DL3 to both of them. These two receptors have a very high degree of homology, are, apparently, allelic forms of the same gene in this region are considered to be interchangeable. Therefore, for the purposes of the present invention is that KIR2DL2/3 is a single molecule inhibitory KIR, and therefore, antibodies that cross-react only with KIR2DL2 and KIR2DL3 and do not react with any of the other inhibitory KIR receptors, are not included in the scope of coverage of the present invention.

Antibodies according to the present invention specifically inhibit the binding of molecules MHC or HLA, at least two inhibitory KIR receptors and contribute to the activity of NK cells. The term "neutralize the inhibitory activity of KIR", as used here, refers to both types of activity. The ability of the antibodies according to the present invention to facilitate the activity of NK cells", "to contribute to the cytotoxicity of NK cells", "to help NK cells, enhance the activity of NK cells, enhance the cytotoxicity of NK cells" or "to give effect to the NK cells in the context of this invention means that the antibody enables NK cells Express on their surface the inhibitory KIR receptor, so they were able to lyse cells expressing on their surface corresponding ligand for this particular Inga is Fornovo receptor KIR (for example, specific antigen HLA). In a particular aspect, the invention provides antibodies that specifically inhibit the binding of molecules HLA-C receptors KIR2DL1 and KIR2DL2/3. In another specific aspect, the invention provides antibodies that contribute to the activity of NK cells in vivo.

Due to the fact that at least about 90% of the human population has at least one receptor KIR2DL1 or KIR2DL2/3, the most preferred antibodies of the present invention can help the activity of NK cells, aimed at the majority of cells associated with allotype HLA-C, respectively, with allotype HLA-C group 1 and allotype HLA-C group 2. Thus, compositions of the present invention can be applied for activation or amplification of NK cells in most human individuals, usually approximately 90% of human individuals or more. Therefore, the only composition with antibodies in accordance with the present invention can be applied for the treatment of most human individuals, and the need to determine allelic groups or to use cocktails of antibodies occurs rarely.

The invention demonstrates for the first time, it is possible to obtain cross-reactive and neutralizing antibodies to inhibitory KIR and that such antibodies provide effective activation of NK cells in a wide the range of human groups.

Therefore a specific object of the present invention are antibodies, and these antibodies specifically associated with the human receptor KIR2DL1, and with the human receptor KID2DL2/3 and cancel mediated these KIR inhibition of cytotoxicity of NK cells. In one of the embodiments of the present invention, the antibodies compete with monoclonal antibodies DF-200, produced by hybridomas DF-200. These antibody that competes with antibody DF-200, themselves may not be antibodies DF-200.

In another embodiment, the antibodies compete with monoclonal antibody NKVSF1, and antibodies that compete with NKVSF1, can (but not necessarily) should not be an antibody NKVSF1.

In another embodiment, the antibody competes with the antibody 1-7F9.

Preferably these antibodies are chimeric antibodies, "humanized" antibodies or human antibodies.

The term "competes with"when it refers to a specific monoclonal antibody (e.g., DF-200, NKVSF1, 1-7F9, EV, GL183), means that the antibody competes with a monoclonal antibody (e.g., DF-200, NKVSF1, 1-7F9, EV, GL183) analysis of binding using either recombinant KIR molecules, either expressed on the surface of the KIR molecules. For example, if the antibody reduces the binding of DF-200 with KIR molecule in the analysis with Azania, antibody "competes" with DF-200. Antibodies that "compete" with DF-200, can compete with the DF-200 for binding to the human receptor KIR2DL1 human receptor KIR2DL2/3, or with both human receptors and KIR2DL1 and KIR2DL2/3.

In a preferred embodiment, the invention provides antibodies that bind to human receptors and KIR2DL1 and KIR2DL2/3, abolishing mediated these KIR inhibition of cytotoxicity of NK cells and competing with DF-200, 1-7F9 or NKVSF1 for binding to the human receptor KIR2DL1 human receptor KIR2DL2/3, or with both human receptors and KIR2DL1 and KIR2DL2/3. At the discretion of, these antibodies are not NKVSF1. At the discretion of, these antibodies are chimeric, human or humanized" antibodies.

In another embodiment, the invention provides antibodies that bind to human receptors and KIR2DL1 and KIR2DL2/3, abolishing mediated these KIR cytotoxicity of NK cells and competing with EV for binding to the human receptor KIR2DL1, competing with GL183 for binding to the human receptor KIR2DL2/3, or competing with EV for binding to the human receptor KIR2DL1 and GL183 for binding to the human receptor KIR2DL2/3. At the discretion of, these antibodies are not NKVSF1; at the discretion of, yasanmamislari are not DF-200. At the discretion of, these antibodies are chimeric, human or humanized" antibodies.

In a useful aspect of the invention provides antibodies that compete with DF-200 and discriminating on the KIR molecule substantially the same or substantially the same, or just the same as on monoclonal antibodies DF-200, epitopes or epitope centers, communicating with them or have them immunospecificity. Preferably specified KIR molecule may be the human receptor KIR2DL1 or human receptor KIR2DL2/3.

A specific object of the invention comprise antibodies, and these antibodies are associated with a common determinant present in the human receptors KIR2DL1 and KIR2DL2/3 and supersede mediated these KIR inhibition of cytotoxicity of NK cells. The more specific antibodies are associated essentially with the same epitope on KIR, as monoclonal antibodies DF-200, produced by hybridomas DF-200, or antibody NKVSF1, produced by hybridomas NKVSF1, and antibodies are not NKVSF1.

In the preferred form of the antibody according to the present invention are monoclonal antibodies. The most preferred antibodies according to the present invention are monoclonal antibodies DF-200, produced by hybridomas DF-200.

Producing antibodies the DF-200 hybridoma were deposited in culture collections CNCM under identification code "DF-200" (DF200), registration number CNCM I-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 produced by the firm Serotec (Cergy Saint-Christophe, France), No. MSA. Antibody NKVSF1 here are the designation pan2D mAb.

The invention also provides functional fragments and derivatives described herein antibodies having essentially the same antigenic specificity and activity (i.e. those that can cross-react with the parent antibodies and to enhance the cytotoxic activity of NK cells expressing inhibitory KIR receptors), including (but not limited to, Fab fragment, the fragment Fab'2, immunoadhesin, dimeric antibody (diabody), and CDRs of ScFv. In addition, the antibodies according to the present invention can be humanitarianism, human or chimeric.

The invention also provides derivatives of antibodies, which are antibodies of the present invention, conjugated or covalently cross-linked to a toxin, a radionuclide, a recognizable part of the molecule (for example, with a fluorophore) or solid media.

The invention also provides pharmaceutical compositions containing the antibodies, as they are described above, fragments or derivatives of any of them. Accordingly the invention relates also is to use antibodies as they are presented here, in the method of manufacture of a medicinal product. In preferred embodiments, the implementation of specified drug or pharmaceutical composition intended for the treatment of cancer or other proliferative disorder, an infection, or for use in transplantation.

In another embodiment, the invention provides a composition comprising antibodies that bind to the products of at least two different genes of the human inhibitory KIR receptors, and these antibodies capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in NK cells expressing at least one of said two different human inhibitory KIR receptors, these antibodies encapsulated inside liposomes. Optionally, this composition may contain an additional substance selected from molecules of nucleic acids for gene delivery for gene therapy; the nucleic acid molecule for the delivery of antisense RNA, interfering RNA (RNAi) or small interfering RNA (siRNA) for supressive gene in NK cells; or toxin or drugs for targeted destruction of NK cells optionally included in these liposomes.

The invention also provides for methods of regulation the act is vnesti NK cells human in vitro, ex vivo or in vivo, comprising contacting cells NK human with an effective amount of the antibody of the present invention, fragments of such antibodies, and derivatives of each or pharmaceutical composition comprising at least one of the above. Preferred methods consist in the introduction of an effective amount of the pharmaceutical compositions according to the present invention and is aimed at increasing the cytotoxic activity of NK cells human, most preferably ex vivo or in vivo, an individual having a cancer, an infectious disease or immune disease.

In further aspects the invention provides hybridoma that contain:

(a) cells of the host mammal (usually the host is a mammal, not a person), immunized with an antigen containing the epitope present on the polypeptide inhibitory KIR, merged with

(b) immortal cells (e.g., myeloma cells), and these hybridoma produce monoclonal antibodies that bind at least two different human inhibitory KIR receptors and is capable of at least substantially neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells expressing the indicated at least two different human inhibitory R is Ceptor KIR.

Optionally, these hybridoma may not produce a monoclonal antibody NKVSF1. Preferably, these antibodies was associated with receptors KIR2DL1 and KIR2DL2/3. Preferably, the specified antibodies bind to a common determinant present on KIR2DL1 and KIR2DL2/3. Preferably these hybridoma produce antibodies that inhibit the binding molecules of the alleles HLA-C with position 80 the residue is Lys, with the human receptor KIR2DL1 and the binding molecules of the alleles HLA-C with position 80 the residue is Asn, with the human receptor KIR2DL2/3. Preferably, these hybridoma was produced antibodies that bind essentially the same epitope as monoclonal antibodies DF-200, produced by hybridomas DF-200, or KIR2DL1 or KIR2DL2/3, or on KIR2DL1 and KIR2DL2/3. An example of such a hybrid - DF-200.

The invention also provides for methods of producing antibodies that cross-react with multiple products KIR2DL gene and which neutralize the inhibitory activity of these KIR, and the method includes the following steps:

a) immunization of a mammal (non-human) immunogen containing the KIR2DL polypeptide;

b) receiving from the immunized mammal antibodies, and antibodies associated with KIR2DL polypeptide;

c) selecting antibodies of step (b) those that cross the PE Giroud, at least two different products KIR2DL gene; and

(d) selecting antibodies of step (C) those that enhance the activity of NK cells.

In one of the embodiments of the present invention indicated mammals (not man) is a transgenic animal engineered to Express a set of human antibodies (for example, a mammal (not people)that contains the loci of the human immunoglobulin and deletion of the native gene for the immunoglobulin, such as Xenomouse™ (Abgenix - Fremont, CA, USA), or mammal (not people)that contains minilogue of the genes encoding the human Ig, such as HuMab-mouse™ (Medarex - Princeton, NJ, USA). Optionally, the method further includes the selection of antibodies that are associated with NK cells or KIR polypeptide of primacy, mainly Cynomolgus monkeys. Optionally, the invention may optionally include the method of evaluation of antibodies, where produced according to the above method, the antibody is administered to a Primate, preferably a Cynomolgus monkey, preferably monkey see on the presence or absence of signs of toxicity antibodies.

The invention also provides a method of producing antibodies that bind at least two different gene products of the human inhibitory KIR receptors, and these antibodies is the contrasted neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells, expressing these at least two different gene product of the human inhibitory KIR receptors, thus this method includes the following steps:

a) immunization of a mammal (non-human) immunogen containing polypeptide inhibitory KIR;

b) obtaining antibodies from the immunized animal, and the antibody binds to a polypeptide KIR;

c) selecting antibodies of step (b), which cross-react with at least two different gene products of the human inhibitory KIR receptors, and

d) selecting antibodies of step (C)is capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells expressing the indicated at least two different gene product of the human inhibitory KIR receptors, and the order of the steps (C) and (d) may be changed at the discretion, and discretion may be repeated one or more times by any number of stages.

Preferably used for immunization polypeptide inhibitory KIR was KIR2DL polypeptide selected in step (C) antibodies cross-reacted with at least KIR2DL1 and KIR2DL2/3. Preferably, these antibodies recognize a common determinant present on at least two different gene products of KIR receptors; most preferably specified KIR t is Auda KIR2DL1 and KIR2DL2/3. Optionally, this method further includes the selection of antibodies binding to NK cells or KIR polypeptide of a Primate, preferably Cynomolgus monkeys. Optionally, the invention further includes a method of evaluation of antibodies, where produced according to the above method, the antibody is administered to a Primate, preferably a Cynomolgus monkey, preferably monkey see on the presence or absence of signs of toxicity antibodies.

Optionally, in the above-described methods, the antibodies selected in step (C) or (d), are not NKVSF1. Preferably, the antibodies obtained in step (b), in the above-described methods was a monoclonal antibody. Preferably, the antibodies selected in step (C) in the above-described methods, inhibited binding of allelic molecules HLA-C with position 80 the residue is Lys, with the human receptor KIR2DL1 and linking allelic molecules HLA-C with position 80 the residue is Asn, with the human receptor KIR2DL2/3. Preferably, the antibodies selected in step (d) in the above-described methods, has increased the cytotoxicity of NK cells - for example, any significant increase or, at least, the enhancement of cytotoxicity of NK cells, at least 5%, 10%, 20%, 30% or more - for example, strengthening addressed to the cytotoxicity of NK cells, m is Nisha least approximately 50% (e.g., at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90% or at least 95% (i.e., the enhancement of cytotoxicity of NK cells, for example, from about 65% to 100%)). Preferably, the antibodies were linked essentially to the same epitope as monoclonal antibody DF-200, KIR2DL1 and/or KIR2DL2/3. Optionally, these methods also or alternatively include an additional step of creating fragments selected monoclonal antibodies, create derivative selected monoclonal antibodies (e.g., by conjugation with a radionuclide, a cytotoxic agent, a reporter molecule or the like) or create derivative fragments of antibodies or derived from such monoclonal antibodies, or those that contain (amino acid) sequences corresponding to the sequences of such monoclonal antibodies.

The invention further provides a method of producing antibodies that bind at least two different gene products of the human inhibitory KIR receptors, and these antibodies capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells, xpressway specified, at least two different gene product of the human inhibitory KIR receptors, the method includes the following steps:

a) selecting, from a library or set of monoclonal antibody or fragment monoclonal antibodies that cross-react with at least two different gene products of the human inhibitory KIR2DL receptor, and

b) selecting antibodies of stage (a)is capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells expressing the indicated at least two different gene product of the human inhibitory KIR2DL receptor. Preferably, the antibodies were associated with a common determinant present in the receptors KIR2DL1 and KIR2DL2/3. At the discretion of the antibodies selected in step (b)may not be NKVSF1. Preferably the antibodies selected in step (b)inhibit the binding of allelic molecules HLA-C with position 80 the residue is Lys, with the human receptor KIR2DL1 and linking allelic molecules HLA-C with position 80 the residue is Asn, with the human receptor KIR2DL2/3. Preferably, the antibodies selected in step (b), has increased the cytotoxicity of NK cells - for example, any significant increase or, at least, the enhancement of cytotoxicity of NK cells, at least 5%, 10%, 20%, 30% or more - for example, strengthening adresovannoi cytotoxicity of NK cells, at least about 50% (e.g., increased cytotoxicity of NK cells, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90% or at least 95% (i.e., for example, approximately 65-100%)). Preferably, the antibodies were linked essentially to the same epitope as monoclonal antibody DF-200, KIR2DL1 and/or KIR2DL2/3. Optionally, the method includes an additional step of creating fragments selected monoclonal antibodies, create derivative selected monoclonal antibody, or create derivatives of the fragments of the selected monoclonal antibodies.

In addition, the invention provides a method of producing antibodies that bind at least two different gene products of the human inhibitory KIR receptors, and these antibodies capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells expressing the indicated at least two different gene product of the human inhibitory KIR receptors, the method includes the following steps:

a) culturing hybridomas according to the present invention under conditions allowing the production of monoclonal anti is l, and

b) separation of monoclonal antibodies from hybridomas. Optionally, the method may include the additional step of creating fragments of these monoclonal antibodies, create derivatives of monoclonal antibodies or create derivatives of such fragments of monoclonal antibodies. Preferably, the antibodies were associated with a common determinant present on KIR2DL1 and/or KIR2DL2/3.

