MXPA05014074A - Methods for the production and cytotoxicity evaluation of kir2dl nk-receptor antibodies - Google Patents

Abstract

The present invention relates to novel compositions and methods for regulating an immune response in a subject. More particularly, the invention relates to specific antibodies that regulate the activity of NK cells and allow a potentiation of NK cell cytotoxicity in mammalian subjects. The invention also relates to fragments and derivatives of such antibodies, as well as pharmaceutical compositions comprising the same and their uses, particularly in therapy, to increase NK cell activity or cytotoxicity in subjects.

Description

METHODS FOR THE PRODUCTION AND EVALUATION OF THE CYTOTOXICITY OF THE NK KIR2DL RECEPTOR ANTIBODIES FIELD OF THE INVENTION The present invention relates to antibodies, antibody fragments and derivatives thereof that react with two or more inhibitory receptors present on the cell surface of NK cells and enhance the cytotoxicity of NK cells in mammalian subjects or in a sample biological The invention also relates to methods of producing the antibodies, fragments, variants and derivatives: pharmaceutical compositions comprising the same; and the use of molecules and compositions, particularly in therapy, to increase NK cell activity or cytotoxicity in subjects. BACKGROUND OF THE INVENTION Natural killer cells (NK) are a sub-population of lymphocytes, involved in unconventional immunity. NK cells can be obtained by various techniques known in the art, such as from blood samples, cytoteresis, collections, etc.

The characteristic and biological properties of NK cells include the expression of surface antigens including CD16, CD56, and / or CD57; the absence of the complex Ref .: 169068 alpha / beta or gamma / delta TCR on the surface of the cell; the ability to bind and kill cells that fail to express "self" MHC / HLA antigens by activating specific cytolytic enzymes; the ability to remove tumor cells or other diseased cells that express a receptor ligand that activates NK; the ability to release cytokines that stimulate or inhibit the immune response; and the ability to subject multiple rounds of cell division and produce daughter cells with biological properties similar to the precursor cell. Within the context of this invention, "active" NK cells designate biologically active NK cells, more particularly NK cells that have the ability to lyse targeting cells. For example, an "active" NK cell is capable of removing cells that express an NK activation receptor ligand and fails to express "self" MHC / HLA antigens (incompatible KIR cells). Based on their biological properties, several therapeutic and vaccine strategies have been proposed in the art, which depend on a modulation of NK cells. However, the activity of NK cells is regulated by a complex mechanism that involves both stimulation signals and djibibers. Accordingly, effective NK-mediated therapy may require both a stimulation of these cells and a neutralization of the inhibitory signals.

NK cells are negatively regulated by specific inhibitory receptors of class I of the major histocompatibility complex (MHC) (Kre et al, 1986, Ohlén et al., 1989). These specific receptors bind polymorphic determinants of class I MHC or HLA molecules present in other cells and inhibit the lysis of NK cells. In humans, certain members of a family of receptors called Ig-terminating receptor-like receptors (KIR) recognize groups of class I HLA alleles. KIRs are a large family of receptors present in certain subsets of lymphocytes, which include NK cells. The nomenclature for the KIRs is based on the number of extracellular domains (KIR2D or KIR3D) and if the cytoplasmic end is either long KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). In humans, the presence or absence of a given KIR is variable from one NK cell to another within the NK population present in a single individual. Within the human population there is also a relatively high level of polymorphism of KIR molecules, with certain KIR molecules that are present in some, but not in all individuals. Certain KIR gene products cause stimulation of lymphocyte activity when bound to an appropriate ligand. All confirmed stimulatory KIRs have a short cytoplasmic end with a loaded transmembrane residue that associates with an adapter molecule that has an immunostimulatory portion (ITAM). Other KIR gene products are inhibitors by nature. All confirmed inhibitory KIRs have a long cytoplasmic end and appear to interact with different subsets of the HLA antigens depending on the KIR subtype. The inhibitory KIRs exhibit in their intracytoplasmic portion one or more inhibitory portions that recruit phosphatases. Known inhibitory KIR receptors include members of the KIR2DL and KIR3DL subfamilies. KIR receptors having two Ig domains (KIR2D) identify allotypes HLA-C: KIR2DL2 (previously designated p58.2) or the closely related gene product KIR2DL3 recognizes an epitope formed by group 2 allotypes HLA-C (Cwl, 3, 7, and 8), considering that KIR2DL1 (p58.1) recognizes an epitope formed by the reciprocal group 1 of HLA-C allotypes (Cw2, 4, 5, and 6). Recognition by KIR2DL1 is dictated by the presence of a Lys residue at position 80 of the HLA-C alleles. The recognition of KIR2DL2 and KIR2DL3 is dictated by the presence of an Asn residue at position 80. Importantly, the vast majority of HLA-C alleles have either an Asn residue or a Lys residue at position 80. A KIR with three domains Ig, KIR3DL1 (p70), recognizes an epitope formed by HLA-Bw4 alleles. Finally, a homodimer of molecules with three Ig domains KIR3DL2 (pl40) recognizes HLA-A3 and -All.

Although KIR inhibitors and other class I inhibitory receptors (Moretta et al., 1997; Valíante et al., 1997a; Lanier, 1998) can be co-expressed by NK cells, in any NK repertoire of the given individual there are cells expressing a unique KIR and thus, the corresponding NK cells are blocked only by cells expressing a specific class I allele group. The population of NK cells or clones that are non-coincident with KIR, that is, population of NK cells expressing KIR that are not compatible with HLA molecules of a host, have been shown to be the most likely mediators of the anti-leukemia effect of graft seen in the allogeneic transplant (Ruggeri et al., 2002). One way to reproduce this effect in a given individual may be to use reagents that block the KIR / HLA interaction.

It has been shown that monoclonal antibodies specific for KIR2DL1 block the interaction of KIR2DL1 with Cw4 (or similar) alleles (Moretta et al., 1993).

Monoclonal antibodies against KIR2DL2 / 3 have also been described that block the interaction of KIR2DL2 / 3 with HLACw3 (or similar) alleles (Moretta et al., 1993). However, the use of reagents in clinical situations may require the development of two therapeutic mAbs to treat all patients, regardless of whether any given patient was expressing class 1 or class 2 HIA-C alleles. Moreover, one could predetermine which type of HLA was expressed by each patient before deciding which therapeutic antibody to use, resulting in much higher cost of treatment. atzl et al., Tissue Antigens, 56, p. 240 (2000) produced cross-reactive antibodies that recognize multiple isotypes of KIR, but those antibodies show no potency of NK cell activity. G. M. Spaggiara et al., Blood, 100, pp. 4098-4107 (2002) carried out experiments using numerous monoclonal antibodies against several KIRs. It is said that one of these antibodies, NKVSF1, recognizes a common epitope of CD158a KIR2DL1), CD158b (KIR2DL2) and p50.3 (KIR2DS4). This does not suggest that NKVSF1 can potentiate the activity of NK cells and there is no suggestion that it can be used as a therapeutic. Consequently, practical and effective approaches in the modulation of NK cell activity have not been made available in the art and still require specific intervention of the HLA allele using specific reagents. BRIEF DESCRIPTION OF THE INVENTION The present invention now provides novel antibodies, compositions and methods that overcome current difficulties in the activation of NK cells and provide additional advantageous features and benefits. In an exemplary aspect, the invention provides a unique antibody that facilitates the activation of human NK cells in virtually all humans. More particularly, the invention provides novel specific antibodies which cross-react with several inhibitory KIR groups and neutralize their inhibitory signals, resulting in potentiation of the cytotoxicity of NK cells in NK cells expressing inhibitory KIR receptors. This ability to cross-react with KIR gene products allows the antibodies of the invention to be effectively used to increase the activity of NK cells in most human subjects, without the charge or cost of predetermination of the HLA type of the subject. In a first aspect, the invention provides antibodies, antibody fragments and derivatives of any of these, wherein the antibody, fragment, or derivative cross-reacts with at least two inhibitory KIR receptors on the surface of NK cells, neutralizes the signals inhibitors of NK cells, and potentiates the activity of NK cells. More preferably, the antibody binds a common determinant of human KIR2DL receptors. Even more specifically, the antibody of this invention binds at least KIR2DL1, KIR2DL2 and KIR2DL3 receptors. For the purposes of this invention, the term "KIR2DL2 / 3" refers to either of the KIR2DL2 and KIR2DL3 receptors. These two receptors have a very high homology, presumably they are allelic forms of the same gene, and are considered by the technique to be interchangeable. Accordingly, KIR2DL2 / 3 is considered to be a simple inhibitory KIR molecule for the purposes of this invention and therefore an antibody that cross-reacts with only KIR2DL2 and KIR2DL3 and no inhibitory KIR receptor notro are within the scope of this invention. The antibody of this invention specifically inhibits the binding of MHC and HLA molecules to at least two inhibitory KIR receptors and facilitates the activity of NK cells. Both activities are inferred by the term "neutralizes the inhibitory activity of KIR", as used here. The ability of the antibodies of this invention to "facilitate the activity of NK cells", "facilitate the cytotoxicity of NK cells", "facilitate NK cells", "enhance NK cell activity", "enhance NK cell cytotoxicity", or " enhancing NK cells "in the context of this invention means that the antibody allows NK cells expressing an inhibitory KIR receptor on their surface to be able to lyse cells expressing on their surface a corresponding ligand for that particular inhibitory KIR receptor ( for example, a particular HLA antigen). In a particular aspect, the invention provides an antibody that specifically inhibits the binding of HLA-C molecules to KIR2DL1 and KIR2DL2 / 3 receptors. In another particular aspect, the invention provides an antibody that facilitates the activity of NK cells in vivo. Because at least one of KIR2DL1 or KIR2DL2 / 3 is present in at least about 90% of the human population, the most preferred antibodies of this invention are capable of facilitating the activity of the NK cell against most of the cells associated with allotype HLA-C, respectively group 1 allotypes HLA-C and group 2 allotypes HLA-C. Thus, the compositions of this invention can be used to effectively activate or enhance NK cells in most human individuals, usually in about 90% of human individuals or more. Accordingly, a single antibody composition according to the invention can be used to treat most human subjects, and there is rarely a need to determine allelic groups or use antibody cocktails. The invention demonstrates, for the first time, that cross reactive and neutralizing antibodies against KIR inhibitors can be generated, and that such antibodies allow for the effective activation of NK cells in a wide range of human groups. A particular objective of this invention thus resides in an antibody, wherein the antibody specifically binds both human receptors KIR2DL1 and KIR2DL2 / 3 and reverses the inhibition of NK cell cytotoxicity mediated by these KIRs. In one embodiment, the antibody competes with the monoclonal antibody DF200 produced by the Hybridoma DF200. Optionally, the antibody that competes with the DF200 antibody is not the same DF200 antibody. In another embodiment, the antibody competes with monoclonal antibody NKVSF1, optionally wherein the antibody competing with the NKVSF1 antibody is not the NKVSF1 antibody. In another embodiment, the antibody competes with the antibody 1-7F9. Preferably the antibodies are chimeric antibodies, humanized antibodies, or human antibodies. The term "competes with" when referring to a particular monoclonal antibody (eg, DF200, NKVSF1, 1-7F9, EB6, FL183) means that an antibody competes with the monoclonal antibody (eg, DF200, NKVSF1, 1-7F9). , EB6, GL183) in a binding assay using any of the recombinant KIR molecules or surface expressed KIR molecules. For example, if an antibody reduces the binding of DF200 to a KIR molecule in a binding assay, the antibody "competes" with DF200. An antibody that "competes" with DF200 may compete with DF200 for binding to the human receptor KIR2DL1, the human receptor KIR2DL2 / 3, or both human receptors KIR2DL1 and KIR2DL2 / 3.

In a preferred embodiment, the invention provides an antibody that binds both human receptors KIR2DL1 and KIR2DL2 / 3, reverses the inhibition of NK cell cytotoxicity mediated by these KIRs, and competes with DF200, 1-7F9, or NKVSF1 for binding the human receptor KIR2DL1, human receptor KSR2DL2 / 3, or both human receptors KIR2DL1 and KIR2DL2 / 3. Optionally, the antibody is not NKVSF1. Optionally, the antibody is a chimeric, human or humanized antibody. In another embodiment, the invention provides an antibody that binds both human receptors KIR2DL1 and KER2DL2 / 3, reverses the inhibition of NK cell cytotoxicity mediated by these KIR, and competes with EB6 for human receptor binding KIR2DL1, competes with GL183 for binding of the human receptor KIR2DL2 / 3, or competes with both EB6 for binding of the human receptor KIR2DL1 and GL183 for binding to the human receptor KIR2DL2 / 3. Optionally, the antibody is not NKVSF1; optionally the antibody is not DF200. Optionally, the antibody is a chimeric, human or humanized antibody. In one advantageous aspect, the invention provides an antibody that competes with DF200 and recognizes, binds, or has immunospecificity for substantially or essentially the same, or the same, epitope or "epitope site" in a KIR molecule as the monoclonal antibody. DF200. Preferably, the KIR molecule is a human KIR2DL1 receptor or a human KIR2DL2 / 3 receptor.