The present invention also provides a method of producing antibodies that bind at least two different gene products of the human inhibitory KIR receptors, and these antibodies capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells expressing the indicated at least two different gene product of the human inhibitory KIR receptors, thus this method includes the following steps:

a) isolation of hybridomas according to the present invention a nucleic acid encoding the indicated monoclonal antibodies;

b) at the discretion of, the modification of the nucleic acid so as to obtain a modified nucleic acid containing a sequence that encodes a modified or modified antibody containing the amino acid sequence, which corresponds to the functioning of the real sequence of the monoclonal antibody or substantially similar (e.g., identical to a sequence at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95% (as of about 70-99%))selected from "gumanitarnogo" antibodies, chimeric antibodies, single-chain antibody, immunoreactive fragment of the antibody or of the fused protein containing such immunoreactive fragment;

c) inserting a nucleic acid or modified nucleic acid (or related nucleic acid that encodes the same amino acid sequence) in expressing vector, and the encoded antibody or encoded by a fragment of the antibody is able to be expressed when expressing the vector is present in the cell host, grown in appropriate conditions;

d) transferowania host cell expressing vector and a host cell does not produce any other way protein immunoglobulin;

e) culturing transtitional host cell under conditions which cause expression of the antibody or antibody fragment; and

f) isolation of the antibody or antibody fragment produced specified the host-cell. Preferably, the antibody is contacted with a common determinant present on KIR2DL1 and KIR2DL2/3.

p> You should take into account that the invention also provides a composition comprising antibodies that bind at least two different gene products of the human inhibitory KIR receptors, and these antibodies capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in NK cells expressing at least one of said two different human inhibitory KIR receptors, antibodies are present in amounts effective to considerably enhance the cytotoxicity of NK cells in a patient or in a biological sample containing NK cells; and a pharmaceutically acceptable carrier or excipient. Preferably, the antibodies were associated with a common determinant present on KIR2DL1 and KIR2DL2/3. The composition may optionally contain a second therapeutic agent selected from, for example, immune-modulating means, hormonal drugs, chemotherapeutic drugs, antiangiogenic funds apoptotic means, the second antibody to bind to the inhibitory KIR receptor and inhibits its action, anti-infectious means, addressing means or tools. Useful immunomodulatory means can be selected from IL-1 alpha, IL-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, TGF-BAA is a, GM-CSF, M-CSF, G-CSF, TNF-alpha, TNF-beta, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-alpha, IFN-beta or IFN-gamma. Examples of these chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, adriamycin, dactinomycin, mitomycin, karminomitsin, daunomycin, doxorubicin, tamoxifen, Taxol, Taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, titera, methotrexate, camptothecin, actinomycin-D, mitomycin C, cisplatin (CIS-DDP), aminopterin, combretastatin(s), the Vinca alkaloids and their derivatives or precursors. Examples of hormonal funds include leiprorelina, goserelin, triptorelin, buserelin, tamoxifen, toremifene, flutamide, nilutamide, cyproterone, bikalutamid, anastrozole, exemestane, letrozole, fadrozole medroxy, chlormadinone, megestrol, other agonists of Gonadotropin-releasing hormone (LHRH, Luteinizing Hormone Releasing Hormone), other anti-estrogens, other anti-androgens, other aromatase inhibitors, and other Progestogens. Preferably, the second antibody to bind to the inhibitory KIR receptor and inhibiting its action, was the antibody or its fragment, which are associated with the same epitope of inhibitory KIR receptor, which is different from the epitope associated with the specified antibody to bind to the about what they determinant, available, at least two different gene products of the human inhibitory KIR receptor.

Further, the invention provides a way noticeable intensification of the activity of NK cells we need in this patient, comprising the step of introducing the patient a composition in accordance with the present invention. A patient in need of increased activity of NK cells may be any patient having a disease or disorder in which such gain may contribute to its therapeutic effect, amplify and/or to induce a therapeutic effect (or contributes to its therapeutic effect, amplifies and/or induces a therapeutic effect for at least a significant portion of patients with the disease or disorder, or individuals basically with the same characteristics as patients, as may be determined, for example, clinical trials). The patient in need of such treatment, can have, for example, cancer, or other proliferative disorder, an infectious disease or an immune disorder. Preferably, the method includes the additional step of introducing the patient is a suitable additional therapeutic agent selected from an immunomodulatory tools, hormonal drugs, chemotherapeutic drugs, antiangiogenic funds apoptotic environments is TBA, second antibody to bind to the inhibitory KIR receptor and inhibits its action, anti-infectious means, addressing means or supporting means, and the additional therapeutic agent is administered to the patient in the form of a single dose together with antibodies or in the form of separate dosages. The dosage of the antibodies (or fragments/derivatives of antibodies) and dosage of the additional therapeutic agent together sufficient to significantly induce, facilitate and/or enhance therapeutic effect in a patient, which requires increased activity of NK cells. If the antibodies, fragments or derivatives and the additional therapeutic agent is administered separately, it is necessary for their introduction in the conditions, such as temporary mode, number of doses, and so on)that make substantial combined therapeutic effect on the patient.

Further, the present invention provides antibodies that can specifically bind to NK cells of a Primate (non-human), preferably monkeys, and/or monkey KIR receptors. The invention provides methods for assessing the toxicity, dosage and/or activity or effectiveness of the antibody according to the present invention, which are candidates for drugs. In one aspect of izobreteny which provides a way of determining the dose of the antibody, toxic to animal or target tissue, through the introduction having cells NK recipient animal is a Primate (non-human) antibodies according to the present invention and evaluation of any toxic or hazardous, or side effects of money on an animal or preferably on the target tissue. In another aspect the invention provides a method of identifying antibodies toxic to animal or target tissue, through the introduction having cells NK recipient animal is a Primate (non-human) antibodies according to the present invention and evaluation of any toxic or hazardous, or side effects of money on an animal or preferably on the target tissue. In another aspect, the invention provides a method of identifying antibodies that are effective in the treatment of infections, diseases or cancer by injecting the antibodies according to the present invention Primate (non-human), modeling the infection, disease or cancer, or symptom. Preferably, the antibody according to the present invention is an antibody that (a) cross-reacts with at least two human inhibitory KIR receptors on the surface of human NK cells and (b) cross-reacts with NK cells or receptor KIR primacy (not a person).

Further, the present invention provides a method of obnarujenia biological sample or in vivo presence of NK cells, having on its cell surface inhibitory KIR, and the method includes the following steps:

(a) bringing a biological sample or a living organism into contact with an antibody according to the present invention, the antibody is conjugated or covalently linked to a recognizable fragment; and

(b) detecting the presence of antibodies in a biological sample or a living organism.

The invention also provides a method of purification from a biological sample of NK cells with cell surface inhibitory KIR, which includes the following steps:

(a) bringing the sample into contact with an antibody according to the present invention under conditions that provide for the binding of NK cells with cell surface inhibitory KIR, antibodies, and antibodies conjugated or covalently bound to a solid carrier (e.g., pellet, matrix, etc.); and

(b) elution associated NK cells with antibodies conjugated or covalently coupled to a solid carrier.

The following aspect of the invention provides an antibody, antibody fragment or derivative of any of them, containing a light variable plot (variable area light chain) or one or more CDRs (complementarity determining regions - areas that define the complementarity of the antibody to the antigen) from the lungs variabeln the x plots antibodies DF-200 or antibodies Pan2D, as shown in Fig. In another aspect, the invention provides an antibody, antibody fragment or derivative of any of them that contains a sequence highly similar to all or substantially all of the light sequence of the variable segment DF-200 or Pan2D or one or more of the CDRs of light variable regions of one or both of these antibodies.

In a further aspect the invention provides an antibody, antibody fragment or derivative of any of them, containing heavy variable plot (variable plot heavy chain) or one or more CDRs of the heavy variable regions of antibodies DF-200, as shown in Fig. In another aspect, the invention provides an antibody, antibody fragment or derivative of any of them that contains a sequence highly similar to the whole or substantially the whole sequence of the variable heavy plot DF-200.

These and additional advantages and features of the present invention can be further described in other sections.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

Figure 1 shows the binding of monoclonal antibodies DF-200 with the General determinants of the various human KIR2DL receptors.

Figure 2 shows the neutralizing monoclonal antibodies DF-200 KIR2DL-mediated inhibition of cytotoxic de the positive effects on KIR2DL1 cells on NK Cw4 positive target cells.

Figure 3 shows monoclonal antibodies DF-200, Fab-fragment DF-200 and specific conventional antibodies to KIR2DL1 or KIR2DL2/3, neutralizing KIR2DL-mediated inhibition of the cytotoxic effect of KIR2DL1 positive NK cells positive for Cw4 target cells and KIR2DL-mediated inhibition of the cytotoxic effect of positive KIR2DL2/3 of NK cells positive for Cw3 target cells.

Figure 4 shows the recovery of cell lysis positive for HLA Cw4 target cells by NK clones in the presence of fragments F(ab')2 antibodies DF-200 and EV.

5 and 6 show monoclonal antibodies DF-200, NKVSF1 (pan2D), human antibody 1-7F9, 1-4F1, 1-6F5 and 1-6F1, as well as conventional antibodies specific to KIR2DL1 or KIR2DL2/3, neutralizing KIR2DL-mediated inhibition of the cytotoxic effect of KIR2DL1 positive NK cells positive for Cw4 target cells (transfetsirovannyh Cw4 cells figure 5 cells and EBV figure 6).

Fig.7 shows the map epitopes, reflecting the results of experiments on competitive binding, obtained by analysis of surface plasmon resonance (BIAcore®) with antibodies to KIR (KIR2DL1), where overlapping circles denote the overlap in binding to KIR2DL1. The results show that antibody 1-7F9 - compete for binding to the KIR2DL1 for EV and 1-4F1, but not for NKVSF1 and DF-200. In turn, antibodies 1-4 F1 compete with EV, DF-200, NKVSF1 1-7 and F9. Antibodies KVSF1 compete for binding to KIR2DL1 with DF-200, 1-4F1 and IV, but not with 1-7F9. DF-200 compete for binding to KIR2DL1 with NKVSF1, 1-4F1 and IV, but not with 1-7F9.

Fig shows map epitopes, reflecting the results of experiments on competitive binding, obtained by the analysis BIAcore® with antibodies to KIR (KIR2DL3), where overlapping circles denote the overlap in binding with KIR2DL3. The results show that antibodies 1-4F1 compete for binding with KIR2DL3 with NKVSF1, DF-200, gl183 and 1-7F9. Antibody 1-7F9 compete for binding with KIR2DL3 with DF-200, gl183 and 1-4F1, but not with NKVSF1. NKVSF1 compete for binding with KIR2DL3 with DF-200, 1-4F1 and GL183, but not with 1-7F9. DF-200 compete for binding with KIR2DL3 with NKVSF1, 1-4F1 and 1-7F9, but not with GL183.

Fig.9 shows the map epitopes, reflecting the results of experiments on competitive binding, obtained by the analysis BIAcore® with antibodies to KIR (KIR2DS1), where overlapping circles denote the overlap in binding with KIR2DS1. The results show that antibodies 1-4F1 compete for binding with KIR2DS1 with NKVSF1, DF-200 and 1-7F9. Antibody 1-7F9 compete for binding with KIR2DS1 with 1-4F1, but do not compete with DF-200 and NKVSF1. NKVSF1 compete for binding with KIR2DS1 with DF-200 and 1-4F1, but not with 1-7F9. DF-200 compete for binding with KIR2DS1 with NKVSF1 and 1-4F1, but not with 1-7F9.

Figure 10 shows the titration NKVSF1 (Pan2D) mAb, demonstrating the binding of mAb with NK cells of Cynomolgus monkeys. The NK cells of Cynomolgus monkeys (total weight of NK cells on the 16th day) were incubated with different amounts of Pan2D mAb with posleduyuschimi conjugated with PE fragments F(ab')2 goat antibodies to mouse IgG (H+L). The percentage of positive cells was determined ezotericheskim control (purified mouse IgG1). Samples in the selection duplicated. MFI - mean fluorescence intensity.

Fig shows comparative parallel alignment of amino acid sequences of the variable regions of light chain and CDR variable regions of light chains of antibodies DF-200 and Pan2D mAb.

Fig variable shows a plot of the heavy chain of the antibody DF-200.

INFORMATION CONFIRMING the POSSIBILITY of carrying out the INVENTION

Antibodies

The present invention provides a new antibodies and their fragments or derivatives that are associated with the General determinants of human inhibitory KIR receptors, preferably with determinant present on at least two different products KIR2DL gene, and cause stimulation of NK cells expressing at least one of these receptors KIR. In the invention, first of all, reveals that such cross-reactive and neutralizing antibodies can be obtained, which is an unexpected result and opens the way for new and effective treatments based on the NK cells, in particular for human individuals. In preferred embodiments, the implementation of the antibodies are monoclonal antibody NKVSF1.

In the context of this image is the shadow of the term "common determinants" denotes the determinant or epitope, available (distributed) in several gene products of the human inhibitory KIR receptors. Preferably, the common determinants had at least two representatives of the group of KIR2DL receptors. More preferably, the determinants were, at least, KIR2DL1 and KIR2DL2/3. Some antibodies of the present invention can, in addition to recognition several products KIR2DL gene, to identify the determinants of the possession of other inhibitory KIR (such as the gene product from the group of receptors KIR3DL). The determinant or epitope can be a peptide fragment or a conformational epitope present in the specified representatives. In a more specific embodiment, antibodies of the present invention specifically bind to essentially the same epitope that is recognized by monoclonal antibodies DF-200. This determinant can be found with KIR2DL1 and KIR2DL2/3.

Within the context of the present invention, the term antibody that "binds" with the General determinants, refers to an antibody that binds to the specified determinants on the basis of the specificity or affinity (affinity).

The term "antibody", as used here, refers to polyclonal and monoclonal antibodies as well as fragments and derivatives of these polyclonal and monoclonal ant the phone, if it is not otherwise defined or does not contradict the obvious context. Depending on the type of the constant domain of heavy chains, antibodies full length usually refers to one of the five major classes: IgA, IgD, IgE, IgG and IgM. Some of them are still divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The constant domains of the heavy chains, corresponding to different classes of immunoglobulins, mean respectively "alpha (α), Delta (δ), Epsilon (ε), gamma (γ)and mu (µ)". Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known. Preferred classes of antibodies used in the present invention are IgG and/or IgM, as this is the most common antibodies in the physiological process and they are easier to prepare in the laboratory. Preferably the antibody according to the present invention are monoclonal antibodies. Since one of the objectives of the invention is to block the interaction in vivo inhibitory KIR receptor and its ligand HLA without prejudice to NK cells, usually prefer corresponding to Fc receptors isotypes, which send a weak function of the effector (such as IgG4).

Antibodies according to the present invention can be obtained by various welding is odik, known in this field. Usually they are produced by immunization of animals (not humans), preferably mice, the immunogen containing polypeptide inhibitory KIR, preferably KIR2DL polypeptide, more preferably KIR2DL polypeptide person. Polypeptide inhibitory KIR may contain the sequence of the full length polypeptide of the human inhibitory KIR or its fragment or derivative, usually immunogenic fragment that is part of a polypeptide containing the epitope exposed on the surface of cells expressing inhibitory KIR receptor. Such fragments typically contain at least about 7 consecutive amino acids from the sequence of the Mature polypeptide and even more preferably at least about 10 consecutive amino acids. Typically, the fragments originate from the extracellular domain of the receptor. Even more preferred polypeptide of the human KIR2DL, including at least one, and preferably both of the extracellular immunoglobulin domains of the polypeptide KIRDL full length and is able to simulate at least one conformational epitope present in the KIR2DL receptor. In other embodiments, implementation of the specified polypeptide contains at least 8 consecutive amino acids of the extracellular immunoglobulin the new domain with the amino acids in positions 1-224 polypeptide KIR2DL1 polypeptide (amino acids are numbered according to the WEB site PROW: http://www.ncbi.nlm.nih.gov/prow/guide/1326018082.htm, describing the family of KIR genes).

In the most preferred embodiment, the immunogen contains the polypeptide of the human KIR2DL wild type in the lipid membrane, usually on the cell surface. In a specific embodiment, the immunogen contains undamaged (intact) NK cells, in particular the intact human NK cells, at the discretion of the processed or lysed.

Stage immunization antigen mammal (not a person) can be carried out by any method well known in this area to stimulate the production of antibodies in mice (see, for example, E. Harlow and D. Lane "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988). Further, the immunogen is suspended or dissolved in a buffer, optionally with an adjuvant such as complete Freund adjuvant. Methods of determining the quantity of immunogen, of buffer types and quantities of adjuvant known to experts in this field and are in no way limiting to the present invention. For different immunogens these parameters can vary, but can easily be defined.

Similarly, in this area is well known for localization and frequency of immunization, sufficient to stimulate the production of antibodies. In a typical mode of immunization of animals (not people) is injected antigen intraperitoneal injection in 1-the day and then again a week later. This is followed by "reminding" the injection of antigen around the 20th day, at the discretion together with adjuvant, such as incomplete Freund adjuvant. Reminiscent of intravenous injections are performed, and can be repeated in the next few days. This is followed by a booster injection on the 40th day, or intravenous, or intraperitoneal, usually without adjuvant. This mode results in approximately 40 days to the appearance of b cells producing antigen-specific antibodies. Can also be used for other modes, if they lead to the production of b cells expressing antibodies to the antigen used in immunization.

For preparation of polyclonal antibodies from the immunized animal (not human) get the serum and allocate present in the antibody using well known methods. The serum can be cleaned affine methods using any of the above immunogenic attached to an insoluble carrier, thereby to obtain antibodies that react with inhibitory KIR receptors.

In an alternative embodiment of the non-immunized animal (not human) was isolated cells, grow them in vitro and then brought into contact with the immunogen in cell culture. Then lymphocytes select and teach stage of the merger described below.

For receiving the Oia monoclonal antibodies, the next step is the selection of the immunized animal (not human) splenocytes and subsequent fusion of the splenocytes with immortal cells to get producing antibodies of hybridoma. The selection of splenocytes from the animal (not human) is well known in this field and, as a rule, involves removal of the shot animal (not human) spleen, cutting it into small pieces and punching of splenocytes from the splenic capsule through the nylon mesh cell strainers in a suitable buffer with obtaining a suspension of individual cells. Cells are washed, centrifuged and resuspended in the buffer, which analyzes all red blood cells. The solution is again centrifuged and the lymphocytes from the precipitate finally resuspended in fresh buffer.

Being isolated and in a state of suspension of single cells, lymphocytes can be fused with immortal line of cells. This is usually a cell line of mouse myeloma, although this region is famous for many other immortal line of cells, suitable for a hybrid. The preferred line of murine hybridomas include (but are not limited to) lines derived from murine tumors MORSE-21 and MPC-11 and provided by the Salk Institute Cell Distribution Center, San Diego, Calif. U.S.A., cells H Ag8653 and SP-2, provided by the American Type Culture Collection, Rockville, Maryland U.S.A. Merger carried out using polyethylene glycol or similar additives. Then the obtained hybridoma grown in selective medium containing one or more, and the substances which inhibit the growth or survival nesmith, parental myeloma cells. For example, if the parental myeloma cells lacking the enzyme gipoksantin-guanine phosphoribosyl-transferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains gipoksantin, aminopterin and thymidine - substances that prevent the growth deficient in HGPRT cells (Wednesday HAT).

Hybridoma grow, as a rule, the nutrient layer of macrophages. Macrophages preferably derived from animals of the same litter, and used to highlight splenocytes mammal (not people). Usually a few days before sowing of splenocytes spend a priming macrophages incomplete Freund adjuvant or anything like that. Merging method described in Goding, "Monoclonal Antibodies: Principles and Practice". Academic Press, 1986. P.59-103. This work is incorporated into this description by reference.

Cells give rise to a selective medium for a time sufficient for the formation of colonies and producing antibodies. It usually takes from about 7 days to about 14 days. Then colonies of hybridomas examined for the production of antibodies that cross-react with many products of the gene inhibitory KIR receptor. Usually used for analysis enzyme-linked immunosorbent assays ELISA with colorimetric determination, although mo is ut to be applied many methods of analysis, adapted to the analysis of cells that grow hybridoma. Other methods of analysis include immunoprecipitation and radioimmunological analysis. Cell, giving a positive reaction for producing the desired antibodies are examined to determine whether in them one or more of visible colonies. If there is more than one colony, the cells can be re-cloned and grown, to ensure that only a single cell has the appearance of colonies that produce the desired antibodies. Positive cells are clearly single colony is usually subjected to repeated cloning and re-analyzed to ensure that only one type of monoclonal antibodies is detected and reproduced. Antibodies can also be obtained by selection of combinatorial libraries of antibodies, as disclosed, for example, in the work of Ward and others // Nature. 1989. T. S.

Antibodies according to the present invention is capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells; in particular the inhibition mediated KIR2DL receptors, and more specifically the inhibition, at least, and KIR2DL1 and KIR2DL2/3. These antibodies are therefore "neutralizing" or "any abscopal" antibodies, in the sense that they block at least partially and significantly, inhibitory signaling pathway mediated Retz what Ptolemy KIR, when they interact with class I molecules MHC. More importantly, this inhibitory activity covers several types of inhibitory KIR receptors, preferably several products KIR2DL receptor gene, and more preferably is at least, and KIR2DL1 and KIR2DL2/3, so that these antibodies can be used with high efficiency in different individuals. Neutralizing KIR mediated inhibition of cytotoxicity of NK cells can be assessed using various assays or tests such as analysis of binding or cellular assays.

Once the antibodies cross-reacting with multiple inhibitory KIR receptors, have been identified, they can be tested for their ability to neutralize the inhibitory activity of these receptors KIR in intact cells NK. In a specific embodiment, the neutralizing activity can be thought of as the ability of these antibodies to restore lysis positive for HLA-C targets positive KIR2DL NK clones. In another specific embodiment, the neutralizing activity of the antibodies is determined by the ability of antibodies to inhibit the binding of molecules HLA-C receptors KIR2DL1 and KIR2DL3 (or closely related KIR2DL2), more preferably as the ability of the antibody to change:

binding molecules HLA-C, selected from Cw1, Cw3, Cw7 and Cw8 (or m is likely HLA-C, having in the position 80 residue Asn), KIR2DL2/3; and

binding molecules HLA-C, selected from Cw2, Cw4, Cw5 and Cw6 (or molecule HLA-C with position 80 the residue Lys), KIR2DL1.

In another embodiment, the inhibitory activity of the antibodies of the present invention can be evaluated in cell test for cytotoxicity, as further disclosed in the Examples.

In another embodiment, the inhibitory activity of the antibodies of the present invention can be evaluated in the test with a cytokine, where NK cells are incubated with the test antibody and with a line of target cells expressing one allele HLA-C, identifiable by KIR molecule populations of NK to stimulate the production of cytokines by NK cells (for example, the production of IFN-γ and/or GM-CSF). In the example Protocol, the production of IFN-γ by cells RVMS assessed by staining cell surface and intracytoplasmic regions and analysis after 4 days of cultivation using flow cytometry. Briefly, Brefeldin A (Brefeldin A (Sigma Aldrich)) can be added at a final concentration of approximately 5 μg/ml, at least 4 hours during the cultivation. Then before permeability (IntraPrep™; Beckman Coulter) cells can be incubated with antibodies anti-D3 and anti-D56 mAb and paint specific indicators PE-anti-IFN-γ or RE-IgG1 (Pharmingen). The production of GM-CSF and IFN-γ in activated floor is clonal NK cells can be measured in the supernatant liquids by the method of ELISA (GM-CSF: DuoSet Elisa, R&D Systems, Minneapolis, MN; IFN-γ: OptE1A set, Pharmingen).

Antibodies according to the present invention can be partially or completely neutralizing KIR mediated inhibition of cytotoxicity of NK cells. The term "neutralizing KIR mediated inhibition of cytotoxicity of NK cells", as used here, means the ability to enhance, at least about 20%, preferably at least about 30%, at least about 40%, at least about 50% or more (for example, from about 25% to 100%) specific lysis obtained in the same ratio with NK cells or cell lines NK, which is not blocked their KIR, measured by the classical test for cytotoxicity the release of chromate, in comparison with the level of specific lysis obtained without antibody, when the population of NK cells expressing this KIR, bring into contact with target cells expressing the appropriate molecules of class I MHC (recognized KIR expressed on NK cells). For example, preferred antibodies according to the present invention is able to induce lysis selected or compatible HLA, or populations of autologous target cells, i.e. populations of cells that do not effectively lyse the NK cells in the absence of said antibody. On the fact of the antibodies according to the present invention can also be defined as contributing to the activity of NK cells in vivo.

The alternative term "neutralize KIR mediated inhibition" means that in the test with chromate using NK cell clone or transfectant expressing one or more of the inhibitory KIR and target cells expressing only one allele HLA, which is recognized as one of KIR on NK cell obtained in the presence of antibodies, the level of cytotoxicity will be at least about 20%, preferably at least about 30%, at least about 40%, at least about 50% (e.g., about from 25% to 100%) or more to exceed the cytotoxicity obtained with known antibodies, blocking molecules of class I MHC (such as antibodies W6/32 to class I molecules MHC).

In a specific embodiment, the antibody binds essentially the same epitope as monoclonal antibodies DF-200 (produced by hybridomas DF-200). Such antibodies here named "DF-200-like antibodies. In a further preferred embodiment, the antibodies are monoclonal antibodies. More preferred "DF-200-like antibody" of the present invention are antibodies, other than the monoclonal antibody NKVSF1. Most preferred are monoclonal antibodies DF-200 (produced by hybridomas DF-200).

The term "binds essentially the same with the th epitope or determinant, as against the interest of the antibody indicates that the antibody "competes" with the specified interest antibody. The term "binds essentially the same epitope or determinant as" to monoclonal antibody DF-200, means that the antibody "competes" with DF-200. Typically, an antibody that "binds essentially the same epitope or determinant as" interest monoclonal antibody (e.g., DF-200, NKVSF1, 17F9), means that the antibody "competes" with interest monoclonal antibody for any one of a larger number of KIR molecules, preferably it is the molecule KIR selected from the group consisting of KIR2DL1 and KIR2DL2/3. In other examples, the antibody that binds essentially the same epitope or determinant on the molecule KIR2DL1, as interest antibody "competes" with interest antibody for binding to KIR2DL1. Antibody that binds essentially the same epitope or determinant on the molecule KIR2DL2/3, as interest antibody "competes" with interest antibody for binding to KIR2DL2/3.

The term "binds essentially the same epitope or determinant as" of interest antibody means that the antibody "competes" with the decree of the tion of interest antibody for any molecule and for all molecules KIR, which specifically binds a specified interest antibody. The term "binds essentially the same epitope or determinant as" monoclonal antibody DF-200, means that the antibody "competes" with DF-200 for any molecule and for all KIR molecule that specifically binds DF-200. For example, the antibody that binds essentially the same epitope or determinant as monoclonal antibodies DF-200 or NKVSF1, "competes" with the specified DF-200 or NKVSF1, respectively, for binding to KIR2DL1, KIR2DL2/3, KIR2DS1 and KIR2DS2.

Identification of one or more antibodies that binds (bind) essentially or substantially the same epitope as described herein, monoclonal antibodies, can be easily implemented using one or more immunological screening methods of analysis, in which it is possible to evaluate the competition of antibodies. A large number of such methods of analysis are widely used in practice and is well known in the art (see, for example, U.S. patent No. 5660827, published August 26, 1997, which is incorporated into this description by reference). It should be understood that the actual definition of the epitope, which binds described here antibody in any way is not necessary to identify the antibody to bind to the same and is and is essentially the same epitope as described here monoclonal antibody.

For example, if you want to analysis the subjects antibodies obtained from different animals, or even have different immunoglobulin isotype, can be applied to a simple competitive analysis, in which the mix (or pre-adsorb) control (for example, DF-200) and the subjects antibodies and add them to the sample containing both KIR2DL1 and KIR2DL2/3, each of which, as is known, is associated with DF-200. For use in such simple research competition suitable protocols, based on different modifications of ELISA, radioimmunoassay, Western-blokirovanie, and can be applied BIACORE analysis (as described, for example, then, in the "Examples"section).

In some embodiments, the implementation can pre-mix the control antibody (e.g., DF-200) with different amounts of the tested antibodies (for example, in a ratio of about 1:10 or about 1:100) at the time to make samples with antigen - inhibitory KIR. In other embodiments, implementation of control and different number of test antibodies can be simply mixed during exposure with the sample antigen KIR. While it is possible to distinguish the bound antibodies from free (for example, using the technique of separation or launder to remove unbound antibodies) and DF-200 from test antibodies (e.g., COI is lsua species-specific or isotype-specific secondary antibodies or specific marking DF-200 recognizable label), you can define, reduce whether subjects antibodies binding DF-200 with two different antigens KIR2DL, which would indicate that the subjects antibodies recognize essentially the same epitope as the DF-200. Binding (labeled) control antibody in the presence of completely unrelated to the case of antibodies may serve as a control upper threshold. The lower the threshold control can be obtained by incubating labeled (DF-200) antibodies with unlabeled antibodies of exactly the same type (DF-200), which may be a competition that reduces the binding of labeled antibodies. In the test analysis a significant decrease in the reactivity of the labeled antibodies in the presence of the tested antibodies indicates the presence of the tested antibodies that recognize essentially the same epitope, i.e., the epitope that is "cross reactive" with a labeled (DF-200) antibodies. Any subjects antibodies that reduce the binding of DF-200 with each of the antigens KIR2DL1 and KIR2DL2/3, at least about 50% (e.g., at least about 60%, or preferably at least about 70% (for example, approximately 65-100%)), for any value of the PR-200 : test antibody in the range of from about 1:10 to about 1:100, are considered as antibody binding essentially to the same epitope or determinant that DF-200. Prefer the Ino, to such subjects antibodies reduced the binding of DF-200 with each of the antigens KIR2DL, at least about 90% (e.g., 95%).

Competition can be assessed, for example, using flow cytometry. In this test, cells carrying this KIR, you can preincubating, for example, DF-200, and then subjects with antibodies labeled fluorochrome or Biotin. We can say that the antibody competes with the DF-200, if the binding obtained with inactivated with a saturating amount of DF-200 is about 80%, preferably about 50%, about 40% or less (for example, approximately 30%) of the binding (measured by fluorescence)obtained with antibodies without inactivated with DF-200. Alternatively, it is believed that the antibody competes with the DF-200, if obtained with labeled (fluorochrome or Biotin) DF-200 binding to cells pre-incubated with saturating amounts of the tested antibodies is approximately 80%, preferably about 50%, about 40% or less (for example, approximately 30%) of the binding obtained without inactivated by antibodies.