A particular objective of this invention resides in an antibody, wherein the antibody binds a common determinant present in both human receptors KIR2DL1 and KIR2DL2 / 3 and reverses the inhibition of NK cell cytotoxicity mediated by these KIRs. The antibody more specifically binds completely the same epitope in KIR as monoclonal antibody DF200 produced by hybridoma DF200 or NKVSF1 antibody produced by hybridoma NKVSF1, where the antibody is not NKVSF1. In a preferred embodiment, the antibody of this invention is a monoclonal antibody. The most preferred antibody of this invention is a monoclonal antibody DF200 produced by hybridoma DF200. The hybridoma that produces the DF200 antibody has been deposited in the CNCM culture collection, as Identification No. "DF200", Registration No. CNCM 1-3224, registered on June 10, 2004, Collection Nationale de Cultures de Microorganismes, Institut Pasteur , 25, Rué du Docteur Roux, F-75724 Paris Cedex 15, France. The NKVSF1 antibody is available from Serotec (Cergy Sainte-Christophe, France), Catalog No. MCA2243. NKVSFl is also referred to as pan2D mAb here. The invention also provides functional fragments and derivatives of the antibodies described herein, which have substantially similar antigenic activity and specificity (eg, which can cross-react with the familial antibody and which enhance the cytotoxicity activity of the NK cells they express inhibitory KIR receptors, including, without limitation, a Fab fragment, a Fab '2 fragment, an immunoadhesin, a diabody, a CDR, and a ScFv In addition, the antibodies of this invention can be humanized, human or chimeric. it also provides antibody derivatives comprising an antibody of the invention conjugated or covalently linked to a toxin, a radionuclide, a detectable portion (eg, a fl oor), or a solid support.The invention also provides pharmaceutical compositions comprising an antibody as described above, a fragment of this, or a derivative or any of these. Accordingly, the invention also relates to the use of an antibody as described herein in a method for the manufacture of a medicament. In preferred embodiments, the medicament or pharmaceutical composition is for the treatment of a cancer or other proliferative disease, an infection, or for use in transplantation. In a modality, the invention provides a composition comprising an antibody that binds at least two different inhibitory KIR receptor gene products, wherein the antibody is capable of neutralizing the KIR mediated inhibition of NK cell cytotoxicity in NK cell expression. at least one of the two different human inhibitory KIR receptors, wherein the antibody is incorporated into a liposome. Optionally the composition comprises an additional substance selected a nucleic acid molecule for the delivery of genes for gene therapy; a nucleic acid molecule for the delivery of RNA, RNAi, or antisense siRNA for suppression of a gene in an NK cell; or a toxin or a drug for the targeted killing of NK cells additionally incorporated in the liposome. The invention also provides methods of regulating activity of human NK cells in vitro, ex vivo, or in vivo, comprising contacting human NK cells with an effective amount of an antibody of the invention, a fragment of such an antibody, a derived from any of these, or a pharmaceutical composition comprising at least one of any of these. Preferred methods comprise the administration of an effective amount of pharmaceutical compositions of this invention and are directed to the increase of the cytotoxic activity of human NK cells, more preferably ex vivo or in vivo, in a subject having cancer, an infectious disease, or an immune disease.

In more aspects, the invention provides a hybridoma comprising: (a) a B cell of a mammalian host (commonly a non-human mammalian host) that has been immunized with an antigen comprising an epitope present on an inhibitory KIR polypeptide, fused to (b) an immortalized cell (eg, a myeloma cell), wherein the hybridoma produces a monoclonal antibody that binds at least two different different human inhibitory KIR receptors and is capable of at least substantially neutralizing the mediated inhibition of KIR of NK cell cytotoxicity in a population of NK cells expressing at least the two different human inhibitor KIR receptors. Optionally, the hybridoma does not produce the monoclonal antibody NKVSF1. Preferably the antibody binds the KIR2DL1 and KIR2DI_2 / 3 receptors. Preferably the antibody binds a common determinant present in KIR2DL1 and KIR2DL2 / 3. Preferably the hybridoma produces an antibody that inhibits the binding of an HLA-c allele molecule having a Lys residue at position 80 to a human KIR2DL1 receptor, and the linkage of the allele molecule HLA-C having an Asn residue in position. 80 to human ER2DL2 / 3 receptors. Preferably the hybridoma produces an antibody that binds to the same epitope substantially as monoclonal antibody DF200 produced by hybridoma DF200 in either KIR2DL1 or KIR2DL2 / 3 or both KIR2D 1 and KIR2DL2 / 3. An example of such a hybridoma is DF200.

The invention also provides methods of producing an antibody, which cross-reacts with multiple KIR2DL gene products and which neutralizes the inhibitory activity of KIRs, the method comprising the steps of: (a) immunizing a non-human mammal with a immunogen comprising a KIR2DL polypeptide; (b) preparation of antibodies from the immunized mammal, wherein the antibodies bind the KIR2DL polypeptide, (c) selection of antibodies from (b) which cross-react with at least two different KIR2DL gene products, and (d) selection of antibodies from (c) that potentiate NK cells. In one embodiment, the non-human mammal is a transgenic animal designed to express a repertoire of human antibodies (eg, a non-human mammal comprising human immunoglobulin sites and deletions of native immunoglobulin gene, such as a Xenomouse ™ (Abgenix- Fremont, Ca, USA) or non-human mammal comprising a miniluge of genes encoding human Ig, such as the HuMab ™ mouse (Medarex-Princeton, NJ, USA)). Optionally, the method further comprises selecting an antibody that binds a primate, preferably a cynomolgus monkey, NK cell or KIR polypeptide. Optionally, the invention further comprises a method of evaluating an antibody, wherein an antibody produced according to the above method is administered to a primate, preferably a cynomolgus monkey, preferably wherein the monkey is observed for the presence or absence of an indication of antibody toxicity. The inventors also provide a method of producing an antibody that binds at least two different human inhibitor KIR receptor gene products, wherein the antibody is capable of neutralizing the mediated KIR inhibition of NK cell cytotoxicity in a population of NK cells expressing at least the two different human inhibitory KIR receptor gene products, the method comprises the steps of: (a) immunizing a non-human mammal with an immunogen comprising an inhibitory KIR polypeptide; (b) preparation of antibodies from the immunized animal, wherein the antibodies bind the KIR polypeptide, (c) selection of antibodies from (b) which cross-react with at least two different human inhibitor KIR receptor gene products, and selection of antibodies of (c) which are capable of neutralizing the KIR mediated inhibition of NK cell cytotoxicity in a population of NK cells expressing at least two different human inhibitor KIR receptor gene products, wherein the order of steps (c) and (d) is optionally reversed and any number of steps is optionally represented 1 or more times. Preferably, the inhibitory KIR polypeptide used for immunization is a KIR2DL polypeptide and the antibodies selected in step (c) cross-react with at least KIR2DL1 and KIR2DL2 / 3. Preferably the antibody recognizes a common determinant present in at least two different KIR receptor gene products; more preferably the KIRs are KIR2DL1 and KIR2DL2 / 3. Optionally, the method further comprises selecting an antibody that binds a primate, preferably a cynomolgus monkey, NK cell or KIR polypeptide. Optionally, the invention further comprises a method of evaluating an antibody, wherein an antibody produced according to the above method is administered to a primate, preferably a cynomolgus monkey, preferably wherein the monkey is observed for the presence or absence of an indication of antibody toxicity. Optionally, in the methods described above, the antibody selected in step c) or d) is not NKVSF1. Preferably, the antibody prepared in step (b) in the above methods is a monoclonal antibody. Preferably the antibody selected in step (c) in the above methods inhibits the binding of an HLA-C allele molecule having a Lys residue at position 80 to a human KIR2DL1 receptor, and the binding of a HLA-C allele molecule. which has an Asn residue in position 80 to human KIR2DL2 / 3 receptors. Preferably, the antibodies selected in step (d) in the above methods cause an enhancement in NK toxicity, for example any substantial potentiation, or at least 5%, 10%, 20%, 30% or greater potentiation in cytotoxicity NK, for example, at least about 50% potentiation of targeted NK cytotoxicity (eg, at least about 60%, at least about 70%, at least about 80%, at least about of 85%, at least about 90%, at least about 95%, (such as, for example about 65-100%) potentiation of NK cell cytotoxicity). Preferably, the antibody binds to substantially the same epitope as monoclonal antibody DF200 in KIR2DL1 and / or KIR2DL2 / 3. Optionally the methods also or alternatively comprise the additional step of producing fragments of the selected monoclonal antibodies, making derivatives of the selected monoclonal antibodies (eg, by conjugation with a radionuclide, cytotoxic agent, or the like), or production of fragment derivatives. of antibodies produced from or comprising sequences corresponding to the sequences of such monoclonal antibodies.

The invention further provides a method of producing an antibody that binds at least two different human inhibitory KIR receptor gene products, wherein the antibody is capable of neutralizing the KIR mediated inhibition of NK cell cytotoxicity in a population of cells. NK expressing at least the two different human inhibitory KIR receptor gene products, the method comprises the steps of: (a) screening, from a library or repertoire, a monoclonal antibody or an antibody fragment that cross-reacts with at least two different human inhibitory KIR receptor gene products, and (b) selection of an antibody from (a) that can neutralize the KIR mediated inhibition of NK cell cytotoxicity in a population of NK cells that express at least the two different human inhibitor KIR receptor gene products. Preferably the antibody binds a common determinant present in KIR2DL1 and KIR2DL2 / 3. Optionally, the antibody selected in step (b) is not NKVSF1. Preferably, the antibody selected in step (b) inhibits the binding of a HLA-c allele molecule having a Lys residue at position 80 to a human KIR2DL1 receptor, and the linkage of a HLA-C allele molecule having a Asn residue in position 80 to human KIR2DL2 / 3 receptors. Preferably, the antibody selected in step (b) causes an enhancement in NK cytotoxicity, for example any substantial potentiation, or at least 5%, 10%, 20%, 30% or greater potentiation in the cytotoxicity of NK, by example, at least about 50% potentiation of targeted NK cytotoxicity (eg, at least about 60%, at least about 70%, at least about 80%, at least about 85% , at least about 90%, at least about 95%, (such as, for example around 65-100%) empowerment of the cytotoxicity of the NK cell). Preferably, the antibody binds to substantially the same epitope as monoclonal antibody DF200 in KIR2DL1 and / or KIR2DL2 / 3. Optionally, the method comprises the additional step of producing fragments of the selected monoclonal antibodies, making derivatives of the selected monoclonal antibodies, or producing monoclonal antibody fragment derivatives. Additionally, the invention provides a method of producing an antibody that binds at least two different human inhibitory KIR receptor gene products, wherein the antibody is capable of neutralizing the KIR mediated inhibition of NK cell cytotoxicity in a population of NK cells expressing at least the two different human inhibitory KIR receptor gene products, the method comprises the steps of: (a) cultivating a hybridoma of the invention under conditions permissive for the production of the monoclonal antibody; and (b) separation of the monoclonal antibody from the hybridoma. Optionally, the method comprises the additional step of production of monoclonal antibody fragments, production of monoclonal antibody derivatives, or production of derivatives of the monoclonal antibody fragments. Preferably the antibody binds to a common determinant present in KIR2DL1 and KIR2DL2 / 3. Also provided by the present invention is a method of producing an antibody that binds at least two different human inhibitory KIR receptor gene products, wherein the antibody is capable of neutralizing the KIR mediated inhibition of NK cell cytotoxicity in a Population of NK cells expressing at least the two different human inhibitory KIR receptor gene products, the method comprises the steps of: (a) isolating a nucleic acid encoding the monoclonal antibody from a hybridoma of the invention; (b) optionally modifying the nucleic acid so as to obtain a modified nucleic acid comprising a sequence encoding a modified antibody or derivative comprising an amino acid sequence corresponding to or substantially similar to a functional sequence of the monoclonal antibody (e.g., is at least about 60%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, (such as, for example, around 70-99%) identical to such a sequence) selected from a humanized antibody, a chimeric antibody, a single chain antibody, an immunoreactive fragment of an antibody, or a fusion protein comprising an immunoreactive fragment such; c) insertion of the nucleic acid or modified nucleic acid (or related nucleic acid encoding the same amino acid sequence) into an expression vector, wherein the encoded antibody or antibody fragment is capable of being expressed when the expression vector is present in a host cell grown under appropriate conditions; d) transfection of a host cell with the expression vector, wherein the host cell does not otherwise produce immunoglobulin protein; e) culturing the transfected host cell under conditions that elicit the expression of the antibody or antibody fragment; and f) isolation of the antibody or antibody fragment produced by the transfected host cell. Preferably the antibody binds a common determinant present in KIR2DL1 and KIR2DL2 / 3.