You can also benefit from using a simple analysis of competition in which subjects antibodies pre-adsorb and applied at saturating concentrations to the surface, n is which immobilized and KIR2DL1, and KIR2DL2/3. The preferred surface for simple analysis of competition is the BIACORE chip (or other medium suitable for analysis based on surface plasmon resonance). Then the control antibody (e.g., DF-200) is brought into contact with the surface at saturating concentrations of KIR2DL1 and KIR2DL2/3 and measure the binding of the control antibody to the surface, carrying KIR2DL1 and KIR2DL2/3. This binding of the control antibody compared to the binding of the control antibody to the surface, carrying KIR2DL1 and KIR2DL2/3, in the absence of the tested antibodies. In the test analysis, a significant reduction of binding of the control antibody to the surface, carrying KIR2DL1 and KIR2DL2/3, in the presence of the tested antibodies indicates that the subjects antibodies recognize essentially the same epitope as the reference antibody, so that the subjects antibodies are cross-reactive in relation to the control antibody. Any subjects antibodies that reduce the binding of control (such as DF-200) antibodies to each of the antigens KIR2DL1 and KIR2DL2/3, at least about 30%, or preferably about 40%, can be considered as antibodies that are associated essentially with the same epitope or determinant as the control antibody (e.g., DF-200). Preferably, these subjects antibodies reduced the binding of the control antibody (e.g., DF-200) with each of the antigens KIR2DL, at least about 50% (e.g., at least about 60%, at least about 70% or more). It should be recognized that the order making the reference and the tested antibodies can be changed: this means that the control antibodies may be associated with the first surface, and thereafter subjects the antibody is brought into contact with the surface in the competition analysis. Preferably, with the surface containing KIR2DL1 and KIR2DL2/3, were first linked antibodies with greater affinity to antigens KIR2DL1 and KIR2DL2/3, since it can be expected that the decrease in binding is detected for the second antibody (assuming that antibodies are cross-reactive)will be more significant. Additional examples of such analyses are given in the "Examples" section and, for example, Saunal and Regenmortel // J. Immunol. Methods. 1995. T. Pp.33-41, information which is incorporated into this description by reference.

Although described in the context of this application means for DF-200 is shown for purposes of example, it should be recognized that the above-described screening methods of immunological analysis can be applied also to identify antibodies that compete with NKVSF1, 1-7F9, EV, GL183, as well as other antibodies of the present invention.

After immunization and production of the antibodies is the spine of the animal or in the cage for the selection of antibodies can be carried out in a special stage of selection, as indicated in the claims. Which, in a specific embodiment, the invention also relates to methods of producing such antibodies, including:

(a) immunizing a mammal (non-human) immunogen containing polypeptide inhibitory KIR;

(b) preparing antibodies from the immunized animal, and the antibody binds to a polypeptide KIR;

(c) selecting antibodies of step (b), which cross-react with at least two different gene products inhibitory KIR; and

(d) the selection of antibodies from step (C), which is capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in a population of NK cells expressing at least two different gene product of the human inhibitory KIR receptors.

Selection of antibodies, which cross-reacts with at least two different gene products inhibitory KIR, can be achieved by screening antibodies against two or more different antigens inhibitory KIR - for example, as described above.

In a more preferred embodiment, the antibody prepared in the step (b)are monoclonal antibodies. Thus, the term "preparation of antibodies from the immunized animal", as used here, includes obtaining from the immunized animal cells and use the use of these cells to obtain hybridomas expressing antibodies, and antibodies directly from the serum of immunized animals. In another preferred embodiment, the antibodies selected in step (C)are those antibodies that cross-react with at least KIR2DL1 and KIR2DL2/3.

In another preferred embodiment, the antibodies selected in step (d), cause target cells expressing the appropriate class I molecule HLA, at least about 10%specific lysis mediated by the action of NK cells that detect at least one recognized by KIR antibodies, and preferably at least about 40%specific lysis at least about 50%specific lysis, or more preferably at least about 70%specific lysis (e.g.approximately 60-100%specific lysis), measured in standard test release of chromate, in comparison with lysis or cytotoxicity obtained with the same ratio of effector/target cells to NK, which is not blocked their receptors KIR. In the alternative, if selected in step (d) antibodies used in the test with chromate using NK cell clone expressing one or more of the inhibitory KIR and target cells expressing the only one allele HLA, which is recognized as one of KIR on NK clone obtained with these antibodies, the level of cytotoxicity should be at least about 20%, preferably at least about 30% or more, the level of cytotoxicity obtained with a blocking monoclonal antibody (mAb) to molecules, class 1 MHC - such as antibodies W6/32 to molecules, class 1 MHC.

The order of the steps (C) and (d) in the above-described method can be changed. Optionally, the method can also or alternatively include additional stages to obtain fragments of the monoclonal antibodies, or derivatives of monoclonal antibodies or fragments (e.g., as described in the present description).

In a preferred embodiment, the animal (not human), used to generate antibodies according suitable for use with the methods of the present invention is a mammal such as a rodent (e.g. mouse, rat, etc.), cattle, pig, horse, rabbit, goat, sheep etc., a Mammal (not people) can also be genetically modified or engineered to produce human antibodies. Such mammals may be, for example, Xenomouse™ (Abgenix) or HuMAb-Mouse™ (Medarex).

In another embodiment, the invention provides a method of producing antibodies, including:

(a) the selection of Bible the Teka or set, monoclonal antibody fragment is a monoclonal antibody or a derivative of any of them, which cross-reacts with the products of at least two different genes of the human inhibitory KIR2DL receptors, and

(b) the selection of the antibody, fragment or derivative (a)is capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells expressing the products of at least two different genes of the human inhibitory KIR2DL receptors.

The set can be any (recombinant) the set of antibodies or their fragments, at the discretion (i.e. not necessarily) provided by any suitable structure (e.g., phage, bacteria, synthetic complex, etc.). The selection of inhibitory antibodies can be produced, as has been disclosed above and will be further illustrated by examples.

According to another variant implementation of the invention provides hybridoma representing a cell of the host, not a person, and b-cell produces antibodies that bind to determinants that are available on the products, at least two different genes of the human inhibitory KIR receptors, and antibody can neutralize the inhibitory activity of the receptors. Preferably, hybridoma according to this aspect of the invention was not hybridomas, producers the th monoclonal antibody NKVSF1. Hybridoma according to this aspect of the invention can be created, as described above, by fusion of splenocytes from the immunized mammal, not a person, with the immortal line of cells. Obtained in this merge hybridoma can be sorted on the presence of such cross-reactive antibodies, as described in the present description. Preferably, hybridoma have produced an antibody that recognizes a determinant present on the products, at least two different KIR2DL gene and enhances the activity of NK cells expressing at least one of these receptors KIR. More preferably, hybridoma was produced antibody that binds essentially the same epitope or determinant that and DF-200, and enhances the activity of NK cells. Most preferably, hybridomas was hybridoma DF-200, producing monoclonal antibody DF-200.

Hybridoma, which confirmed the production of monoclonal antibodies of the present invention, can be grown in larger quantities in a suitable medium such as DMEM or RPMI-1640. Alternatively, cells hybridomas can be grown in vivo as ascitic tumors in the animal.

Once achieved sufficient growth to produce the necessary antibodies containing monoclonal is ntitle growth environment (or ascitic fluid) is separated from the cells and purify present in the monoclonal antibodies. Cleaning is usually performed by means of electrophoresis in a gel, dialysis, chromatography on sepharose with sewn protein And or separate with sewn protein G, or to mouse immunoglobulin antibodies associated with an insoluble carrier such as agarose pellets or sepharose (all of these methods are described, for example, in Antibody Purification Handbook", Amersham Biosciences, publication No. 18-1037-46, edition AC, whose contents are incorporated into this description by reference). Bound antibodies, typically elute from the column with protein a or protein G buffer with low pH (glycine or acetate buffer of pH 3.0 or below). Containing antibody fractions are immediately neutralized. These fractions are combined cialiswhat and concentrate as needed.

According to alternative implementation, the DNA encoding the antibody that binds to a determinant present on the products, at least two different genes of the human inhibitory KIR receptors, isolated from the hybridomas of the present invention and placed in a suitable expressing vector for transfection of a suitable host. Then the master used for the recombinant production of antibodies or their variants such as "humanized" versions of such monoclonal antibodies, active fragments of antibodies or chimeric antibodies containing recognize the antigen part of antit the La. Preferably used in this embodiment, the DNA encodes an antibody that recognizes a determinant present in the products of at least two different KIR2DL gene, and enhances the activity of NK cells expressing at least one of these receptors KIR. More preferably, the DNA encodes the antibody binding essentially to the same epitope or determinant that and DF-200, and increased activity of NK cells. Most preferably, the DNA encodes a monoclonal antibody DF-200.

DNA encoding the monoclonal antibodies of the present invention, can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specific contact with the genes encoding the heavy and light chains of murine antibodies). After DNA extraction can be placed in expressing vectors, which are then transferout in cell host such as E. coli cells, simian COS cells, ovarian cells of the Chinese hamster (Cho) or myeloma cells that do not otherwise produce protein immunoglobulin, to obtain the synthesis of monoclonal antibodies in the recombinant cell host. Recombinant expression in bacteria of DNA encoding the antibodies, are well known in the art (see, for example, Skerra, etc. // Curr. Opinion in Immunol. 1993. T.. S; and Pluckthun // Immunol. Revs. 1992. T. S.

Fragments and derivatives of monoclonal antibodies

Fragments and derivatives of the antibodies of the present invention (which are covered by the terms "antibody" or "antibodies"as used in this application, unless otherwise agreed or unless contrary to the context), preferably DF-200-like antibodies can be obtained using methods known in this field. "Immunoreactive fragments contain a portion of an intact antibody, generally the antigen binding site or variable plot. Examples of fragments of antibodies include Fab fragments, Fab', Fab'-SH, F(ab')2and Fv; dia-antibodies (double antibody, diabodies); any fragment of the antibody, which is a polypeptide having a primary structure consisting of one unbroken sequence of adjacent amino acid residues (which here are called "single-chain fragment antibodies or single-chain polypeptide"), including (but not limited to) (1) single-chain Fv molecules (scFv), (2) single-chain polypeptides containing only one variable domain light chain or fragments containing the three CDRs of the variable domain of the light chain, without an attached part of the heavy chain, and (3) single-chain polypeptides containing only one variable plot heavy chain or a fragment containing t and CDR of the variable segment of the heavy chain, without an attached part of the light chain; and polyspecific antibodies formed by fragments of antibodies. For example, Fab fragments or F(ab')2 can be obtained proteases splitting of the selected antibodies according to conventional methods. Admittedly, immunoreactive fragments can be modified using known methods, for example, to slow excretion in vivo and more acceptable pharmacokinetic profile fragment can be modified by polyethylene glycol (PEG). Methods of binding and site-specific conjugation of the PEG with the fragment Fab' described, for example, in the works Leong, etc. // Cytokine. 2001. So 16, No. 3. P.106-119; and Delgado and others // Brit. J. Cancer. 1996. T. No. 2. S-182, the contents of which are incorporated into this description by reference.

In the private aspect, the invention provides antibodies, antibody fragments and derivatives of antibodies containing the sequence of the variable segment light chain DF-200, as shown below in Fig. In another private aspect, the invention provides antibodies, antibody fragments and derivatives of antibodies containing the sequence of the variable segment light chain Pan2D, as shown below in Fig. In another aspect, the invention provides antibodies, antibody fragments and derivatives of antibodies containing one or more of the CDRs of the variable segment light chain DF-200,as shown below in Fig. In another aspect, the invention provides antibodies, antibody fragments and derivatives of antibodies containing one or more of the CDRs of the variable segment light chain Pan2D, as shown below in Fig. Functional variants/analogs of such sequences can be created by appropriate substitutions, insertions and/or deletions in these amino acid sequences were made using standard methods, which can contribute to the mapping of sequences. For example, the CDR residues that are conservative in comparison Pan2D and DF-200, may be suitable targets for modification, as these residues can not give contribution to the different profiles of the competition of these antibodies with other antibodies disclosed in the present invention (although Pan2D and DF-200 are not competitors), and therefore may not give a contribution to the specificity of these antibodies to their respective specific epitopes. In another aspect of deletions, substitutions and/or insertions may be suitable position, which amino acid residue occupies in the sequence of one of these antibodies, but not others.

In a separate aspect, the invention provides antibodies, antibody fragments and derivatives of antibodies containing the sequence of the variable segment of the heavy chain DF-200, as shown in Fig. In another aspect from retina provides antibodies, antibody fragments and derivatives thereof that contain one or more of the CDRs of the variable segment of the heavy chain DF-200, as shown in Fig. Functional variants/analogs of such sequences can be created by appropriate substitutions, insertions and/or deletions in these disclosed amino acid sequences made using standard methods, which can contribute to the mapping of sequences. In another aspect of deletions, substitutions and/or insertions may be suitable position, which amino acid residue occupies in the sequence of one of these antibodies, but not others.

Alternatively, the DNA hybridoma producing the antibody according to the present invention, preferably DF-200-like antibody, can be modified so that it encodes the antibody fragment according to the present invention. The modified DNA is then inserted into expressing vector and used for transformation or transfection of a suitable cell, which then expresses the desired fragment.

In an alternative implementation of the DNA hybridoma producing the antibody according to the present invention, preferably DF-200-like antibody can be modified before insertion in expressing vector - for example, by replacing the coding sequence glacialoides constant domains of the heavy and light chains are not homologous human sequences (e.g., Morrison and others// Proc. Natl. Acad. Sci. U.S.A. 1984. T. S), or by joining a sequence that encodes an immunoglobulin, all or part of the coding sequence for nimmanahaeminda polypeptide. Thus, preparing a "chimeric" or "hybrid" antibodies having the binding specificity of the original antibody. Typically, such nimmanahaeminda polypeptides replace the constant domains of the antibodies according to the present invention.

Thus, according to another variant implementation of the antibodies, preferably DF-200-like antibodies, are "humanization". "Humanized" (humanized forms of the antibodies according to the present invention are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2or other antigen-binding sequences of antibodies)that contain minimal sequence derived from murine immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody)in which residues from a complementarity determining areas (complementary-determining region CDR) of the recipient are replaced by residues from a CDR of the original antibody (donor antibody), but which retain the desired specificity, affinity (affinity) and the capacity of the original antibody. In kotoryj cases, the amino acid residues of the skeleton of the Fv human immunoglobulin can be replaced with the appropriate not human remains. Furthermore, humanized antibodies may contain residues which are not found in the recipient antibody nor in the inserted sequences, CDR or framework. These modifications do to improve and optimize the quality of the antibodies. As a rule, humanitariannet antibody essentially contains at least one, and typically two, variable domains, in which all or essentially all of the sites CDRs correspond to those parts of the original antibody, and all or essentially all of the sites of the skeleton (framework, FR) are areas of consensus sequence of human immunoglobulin. Humanized antibody optimally also contain at least part of the constant part of the immunoglobulin (Fc), typically area of the human immunoglobulin. Detailed in: Jones, etc. // Nature. 1986. T. S; Reichmann and others // Nature. 1988. T. S; and Presta // Curr. Op. Struct. Biol. 1992. Vol.2. S.