It will be appreciated that the invention also provides a composition comprising an antibody that binds at minus two different human inhibitory KIR receptor gene products, wherein the antibody is capable of neutralizing the KIR mediated inhibition of NK cell cytotoxicity in NK cells expressing at least one of the two KIR receptor gene products human inhibitor, the antibody that is present in an amount effective to detectably enhance the cytotoxicity of the NK cell in a patient or in a biological sample comprising NK cells; and a pharmaceutically acceptable carrier or excipient. Preferably the antibody binds a common determinant present in KIR2DL1 and KIR2DL2 / 3. The composition optionally may further comprise a second therapeutic agent selected from, for example, an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, a second antibody that binds and inhibits an inhibitory KIR receptor. , an anti-infective agent, a targeting agent, or an adjunct. Advantageous immunomodulatory agents can be selected from IL-lalfa, IL-lbeta, 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-beta, 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 the chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, adriamycin, dactinomycin, mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil ( 5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, captothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatin (s), other vinyl alkyloids and derivatives or prodrugs thereof. Examples of hormonal agents include leuprorelin, goserelin, triptorelin, buserelin, tamoxifen, toremifene, fultamide, nilutamide, cyproterone bicalutamid anastrozole, exemestane, letrozole, medroxy fadrozole, chlormadinone, megestrol, other LHRH agonists, other anti-estrogens, other anti-androgens, other aromatase inhibitors, and other progestogens. Preferably, the second antibody that binds and inhibits an inhibitory KIR receptor is an antibody or a derivative or fragment thereof that binds an epitope of an inhibitory KIR receptor that differs from the epitope bound by the antibody binding a common determinant present in at least two different human inhibitor KIR receptor gene products. The invention further provides a method of detectably enhancing the activity of NK cells in a patient in need thereof, comprising the step of administering to the patient a composition according to the invention. A patient in need of potentiation of NK cell activity can be any patient having a disease or disorder wherein the potentiation can promote, ameliorate and / or induce a therapeutic effect (or promote, improve, and / or induce such an effect). in at least a substantial portion of patients with the disease or disorder and substantially similar characteristics as the patient - as may be determined, for example, by clinical tests). A patient in need of such treatment may be suffering from, for example, cancer, another proliferative disorder, an infectious disease or an immune disorder. Preferably the method comprises the additional step of administering to the patient an appropriate additional therapeutic agent selected from an immunomodulatory agent, a hormonal agent, a chemotherapeutic agent, an anti-angiogenic agent, an apoptotic agent, a second antibody that binds and inhibits a receptor KIR inhibitor, an anti-infective agent, a targeting agent or an adjunct compound wherein the additional therapeutic agent is administered to the patient as a single dosage form together with the antibody, or as a separate dosage form. The dosage of the antibody (or fragment / antibody derivative) and the dosage of the additional therapeutic agent are collectively sufficient to induce, promote, and / or detectably improve a therapeutic response in the patient comprising enhancing the activity of NK cells. Where administered separately, the antibody, fragment, or derivative and the additional therapeutic agent are desirably administered under conditions (eg, with respect to times, number of doses, etc.) that result in a combined therapeutic benefit detectable for the patient. In addition, antibodies of the invention that are capable of binding specifically to NK cells of non-human primate, preferably of monkey, and / or to KIR receptors of monkey are encompassed by the present invention. Also included are methods for evaluating the toxicity, dose and / or activity or efficacy of antibodies of the invention that are candidate drugs. In one aspect, the invention encompasses a method for determining a dose of an antibody that is toxic to a target animal or tissue by administering an antibody of the invention to a non-human primate recipient animal having NK cells, and evaluating any effect toxic or harmful or adverse agent in the animal, or preferably in a target tissue. In another aspect, the invention is a method for identifying an antibody that is toxic to a target animal or tissue by administering an antibody of the invention to a non-human primate recipient animal having NK cells, and evaluating any toxic effect or of elimination or adverse agent in the animal, or preferably in a target tissue. In another aspect, the invention is a method for identifying an antibody that is effective in the treatment of an infection, disease or tumor by administering an antibody of the invention to a non-human primate model of infection, disease or cancer, and identification of the antibody that decreases the infection, disease or cancer, or a symptom of these. Preferably the antibody of the invention is an antibody that (a) cross-reacts with at least two inhibitory human KIR receptors on the surface of human NK cells, and (b) cross-reacts with NK cells or a KIR receptor of the non-human primate. Further encompassed by the present invention is a method of detecting the presence of NK cells carrying an inhibitory KIR on their cell surface in a biological sample or a living organism, the method comprising the steps of: a) contacting the biological sample or living organism with an antibody of the invention, wherein the antibody is conjugated or covalently linked to a detectable portion; and b) detecting the presence of the antibody in the biological sample or living organism.

The invention also provides a method of purifying sample NK cells that carry an inhibitory KIR on their cell surface comprising the steps of: a) contacting the sample with an antibody of the invention under conditions that allow NK cells to give a KIR inhibitor on its cell surface for binding the antibody, wherein the antibody is conjugated or covalently linked to a solid support (e.g., a bead, a matrix, etc.); b) elution of the bound NK cells of the antibody conjugated or covalently linked to a solid support. In a further aspect, the invention provides an antibody, antibody fragment, or derivative of any of these, comprising the light variable region or one or more light variable region CDR of antibody DF200 or Pan2D antibody as illustrated in FIG. 12. In still another aspect, the invention provides an antibody, antibody fragment, or derivative of any of these comprising a sequence that is mostly similar to all or substantially all of the light variable region sequence of DF200 or Pan2D or one or more CDR of light variable region of one or both of these antibodies. In a further aspect, the invention provides an antibody, antibody fragment, or derivative of any of these, comprising the heavy variable region or one or more light variable region CDR of antibody DF200 as illustrated in FIG. 13. In yet another aspect, the invention provides an antibody, antibody fragment, or derivative of any of these comprising a sequence that is highly similar to all or substantially all of the heavy variable region sequence of DF200. These and additional advantageous aspects and features of the invention may also be described elsewhere herein. BRIEF DESCRIPTION OF THE FIGURES Figure 1 represents the monoclonal antibody DF200 that binds to a common determinant of several human KIR2DL receptors. Figure 2 depicts a monoclonal antibody DF200 that neutralizes the mediated KIR2DL inhibition of cytotoxicity of KIR2DL1 NK positive cells in C4 positive targeting cells. Figure 3 depicts a monoclonal antibody DF200, a Fab fragment of DF200 and conventional specific antibodies KIR2DL1 or KIR2DL2 / 3 that neutralize KIR2DL mediated inhibition of positive NK cell cytotoxicity KIR2DL1 in positive targeting cells Cw4 and the mediated jjliibition of _IR2DL of NK cell cytotoxicity K__R2DL2 / 3 in positive direction cells Cw3.

Figures 4A and 4B depict the reconstitution of cell lysis by NK clones of Cw4 positive targeting cells in the presence of F (ab ') 2 fragments of antibodies DF200 and EB6. Figures 5A and 5B and 6A and 6B represent monoclonal antibodies DF200, NKVSF1 (pan2D), human antibody 1-7F9, 1-4F1, 1-6F5 and 1-6F1, and conventional antibodies specific KIR2DL1 or KIR2DL2 / 3 that neutralize the inhibition KIR2DL mediated cytotoxicity of KIR2DL1 positive NK cell cytotoxicity in Cw4 positive cells (transfected Cw4 cells in Figure 5 and EBV cells in Figure 6). Figure 7 depicts an epitope map showing results of competitive binding experiments obtained by plasma surface resonance analysis (BIAcore®) with anti-KIR antibodies to KIR2DL1, where the overlapping circles designate link overlap to KIR2DL1. The results show that 1-7F9 is competitive with EB6 and 1-4F1, but not with NKVSFl and DF200, in KIR2DL1. The 1-4F1 antibody in turn is competitive with EB6, DF200, NKVSF1, and 1-7F9. The NKVSF1 antibody competes with DF200, 1-4F1, and EB6, but not with 1-7F9, in KIR2DL1. DF200 competes with NKVSFl, 1-4F1, and EBß, but not with 1-7F9, in KIR2DL1. Figure 8 depicts an epitope map showing results of competitive binding experiments obtained by BIAcore® analysis with anti-KIR antibodies for KIR2DL3, where the overlapping circles designate link overlap to KIR2DL3. The results show that 1-4F1 is competitive with NKVSFl, DF200, gll83, and 1-7F9 in KIR2DL3. 1-7F9 is competitive with DF200, g! 183, and 1-4F1, but not with NKVSF2, in KIR2DL3. NKVSFl competes with DF200, 1-4F1, and FL183, but not with 1-7F9, in KIR2DL3. DF200 competes with NKVSFl, 1-4F1, and 1-7F9, but not with FL183, in KIR2DL3. Figure 9 depicts an epitope map showing results of competitive binding experiments obtained by BIAcore® with anti-KIR antibodies to KIR2DS1, where the overlapped circles designate link overlap to KIR2DS1. The results show that the 1-4F1 antibody is competitive with NKVSF1, DF200, and 1-7F9 in KIR2DS1. The antibody 1-7F9 is competitive with 1-4F1, but not competitive with DF200 and NKVSFl in KIR2DS1. NKVSFl competes with DF200 and 1-4F1, but not with 1-7F9, in KIR2DS1. DF200 competes with NKVSF12 and 1-4F1, but not with 1- 7F9, in KIR2DS1. Figure 10 represents the titration of NKVSF1 (pan2D) demonstrating the binding of the mAb to NK cells of cinomolgus. The NK cells of Cinsmolgus (NK Volume 16 day) were incubated with different amount of Pan2D mAb followed by IgG anti-mouse antibodies (H + L) from goat F (ab ') 2 fragments conjugated with PE. Figure 11b shows the percentage of positive cells was determined with an isotopic control (purified mouse IgGl). The samples were made in duplicate. Fluorescence intensity of the medium = MFI.

Figure 12 provides a comparative alignment of the amino acid sequences of the light variable regions and the light variable region CDRs of antibodies DF200 and Pan2D mAb. Figure 13 provides the heavy variable region of the DF200 antibody. DETAILED DESCRIPTION OF THE INVENTION Antibodies The present invention provides antibodies and fragments or derivatives of these novel ones that bind common determinants of human inhibitory KIR receptors, preferably a determinant present in at least two different KIR2DL gene products, and cause cell enhancement NK expressing at least one of these KIR receptors. The invention describes, for the first time, that cross reaction and neutralization of antibodies can occur, which represents an unexpected result and opens a door to novel and effective NK-based therapies, particularly in human subjects. In a preferred embodiment, the antibody is not the monoclonal antibody NKVSF1. Within the context of this invention a "common determinant" is designated a determinant or epitope that is formed by several gene products of human inhibitory KIR receptors. Preferably, the common determinant is shared by at least two members of the KIR2DL receptor group. More preferably, the determinant is shared by at least KIR2DL1 and KIR2DL2 / 3. Certain antibodies of this invention can in addition to recognizing multiple gene products of KIR2DL, also recognize determinants present in other KIR inhibitors, such as gene product of the KIR3DL receptor group. The determinant or epitope may represent a peptide fragment or a conformational epitope shared by the members. In a more specific embodiment, the antibody of this invention specifically binds substantially the same epitope recognized by the monoclonal antibody DF200. This determinant is present in both KIR2DL1 and KIR2DL2 / 3. Within the context of this invention, the term "antibody" that "binds" a common determinant designates an antibody that binds the determinant with specificity and / or affinity. The term "antibody", as used herein, refers to polyclonal and monoclonal antibodies, in addition to fragments and derivatives of polyclonal and monoclonal antibodies unless otherwise stated or clearly contradicted by context. Depending on the type of constant domain in heavy chains, very long antibodies are commonly assigned to one of five classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, and the like. The heavy chain constant domains corresponding to the immunoglobulin difference classes are referred to as "alpha", "delta", "epsilon", "gamma" and "mu", respectively. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG and / or IgM are the preferred classes of antibodies used in this invention because they are the most common antibodies in the physiological situation and because they are more easily produced in a laboratory environment. Preferably the antibody of this invention is a monoclonal antibody.

Because one of the goals of the invention is to block the interaction of an inhibitory KIR and its corresponding HLA ligand in vivo without suppressing the cells, the isotypes corresponding to Fe receptors that mediate low effector function, such as IgG, they are commonly preferred. The antibodies of this invention can be produced by a variety of techniques known in the art. Commonly, they are produced by immunization of a non-human animal, preferably a mouse, with an immunogen comprising an inhibitory KIR polypeptide, preferably a KIR2DL polypeptide, more preferably a human KIR2DL polypeptide. The inhibitory KIR polypeptide may comprise the full length sequence of a human inhibitory KIR polypeptide, or a fragment or derivative thereof, commonly an immunogenic fragment, that is, a portion of the polypeptide comprising an epitope exposed on the surface of the cell expressing an inhibitory KIR receptor. Such fragments commonly contain at least about 7 consecutive amino acids of the mature polypeptide sequence, even more preferably at least about 10 consecutive amino acids thereof. The fragments are commonly derived essentially from the extracellular domain of the receptor. Even more preferred is a human KIR2DL polypeptide which includes at least one, more preferably both, extracellular Ig domains, of the full-length KLRDL polypeptide and is capable of mimicking at least one conformational epitope present in a KIR2DL receptor. In other embodiments, the epitope comprises at least about 8 consecutive amino acids of an extracellular Ig domain of 1-22 ainino acid positions of the KIR2DL1 polypeptide (the amino acid numbering according to the PRO website that describes the KIR gene family, http: // www. ncbi.nlm.nih, gov / prow / guide / 132618082.htm) In a more preferred embodiment, the immunogen comprises a wild-type human KIR2DL polypeptide in a lipid membrane, commonly on the surface of a cell In a specific embodiment, the immunogen comprises intact NK cells, particularly intact, optionally treated or lysed human NK cells.

The immunization step in non-human mammal with an antigen can be carried out in any manner well known in the art for stimulation of antibody production in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory PRess, Cold Spring Harbor, NY (1988)). The immunogen is then suspended or dissolved in a solution buffer, optionally with an adjuvant, such as complete Freund's adjuvant. Methods for determining the amount of immunogen, types of buffer solution and amounts of adjuvant are well known to those skilled in the art and are not limited in any way in the present invention. These parameters may be different for different immunogens, but they are easily elucidated. Similarly, the location and frequency of immunization sufficient to stimulate antibody production is also well known in the art. In a typical immunization protocol, non-human animals are injected intraperitoneally with antigen on day 1 and again about a week later. This is followed by recurrent antigen injections around day 20, optionally with adjuvant such as incomplete Freund's adjuvant. Recurrent injections are done intravenously and can be repeated for several consecutive days. This is followed by a booster injection on day 40, either intravenously or intraperitoneally, typically without adjuvant. This protocol results in the production of B cells that produce antigen-specific antibodies after about 40 days. Other protocols may also be used as long as they result in the production of B cells expressing an antibody directed to the antigen used in the immunization. For the preparation of polyclonal antibodies, serum from an immunized non-human animal is obtained and the antibodies present therein are isolated by well-known techniques. The serum can be purified by affinity using any of the previously established immunogens bound to a solid support, in order to obtain antibodies that react with inhibitory KIR receptors. In an alternative embodiment, lymphocytes from non-immunized non-human mammals are isolated, grown in vitro, and then exposed to the immunogen in the cell culture. The lymphocytes are then harvested and the melting step described below is carried out. For monoclonal antibodies, the next step is the isolation of splenocytes from the immunized non-human mammal and the subsequent fusion of these splenocytes with an immortalized cell in order to form a hybridoma that produces antibodies. The isolation of splenocytes from a non-human mammal is well known in the art, and typically involves the removal of the spleen from an anesthetized non-human mammal, cutting into small pieces and squeezing the splenocytes from the splenic capsule through a Nylon mesh from a cell strainer in an appropriate buffer solution, to produce a suspension of single cells. The cells are washed, centrifuged and re-suspended in a buffer solution that lyses some red blood cells. Again the solution is centrifuged and the remaining lymphocytes in the pellet are finally resuspended in fresh buffer solution. Once isolated and presented in a single cell suspension, the lymphocytes can be fused to an immortal cell line. This is typically a mouse myeloma cell line, although many other immortal cell lines useful for creating hybridomas are known in the art. Preferred murine myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. E.U.A., X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland, E.U.A. The melting is carried out polyethylene glycol or the like. The resulting hybridomas are then grown in selective media containing one or more substances that inhibit the growth or survival of the unfused precursor myeloma cells. For example, if the precursor myeloma cells lack the hypoxanthine guanine phosphoribosyl transferase enzyme (HGPRT or PRET), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium), which substances prevent the growth of deficient cells. in HGPRT. Hybridomas are typically grown in a macrophage feeder layer. The macrophages are preferably from the bait of the non-human mammal used to isolate the splenocytes and are typically primed with incomplete Freund's adjuvant or the like, several days before plating the hybridomas. The methods of fusion are described in Goding, "Monoclonal Antibodies: Principles and Practice," pp. 59-103 (Academic Press, 1986), the description of which is incorporated herein by reference. The cells are allowed to grow in the selection media for a sufficient time for the formation of colonies and the production of antibodies. This is usually between around 7 and about 14 days. The hybridoma colonies are then tested for the production of antibodies that cross-react with the gene products of the multiple inhibitor KIR receptor. This assay is typically a colorimetric assay of the ELISA type, although any assay that can be adapted to the wells in which the hybridomas grow can be used. Other assays include immunoprecipitation and radioimmunoassay. Positive wells for the desired production of antibodies are examined to determine if one or more different colonies are present. If more than one colony is present, the cells can be cloned and grown to ensure that only a single cell has given rise to the colony that produces the desired antibody. Positive wells with a single apparent colony are typically cloned and retested to ensure that only one monoclonal antibody is detected and produced. The antibodies can also be produced by the selection of combinatorial collections of immunoglobulins as described for example in ard et al., Nature, 341 (1989) p.544). The antibodies of this invention can neutralize the KIR-mediated inhibition of NK cell cytotoxicity, particularly the inhibition mediated by KIR2DL receptors and more particularly at least the KIR2DL1 and KIR2DL2 / 3 inhibition. These antibodies are thus, "neutralizing" or "inhibitory" antibodies, in the sense that they block, at least partially and detectably, the inhibitory signaling pathway mediated by KIR receptors when they interact with MHC class I molecules. More importantly, this inhibitory activity is deployed with respect to several types of KIR inhibitory receptors, preferably several gene products of the KIR2DL receptor, and more preferably at least both KIR2DL1 and KIR2DL2 / 3 so that these antibodies can be used in various subjects with high efficiency Inhibition of KIR-mediated inhibition of NK cell cytotoxicity can be assessed by various assays or tests, such as cell or binding assays. Once an antibody that cross-reacts with multiple receptors of the KIR inhibitor is identified, it can be tested for its ability to neutralize the inhibitory effect of those KIR receptors on intact NK cells. In a specific variant, the neutralizing activity can be illustrated by the ability of the antibody to reconstitute lysis by NK-positive clones of KIR2DL of specific HLA-C targets. In another specific embodiment, the neutralizing activity of the antibody is defined by the ability of the antibody to inhibit the binding of HLA-C molecules to the KIR2DL1 and KIR2DL3 receptors (or the closely related KIR2DL2), more preferably since it is the ability of the antibody to alter: - the binding of the HLA-C molecule selected from Cwl, Cw3, Cw7 and Cw8 (or from an HLA-C molecule having an Asn residue at position 80) for KIR2DL2 /3; and - the linkage of the HLA-C molecule selected from Cw2, Cw4, Cw5 and Cw6 (or from an HLA-C molecule having a Lys residue at position 80) for KIR2DL1. In another variant, the inhibitory activity of an antibody of this invention can be evaluated in a cell-based cytotoxicity assay, as described in the Examples provided herein. In another variant, the inhibitory activity of an antibody of this invention can be evaluated in a cytokine release assay, wherein the NK cells are incubated with the test antibody and a targeting cell line expressing an HLA-C allele recognized by a KIR molecule of the NK population, to stimulate the production of cytokines from NK cells (for example, IFN-α and / or production of GM-CSF). In an exemplary protocol, the production of IFN-? of PBMC is evaluated by intracytoplasmic staining on the cell surface and analysis by flow cytometry after about 4 days in the culture. Briefly, Brefeldin A (Sigma Aldrich) can be added in a final concentration of about 5 ug / ml for at least about 4 hours of culture. The cells can then be incubated with anti-CD3 and anti-CD56 Ab before permeabilization (IntraPrep ™, Beckman Coulter) and stained with PE-anti-IFN-? or PE-IgGl (Pharmingen). The production of GM-CSF and IFN-? of polyclonal activated NK cells can be measured in supernatants using ELISA (GM-CSF: DuoSet Elisa, R &D Systems, Minneapolis, MN; IFN-α: Set OptElA, Pharmingen). The antibodies of this invention can partially or totally neutralize the inhibition of NK cell cytotoxicity. The term "neutralizing the KIR-mediated inhibition of NK cell cytotoxicity", as used herein, means the ability to increase at least about 20%, preferably at least about 30%, at least about 40%, at least about 50% or more (eg, about 25-100%) of specific lysis obtained in the same proportion with NK cells or NK cell lines that are not blocked by their KIR, as measured by a release test of classical chromium of cytotoxicity, compared to the level of specific lysis obtained without the antibody when a population of NK cells expressing a given KIR is placed in contact with a targeting cell that expresses the similar MHC class I molecule ( recognized by the KIR expressed in the NK cell). For example, preferred antibodies of this invention have the ability to induce lysis of HLA-compatible or matching populations of targeting cells, ie, cell populations that would not be efficiently lysed by NK cells in the absence of said antibody. Accordingly, the antibodies of this invention can also be defined to facilitate the activity of NK cells in vivo. Alternatively, the term "neutralizing KIR-mediated inhibition" means that in a chromium assay using a transfectant or clone of NK cells expressing one or several inhibitory KIRs and a targeting cell expressing only one HLA allele that is recognized by one of the KIRs in the NK cells, the level of cytoxicity obtained with the antibody should be at least about 20%, preferably at least about 30%, at least about 40%, at least about 50 % (for example, about 25-100%), or more of the cytotoxicity obtained with an anti-MHC block class I molecule known such as the class I anti MHC antibody W6 / 32. In a specific embodiment, the antibody binds substantially the same epitope as the monoclonal antibody DF200 (produced by the hybridoma DF200). Such antibodies are referred to as "DF200 type antibodies". In a further preferred embodiment, the antibody is a monoclonal antibody. The most preferred "DF200 type antibodies" of this invention are antibodies other than the NKVSF1 of the monoclonal antibody. Monoclonal antibody DF200 (produced by hybridoma DF200) is most preferred.

The term "substantially binds to the same epitope or determinant as" an antibody of interest means that an antibody "competes" with said antibody of interest. The term "binds substantially to the same epitope or determinant as" the monoclonal antibody DF200 means that an antibody "competes" with DF200. Generally, an antibody that "binds substantially the same epitope or determinant as" the monoclonal antibody of interest (eg, DF200, NKVSF1, 17F9) means that the antibody "competes" with said antibody of interest for any one or more KIR molecules. , preferably a KIR molecule selected from the group consisting of KIR2DL1 and KIR2DL2 / 3. In other examples, an antibody that binds substantially the same epitope or determinant in a KIR2DL1 molecule when the antibody of interest "competes" with the antibody of interest to bind to KIR2DL1. An antibody that binds to substantially the same epitope or determinant in a KIR2DL2 / 3 molecule when the antibody of interest "competes" with the antibody of interest to bind to KIR2DL2 / 3. The term "binds essentially the same epitope or determinant as" an antibody of interest means that an antibody "competes" with said antibody of interest for any and all KIR molecules to which said antibody of interest specifically binds. The term "binds to essentially the same epitope or determinant as" the monoclonal antibody DF200 means that an antibody "competes" with DF200 for any and all KIR molecules in which DF200 binds specifically. For example, an antibody that binds to essentially the same epitope or determinant as the monoclonal antibodies DF200 or NKVSF1"compete" with said DF200 or NKVSF1 respectively to bind to KIR2DL1, KIR2DL2 / 3, KIR2DS1 and KIR2DS2. The identification of one or more antibodies that bind substantially or essentially the same epitope as the monoclonal antibodies described herein can easily be determined using any of a variety of immunological exclusion screening assays in which antibody competition can be assessed. A variety of such assays are routinely practiced and well known in the art (see for example, U.S. Patent No. 5,660,827, issued August 26, 1997 which is specifically incorporated herein by reference). It will now be understood that the determination of the epitope in which the antibody described herein does not bind in any manner required to identify an antibody that binds to the same or substantially the same epitope as the monoclonal antibody described herein. For example, when the test antibodies to be examined are obtained from different animal sources or even from different Ig isotypes, a simple competition assay can be used where the test and control antibodies (eg, DF200) are tested. they mix (or pre-adsorb) and apply to the sample containing both, KIR2DL1 and KIR2DL2 / 3 each of which are known to be linked by DF200. The protocols based on ELISA, radioimmunoassays, Western Blotting and the use of BIAGORE analysis (as established for example in the Examples section) are suitable for use in such simple competition studies. In certain embodiments, the control antibodies (eg, DF200) would be pre-mixed with varying amounts of the test antibodies (eg, about 1: 10 or about 1: 100) over a period of time. prior to the application of the inhibitor KIR antigen sample. In other embodiments, the control and varying amounts of test antibodies can simply be mixed during exposure to the KIR antigen sample. While it is possible to distinguish binding of free antibodies (for example, using separation or washing techniques to eliminate unbound antibodies) and DF200 of the test antibodies (for example, using secondary antibodies of specific isotypes or specific species or by specific labeling of DF200 with a detectable label) it will be possible to determine whether the test antibodies reduce the ligation of DF200 to the two different KIR2DL antigens indicating that the test antibody substantially recognizes the same epitope as DF200. The binding of control antibodies (labeling) in the absence of a completely irrelevant antibody can serve as the high control value. The low control value can be obtained by incubating the labeled antibodies (DF200) with unlabeled antibodies of exactly the same type (DF200), when competition would occur and reduce the binding of the labeled antibodies. In a test assay, a significant reduction in the reactivity of the labeled antibody in the presence of a test antibody is indicative of a test antibody that recognizes substantially the same epitope, i.e., one that "cross-reacts" with the antibody labeled (DF200). Any test antibody that reduces the DF200 binding in each of the KIR2DL1 and KIR2DL2 / 3 antigens by at least about 50%, such as at least about 60%, or more preferably at least about 70% (per example, about 65-100%), in any proportion of DF200: test antibody between about 1:10 and about 1: 100 is considered to be an antibody that binds to substantially the same epitope or determinant as DF200. Preferably, such a test antibody will reduce the binding of DF200 in each of the KIR2DL antigens by at least about 90% (e.g., about 95%).