Methods of humanizing antibodies according to the present invention are well known in this field. As a rule, humanitariannet antibody according to the present invention has included one or more amino acid residues from the original antibody. These murine or other non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization essentially can the be performed according to the method of Winter and co-authors (Jones and others // Nature. 1986. T. S; Riechmann, etc. // Nature. 1988. T. S; Verhoeyen, etc. // Science. 1988. T. S). In accordance with this such humanitarianism" antibodies are chimeric antibodies (Cabilly et U.S. Patent No. 4816567), where part, substantially less intact human variable domain has been substituted by the corresponding sequence from the initial antibody. In practice, humanized antibodies according to the present invention is typically human antibodies in which some CDR residues and possibly some FR residues substituted by residues from analogous sites in the original antibody.

To reduce the antigenicity very important choice of human variable domains, and light and heavy, to be used in making a humanized antibody. According to the so-called method of "best fit" "best fit"), screening the sequence of the variable domain of the antibody of the present invention in comparison with a full library of known sequences of human variable domain. The human sequence which is closest to that of the mouse, then accepted as the human skeleton (framework, FR) for gumanitarnogo antibody (Sims and others// J. Immunol. 1993. T. S; Chothia and Lesk // J. Mol. Biol. 1987. T. S. In another method uses a special frame of consensus sequences of vs the x human antibodies specific subgroup of light or heavy chains. The same frame can be used for several different humanized antibodies (Carter and others // Proc. Natl. Acad. Sci. U.S.A. 1992. T. S; Presta et // J. Immunol. 1993. T).

Further important that antibodies were humanitarian with retention of high affinity to many inhibitory KIR receptors and other useful biological properties. To achieve this goal, according to the preferred method, humanized antibodies are prepared in the process of analysis of the parental sequences and various speculative humanized products using three-dimensional models of the source and humanized sequences. Three-dimensional models of immunoglobulin widely available and well-known experts in this field. There are computer programs that illustrate and design the possible three-dimensional conformational structures of selected promising sequences of immunoglobulin. Examination of these structures allows to consider the likely role of amino acid residues in the functioning of a sequence of perspective immunoglobulin, i.e. identify residues that influence the ability of the immunoglobulin candidate to bind its antigen. This way, FR residues can be selected and composed of consensus and import sequences so that achieves the required characteristics of n is necessary antibodies - such as increased affinity for the target antigen (antigens target). Typically, the CDR residues are directly and most substantially affect binding to the antigen.

Another way to get a "humanized" monoclonal antibody is used in the role of mice for immunization of transgenic animal is a XenoMouse® (Abgenix, Fremont, CA). XenoMouse is a mouse host according to the present invention, in which the genes of the immunoglobulin replaced by a functional human immunoglobulin genes. Therefore, antibodies produced by this or mouse hybridomas made from b-cells of this mouse already humanitarian. XenoMouse described in U.S. patent No. 6162963, which is incorporated into the present application by reference in its entirety. A similar method can be carried out with the use of transgenic animal - HuMAb-Mouse™ (Medarex).

Human antibodies can also be obtained using various other techniques - for example, using other immunization of transgenic animals engineered to Express a set of human antibodies (Jakobovitz, etc. // Nature. 1993. T. S.255), or by selection of a set of antibodies using phage design. Such techniques are well known to experienced professionals and can be carried out, starting with monoclonal antibodies as a source, as disclosed in this application.

Antibodies which according to the present invention, preferably DF-200-like antibodies, may also be modified in a "chimeric" antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in the original antibody, while the remainder of the chain (chain) is identical with or homologous 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 they exhibit the desired biological activity (Cabilly, and others, see above; Morrison and others // Proc. Natl. Acad. Sci. U.S.A. 1984 T. S).

Other derivatives within the scope of the present invention include functionalized antibodies, i.e. antibodies, conjugated or covalently connected to a toxin such as ricin, diphtheria toxin, abrin and Pseudomonas exotoxin; with a recognizable part of the molecule such as a fluorescent probe, a radioisotope label or imaging agent; or a solid carrier such as agarose pellets and other Methods of conjugation or covalent compounds other agents to antibodies are well known in this field.

Conjugation with toxin useful for targeted destruction of NK cells expressing on their cell surface of one of the cross-reactive receptors KIR. When this antibody is the future of the invention binds to the cell surface of such cells, it internalized, and the toxin is released into the cell, selectively killing her. This application is an additional option of implementing the present invention.

Conjugation with a recognizable part of the molecule is useful when the antibodies of the present invention are used for diagnostic purposes. These goals include (but are not limited to the examination of biological samples for the presence of NK cells, bearing on its cell surface cross-reactive KIR receptor, and detecting in vivo the presence of NK cells bearing cross-reactive KIR. Such methods of examination and discovery are also additional embodiment of the present invention.

Conjugation of antibodies according to the present invention with the solid carrier is useful as a means of affinity purification from a source such as a biological fluid, NK cells, bearing on its cell surface cross-reactive receptor KIR. This method of cleaning is yet another additional embodiment of the present invention, since it yields a purified population of NK cells.

In an additional embodiment, antibodies of the present invention, including NKVSF1, communicating with a common determinant present on the gene products of at least two different human inhibitory KIR receptors, where these antibodies are capable of neutralizing KIR mediated inhibition of cytotoxicity of NK cells in NK cells expressing at least one of said two different human inhibitory KIR receptors, can be incorporated into liposomes ("immunoliposome"), either alone or together with another substance addressed for delivery to the animal. Such other substances include nucleic acids for gene delivery in gene therapy or for the delivery of antisense RNA, interfering RNA or small interfering RNA for the purpose of suppression of genes in NK cells, or toxins or poisons addressed to the destruction of NK cells.

Computer modeling of the extracellular domains of the KIR2DL1, KIR2DL2, and KIR2DL3 (KIR2DL1-3), based on published crystal structures (Maenaka and others (1999), Fan and others (2001), Boyington and others (2000)), predicted the participation of certain sections of KIR2DL1, KIR2DL2, and KIR2DL3 in the interaction between KIR2DL1 and cross-reacting with KIR2DL1-3 mouse monoclonal antibody DF-200 and NKVSF1. So, in one of the embodiments the present invention provides antibodies that bind to KIR2DL1 only within the area defined by amino acids(105, 106, 107, 108, 109, 110, 111, 127, 129, 130, 131, 132, 133, 134, 135, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 181, 192). In another embodiment, the invention provides antibodies, binding is to be with KIR2DL1 and KIR2DL2/3 without interaction with amino acid residues outside of the site, defined amino acid residues(105, 106, 107, 108, 109, 110, 111, 127, 129, 130, 131, 132, 133, 134, 135, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 181, 192).

In another embodiment, the invention provides antibodies that bind to KIR2DL1 and does not bind to the mutant KIR2DL1, in which R131 is l.

In another embodiment, the invention provides antibodies that bind to KIR2DL1 and does not bind to the mutant KIR2DL1, in which R157 is l.

In another embodiment, the invention provides antibodies that bind to KIR2DL1 and does not bind to the mutant KIR2DL1, in which R158 is l.

In another embodiment, the invention provides antibodies that bind with amino acid residues (131, 157, 158) KIR2DL1.

In another embodiment, the invention provides antibodies that bind with KIR2DS3(R131W), but not with wild type KIR2DS3.

In another embodiment, the invention provides antibodies that bind with KIR2DL1 and KIR2DL2/3, and with KIR2DS4.

In another embodiment, the invention provides antibodies that bind with KIR2DL1 and KIR2DL2/3, but not with KIR2DS4.

To determine whether the associated antibody within one of the sections of the epitope defined above, by methods known to experts in this field. As one example of such mapping techniques/characteristics area of the epitope for antibodies to KIR, you can define method decode traces" (foot-printing) by chemical modification of the exposed amines/carboxy in the protein KIR2DL1 or KIR2DL2/3. One of the concrete examples of this technique decryption traces is to use recorded using mass spectrometry exchange of hydrogen/deuterium (hydrogen-deuterium exchange detected by mass spectrometry, HXMS)where the exchange of hydrogen/deuterium in Amidah proteins and receptor ligand binding and reverse exchange, and amide groups of the skeleton involved in the binding of the protein, protected from reverse exchange and remain so deuterated. At this stage related to business areas can be identified using peptide proteolysis, fast separation microprobe high-performance liquid chromatography and/or mass spectrometry with electrospray ionization (see, for example: N. Ehring. // Analytical Biochemistry. 1999. T. No. 2. S-259; and/or J.R. Engen and Smith D.L. //Anal. Chem. 2001. T. SA-265A). Another example of suitable techniques identify the epitope is an epitope mapping using nuclear magnetic resonance (NMR), which usually match the position of the signals in the spectra of two-dimensional NMR of the free antigen and antigen in complex with binding to the antigen peptide such as an antibody. Typically, the antigen is selectively injected isotope tag15N, so that the NMR spectrum visible only signals corresponding to the antigen, and the signals from communicating with the antigen peptide is not visible. Typically, the position signal is Alov antigen from amino acids, involved in the interaction with the antigen-binding peptide which in the spectra of the complex in comparison with the spectra of free antigen, allowing thus to identify amino acids involved in the binding. For examples, see: // Ernst Schering Res. Found. Workshop. 2004. T. S-167; Huang and others // J. Molec. Biol. 1998. T. No. 1. Pp.61-67; and Saito and Patterson // Methods. 1996. V.9, No. 3. S-524. Mapping characterization of epitopes can also be carried out using methods of mass spectrometry (see, for example, Downward // J. Mass Spectrom. 2000. T.35. No. 4. S-503; and Kiselar and Downard // Anal Chem. 1999. T, No. 9. S-1801.

Technique proteasome splitting can also be useful in the context of mapping and identification of epitopes. Associated with antigenic determinant sites/sequences can be determined using proteasome splitting - for example, cleavage at 37°C and pH 7-8 using trypsin in a ratio of about 1:50 to KIR2DL1 or KIR2DL2/3, with subsequent mass spectrometric (MS) analysis for the identification of peptides. The peptides protected from cleavage by trypsin, bound by antibody to KIR, can then be identified by comparing the samples subjected to cleavage by trypsin, and the samples incubated with antibodies and then subjected to cleavage, for example, trypsin (thus revealing the "footprint" of the light of the data component). In such simple methods to characterize epitopes can be also or alternatively used other enzymes such as chymotrypsin, pepsin, etc. in Addition, enzymatic cleavage can provide a quick way analysis of whether the sequence of potential antigenic determinants within the site KIR2DL1, in the sense that the polypeptide anti-KIR, which is not apparent on the surface, as a consequence likely will not be detected by methods based on immunogenicity/antigenicity. For a discussion of such techniques see, for example, Manca // Ann. Ist. Super Sanita. 1991. V.27, No. 1. P.15-19.

Cross-reactivity to Cynomolgus monkeys

It was found that antibody NKVSF1 contact with NK cells from Cynomolgus monkeys (see example 7). Therefore, the invention provides antibodies and their fragments and derivatives, where the antibodies, fragments or derivatives cross-react with at least two human inhibitory KIR receptors on the surface of human NK cells and, in addition, contact with NK cells from Cynomolgus monkeys. In one embodiment, executing antibodies are antibody NKVSF1. The invention also provides a method for determining the toxicity of the antibodies and their fragments and derivatives, where the antibodies, fragments or derivatives Perekrest the react at least two human inhibitory KIR receptors on the surface of human NK cells, and the method includes the test antibodies in Cynomolgus monkeys.

Composition and introduction

The invention also provides pharmaceutical compositions containing the antibodies and their fragments or derivatives, where these antibodies, fragments or derivatives cross-react with at least two inhibitory KIR receptors on the surface of NK cells, neutralize inhibitory signals and amplify the activity of these cells in a suitable medium in a quantity effective to considerably increase the cytotoxicity of NK cells in a patient or in a biological sample containing cells NK. Compositions additionally contain a pharmaceutically acceptable carrier. Such compositions are also called "songs with antibodies according to the present invention. In one embodiment, implementation of the composition with antibodies according to the present invention contain antibodies disclosed in the above embodiments, implementation. Antibody NKVSF1 included in the scope of antibodies that may be present in compositions with antibodies according to the present invention.

The term "biological sample", as used in this application, including but not limited to) biological liquid is (for example, serum, lymph, blood), cell sample, or tissue sample (e.g., bone marrow).

Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alum, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as preteenslut, disubstituted sodium phosphate, acid phosphate of potassium, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, substances on the basis of cellulose, the polyethylene glycol, sodium salt of carboxymethylcellulose, polyacrylates, waxes, block copolymers of polyethylene with polyoxypropylene, polyethylene glycol and lanolin.

Compositions of the present invention can be applied in the method of increasing the activity of NK cells in a patient or in a biological sample. This method includes the step of contacting the composition with a patient or a biological sample. This method will be useful both for diagnostic and for therapeutic purposes.

For use with the biological sample composition with antibodies can be entered is Rostam mixing with the sample or changes made directly in the sample, depending on the nature of the sample (liquid or solid). The biological sample may be brought into contact directly with the antibody in a suitable fixture (Cup, package, bottle etc). For use in a patient, the composition should be formulated for administration to a patient.

Compositions of the present invention can be administered orally, parenterally, by inhalation spray, surface, rectal, intranasal, cheek, intrawaginalno or via an implanted reservoir. The term "parenteral"as used here, includes subcutaneous, intravenous, intramuscular, intra-articular, vnutrigrudne, vnutriobolochechnoe, intrahepatic, inside the affected places and intracranial way of injections or infusions. Preferably oral, intraperitoneal or intravenous compositions.

Sterile suitable for injection forms of the compositions of the present invention can be aqueous or oil suspensions. These suspensions can be formulated according to known in the field methods using suitable dispersing or wetting agents and suspendida agents. Sterile suitable for injection drug may also be suitable for sterile injection solution or suspension in a non-toxic suitable for parenteral introduction the Oia diluent or solvent, as, for example, a solution in 1,3-butanediol. Suitable for use acceptable carriers and solvents are water, ringer's solution and isotonic sodium chloride solution. In addition, as a solvent or suspendida environment usually apply sterile non-volatile oil. For this purpose, can be applied to any slight non-volatile oils, including synthetic mono - or diglycerides. For the preparation of injectable preparations suitable fatty acids such as oleic acid and its glyceride derivatives, and natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially their polyoxyethylene derivatives. These oil solutions or suspensions may also contain long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the preparation of pharmaceutically acceptable dosage form, including emulsions and suspensions. For the purposes of the compositions can also be used with other commonly used surfactants such as twins, spiny and other emulsifying agents or amplifiers biological availability, which is typically used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage the ORM.

The compositions of this invention can be administered orally in any suitable for oral administration dosage form, including (but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral administration commonly used carriers include lactose and corn starch. Usually add lubricants, such as magnesium stearate. For oral administration in capsule form suitable diluents include lactose and dried corn starch. If for oral administration requires water suspension, the active ingredient is combined with emulsifying and suspendresume agents. If necessary, can be also added some sweetening, flavoring or coloring agents.

Alternatively, the compositions of this invention can be introduced in the form of suppositories for rectal use. They can be prepared by mixing the agent with a suitable not causing irritation excipient that is solid at room temperature, but liquid at the rectal temperature. It will therefore melt in the rectum, releasing the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The compositions of this invention can be entered locally, especially when the target of treatment includes areas or organs, is egco available for local use. These include diseases of the eye, skin, or the lower part of the intestinal tract. For each of these areas or organs can easily prepare suitable compositions for local application.