The competition can be evaluated by, for example, a flow cytometry test. In such a test, cells carrying a given KIR can be first incubated with DF200, for example, and then with the labeled test antibody with a fluorochrome or biotin. The antibody is said to compete with DF200 if the link obtained after preincubation with the saturation amount of DF200 is about 80%, preferably about 50%, about 40% or less (eg, about 30%) of the binding (measured by fluorescence media) obtained by the antibody without preincubation with DF200. Alternatively, an antibody is said to compete with DF200 if the binding obtained with a labeled DF200 (by a fluorochrome or biotin) in preincubated cells with a saturation amount of the test antibody is about 80%, preferably about 50%, around of 40%, or less (for example, about 30%) of the link obtained without preincubation with the antibody. A simple competition assay in which a test antibody is pre-absorbed and applied to the saturation concentration on a surface on which both KIR2DL1 and KIR2DL2 / 3 are immobilized can be advantageously employed. The surface in the simple competition test is preferable to a BIACORE chip (or other suitable means for surface plasmon resonance analysis). The control antibody (e.g., DF200) is then brought into contact with the surface at a saturation concentration of KIR2DL1 and KIR2DL2 / 3 and the binding surface KIR2DL1 and KIR2DL2 / 3 of the control antibody is measured. This binding of the control antibody is compared to the binding of the control antibody to the surface containing KIR2DL1 and KIR2DL2 / 3 in the absence of the test antibody. In a test assay, a significant reduction in the binding of the surface containing KIR2DL1 and KIR2DL2 / 3 by the control antibody in the presence of a test antibody indicates that the test antibody substantially recognizes the same epitope as the control antibody such that the test antibody "cross-reacted" with the control antibody. Any test antibody that reduces the binding of the control antibody (such as DF200) in each of the KIR2DL1 and KIR2DL2 / 3 antigens by at least about 30% or more, preferably around 40%, can be considered co or an antibody which binds substantially to the same epitope or determinant as a control (e.g., DF200). Preferably, such a test antibody will reduce the binding of the control antibody (e.g., DF200) in each of the KIR2DL antigens by at least about 50% (e.g., at least about 60%, at least about 70%, or more). It will be appreciated that the control order and test antibodies can be reversed: that is, the control antibody can first be ligated to the surface and the test antibody is then brought into contact with the surface in a competition assay. Preferably, because the antibody having higher affinity for the KIR2DL1 and KIR2DL2 / 3 antigens is first linked to the surface containing KIR2DL1 and KIR2DL2 / 3, the decrease in the bond observed in the secondary antibody (assuming that the antibodies react in a cross way) is of greater magnitude. Additional examples of such assays are provided in the examples and in for example, Saunal and Regenmortel, (1995) J. Immunol. Méthods 183: 33-41, the description of which is incorporated herein by The reference. Although described in the context of DF200 for purposes of exemplification, it will be appreciated that the immunological exclusion screening assays described above can also be used to identify antibodies that compete with NKVSF1, 1-7F9, EB6, GL183, and other antibody according to the invention. Following immunization and production of antibodies in a vertebrate or cell, particular selection steps can be performed to isolate the antibodies as claimed. In this regard, in a specific embodiment, the invention also relates to methods for producing such antibodies comprising: (a) immunizing a non-human mammal with an immunogen comprising an inhibitory KIR polypeptide; (b) preparing antibodies from said immunized animals, wherein said antibodies bind to said KIR polypeptide, (c) selecting antibodies from (b) that cross-react with at least two different products of the inhibitor KIR gene, and (d) ) selecting antibodies from (c) which are capable of neutralizing KIR-mediated inhibition from the cytotoxicity of NK cells in a population of NK cells expressing at least two different products of the human inhibitor KIR receptor gene. The selection of an antibody that cross-reacts with at least two different products of the KIR inhibitor gene can be obtained, for example, by separation by exclusion of the antibody against two or more different inhibitory KIR antigens, as described above. In a more preferred embodiment, the antibodies prepared in step (b) are monoclonal antibodies. Thus, the term "antibodies prepared from said immunized animal" as used herein, includes obtaining B cells from an immunized animal and using those B cells to produce a hybridoma that expresses the antibodies, as well as obtaining antibodies. directly from the serum of an immunized animal. In another preferred embodiment, the antibodies selected in step (c) are those that cross-react with at least KIR2DL1 and KIR2DL2 / 3. In yet another preferred embodiment, the antibodies selected in step (d) elicit at least about 10% specific lysis mediated by NK cells exhibiting at least one KIR recognized by the antibody, and preferably at least about 40% of specific lysis, at least about 50% specific lysis, or more preferably, at least about 70% specific lysis (e.g., about 60-100% specific lysis), as measured in a release assay from standard chromium to a targeting cell that expresses the similar of the class I molecule compared to the lysis or cytotoxicity obtained in the same ratio of ef ect / targeting with the NK cells that are obtained by its KIR. Alternatively, when the antibodies selected in step (d) are used in a chromium assay employing a clone of the NK cell, which expresses one or several inhibitory KIRs and a targeting cell expressing only one HLA allele that is recognized by one of the KIR in the NK clone, the level of cytotoxicity obtained with the antibody, must be at least about 20%, preferably at least about 30%, or more of the cytotoxicity obtained with the mAB class I anti MHC of blocking such as the class I anti MHC antibody W6 / 32. The order of steps (c) and (d) of the method immediately described above can change. Optionally, the method also or alternatively may further comprise additional steps for making monoclonal antibody fragments or monoclonal antibody derivatives or such fragments, for example, as described elsewhere herein. In a preferred embodiment, the non-human animal used to produce antibodies according to the applicable methods of the invention, is a mammal, such as a rodent (e.g., mouse, rat), etc.), bovine, porcine, horse, rabbit, goat, sheep, etc., Also, the non-human mammal can be genetically modified or designed to produce "human" antibodies such as Xenomouse ™ (Abgenix) or HuMAb-Mouse ™ (Medarex). In another variant, the invention provides a method for obtaining an antibody comprising: (to be selected from a library or repertoire, a monoclonal antibody, a fragment of a monoclonal antibody or a derivative of any of them that cross-react with at least two products of the human inhibitor KIR2DL gene, and (b) selecting an antibody, fragment, or derivative of (a) that is capable of neutralizing NK-mediated inhibition of NK cell cytotoxicity in a population of cells NK expressing the at least two different products of the gene KIR2DL human inhibitor receptor. The repertoire can be any repertoire (recombinant) of antibodies or fragments thereof, which optionally exhibited by any suitable structure (e.g., phage, bacteria, synthetic complex, etc.). The selection of the inhibitory antibodies can be carried out as described above and is further illustrated in the examples. According to another embodiment, the invention provides a hybridoma comprising a B cell of a non-human host, wherein said B cell produces an antibody that binds to the determinant present in at least two different products of the human inhibitory KIR receptor gene and said antibody is capable of neutralizing the inhibitory activity of said receptors. More preferably, the hybridoma of this aspect of the invention is not a hybridoma that produces the monoclonal antibody NKVSF1. The hybridoma according to this aspect of the invention can be created as described above by fusion of splenocytes from the non-human mammal immunized with an immortal cell line. Hybridomas produced by this fusion can be removed by exclusion for the presence of such an antibody that cross-reacts as described elsewhere herein. Preferably, the hybridoma produces an antibody that recognizes a determinant present in at least two products of the KIR2DL gene and that cause an enhancement of NK cells expressing at least one of the KIR receptors. Even more preferably, the hybridoma produces an antibody that binds to substantially the same epitope or determinant as DF200 and potentiates the activity of NK cells. More preferably, that hybridoma is hybridoma DF200 which produces the monoclonal antibody DF200. Hybridomas that are confirmed to produce a monoclonal antibody of this invention can be grown in large amounts in an appropriate medium, such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can be grown in vivo as ascites tumors in an animal. After sufficient growth to produce the desired monoclonal antibody, the growth medium containing the monoclonal antibody (or the ascites fluid) is separated out of the cells and the monoclonal antibody present there is purified. The purification is typically accomplished by gel electrophoresis, dialysis, chromatography using protein A or protein G-Sepharose, or an anti-mouse Ig linked to a solid support such as agarose beads or Sepharose (all described for example, in US Pat. Antibody Purification Manual, Amersham Biosciences, publication No. 18 -1037-46, Edition AC, the description of which is incorporated herein by reference). The bound antibody is typically eluted from protein A / protein G columns using buffer solutions of low pH (glycine or acetate buffer solutions of pH 3.0 or less) with immediate neutralization of fractions containing the antibody. These fractions are pooled, dialyzed and concentrated as necessary. According to an alternative embodiment, the DNA encoding an antibody binding to a determinant present in at least two different products of the human inhibitory KIR receptor gene is isolated from the hybridoma of this invention and placed in a vector of expression appropriate for transfection in an appropriate host. The host is then used for the recombinant production of the antibody, or variants thereof such as humanized versions of the monoclonal antibody, active fragments of the antibody, or chimeric antibodies comprising the antibody antigen recognition portion. Prefferently, the DNA used in this embodiment encodes an antibody that recognizes a determinant present in at least two different products of the KIR2DL gene and causes the enhancement of NK cells that express at least one of the KIR receptors. Even more preferably, the DNA encodes an antibody that binds to substantially the same epitope or determinant as DF200 and that enhances the activity of the NK cells. More preferably, that DNA encodes the monoclonal antibody DF200. The DNA encoding the monoclonal antibodies of the invention is easily isolated and formed in a sequence using conventional methods (for example, using oligonucleotide probes that are capable of binding specifically to the genes encoding the heavy and light chains of the murine antibodies). Once isolated, the DNA can be placed in the expression vectors which are then transfected into the host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not produce the immunoglobulin protein, to obtain the synthesis of the monoclonal antibodies in the recombinant host cells. Recombinant expression in the DNA bacteria encoding the antibody is well known in the art (see, for example, Skerra et al., Curr Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol. Revs. , 130, pp. 151 (1992).