Local introduction in the case of the lower section of the intestinal tract can be carried out using the composition in the form of rectal suppositories (see above) or suitable compositions for enemas. You can also use a percutaneous tampons.

Compositions for local injection can be formulated as a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include (but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene or polyoxypropylene compound, emulsifying wax and water. Alternatively, compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more of pharmaceutically acceptable carriers. Suitable carrier materials include (but are not limited to, mineral oil, sorbitan-monostearate, Polysorbate 60, wax atilovykh esters, Cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Compositions for oftalmologiches the CSOs application can be compiled as-ionized suspensions in isotonic sterile saline solution with a defined pH or preferably, as solutions in isotonic sterile saline solution with a defined pH, or a preservative such as benzalconi-chloride, or without it. Alternatively, compositions for ophthalmic applications can be made in ointments such as vaseline.

Compositions of the present invention can also enter intranasal spray aerosol or inhalation. Such compositions are prepared according to the well-known in the pharmaceutical prescription field methods. They can be prepared as solutions in physiological saline with benzyl alcohol or other suitable preservatives, enhancers of absorption to enhance bioavailability, fluorocarbons, and/or other conventional solubilizers or dispersing agents.

It was found that in clinical situations effective some monoclonal antibodies such as Rituxan (Rituximab), Herceptin (Trastuzumab) or Xolair (Omalizumab), and antibodies of the present invention can be used in the same modes of administration (i.e. composition and/or dose and/or graphics introduction). Modes of administration and dosage for antibodies in the pharmaceutical compositions of the present invention can be determined by known methods for these products - for example, using the manufacturer's instructions. For example, the Pris is relevant in the pharmaceutical compositions of the present invention antibodies can be entered at a concentration of 10 mg/ml in vials single use packaging 100 mg (10 ml) or 500 mg (50 ml). Compositions for intravenous administration are in a solution of 9.0 mg/ml sodium chloride, of 7.35 mg/ml sodium citrate, 0.7 mg/ml Polysorbate 80 in sterile water for injection. Bring the pH to 6.5. For example, a suitable dosage interval for the antibody in the pharmaceutical composition of the present invention may be from about 10 mg/m2up to 500 mg/m2. However, it should be recognized that these recipes are for example and the best recipe and the mode of administration can be developed, taking into account the affinity and tolerability of specific antibody in the pharmaceutical composition, which can be determined in clinical trials. The number and mode of injection of the antibody in the pharmaceutical composition of the present invention, which are saturated NK cells within 24 h, 48 h, 72 h or weeks, should be determined, taking into account the affinity of the antibodies and their pharmacokinetic parameters.

According to another variant implementation of the composition with antibodies of the present invention may further comprise another therapeutic agent, including tools that are usually used for a specific therapeutic purpose for which the injected antibodies. Standard additional therapeutic agent is present in the composition in amounts commonly used for this tool when alone subject Leche is of particular diseases or conditions. Such therapeutic agents include, but are not limited to) used to treat cancer therapeutic drugs used for the treatment of infectious diseases therapeutic tools used in other types of immunotherapy therapeutic agents, cytokines (such as IL-2 or IL-15), other antibodies and fragments of other antibodies.

For example, for the treatment of cancer there are a large number of therapeutic agents. Composition with antibodies and methods of the present invention can be combined with any other methods commonly used for the treatment of specific diseases, in particular tumor, cancer or another disease or disorder that affects the patient. Until then, there is no evidence that a specific therapeutic approach in itself is harmful to the health of the patient, and he does not counteract the activity of the antibody in the pharmaceutical composition of the present invention, considered in combination with the present invention.

In connection with the treatment of dense (solid) tumors of the pharmaceutical compositions of the present invention can be used in combination with classical approaches such as surgery, radiotherapy, chemotherapy and the like. Therefore, the invention provides for the combined treatments, in which formats viteska composition of the present invention is used simultaneously with, before or after surgery or radiation; or is administered to patients simultaneously with, before, or after conventional chemotherapeutic, radiotherapeutic or anti-angiogenic funds or addressed immunotoxins or coagulating ligands.

If in therapeutic scheme in combination with containing the antibody composition of the present invention are applied to one or more funds, there is no requirement that the total results were additive with respect to the effects obtained when each treatment is applied separately. Although it is generally desirable to at least additive effects, it would be useful any increase in anticancer activity compared with monotherapy. Similarly, there was no specific requirement that the combined treatment gave synergenic effect, although it is possible and useful.

To practice combined anti-cancer therapy, you can simply enter the animal a composition with antibodies of the present invention in combination with other anti-cancer method is effective in obtaining the animal combined anticancer actions. Therefore, means must be provided in such quantities and for such period of time to ensure that their combined presence within the tumor vascular network and a combination of the s impact on the environment of the tumor. To achieve this goal, a composition with antibodies of the present invention and anti-cancer tool, you can enter the animal at the same time - or as one unified composition, or as two distinct compositions, inserted in different ways.

Alternatively, the introduction of the composition with antibodies of the present invention may precede the introduction of anti-cancer remedies or to follow his introduction with time intervals from minutes to weeks and months. Should ensure that anti-tumor agent and the antibody in combination with antibodies of the present invention has provided a useful combined effect on the cancer process.

Most anticancer funds in case protivoallergennoy therapy should be introduced to composition with inhibitory KIR antibodies to the present invention. However, if in combination with antibodies of the present invention are used immunoconjugate antibodies, various anticancer funds can be administered simultaneously or sequentially.

In some cases, it may even be necessary to significantly extend the duration of treatment, interval between successive introduction of anti-cancer remedies or anti-cancer treatment and the introduction of the composition with antibodies of the present invention in a few days(2, 3, 4, 5, 6 or 7), and W which are weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even a few months(1, 2, 3, 4, 5, 6, 7 or 8). This can be useful in such circumstances, when the target of anti-cancer treatments (such as surgery or chemotherapy) - much to destroy the tumor and the composition with antibodies of the present invention is to prevent micrometastasis or re-growth of the tumor.

It is also envisaged that it will be necessary to apply more than one injection or compositions on the basis of antibodies to inhibitory KIR present invention, or anti-cancer tool. These agents can be administered sequentially, in alternate days or weeks; or a cycle of treatment composition with antibodies to inhibitory KIR present invention, followed by a cycle therapy anti-cancer agent. In any case, all that is required to achieve tumor regression when combined therapy is to enter both the agent in the total number, which effectively exhibits antitumor activity, regardless of the points of introduction.

In terms of surgery any surgical intervention may be implemented in combination with the present invention. In connection with radiotherapy, can be considered any mechanism for local induction of DNA breaks inside of cancer cells such as gamma-irradiation, x-ray beam is, UV radiation, microwave radiation, and even electron emission, and the like. Considered also directed delivery of radioisotopes to tumor cells, and this may be applied in connection with targeted antibodies or other targeting means.

In other aspects in combination with the compositions with antibodies or as part of a composition with antibodies of the present invention can be introduced immunomodulatory compounds or modes. Preferred examples of immunomodulatory compounds include cytokines. In such combined approaches may involve different cytokines. Examples of cytokines that are useful in the combinations covered by the present invention include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ. Used in combinational treatment or compositions of the present invention, the cytokine is administered according to the standard modes, consistent with clinical indicators - such as the patient's condition, and the relative toxicity of the cytokine.

In some embodiments, the implementation of therapeutic compositions of the present invention containing cross-reacting antibodies to inhibitory KIR, can be administered in combination with a chemotherapeutic agent or th is monalnoy therapy, or can optionally contain such a tool. In the disclosed here are methods of combined treatment can be applied to various means of hormonal therapy and chemotherapy. Chemotherapeutic consider, for example, include (but are not limited to, alkylating agents, antimetabolites, cytotoxic antibiotics, alkaloids from Vinca - for example adriamycin, dactinomycin, mitomycin, karminomitsin, daunomycin, doxorubicin, tamoxifen, Taxol, Taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, titera, methotrexate, camptothecin, actinomycin-D, mitomycin C, cisplatin (CIS-DDP), aminopterin, combretastatin(s) and their derivatives and precursors of drugs.

Hormonal agents include (but are not limited to, for example, agonists of gonadotropin-releasing hormone (LHRH) - such as leiprorelina, goserelin, buserelin; anti-estrogens such as tamoxifen and toremifene; anti-androgens such as flutamide, nilutamide, cyproterone and bikalutamid; aromatase inhibitors such as anastrozole, exemestane, letrozole and fadrozole; and Progestogens such as medroxy, chlormadinone and megestrol.

As will be clear to experts in the art, the appropriate doses of chemotherapeutic agents should be close to the eat, that have been used in clinical courses of therapy, when chemotherapeutic agents were administered alone or in combination with other chemotherapeutics. Just for example, can be used such agents as cisplatin and other alkylating DNA agents. Cisplatin has been widely used for cancer treatment, and clinical application used effective dose of 20 mg/m2for 5 days every 3 weeks, only 3 courses. Cisplatin is not absorbed with oral administration and should therefore be given by injection intravenously, subcutaneously, into the tumour or intraperitoneally.

Other useful chemotherapeutic agents include compounds that disrupt the DNA replication, mitosis and chromosome segregation, and agents that violates the synthesis and the correctness of the predecessors of polynucleotides. A large number here, for example, chemotherapeutic agents for combination therapy are shown in the table of U.S. Patent No. 6524583 disclosed in which agents and instructions included in this description by reference. Listing agents list is made for example and is not limiting. An experienced specialist should refer to "Remington''s Pharmaceutical Sciences, 15th edition, Chapter 33, especially p. 624-652. Depending on the subject to the treatment of painful conditions may be changes in dozer is vcah. Producing the treating physician should be able to determine for a particular individual the right dosage.

Compositions of the present invention with cross-reacting antibodies to inhibitory KIR can be used in combination with any one or more of the other anti-angiogenic drugs, or may contain additional anti-angiogenic agents. Examples of such agents include neutralizing antibodies, antisense RNA, a small interfering RNA (siRNA), interfering RNA (RNAi), RNA aptamers and ribozymes, each of which is directed against growth factor vascular endothelial (VEGF) or receptor growth factor vascular endothelial (U.S. Patent No. 6 524 583, the content of which is incorporated into this description by reference). Can also be used variants of factor a vascular endothelial growth with antagonistic properties, as described in patent document WO 98/16551 included in the present description by reference. Additional examples of anti-angiogenic agents that may be useful in connection with combined therapy are listed in table D of U.S. Patent No. 6524583 disclosed in which agents and instructions included in this description by reference.

Compositions of the present invention with antibodies to inhibitory KIR can also be used in combination with the methods induced by the I of apoptosis, or may further comprise inducing apoptosis agents. For example, it was identified a large number of oncogenes, which inhibit apoptosis, or programmed cell death. Here, for example, oncogenes this category include (but are not limited to, bcr-abl, bcl-2 (distinct from bcl-1), cyclin D1 (access number in GenBank: M14745, H; U.S. Patent No. 5650491 and 5539094; each of which is incorporated into this description by reference) and representatives of the family, including l-x1, Mcl-1, Bak, A1, A20. Over-expression of bcl-2 was originally opened in lymphoma T-cells. The function of the oncogene bcl-2 is the binding of Bax protein (protein pathway of apoptosis) and its inactivation. Inhibition of the function of bcl-2 prevents Bax inactivation and allows you to be the way of apoptosis. Inhibition of this class of oncogenes - for example, using antisense nucleotide sequences, RNAi, siRNA or a low molecular weight chemical compounds considered in terms of the use of the present invention to enhance apoptosis (U.S. Patent No. 5650491, 5539094 and 5583034, each of which is incorporated into this description by reference).

Compositions of the present invention with antibodies to inhibitory KIR may also include molecules or be used in combination with molecules that contain addressing part - for example an antibody, ligand or conjugate, aimed at specific marker of the target cell ("targeting agent") - n is an example of the target cell tumors. Generally speaking, the target agents for use in these additional aspects of the invention, should preferably recognize the available tumor antigens that are preferentially or specifically expressed in the site of tumor development. Targeting agents, as a rule, the contact expressed on the surface available on the surface or localized on the surface of the component of the tumor cells. Target agents will also preferably exhibit properties of high affinity and will not cause significant side effects in vivo no relation to life-sustaining normal tissues, such as one or more tissues selected from the tissues of the heart, kidneys, brain, liver, bone marrow, colon, breast, prostate, thyroid, gall bladder, lung, adrenals, muscle, nerve fibers, pancreas, skin, or other life-sustaining organs or tissues of the human body. The term "do not cause significant side effects, as it is used here, refers to the fact that the target agent when he entered in vivo, will cause only negligible or clinically controlled side effects - such as those that typically occur with chemotherapy.

In the treatment of tumors composition with antibodies of this izaberete the Oia may optionally contain auxiliary connection or can be used in combination with auxiliary compounds. An ancillary compound may include as an example of an antiemetic such as serotonin antagonists, and drugs such as phenothiazines, substituted benzamide, antihistamines, butyrophenones, corticosteroids, benzodiazepines and cannabinoids; bisphosphonates such as zoledronicaa acid and pamidronate acid; and haematopoietic growth factors such as erythropoietin and G-CSF (e.g., filgrastim, lenograstim and darbepoietin).

In another embodiment, two or more types of antibodies of the present invention having different cross-reactivity, including NKVSF1, can be combined in a single composition to neutralize the inhibitory action of a greater number of genes inhibitory KIR. Compositions containing a combination of cross-reactive antibodies to inhibitory KIR present invention or their fragments or derivatives, provide even wider application, as is likely, there is a very small part of the human population that doesn't have each of the products of genes inhibitory KIR recognized a single type of cross-reacting antibodies. Similarly, the composition with antibodies of the present invention can optionally contain one or more of the types of antibodies that races will Osnat single subtypes of inhibitory KIR. Such combinations will also have a broader applicability in therapeutic prescriptions.

The invention also provides a method of enhancing the activity of NK cells we need in this patient, containing the step of introducing the composition according to the present invention the specified patient. More specifically, the method is aimed at increasing the activity of NK cells in patients having a disease in which useful increase in the activity of NK cells, which are involved that are influenced or caused by cells susceptible to lysis by NK cells cells, or which is caused or characterized by deficient activity of NK cells. It's such diseases as cancer and other proliferative disease, an infectious disease or an immune disorder. More specifically, the methods of the present invention are used to treat various types of cancer and other proliferative diseases, including (but not limited to carcinoma (including carcinoma of the bladder, breast, colon, kidney, liver, lung, ovarian, prostate, pancreas, stomach, cervix, thyroid and skin, including squamous carcinoma cells); tumors of the hematopoietic organs of lymphoid origin (including leukemia, acute limfotsity leukemia, acute lymphocytic leukemia, lymphoma B-cell lymphoma is T-cells, Hodgkin's lymphoma, non Hodgkin's lymphoma, lymphoma villous cells and Burkitt's lymphoma); hematopoietic tumor of myeloid bodies of origin (including acute and chronic myelogenous leukemia and promyelocytic leukemia); tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the Central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and neurinomas; and other tumors, including melanoma, pigmentosa xerodermia, keratoakantoma, seminoma, thyroid follicular cancer and teratocarcinoma.