Fragments and Derivatives of a Monoclonal Antibody The fragments and antibody derivatives of this invention (which is encompassed by the term "antibody" or "antibodies" as used in this application, unless otherwise stated or clearly contradict in the context), preferably an antibody type DF-200 can be produced by techniques that are known in the art. "Immunoreactive fragments" comprise a portion of the intact antibody, generally the antigen or variable region binding site. Examples of antibody fragments include Fab, Fab, Fab'-SH, F (ab ') 2, and Fv fragments, diabodies, any fragment of the antibody which is a polypeptide having a primary structure consisting of an uninterrupted sequence of residues of immediate amino acids (referred to herein as a "single chain antibody fragment" or "single chain polypeptide"), which includes without limitation (1) single chain Fv (scFv) molecules (2) single chain polypeptides containing only a variable domain light chain or a fragment thereof containing the three CDRs of the light chain variable domain, without an associated heavy chain portion and (3) single chain polypeptides containing only one heavy chain variable region or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain portion and multispecific antibodies formed from the fragments of the antibody. For example, Fab or F (ab ') 2 fragments can be produced by digestion of the protease from the isolated antibodies, according to conventional techniques. It will be appreciated that the immunoreactive fragments can be modified using the known methods, for example, by slow cleaning in vivo and obtaining a desirable pharmacokinetic profile, the fragment can be modified with polyethylene glycol (PEG). Methods for coupling and conjugating PEG at a specific site to a Fab 'fragment are described for example in Leong et al, Cytokine 16 (3): 106-119 (2001) and Delgado et al, Br. J. Cancer 73 (2). ): 175-182 (1996), the description of which is incorporated herein by reference. In a particular aspect, the invention provides antibodies, antibody fragments and antibody derivatives comprising the light chain variable region of DF-200 as set forth in Figure 12. In another particular aspect, the invention provides antibodies, fragments of the antibody and antibody derivatives comprising the Pan2D light chain variable region sequence set forth in Figure 12. In another aspect, the invention provides the antibodies, antibody fragments and derivatives thereof comprising one or more of the light variable region CDRs of DF-200 as set forth in Figure 12. In yet another aspect, the invention provides antibodies, antibody fragments and derivatives thereof comprising one or more light variable region CDRs of Pan2D as set forth in Figure 12. Functional variants / analogues of such sequences can be generated by making appropriate substitutions, additions and / or deletions in these amino acid sequences described using standard techniques that can be aided by comparing the sequences. Thus, for example, CDR residues that are conserved between Pan2D and DF-200 can be suitable targets for modification considering that such residues can not contribute to the different profiles in the competition of these antibodies with respect to other antibodies discovered in the present (although Pan2D and DF-200 compete) and can not, therefore, contribute to the specificity of these antibodies for their respective particular epitopes. In another aspect, positions where a residue is within a sequence of one of these antibodies, but not another, may be convenient for deletions, substitutions and / or insertions. In a particular aspect, the invention provides antibodies, antibody fragments and antibody derivatives comprising the heavy chain variable region sequence of DF-200 as set forth in Figure 13. In another aspect, the invention provides antibodies, fragments of antibodies and derivatives thereof comprising one or more of the heavy variable CDR region of DF-200 as set forth in Figure 13. Functional variants / analogues of such sequences can be generated by making suitable substitutions, additions and / or deletions in these amino acid sequences described using standard techniques that can be aided by comparing the sequences. In another aspect, positions where a sequence of one of these antibodies is present, but not another, may be suitable for deletions, substitutions and / or insertions. Alternatively, the DNA of a hybridoma that produces an antibody of this invention, preferably an antibody type DF-200 can be modified to encode a fragment of this invention. The modified DNA is then inserted into an expression vector and transformed or transfected into an appropriate cell which then expresses the desired fragment. In an alternate embodiment, the DNA of a hybridoma producing an antibody of this invention, preferably an antibody type DF-200, can be modified prior to insertion into an expression vector, for example, by substituting the coding sequence for the chain constants. light and heavy human instead of the non-human homologous sequences (eg, Morrison et al., Proc.

Nati Acad. Sci. U. S. A., 81, pp. 6851 (1984)), or by the covalent attachment of all or part of the immunoglobulin coding sequence for a polypeptide without immunoglobulin. In that way, "chimeric" or "hybrid" antibodies are prepared so that there is a specificity of binding in the original antibody. Typically, such polypeptides without immunoglobulin are substituted for the constant domains of an antibody of the invention. Thus, according to another embodiment, the antibody of this invention, preferably is an antibody type DF-200 is humanized. The "humanized" forms of antibodies according to this invention are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab ', F (ab') 2 or other subsequences that bind antigens of the antibodies ) that contain a minimal sequence derived from murine immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibody) whose residues from a region that determines complementarity (CDR) of the receptor are replaced by the residues of a CDR of the original antibody (donor antibody) while maintaining the desired specificity, affinity, and capacity of the original antibody. In some cases, the residues of the Fv structure of the human immunoglobulin can be replaced by corresponding non-human residues. In addition, the humanized antibodies may comprise residues that are not found in the antibody of the recipient or in the sequences of the structure or CDRs imported. These modifications are made to further refine and optimize the development of the antibody. In general, the humanized antibody will comprise substantially all or at least one, and typically two variable domains wherein all or substantially all of the CDR regions correspond to those original antibodies and all or substantially all of the FR regions are those of an immunoglobulin consensus sequence. human The humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fe), typically a human immunoglobulin. For additional details see Jones et al., Nature, 321, pp. 522 (1986); Reichmann et al., Nature, 332, pp. 323 (1988); and Presta, Curr. Op. Struct. Biol., 2, pp. 593 (1992). Methods for humanizing the antibodies of this invention are well known in the art. Generally, a humanized antibody according to the present invention has one or more amino acid residues introduced into the original antibody. These murine residues or other non-human amino acid residues are frequently referred to as "import" residues that typically occur from a variable "import" domain. Humanization can be performed essentially following the method of Winter and co-workers (Jones et al., Nature, 321, pp. 522 (1986); Riech ann et al., Nature, 332, pp. 323 (1988); Verhoeyen et al., Science, 239, pp. 1534 (1988)). Accordingly, such "humanized" antibodies are chimeric antibodies (Cabilly et al., U.S. Pat. No. 4,816,567), wherein substantially less than a human intact variable domain has been replaced by the corresponding sequence of the original antibody. In practice, the humanized antibodies according to this invention are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues of analogous sites in the original antibody. The selection of human variable domains, both light and heavy, to be used in the preparation of humanized antibodies is very important to reduce antigenicity. According to the so-called "most appropriate" method, the variable domain sequence of an antibody of this invention is separated by exclusion against the entire collection of known human variable domain sequences. The human sequence that is most closely related to that of the mouse is then accepted as the human structure (FR) for the humanized antibody (Sims et al.,. Immunol., 151, pp. 2296 (1993).; Chothia and Lesk, J., Mol. Biol. , 196, pp. 901 (1987)). Another method uses a particular structure from the consensus sequence for all human antibodies of a particular subgroup of heavy or light chains. The same structure can be used for different humanized antibodies (Cárter et al., Proc Nati Acad Sci USA, 89, pp. 4285 (1992), Presta et al., J. Immunol., 51, pp. 1993 )). Additionally, it is important that the antibodies are humanized with high affinity retention for the KIR receptors of multiple inhibition and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art. Computer programs are available that illustrate and probably exhibit three-dimensional conformation structures of selected candidate immunoglobulin sequences. The inspection of these exposures allows the analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, that is, the analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this manner, the FR residues can be selected and combined from consensus and import sequences so that the characteristics of the desired antibody are obtained, such as increased affinity for the targeting antigens. In general, the CDR residues are directly and substantially involved with the antigen binding. Another method for making "humanized" monoclonal antibodies is to use a XenoMouse® (Abgenix, Fremont, CA) as the mouse used for immunization. A XenoMouse is a murine host according to this invention that has its immunoglobulin genes replaced by the immunoglobulin genes replaced by the functional human immunoglobulin genes. Thus, the antibodies produced by this mouse or in the hybridomas elaborated from B cells of this mouse, are already humanized. The XenoMouse is described in U.S. Patent No. 6,162,963 which is incorporated herein by reference in its entirety. An analogous method using a HuMAb-Mousem ™ (Medarex) can be obtained. Human antibodies can also be produced according to various techniques, which are used for immunization, other transgenic animals that have been designed to express a repertoire of the human antibody (Jakobovitz et al., Nature 362 (1993) 255), or by the selection of antibody repertoires using methods that display phages. Such techniques are known to persons of skill and can be implemented starting from the monoclonal antibodies described in the present application. The antibodies of the present invention, preferably an antibody type DF-200 can also be derived from "chimeric" antibodies (immunoglobulins) whose heavy and / or light chain portion is identical with or homologous to the corresponding sequences in the original antibody, while that the rest of the chain (s) is identical with or homologous to the corresponding sequences in the antibodies derived from other species or belonging to another class or subclass of the antibody, as well as fragments of such antibodies, as long as they exhibit the activity desired biological (Cabilly et al., supra; Morrison et al., Proc. Nati. Acad. Sci. USA, 81, pp. 6851 (1984)). Other derivatives within the scope of this invention include functionalized antibodies, i.e., antibodies that are conjugated or covalently linked to a toxin such as ricin, diphtheria toxin, abrin and Pseudomonas etotoxin.; to a detectable portion such as a fluorescence moiety, a radioisotope or an imaging agent, a solid support such as agarose beads or the like. Methods for the conjugation or covalent attachment of these other binding agents or the covalent attachment of these and other agents to the antibodies are well known in the art. The conjugation of a toxin is useful for death by targeting NK cells that exhibit one of the cross-reactive KIR receptors on their cell surface. Once the antibody of the invention binds to the cell surface of such cells, it is internalized and the toxin is released into the cell, selectively killing the cell. Such use is an alternate embodiment of the present invention. Conjugation to a detectable portion is useful when the antibody of this invention is used for diagnostic purposes. Such purposes include, but are not limited to, assaying biological samples for the presence of NK cells that produce KIR that cross-reacts on their cell surface and detecting the presence of NK cells that produce KIR that cross-reacts in a living organism Such assay and detection methods are also alternate embodiments of the present invention. The conjugation of an antibody of this invention to a solid support is useful as a tool for an affinity purification of NK cells that produce KIR which cross-reacts on its cell surface from a source, such as a biological fluid. This purification method is another alternate embodiment of the present invention, as is the resulting purified population of NK cells. In an alternate embodiment, an antibody that binds to a determinant present in at least two different products of the human inhibitory KIR receptor gene, wherein said antibody is capable of neutralizing the KIR-mediated inhibition of NK cell cytotoxicity in NK cells expressing at least one of said two different human inhibitory KIR receptors of this invention, including NKVSF1, can be incorporated into liposomes ("immunoliposomes"), alone or together with another substance for the delivery of targeting to an animal. Such other substances include nucleic acids for the delivery of genes for gene therapy or for the delivery of antisense RNA, RNAi or siRNA to suppress a gene in an NK cell, toxins or drugs for targeted killing of NK cells. Computer modeling of the extracellular domains of KIR2DL1, -2 and -3 (KIR2DL1-3), based on their published crystalline structures (Maenaka et al. (1999), Fan et al. (2001), Boyington et al., (2000)), predicted in the involvement of certain regions or KIR2DL1, -2 and -3 in the interaction between KIR2DL1 and the cross-reactive mouse monoclonal antibodies KIR2DL1-3-DF200 and NKVSFl. Thus, in one embodiment, the present invention provides antibodies that bind exclusively to KIR2DL1 within a region defined by the 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 KIR 2DL2 / 3 without interacting with the amino acid residues outside the region defined by the 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 that do not bind to a KIR2DL1 mutant in which R131 is Ala. In another embodiment, the invention provides antibodies that bind to KIR2DL1 and that do not bind to a KIR2DL1 mutant in which R157 is Ala. In another embodiment, the invention provides antibodies that bind to KIR2DL1 and which do not bind to a KIR2DL1 mutant in which R158 is Ala. In another embodiment, the invention provides antibodies that bind to the KIR2DL1 residues (131, 157, 158). In another embodiment, the invention provides antibodies that bind to KIR2DS3 (R131W), but not to wild type CR2DS3. In another embodiment, the invention provides antibodies that bind to KIR2DL1 and KIR2DL2 / 3 as well as KIR2DS4.

In another embodiment, the invention provides antibodies that bind to KIR2DL1 and KIR2DL2 / 3, but not to KIR2DS4. The determination of whether an antibody binds to one of the epitope regions defined above can be carried out in ways known to one skilled in the art. As an example of such mapping / characterization methods, a region of the epitope for an anti-KIR antibody can be determined by a "finger print" epitope using a chemical modification of the amines / carboxyls exposed in the KIR2DL1 or KIR2DL2 / 3 protein. . A specific example of such a finger print technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry) where there is a hydrogen / deuterium exchange of the receptor and amide protons of the ligand protein, which they bind and fall back, where the amide groups in the structure that participate in the binding protein are protected from the exchange of backsliding and will therefore continue to be deuterated. Relevant regions can be identified at this point by peptide proteolysis, separation by high performance liquid chromatography, and / or mass spectrometry by electro-ionization ionization. See for example, Ehring H, Analytical Biochemistry, Vol., 267 (2) pp. 252-259 (1999) and / or Engen, J., R. and Smith, D., L. (2001) Anal. Chem. 73, 256A-265A. Another example of a suitable epitope identification technique is a mapping of the epitope by nuclear magnetic resonance (NMR), typically, the position of the signs in the two-dimensional NMR spectra of the free antigen and the antigen forming complexes with the peptide. which binds to the antigen such as an antibody. The antigen is characteristically labeled in an isotopically selective manner with 15N so that only the signals corresponding to the antigen are observed and not the peptide signals that binds antigens in the NMR spectrum. The antigen signals that originate from the amino acids involved in the interaction with the peptide that bind antigens, will typically change the position of the spectra of the complex compared to the spectra of the free antigen, and the amino acids involved in the linkage can be identified thus. See for example, Ernst Schering Res Found Workshop 2004; (44): 149-67; Huang et al, Journal of Molecular Biology, Vol. 281 (1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 June 9 (3): 516-24. Epitope mapping / characterization can also be performed using mass spectrometry methods. See for example, Downward, J Mass Spectro. 2000 Apr; 35 (4): 493-503 and Kiselar and Downard, Chem Anal. 1999 on May 1; 71 (9) .1792-801. Protease digestion techniques may also be useful in the context of epitope mapping and identification.

The sequences / regions of relevant antigenic determination can be determined by digestion of the protease, for example, using trypsin in a ratio of about 1: 50 to KIR2DL1 or KIR2DL2 / 3 or / n digestion at 37 ° C and pH 7- 8, followed by a mass spectrometry (MS) analysis for the identification of the peptide. The peptides protected from the cleavage of trypsin by the anti-binding agent KIR can subsequently be identified by comparing samples that undergo trypsin digestion and samples incubated with the antibody and then subjected to digestion by eg trypsin (revealing thus, a print on foot for the binder). Other enzymes such as chymotrypsin, pepsin, etc. , also or alternatively they can be used in similar epitope characterization methods. On the other hand, enzymatic digestion may provide a rapid method to analyze whether a potential antigenic determinant sequence lies within a region of KIR2DL1 in the context of an anti-KER polypeptide that is not exposed to the surface and, consequently, probably unimportant in djpimingenicity / antigenicity. See for example, Manca, Ann Ist Super Sanita. 1991; 27 (1): 15-9 for a discussion of similar techniques. Cross reactivity with Cinomolgus monkeys It has been found that the NKVSF1 antibody also binds to NK cells from cynomolgus monkeys, see example 7. The invention therefore provides an antibody, as well as fragments and derivatives thereof, wherein said antibody, fragment or derivative cross-reacts with at least two human KIR receptors on the surface of human NK cells, which additionally bind to NK cells of cynomolgus monkeys. In one embodiment of this, the antibody is not an NKVSF1 antibody. The invention also provides a method for testing the toxicity of an antibody, as well as fragments and derivatives thereof, wherein said antibody, fragment or derivatives cross-react with at least two inhibitory human KIR receptors on the surface of NK cells. human, wherein the method comprises testing the antibody in a cynomolgus monkey. Compositions and Administration The invention also provides pharmaceutical compositions comprising an antibody, as well as fragments and derivatives thereof, wherein said antibody, fragment or derivative cross-reacts with at least two inhibitory KIR receptors on the surface of NK cells, neutralizes its inhibitory signals and enhance the activity of those cells, in any suitable vehicle in an amount effective to detectably enhance the cytotoxicity of the NK cell in a patient or in a biological sample of NK cells. The composition further comprises a pharmaceutically acceptable carrier. Such compositions are also referred to as "antibody compositions of this invention". In one embodiment, the antibody compositions of this invention comprise an antibody described in the antibody of the above embodiments. The NKVSF1 antibody is included within the scope of the antibodies that may be present in the antibody compositions of this invention. The term "biological sample" as used herein includes but is not limited to a biological fluid (e.g., serum, lymph, blood), cell samples or tissue samples (e.g., bone marrow). Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, whey proteins, such as human serum albumin, buffer substances such as phosphates, glycine, acid sorbic, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, sodium acid diphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, trisilicate magnesium, polyvinyl pyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool grease.

The compositions of this invention can be employed in a method for enhancing the activity of NK cells in a patient or a biological sample. This method comprises the step of contacting said composition with said biological or patient sample. Such a method will be useful for both diagnostic and therapeutic purposes. To be used in conjunction with a biological sample, the antibody composition can be administered by simply mixing with or applying directly to the sample, depending on the nature of the sample (fluid or solid). The biological sample can be contacted directly with the antibody in any suitable device (dish, bag, flask, etc.). To be used in conjunction with a patient, the composition must be formulated to be administered to a patient. The compositions of the present invention can be administered orally, parenterally, by spray inhalation, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes hypodermic, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, intraperitoneally or intravenously.

The sterile injectable forms of the compositions of this invention may be aqueous or in an oil suspension. These suspensions can be formulated according to techniques known in the art using wetting and dispersing agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable solvent or diluent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and an isotonic sodium chloride solution. In addition, fixed sterile oils are conventionally employed as a solvent or suspension medium. For this purpose, any soft fixed oil can be employed that include mono or synthetic diglycerides. Fatty acids, such as oleinic acid and its glyceride derivatives are useful in the preparation of injectables, such as natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oily solutions or suspensions may also contain a long chain diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents that are normally used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants such as Tweens, Apans and other emulsifying agents or bioavailability enhancers are used are commonly used in the manufacture of liquid, solid or other pharmaceutically acceptable dosage forms that can be used for formulation purposes. The compositions of this invention can be administered orally in any orally acceptable dosage form, but are not limited to capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, the carriers normally used include lactose and corn starch. Lubricating agents such as magnesium stearate are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, flavors or coloring agents can be added. Alternatively, the compositions of this invention can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a non-irritating excipient that is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.

Such materials include cocoa butter, beeswax and polyethylene glycol. The compositions of this invention can be administered topically, especially when the purpose of the treatment includes easily accessible areas or organs by topical application, which includes diseases of the eyes, skin or lower intestinal tract. Topical formulations suitable for each of these areas or organs are easily prepared. Topical application to the lower intestinal tract can be done in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical transdermal patches can also be used. For topical applications, the compositions may be formulated in 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, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the compositions may be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester waxes, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. For ophthalmic use, the compositions can be formulated as micronized suspensions in sterile saline with adjusted isotonic pH, or preferably, as solutions in sterile saline with adjusted pH either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the compositions may be formulated in an ointment such as petrolatum. The compositions of this invention can also be administered by nasal spray or by inhalation. Such compositions are prepared according to techniques well known in the art in the pharmaceutical formulation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons and other conventional solubilization agents or dispersion. Several monoclonal antibodies have been shown to be efficient in clinical situations, such as Rituxan (Rituximab), Herceptin (Trastuzumab) or Xolair (Omalizumab) and similar administration regimens (ie, formulations and / or dosages and / or administration protocols) can be used with the antibodies of this invention. The schedules and doses for administration of the antibody in the pharmaceutical compositions of the present invention can be determined according to known methods for those products, for example, using the manufacturer's instructions. For example, an antibody present in a pharmaceutical composition of this invention can be delivered at a concentration of 10 mg / mL in either single-use vials of 100 mg (10 mL) or 500 mg (50 mL). The product is formulated for intravenous administration in sodium chloride 9.0 mg / mL, sodium citrate dihydrate 7.35 mg / mL, polysorbate 80, 0.7 mg / mL, and sterile water for injection. The pH is adjusted to 6.5. An exemplary suitable dose range for an antibody in a pharmaceutical composition of this invention may be between about 10 mg / m2 and 500 mg / m2. However, it will be appreciated that these programs are exemplary and that the optimal program and regimen can be adapted taking into account the affinity and tolerance of the particular antibody in the pharmaceutical composition to be determined in clinical trials. The amounts and schedule of injection of an antibody into a pharmaceutical composition of this invention that saturates the NK cells for 24 hours, 48 hours and 72 hours or a week or month, will be determined by considering the affinity of the antibody and its armacokinetic parameters f.

According to another embodiment, the antibody compositions of this invention may further comprise another therapeutic agent including agents normally used for the particular therapeutic purpose for which the antibody is administered. The additional therapeutic agent will normally be present in the composition in amounts typically used for that agent in a monotherapy for a particular disease or condition to be treated. Such therapeutic agents include but are not limited to, therapeutic agents used in the treatment of cancers, therapeutic agents used to treat infectious diseases, therapeutic agents used in other immunotherapies, cytokines (such as IL-2 or IL-15), other antibodies and fragments of other antibodies. For example, various therapeutic agents are available for the treatment of cancers. The antibody compositions and methods of the present invention can be combined with some other methods generally employed in the treatment of the particular disease, particularly a tumor, cancer disease or other disease or disorder that the patient exhibits. Provided that a particular therapeutic method is not known to be detrimental in itself to the condition of the patient, and does not significantly counteract the activity of the antibody in a pharmaceutical composition of this invention, its combination with the present invention is contemplated.

In conjunction with the treatment of solid tumors, the pharmaceutical compositions of the present invention can be used in combination with classical methods such as surgery, radiotherapy and chemotherapy and the like. The invention therefore provides combination therapy in which a pharmaceutical composition of this invention is used simultaneously with before or after surgery or radiation treatment, or is administered to patients with before or after conventional chemotherapeutic, radiotherapeutic or antiangiogenic agents or immunotoxins or coaguligandos addressed. When one or more agents are used in combination with a composition containing antibodies of this invention in a therapeutic regimen, there is no requirement that the combined results be additive to the effects observed when each treatment is carried out separately. Although at least the additive effects are generally desirable, some anti-cancer effect increased above one of the simple therapies would be of benefit. Also, there is no particular requirement for the combined treatment to show synergistic effects, although it is certainly possible and advantageous. To practice a combined anticancer therapy, an antibody composition of this invention would simply be administered to an animal in combination with another anticancer agent in an effective manner to result in its anticancer actions combined within the animal. The agents would therefore be delivered in effective amounts and for effective periods of time to result in their combined presence within the tumor vascularity and their combined actions in the tumor environment. To achieve this goal, an antibody composition of this invention and anticancer agents can be administered simultaneously to the animal, either in a single combined composition or as two different compositions using different administration routes. Alternatively, the administration of an antibody composition of this invention can precede or follow the treatment of the anticancer agent by, for example, intervals ranging from minutes to weeks and months. Someone would ensure that the anti-cancer agent and an antibody in the antibody composition of these invention exert an advantageously combined effect on cancer. Most anticancer agents would be given prior to an KIR antibody inhibitor composition of this invention in an anti-angiogenic therapy. Nevertheless, when the immunoconjugates of an antibody are used in the antibody composition of this invention, various anticancer agents may be administered simultaneously or subsequently. In some situations, it may be desirable to extend the period of time for treatment significantly, where several days (2, 3, 4, 5, 6, or 7), several weeks (1, 2, 3, 4, 5, 6 , 7 or 8) or even several months (1, 2, 3, 4, 5, 6, 7 or 8) take place between the respective administration of the anticancer agent or the anticancer treatment and the administration of an antibody composition of this invention. This would be advantageous in circumstances where anti-cancer treatment is intended to substantially destroy the tumor such as surgery or chemotherapy and the administration of an antibody composition of this invention is intended to prevent micrometastasis or tumor regrowth. It is also envisioned that more than one administration of an inhibitory KIR antibody-based composition of this invention or the anticancer agent will be used. These agents may be administered interchangeably, or in alternate days or weeks, or a course of treatment with an KIR antibody inhibitor composition of this invention followed by a cycle of anti-cancer agent therapy. In any case, to achieve regression of the tumor using a combination therapy, all that is required is to deliver both agents in an effective combined amount to exert an antitumor effect regardless of the administration times. In terms of surgery, any surgical intervention in combination with the present invention can be practiced. In conjunction with radiotherapy, any mechanism for inducing DNA damage locally within cancer cells is contemplated such as gamma radiation, X-rays, UV radiation, microwaves and even electronic emissions and the like. Targeted delivery of radioisotopes to cancer cells is also contemplated, and this may be used in conjunction with a targeting antibody or other targeting means. In other aspects, the immunomodulatory compounds or regimens may be administered in combination with or as part of antibody compositions of the present invention. Preferred examples of immunomodulatory compounds include cytokines. Various cytokines may be employed in such combined methods. Examples of useful cytokines in the combinations contemplated in this invention include IL-lalfa, IL-lbeta, 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-beta, GM-CSF, M-CSF, G-CSF, TNF-alpha, TNF-beta , LAF, TCGF, BCGF, TRF, BAF, BDG, MP, PIL, OSM, TMF, PDGF, IFN-alpha, IFN-beta, IFN-gamma. The cytokines used in the treatment or combination compositions of this invention are administered according to standard regimens consistent with clinical indications such as patient conditions and the relative toxicity of the cytokine. In certain embodiments, therapeutic compositions comprising cross-reacting inhibitory KIR antibodies of the present invention may be administered in combination with or may further comprise a hormonal or chemotherapeutic agent. A variety of chemotherapeutic and hormonal therapy agents can be used in the combined treatment methods described herein. Chemotherapeutic agents contemplated as examples include, but are not limited to alkylating agents, antimetabolites, cytotoxic antibiotics, vinca alkaloids, for example adriamycin, dactinomycin, mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine , etoposide (VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatins and derivatives and prodrugs thereof. Hormone agents include, but are not limited to, for example LHRH agonists such as leuprorelin, goserelin, triptorelin and buserelin, antiestrogens such as tamoxifen and toremifene, antiandrogens such as flutamide, nilutamide, cyproterone and bicalutamide; aromatase inhibitors such as anastrozole, exemestane, letrozole and fadrozole; and progestagens such as medroxy, chlormadinone and megestrol. As will be understood by those of ordinary skill in the art, suitable doses of chemotherapeutic agents will approximate those already employed in clinical therapies wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics. By way of example only, agents such as cisplatin and other DNA alkylating agents can be used. Cisplatin has been widely used to treat cancer with effective doses used in clinical applications of 20 mg / m2 for 5 days every 3 weeks for a total of 3 courses. Cisplatin is not absorbed orally and therefore can be delivered by injection intravenously, subcutaneously, intratumorally or intraperitoneally. Additional useful chemotherapeutic agents include compounds that interfere with DNA replication, mitosis and chromosomal segregation, and agents that fragment the synthesis and fidelity of polynucleotide precursors. Various exemplary chemotherapeutic agents for combination therapy are listed in Table C of US Patent No. 6,524,583, the description of which agents and indications are specifically incorporated herein by reference. Each of the agents listed are exemplary and not limiting. The experienced technician addresses "Remington's Pharmaceutical Sciences" 15a. Edition, chapter 33, in particular pages 624-652. The variation in the dose will probably happen depending on the condition to be treated. The doctor who administers the treatment may determine the appropriate dose for the subject individually. The present cross-reaction inhibitory KIR antibody compositions of this invention can be used in combination with any one or more other anti-angiogenic therapies or can further comprise the anti-angiogenic agents. Examples of such agents include neutralizing antibodies, antisense RNA, siRNA, RNAi, RNA aptamers and ribozymes each directed against VEGF or VEGF receptors (US Patent No. 6,524,583, the disclosure of which is incorporated herein by reference. VEGF variants with antagonistic properties can also be employed as described in WO 98/16551, specifically incorporated herein by reference, The additional exemplary antiangiogenic agents which are useful in conjunction with the combination therapy are listed in Table D of the US Patent No. 6,524,583, the description of whose agents and indications is specifically incorporated herein by reference, The KIR antibody inhibitor compositions of this invention can also be advantageously used in combination with methods for inducing apoptosis or can comprise agents For example, several oncogenes have been identified that inhibit n apoptosis or programmed cell death. Exemplary oncogenes in this category include, but are not limited to bcr-abl, bcl-2 (different from bcl-1, cyclin DI, access numbers to GenBank M14745, X06487; US Patent NOS. 5,650,491; and 5,539,094; each incorporated herein by reference) and family members including Bcl-xl, Mcl-1, Bal, Al and A20. Overexpression of bcl-2 was first discovered in T-cell lymphomas. The bcl-2 oncogene works by the binding and inactivation of Bax, a protein in the apoptotic pathway. The inhibition of the bcl-2 function prevents the inactivation of Bax and allows the apoptotic path. The inhibition of this type of oncogenes for example using antisense nucleotide sequences, RNAi, siRNA or small molecule chemical compounds is contemplated for use in the present invention to give an improvement of apoptosis (US Patent Nos. 5,650,491; 5,539,094; and 5,583,034, each incorporated herein by reference). The inhibitory KIR antibody compositions of this invention may also comprise or be used in combination with molecules comprising a targeting moiety for example antibody, ligand or conjugate thereof, directed to a specific marker of a target cell ("targeting agent") , for example a target tumor cell. Generally speaking, the agents of address for use in these additional aspects of The invention will preferably recognize accessible tumor antigens that are preferably or specifically expressed at the site of the tumor. The targeting agents will generally bind to a component located on the surface, expressed on the surface or accessible on the surface of a tumor cell. The targeting agents will also preferably show high affinity properties, and will not exert significant in vivo side effects against normal life supporting tissues such as one or more selected tissues of heart, kidney, brain, liver, bone marrow, colon, breast, prostate, thyroid, gall bladder, lung, adrenal, muscle, nerve fibers, pancreas, skin, or other tissue or organ that sustains life in the human body. The term "having no significant side effects" as used herein, refers to the fact that a targeting agent when administered in vivo will produce only negligible or clinically administrable side effects such as those normally encountered during chemotherapy. . In the treatment of tumors, an antibody composition of this invention may additionally comprise or be used in combination with adjuvant compounds. Adjuvant compounds may include, for example, antiemetics such as serotonin antagonists and therapies such as phenothiazines, substituted benzamides, antihistamines, butyrophenones, corticosteroids, benzodiazepines and cannabinoids.; bisphosphates such as zoledronic acid and pamidronic acid; and hematopoietic growth factors such as erythropoietin and G-CSF, for example filgrastim, lenograstim and darbepoetin. In another embodiment, two or more antibodies of this invention having different cross-reactivities, including NKVSF1 can be combined in a single composition so as to neutralize the inhibitory effects of as many KER inhibitor gene products as possible. The compositions comprising combinations of cross-reactive inhibitor KIR antibodies of this invention or fragments or derivatives thereof will allow even wider utility because there is likely to be a small percentage of the human population that may lack each of the gene products. KIR inhibitors recognized by a single cross-reactive antibody. Similarly, an antibody composition of this invention may further comprise one or more antibodies that recognize the simple inhibitor KIR subtypes. Such combinations would again provide greater utility in a therapeutic environment.