Preferred diseases which can be treated in accordance with the present invention include tumors of the hematopoietic organs of lymphoid origin, such as tumor T-cells and b-cells, including (but not limited to) the violation of T-cells such as T-prolymphocytic leukemia (T-PLL), including such small cells and cerebriform type cells; leukemia large granular lymphocytes (LGL), primarily in T-cells; Sezary syndrome (SS); leukemia adult T-cell (ATLL); hepato-splenic lymphoma T-NHL; peripheral/positionnow lymphoma T cells (pleomorphic and immunoblastic subtypes); angio-immunoblastic lymphoma T-cells; angiocentricity nasal) lymphoma T-cells; anaplastic (Ki 1+) lymphoma of large cells; intestinal lymphoma T-cells; lymphoblasts and lymphoblastic T-cells (T-Lbly/T-ALL).

According to the present invention can be treated and also other proliferative disorders, including, for example, hyperplasia, fibrosis (especially pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and growth of smooth muscle in blood vessels, such as stenosis or restenosis after plastic surgery on the blood vessels. Cross-reacting antibodies to inhibitory KIR present invention can be used to treat or prevent infectious diseases, including mainly any infection caused by viruses, bacteria, protozoa, mold fungi or fungus. Such viral infections include, but are not limited to, hepatitis a, hepatitis b, hepatitis C, influenza, varicella, adenovirus, herpes simplex 1 type (HSV-1), herpes simplex 2nd type (HSV-2), the plague of cattle, rhinovirus, Echovirus, rotavirus, respiratory syncytial virus, human papilloma virus, cytomegalovirus, echinodorus, arbovirus, Hantavirus causing hemorragic fever, mumps virus, measles virus, rubella virus, polio, and the human immunodeficiency virus 1-St or 2-nd type (HIV-1, HIV-2).

Bacterial infection, which can be affected in accordance with the laws the AI with the present invention, include (but are not limited to, infections caused by the following microorganisms: Staphylococcus; Streptococcus, including S. pyogenes; Enterococci; Bacillus, including Bacillus anthracis, and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella including G. vaginalis; Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponerna; Camplyobacter, Pseudomonas, including P. aeruginosa; Legionella; Neisseria including N. gonorrhoeae and N. meningitides; Flavobacterium including F. meningosepticum and F. odoraturn; Brucella; Bordetella, including pertussis Century and B. bronchiseptica; Escherichia, including E. Li, Klebsiella; Enterobacter, Serratia, including S. marcescens and S. liquefaciens; Edwardsiella; Proteus, including P. mirabilis and P. vulgaris; Streptobacillus; Rickettsiaceae, including R fickettsfi; Chlamydia, including C. psittaci and C. trachomatis; Mycobacterium, including M. tuberculosis, M. intracellulare, M. folluiturn, M. laprae, M. avium, M. bovis, M. africanum, M. kansasii, M. Intracellulare and M. lepraernurium; and Nocardia.

Protozoal infections, which can be affected in accordance with the present invention, include, but are not limited to, infections caused by Leishmania, coccidia and trypanosomes. Full list of infectious diseases, which is included in the present description by reference, can be found on the website of the National center National Center for Infectious Disease (NCID) at the Center for disease control Center for Disease Control (CDC) (http://www.cdc.gov/ncidod/diseases/). All of these diseases are candidates for treatment with the use of cross-reacting antibodies to inhibitory KIR according to the present invention.

In such ways the Ah treatment of various infectious diseases can be applied composition with antibodies of the present invention, either alone, or in combination with other treatments and/or therapies known to treat such diseases, including antiviral agents, antifungal agents, antibacterial remedies, antibiotics, antiparasitic tools and Antiprotozoal drugs. If these methods include additional treatment with additional therapeutic tools, these tools can be administered with the antibodies of the present invention either in the form of a single dosage or in the form of separate, multiple dosage. Additional means, if it is injected in the form of separate dosages, can be administered before, concurrently or after administration of the antibodies of the present invention.

Additional aspects and advantages of the present invention will be disclosed in the following experimental section, which should be viewed as illustrative, but not limiting scope of the present invention.

DESCRIPTION of embodiments of the INVENTION

Example 1

Purification of peripheral blood lymphocytes (PBL) and the creation of a polyclonal or clonal cell lines NK

Lymphocytes PBL were obtained from healthy donors using Ficoll gradients Hypaque and removal of the adjacent plastic cells. To obtain enriched NK cells, PBL were incubated (30 min at 4°C) with mo is olonline antibodies mAb to CD3 (anti-D3), to CD4 (anti-CD4) and HLA-DR (anti-HLA-DR), then the magnetic granules (Dynal) with goat antibody to mouse immunoglobulin (30 min at 4°C), followed by immunomagnetic selection using known in the field of methods (Pende and others 1999). Cells CD3-, CD4-DR-were cultured on irradiated nourishing the cells with 100 U/ml interleukin-2 (Proleukin, Chiron Corporation) and 1.5 ng/ml phytohemagglutinin A (Gibco BRL) to obtain polyclonal populations of NK cells. The NK cells were cloned by limiting dilution and determined the expression of cell surface receptors in NK cell clones using flow cytometry.

Used monoclonal antibodies were JT3A (IgG2a, aHTH-CD3), EV and GL183 (IgG1 to KIR2DL1 and KIR2DL3, respectively), HA-141 (IgM to KIR2DL1 with the same specificity as EV), anti-CD4 (HP2.6) and anti-DR (D1.12, IgG2a). Instead HP2.6 and R1.12, which were obtained by the applicants, it is possible to use commercially available monoclonal antibodies with the same specificnosti (Beckman Coulter Inc., Fullerton, CA). EV and GL183 are commercial products (Beckman Coulter Inc., Fullerton, CA). HA-141 is not a commercial product, but to control the recovery of lysis can be used EV, as described in floors are only and others (1993).

Cells were stained with appropriate antibodies (30 min at 4°C, then added conjugated with PE or FITC polyclonal antibody to mouse immunoglobulin (Suthern Biotechnology Associates Inc.). The samples were analyzed by the method of cytofluorometry on installing FACSAN apparatus (Becton Dickinson, Mountain View, CA).

In this research, we used the following clones. CP11, CN5 and CN505 is KIR2DL1 positive clones were stained with antibodies EV (IgG1 to KIR2DL1) or HA-141 (IgM to KIR2DL1 with the same specificity as the antibody EV). CN12 and SR is positive for KIR2DL3 clones were stained with antibodies GL183 (IgG1 to KIR2DL3).

The cytolytic activity of NK clones was assessed by the standard method for the release of51SG after 4 h, in which the activity of effector NK cells was measured by positive Cw3 or Cw4 cell lines, about which we know that they are sensitive to lysis by NK cells. All target cells were taken in the amount of 5000 cells per cell in microtiter tablet, the ratio of effector: target is indicated on the figures (usually 4 effector cells one cell-target). Analysis of cytotoxicity was performed in the presence or without supernatant with the specified monoclonal antibody with dilution1/2. The procedure was basically the same as described floors are only and others (1993).

Example 2

Getting new monoclonal antibodies

Monoclonal antibodies (mAbs) were obtained by immunization with 5-week-old mice of Balb lines activated polyclonal or monoclonal NK cells, as described by floors are only and others (1990). After the merger of different cells, mAbs in which the Achal were selected on their ability to cross-reactions with positive EV and GL183-line cells and NK clones. Then there was some positive screening of monoclonal antibodies for their ability to restore lysis Cw4 positive or Cw3 target cells positive for EV or GL183 NK clones.

Staining of cells was produced as follows. Cells were stained with a set of antibodies (1 μg/ml or 50 ál of the supernatant liquid, 30 min at 4°C), after which contributed conjugated with PE fragments F(ab')2 goat antibodies to mouse IgG (H+L) or conjugated with PE fragment F(ab')2 antibody to human IgG (Fc-gamma) (Beckman Coulter). Cytofluorometric analysis was carried out on the installation of Epics XL.MCL (Beckman Coulter).

It was found that one of the monoclonal antibodies, DF-200 takes, reacts with various representatives of the KIR family, including KIR2DL1, KIR2DL2/3. Cells NK and KIR2DL1+, and KIR2DL2/3+ brightly colored DF-200 mb (figure 1).

The NK clones expressing one or the other (or even both) of these specific to the class I HLA inhibitory receptors, were used as effector cells against target cells, expressively one or more of the alleles HLA-C. Analysis of cytotoxicity was carried out as follows. The cytolytic activity of cell lines YTS-KIR2DL1 or YTS-Eco assessed standard method for the release of51SG after 4 hours Effector cells were tested positive for HLA-Cw4 or negative for EBV cell lines and transfected with HLA-Cw4 cells 721.221. All cells Mish is neither used in tablets for micrometrology at 3000 cells per cell. The ratio of effector/target indicated on the drawings. Analysis of the cytolytic action was performed, as indicated, with mouse or human monoclinal antibodies to full length or fragments F(ab')2, or without them. As expected, NK clones KIR2DL1+showed a small, if not exercised, the cytolytic activity against target cells expressing HLA-Cw4 and NK clones KIR2DL3+showed a small, if not exercised, the cytolytic activity against target cells positive for Cw3. However, in the presence of monoclonal antibodies DF-200mAb (used to mask their KIR2DL receptors) NK clones have acquired the ability to recognize their ligands HLA-C and showed a strong cytolytic activity against target cells with Cw3 or Cw4.

For example, the line C1R cells (cell line CW4+ADS n°CRL 1993) could not be killed by NK clones KIR2DL1+(CN5/CN505), but the inhibition could be effectively changed by using either antibodies DF-200, or conventional monoclonal antibodies KIR2DL1. On the other hand, NK clones expressing the phenotype KIR2DL2/3+KIR2DL1-(CN12), effectively destroyed C1R cells, and this destruction is not affected DF-200mAb (figure 2). Similar results were obtained with NK clones: KIR2DL2 or positive KIR2DL3 targets, positive in Cw3.

Similarly, cell lines Cw4+ 221 EBV was not destroyed tra is sizeranne by KIR2DL1 +the NK cells, but the inhibition could effectively change when using either DF-200, or fragment DF-200 Fab or monoclonal antibodies to KIR2DL1 - EV or HA. Cell lines Cw3+ 221 EBV also could not be killed by NK cells KIR2DL2+but this inhibition can be modified using either antibodies DF-200 or their fragments DF-200 Fab. Finally, the cells last line Cw3+ 221 EBV could not be killed by NK cells KIR2DL3*, but this inhibition can be modified by using a piece of DF-200 Fab or conventional monoclonal antibodies to KIR2DL3 - GL183 or Y249. The results are shown in figure 3.

Fragments F(ab')2 was tested also on their ability to recover lysis Cw4 positive target cells. Fragments F(ab')2 antibodies DF-200 and EV Abs were both able to modify the inhibition of lysis transtitional Cw4 cell line 221 and cell line Cw4+ TUBO EBV transfitsirovannykh by KIR2DL1 cells NK. The results are presented in figure 4.

Example 3

Receive new human monoclonal antibodies

Human monoclonal antibodies to KIR was obtained by immunization of transgenic mice, engineered to Express a set of human antibodies with recombinant protein KIR. After the merger of different cells were first selected monoclonal antibodies for their ability to cross-react with the immobilized protein KIR2DL1 and IR2DL2. It was found that some monoclonal antibodies, including 1-7F9, 1-4F1, 1-6F5 and 1-6F1, react with KIR2DL1 and KIR2DL2/3.

This was followed by further screening positive monoclonal antibodies for their ability to restore lysis Cw4 positive target cells positive for EV the transfectants NK expressing KIR2DL1. Of NK cells expressing inhibitory receptors specific for the class I HLA, were used as effector cells against target cells expressing one or more of the alleles HLA-C (figures 5 and 6). Analysis of cytotoxicity was performed as described above. The ratio of effector/target indicated on the drawings, antibodies were used at a concentration of 10 μg/ml or 30 μg/ml.

As expected, NK cells KIR2DL1+showed low, if not exercised, the cytolytic activity against target cells expressing HLA-Cw4. However, in the presence of monoclonal antibody 1-7F9 mb NK cells lose their ability to recognize their ligands HLA-C and showed a strong cytolytic activity against target Cw4. For example, two tested cell line (cell line HLA-Cw4, transfusiona 721.221, and CW4+EBV) could not be killed by NK cells KIR2DL1+however , the inhibition can be effectively modified using either monoclonal antibodies mAb 1-7F9 or conventional antibodies anti-KIR2DL1 mAb EB6. Antibody F-200 and panKIR (also denoted NKVSF1) were comparable with 1-7F9. On the other hand, antibodies 1-4F1, 1-6F5 and 1-6F1 has not been able to restore lysis by NK cells target cells positive for Cw4.

Example 4

Analysis by the method of surface plasmon resonance on the installation BIAcore interaction DF-200 mAb/KIR2DL1 and DF-200 mAb/KIR2DL3

Production and purification of recombinant proteins

Recombinant proteins KIR2DL1 and KIR2DL3 was developed in E. Li. cDNA encoding the entire extracellular domain of KIR2DL1 and KIR2DL3 amplified by polymerase chain reaction (PCR) from the vectors, respectively, pCDM8 clone 47.11 (Biassoni and others, 1993) and RSVS(gpt)183 clone 6 (Wagtman and others, 1995) using the following primers:

sense: 5'-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3'

antisense: 5'-CGGGATCCCAGGTGTCTGGGGTTACC-3'

They were cloned in expressing vector pML1 in one frame with the sequence encoding the signal biotinidase (Sauln and others, 2003).

The protein expression was carried out in the bacterial strain BL21(DE3) (Invitrogen). Transfetsirovannyh bacteria were grown to an optical density OD600or =0.6 at 37°C in medium supplemented with ampicillin (100 μg/ml), expression was induced 1 mm isopropyl-thiogalactoside (IPTG).

Proteins were isolated from Taurus inclusion in denaturing conditions (8 M urea). Return the folding of recombinant proteins was carried out at room temperature in buffer containing 20 mm Tris, pH of 7.8, 150 mm NaCl, containing L-arginine (400 mm, Sigma) and β (1 mm), decreasing concentrations of urea 6-speed dialysis(4, 3, 2, 1, 0,5 and 0 M urea, respectively). During stages dialysis against 0.5 M and 0 M urea was added reduced and oxidized glutathione (5 mm and 0.5 mm, Sigma). Finally, carefully proteins were dialyzed against buffer containing 10 mm Tris, pH 7.5 and 150 mm NaCl, Soluble, re-folded proteins were concentrated and then purified gel permeation chromatography on a column of Superdex 200 (Pharmacia; ACT system).

Measurement of surface plasmon resonance was performed on installing a Biacore apparatus (Biacore). In all experiments on a Biacore assay buffer was HBS buffer with the addition of 0.05% surfactant P20.

Immobilization of proteins

Produced, as described above, proteins KIR2DL1 and KIR2DL3 was immobilized by covalent binding with carboxyl groups in the dextran layer on the sensor chip, the Sensor Chip CM5 (Biacore). The surface of sensor chip were activated by EDC/NHS (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide-hydrochloride and N-oxysuccinimide, Biacore). Proteins were injected in the buffer for binding (10 mm acetate, pH 4.5). The remaining activated groups iactiveaware with 100 mm ethanolamine pH 8 (Biacore).

Measurement of affinity (affinity)

For kinetic measurements on immobilized sample was applied various concentrations of soluble antibodies (1×10-7up to 4×10-10 M). The measurements were carried out at constant speed, flow 20 ml/min For each cycle of the surface of sensor chip was regenerated by injection of 5 μl of 10 mm NaOH pH 11. Data analysis used the program BIAlogue Kinetics Evaluation program (BIAevaluation 3.1, Biacore). On the layers of dextran containing 500 or 540 units of reflectance (reflectance units, RU) and 1000 or 700 EN respectively KIR2DL1 and KIR2DL3, introduced a soluble analyte (40 μl of different concentration) when the velocity of the flow of 20 μl/min in HBS buffer. Data represent 6 independent experiments. The results are presented below in table 1.

Table 1
The BIAcore analysis of binding of DF-200 mb with immobilized KIR2DL1 and KIR2DL3
ProteinToD(10-9M)
KIR2DL110,9±3,8
KIR2DL32,0±1,9
ToD: dissociation constant.

Example 5

The analysis BIAcore (surface plasmon resonance) competitive binding of mouse and human antibodies to KIR

Analysis of epitope mapping was performed on immobilized KIR2DL1 (900 RU), KIR2DL3 (2000) and KIR2DS1 (1000 RU) with murine antibodies is to KIR2D: DF-200, Pan2D, gl183 and EV, and human antibodies to 2D KIR: 1-4F1, 1-6F1, 1-6F5 and 1-7F9, as described previously (Gauthier and others, 1999, Saunal and van Regenmortel, 1995).

All experiments were carried out with the velocity of the flow of 5 μl/min in HBS buffer with injection within 2 min of different antibodies at a concentration of 15 µg/ml For each pair of antibodies analysis of competitive binding was performed in 2 stages. In the first phase squirrel-target KIR2D introduced the first monoclonal antibody (mAb), then the second monoclonal antibody (without uninstalling first) and determined the value of EN for the second antibody (RU2). In the second stage first introduced the second monoclonal antibody directly to the exposed protein, and determined the value of EN for monoclonal antibodies (RU1). The percentage inhibition of binding of the second mAb protein KIR2D first mAb was calculated as 100x(1-RU2/RU1).

The results are presented in tables 2, 3 and 4, where the antibody is labeled "first antibody"are listed in a vertical column, and the "second antibody" is shown in a horizontal line. For each tested combination of antibodies in the table shows the values for the direct binding (RU) antibody chip, where the direct binding of the second antibody with a chip KIR2D shown in the upper part of the cell, and the value for binding of the second antibody with a chip KIR2D where already present the first antibody, is shown in the lower part of the cell. In the right part of each cell shows recent inhibition of binding of the second antibody. Table 2 shows the binding on the chip KIR2DL1, table 3 shows the binding of the antibody on the chip KIR2DL3, and table 4 shows the binding of the antibody on the chip KIR2DS1.

Were evaluated by competitive binding to immobilized KIR2DL1, KIR2DL2/3 and KIR2DS1 mouse antibodies DF-200, NKVSF1 and EV and human antibodies 1-4F1, 1-7F9 and 1-6F1. Mapping of epitopes (7) in the experiments with the binding of the antibody to KIR with KIR2DL1 showed that

(a) antibody 1-7F9 compete with EV and 1-4F1, but not with NKVSF1 and DF-200;

(b) antibodies 1-4 F1, in turn, compete with EV, DF-200, NKVSF1 and 1-7 F9;

(c) antibody NKVSF1 compete with DF-200, 1-4F1 and IV, but not with 1-7F9; and

(d) antibodies DF-200 compete with NKVSF1, 1-4F1 and IV, but not with 1-7F9.

Mapping of epitopes (Fig) in experiments with the binding of the antibody to KIR with KIR2DL3 showed that

(a) antibodies 1-4F1 compete with NKVSF1, DF-200, gl183 and 1-7F9;

(b) antibody 1-7F9 compete with DF-200, gl183 and 1-4F1, but not with NKVSF1;

(c) antibody NKVSF1 compete with DF-200, 1-4F1 and GL183, but not with 1-7F9; and

(d) antibodies DF-200 compete with NKVSF1, 1-4F1 and 1-7F9, but not with GL183.

Mapping of epitopes (Fig.9) in experiments with the binding of the antibody to KIR with KIR2DS1 showed that

(a) antibodies 1-4F1 compete with NKVSF1, DF-200 and 1-7F9;

(b) antibody 1-7F9 compete with 1-4F1, but do not compete with DF-200 and NKVSF1;

(c) antibody NKVSF1 compete with DF-200 and 1-4F1, but not with 1-7F9; and

(d) antibodies DF-200 compete with NKVSF1 and 1-4F1, but not with 1-7F9.

Example 6

Titration of antibodies (mAb) to KIR NK cells Cynomogus

Antibody NKVSF1 to KIR tested for their ability to bind to NK cells from Cynomolgus monkeys. The binding of the antibody to monkey NK cells presented in figure 10.

Purification of murine peripheral blood mononuclear (RVMS) and obtaining the mass of polyclonal NK cells

Mononuclear cells peripheral blood of Cynomolgus macaques were obtained from CPT tubes with sodium citrate (Becton Dickinson). Purification of NK cells was performed by the method of negative depletion (set Macaque NK cell enrichment kit, Stem Cell Technology). To obtain populations of polyclonal NK cells were cultured NK cells irradiated on the supply of human cells with 300 u/ml interleukin 2 (Proleukin, Chiron Corporation) and 1 ng/ml of phytohemagglutinin A (Invitrogen, Gibco).

Titration Pan2D mAb with NK cells Cynomolgus

Cells NK Cynomolgus (weight of NK cells on the 16th day) were incubated with various amounts of antibodies Pan2D mAb and then conjugated with PE fragments F(ab')2 goat antibodies to mouse IgG (H+L). The percentage of positive cells was determined ezotericheskim control (purified mouse lgG1). Samples were collected and processed for 2 in parallel. MFI - mean fluorescence intensity (mean fluorescence intensity).

Example 7

Mapping of epitopes binding DF-200 and Pan2D with KIR2DL1

Computer modeling of the extracellular to the ENES KIR2DL1, KIR2DL2 and KIR2DL3 (KIR2DL1-3) based on published crystal structures (Maenaka and others (1999), Fan and others (2001), Boyington and others (2000)predicted participation amino acids R1311(1Single-letter code of amino acids) in the interaction between cross-reactive with respect to KIR2DL1 and KIR2DL1-3 mouse monoclonal antibody (mb) DF-200 and pan2D. To check this, were prepared fused protein consisting of the full extracellular domain KIR2DL1 (amino acids N1-N), either wild-type or having a point mutation (for example, R131W2) (2Replacing R by W in position 131 (from N-Terminus) amino acid sequence of KIR2DL1)), fused to human Fc (hFc). Materials and methods used for production and evaluation of various fused proteins KIR2DL1-hFc were described previously (Winter and Long (2000)). Briefly, encoding KIR2DL1 (R131W)-hFc cDNA vectors were obtained by PCR mutagenesis (Quickchange II, Promega) vector CL42-lg - published cDNA-vector to obtain KIR2DL1-hFc wild-type (Wagtmann and others (1995)). KIR2DL1-hFc and KIR2DL1 (R131W)-hFc was received in COS7 cells and was isolated from the culture medium, basically as described previously (Wagtmann and others, 1995). To verify the correct folding, KIR2DL1-hFc and KIR2DL1(R131W)-hFc were incubated with cells LCL721.221 that Express either HLA-Cw3 (which is not a ligand for KIR2DL1), or HLA-Cw4 (ligand KIR2DL1), and analyzed the interaction between the fused proteins KR-Fc and cells by the method of FACS - standard method for investigating protein interactions at the cell surface. Example independent experiments are shown figure 11, block A. As expected based on literature data, none of the fused proteins KIR2DL1-hFc was not associated with cells LCL721.221 expressing HLA-Cw3. On the contrary, and KIR2DL1-hFc, and KIR2DL1 (R131W)-hFc was associated with cells LCL721.221 expressing HLA-Cw4, which confirmed their correct folding.

The binding of KIR2DL1 (R131W)-hFc and KIR2DL1-hFc with specific KIR antibodies mb (DF-200, pan2D, EV and GL183) was studied using ELISA method is a standard method for studying protein interactions. Briefly, KIR2DL1 (R131W)-hFc and KIR2DL1-hFc sewn to the cell plate of 96 cells using goat antibodies to human immunoglobulin, after which was added in different concentrations of specific KIR monoclonal antibodies (0-1 μg/ml in PBS). Interaction between KIR2DL1-hFc and antibodies mAb was visualized using spectrophotometry (450 nm), using the associated with peroxidase secondary antibody specific to murine antibodies, for conversion of TMB substrate. Examples of independent experiments are shown figure 11, block Century. while specific to KIR2DL2-3 antibodies GL183 mAb were not able to contact any one of the fused proteins KIR2DL1-hFc specific to KIR2DL1 antibody mAb EB6, DF-200 and Pan2D was associated with options KIR2DL1-hFc with dose dependence. Point mutation (R31W) affected the binding of DF-200 and pan2D, reducing binding to ~10% of the binding of wild-type at higher concentrations of antibodies mAb (1 μg/ml). This confirmed that R131 is part of the binding site of antibodies DF-200 and Pan2D in the extracellular domain 2 KIR2DL1.

All cited publications, patent applications and patents included in the present work by reference to the extent as if it had been specified that each reference was individually incorporated by reference and was given here in its entirety.

All headings and subheadings used in this description for convenience and should not be construed as limiting in any way the present invention.

Any combination of the above-described elements in all possible variations covered by the present invention, unless otherwise stated, or if it is in any other way contrary to clearly sense.

Delineation of intervals of values is the only way to be concise tool for relating individually to each separate value falling within this interval, unless specified, and each separate value is included in the description as if it is listed separately. Unless otherwise specified, all presented here the exact values represent the corresponding approximate values (EmOC is emer, all the exact sample values submitted in connection with a specific factor or dimension can be thought of as representing a corresponding approximate value, and accompanied, where necessary, by the addition of "approximately").

All described in the present description, the methods may be performed in any suitable order unless otherwise indicated, and if it doesn't come in clear contradiction with the context.

Provided for the use of any and all examples, or characteristic constructions (e.g., "such as") are intended only to better illuminate the invention and is not intended to limit the invention, unless otherwise specified. No expressions in the description should not be construed as indicating that any element is necessary for the practice of application of the invention, while it is not precisely specified.

Citation and inclusion in this patent document is for convenience only and do not reflect an opinion on the validity, patentability and/or the imposition of such patent documents.

Describe any aspect or variant of the invention, using such terms as "representing", "having", "including" or "containing", in relation to the element or elements, is the support in order to avoid such an aspect or variant of the invention, which "consist of", "consisting essentially of" or "essentially contain these specific item or specific items, unless otherwise specified or if this is not in obvious contradiction with the context (for example, the composition described herein as containing a particular element should be understood as describing the composition consisting of that element, unless otherwise specified or if it is not clearly contrary to the context).

The present invention in the maximum extent permitted by applicable law includes all modifications and equivalents of the subject matter set forth in these aspects or claim.

1. A method of obtaining antibodies to bind to NK cells, which cross-reacts with the products of the gene KIR2DL1 and KIR2DL2/3 and which neutralizes the inhibitory activity of these KIR, which includes the following steps:
(a) immunization of a mammal, not a person, immunogen containing the KIR2DL polypeptide;
(b) obtaining the antibody from the specified immunized mammal, and these antibodies bind with the specified KIR2DL polypeptide;
(c) the selection of antibodies obtained in step (b), which cross-react what about the least with gene products KIR2DL1 and KIR2DL2/3, and (a) the selection of antibodies obtained in step (C), which is able to restore the lysis of NK cells Cw3+ or Cw4+ target cells;
(e) selecting antibodies that bind to NK cells or KIR polypeptide of primacy.

2. The method according to claim 1, where the primacy in step (e) is the Cynomolgus monkey.

3. The antibody to bind to NK cells obtained by the method according to claim 1, which is associated with KIR2DL1 and KIR2DL2/3, but not with KIR2DS4.

4. The antibody according to claim 3, representing a monoclonal antibody.

5. The antibody according to claim 3, represents a fragment of the antibody.

6. The antibody according to claim 5, representing the Fab, Fab', Fab'-SH, F(ab')2; dianthicola, single-chain fragment antibodies are polyspecific antibody, formed by fragments of antibodies.

7. Derived antibodies for binding to NK-cells containing the antibody according to claim 3, in which the antibody is conjugated or covalently cross-linked to a toxin, a radionuclide, a recognizable group, with a solid carrier or PEG.



 

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EFFECT: increased physiological activity in vivo construction in comparison with native physiologically active polypeptide and increase of half-life in serum of physically active polypeptide with minimal risk of inducing undesirable immune response.

17 cl, 18 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: method of augmentation of duration of action of physiologically active polypeptide in vivo is described. Physiologically active polypeptide is conjugated with Fc fragment of immunoglobulin by means of PEG. Invention use, in comparison with native physiologically active polypeptide, provides the raised physiological activity in vivo designs and-or augmentation of time of a semilife in Serum of physiologically active polypeptide with the minimum risk of induction of undesirable immune responses.

EFFECT: possibility of application of bond at manufacturing of various polypeptides medicinal preparations of the prolonged action.

11 cl, 18 dwg, 8 ex

FIELD: medicine; pharmacology.

SUBSTANCE: immunogenic hybrid polypeptide includes mimetic peptide of V-cellular epitope of apolypoprotein B-100 in which C-end of mimetic peptide is merged with N-end of T-helper epitope. Amino acid sequences of polypeptide variants are presented in description. Described is method of specified polypeptide production providing application of host cell transformed with recombinant express vector including gene coding specified polypeptide. Besides, invention concerns vaccine composition including specified immunogenic hybrid polypeptide for obesity prevention or treatment, recombinant express vector and host cell.

EFFECT: excellent anti-obesity activity without induction of immune response or severe by-effects.

15 cl, 25 dwg, 4 tbl, 15 ex

FIELD: medicine; pharmacology.

SUBSTANCE: it is obtained a chimerous photo protein (photin), presented by amino-acid sequence of obeline protein, which part (from the rest in position 50 to the rest in position 94) is replaced by a homologous site of amino-acid sequence of clitine protein (the rests 53-97). The method of obtaining new chimerous photo protein by the method of recombinant DNA and vectors applied to it and cells is described. It is offered to use photo protein under the invention as the calcium indicator in various test systems in vitro and in vivo.

EFFECT: increased level of bioluminescence in comparison with natural protein.

11 cl, 6 dwg, 2 tbl, 3 ex

FIELD: medicine; pharmacology.

SUBSTANCE: variants of the combined protein which contain the extracellular domain of a human receptor of a hormone of growth and the domain which includes alarm sequence for joining glycosylphosphatidylynozyte (GPI) anchors are offered.

EFFECT: effective medical product for acromegalia and gigantism treatment.

8 cl, 16 dwg

FIELD: medicine.

SUBSTANCE: there is described a humanised CD4 antibody or its fragment able to activate regulatory CD25+CD4+ T-cells, which includes complementarity-determining regions (CDRs) of mice monoclonal CD4 B-F5antibody. There is offered medicinal composition for prevention and/or treatments of dysimmunity containing described antibody or its fragment in effective amount. There is disclosed method of treating a person that implies introduction of the offered composition to said person. The described antibody is able to activate regulatory CD25+CD4+ T-cells.

EFFECT: described antibody can be used for preparing immunosuppressive compositions.

16 cl, 14 dwg, 4 tbl, 4 ex

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