The invention also provides a method for enhancing the activity of NK cells in a patient in need of the same, comprising the step of administering a composition according to this invention to the patient. The method is directed more specifically to increased NK cell activity in patients who have a disease in which increased NK cell activity is beneficial, involving, affecting or being caused by cells susceptible to NK cell lysis, or causes or characterizes by an insufficient NK cell activity such as cancer, another proliferative disorder, an infectious disease or an immune disorder. More specifically, the methods of the present invention are used for the treatment of a variety of cancers and other proliferative diseases including, but not limited to, carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary , prostate, pancreas, stomach, womb, thyroid and skin including squamous cell carcinoma, hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, lymphoma that is not from Hodgkins, hairy cell lymphoma and Burketts lymphoma; haematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias 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 schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyoscaroma and osteosarcoma; and other tumors including melanoma, xeroderma pigmentosum, kertaocanthoma, seminoma, follicular thyroid cancer, and teratocarcinoma. Preferred disorders that can be treated according to the invention include hematopoietic tumors of lymphoid lineage, for example B cell or T cell tumors, including, but not limited to, T cell disorders such as T-prolymphocytic leukemia (T-PLL) , which includes the cerebriform and small cell type; Large granular lymphocyte leukemia (LGL), preferably of the T cell type, Sezary syndrome (SS); Adult T cell leukemia lymphoma (ATLL); a / d hepatosplenic lymphoma T-NHL; peripheral / post-thymic T cell lymphoma (pleomorphic and immunoblastic subtypes); angio-immunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic large cell lymphoma (Ki 1+); intestinal T cell lymphoma; lymphoblastic T; and lymphoma / leukemia (T-Lbly / T-ALL).

Other proliferative disorders can also be treated according to the invention including for example hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in blood vessels, such as as stenosis or restenosis after angioplasty. The KIR cross-reacting inhibitor antibody of this invention can be used to treat or prevent infectious diseases, preferably including some infections caused by viruses, bacteria, protozoa, molds or fungi. Such viral infectious organisms include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, influenza, varicella, adenovirus, herpes simplex type I (HSV-1), herpes simplex type 2 (HSV-2), morriña , rhinovirus, ecovirus, rotavirus, respiratory syncytial virus, papilloma virus, cytomegalovirus, equinovirus, arbovirus, huntavirus, coxsackievirus, mumps virus, measles virus, rubella virus, poliovirus, human immunodeficiency virus type 1 or type 2 (HIV-1, HIV-2). Bacterial infections that can be treated in accordance with this invention include but are not limited to infections caused by the following: Staphylococcus; Streptococcus, including S. pyogenes; Enterococci; Bacillus, including Bacillus anthracis, and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella including G. vaginalis; Nocardia; Streptomyces; Thermoactinomyces vulgaris; Treponena; Campiyobacter, Pseudomonas including Raeruginosa; Legionella; Neisseria including N. gonorrhoeae and N. meningitides; Flavobacterium including F. Meningosepticum and F. Odoraturn; Brucella; Bordetella including B. pertussis and B. bronchiseptica; Escherichia including E. coli, Klebsiella Enterobacter, Serratia including S. marcescens and S. liquefaciens Edwardsiella; Proteus including P. mirabilis and P. vulgaris Streptobacillus; Rickettsiaceae including R. fickettsfi, Chla-Ttydia including C. psittaci and C. trachornatis; Mycobacterium including M. Tuberculosis, M. Intracellulare, M. Folluiturn, M. Laprae, M. Avium, M. Bovis, M. Africanum, M. Kansasii, M. Intracellulare, and M.] _ep_ .ernurium; and Nocardia. Infections by protozoa that can be treated in accordance with this invention include, but are not limited to, infections caused by leishmania, kokzidioa, and trypanosome. A complete list of infectious diseases can be found on the website of the National Center for Infectious Diseases (NCID) at the Center for Disease Control (CDC) (http: // www. Cdc. Gov / ncidod / diseases /), whose list is incorporated herein by reference. All of those diseases are candidates for treatment using KIR inhibitors cross-reacting inhibitors of the invention.

Such methods of treating various infectious diseases can employ the antibody composition of this invention, either alone or in combination with other treatments and / or therapeutic agents known for the treatment of such diseases, including anti-viral agents, anti-fungal agents , antibacterial agents, antibiotics, anti-parasitic agents and anti-protozoan agents. When these methods involve additional treatments with additional therapeutic agents, those agents can be administered together with the antibodies of this invention either in single dose form or as separate multiple dose forms. When administered as a separate dosage form, the additional agent can be administered prior to, concurrent with, or after administration of the antibody of this invention. Additional aspects and advantages of this invention will be disclosed in the following experimental section, which should be referred to as illustrative and not limiting of the scope of this application. Example 1 Purification of PBLs and generation of polyclonal or clonal NK cell lines PBLs are derived from healthy donors by Ficoll Hypaque gradients and plastic adherent cell removal.

To obtain enriched NK cells, PBLs CON anti-CD3, anti-CD4 and anti-HLA-DR mAbs are incubated (30 minutes at 4 ° C), followed by goat anti-mouse magnetic beads (Dynal) (30 minutes at 4 ° C) and immunomagnetic selection by methods known in the art (Pende et al., 1999). CD3", CD4", DR "cells are cultured in irradiated feeder cells and 100 U / ml of Interleukin 2 (Proleukin, Chiron Corporation) and 1.5 ng / ml of Phytohemagglutinin A (Gibco BRL) to obtain populations of polyclonal NK cells. NK cells are cloned by limiting dilution and the NK cell clones are characterized by flow cytometry for expression of cell surface receptors.The mAbs used were JT3A (IgG2a, anti CD3), EB6 and GL183 (IgGl anti KIR2DL1 and KIR2DL3 respectively) , XA-141 IgM (anti KIR2DL1 with the same specificity as EB6), anti CD4 (HP2.6), and anti DR (DI.12, IgG2a) instead of JT3A, HP2.6, and DR1.12, which are produced by the applicants, commercially available mAbs of the same specificities can be used (Beckman Coulter Inc., Fullerton, CA) EB6 and GL183 are commercially available (Beckman Coulter Inc., Fullerton, CA. XA-141 is not commercially available , but EB6 can be used for the reconstitution of cont role of lysis as described in (Moretta et al., 1993). The cells are stained with the appropriate antibodies (30mns at 4 ° C) followed by polyclonal anti-mouse antibodies conjugated PE or FITC (Southern Biotechnology Associates Inc). The samples are analyzed by cytofluorometric analysis in a FACSAN apparatus (Becton Dickinson, Mountain View, CA). The following clones are used in this study. CP11, CN5 and CN505 are positive clones KIR2DL1 and stained by EB6 ((IgGl anti KIR2DL1) or XA-141 (IgM anti KIR2DL1 with the same specificity compared to EB6 antibodies).

CP502 are KIR2DL3 positive clones and are stained by antibody GL183 (IgGl anti KIR2DL3). The cytolytic activity of NK clones was evaluated by a standard 4 hour 51 Cr release assay in which the effector NK cells are tested on Cw3 or Cw4 positive cell lines for sensitivity to NK cell lysis. All the targets are used at 5000 cells per well in microtiter plate and the effector: target ratio is indicated in the figures (usually 4 effectors per target cells). The cytolytic assay is performed with or without supernatant of the indicated monoclonal antibodies at a 3_ dilution. The procedure is essentially the same as described in (Moretta et al., 1993). Example 2 Generation of new mAbs Mabs were generated by immunizing 5-week-old Balb C mice with activated polyclonal or monoclonal NK cell lines as described in (Moretta et al., 1990). After different cell fusions, mAbs are first selected for their ability to cross-react with positive NK EB6 and GL183 cell lines and clones. The positive monoclonal antibodies are further separated by exclusion for reconstitutive lysis capacity by NK positive EB6 clones or positive GL183 clones of positive Cw4 or Cw3 targets respectively. The cell dyeing is carried out as follows. Cells are stained with a panel of antibodies (1 μg / ml or 50 μl of supernatant, 30mns at 4 ° C) followed by anti-mouse IgG (H + L) antibodies from goat F (ab ') 2 fragments conjugated to PE or anti-human IgG (Fe gamma) from goat F (ab ') 2 fragment conjugated to PE (Beckman Coulter). The cytofluorometric analysis was carried out in an Epics XL.MCL device (Beckman Coulter). One of the monoclonal antibodies, the DF200 mAb, is found to react with several members of the KIR family including KIR2DL1, KIR2DL2 / 3. The NK cells, both KIR2DL1 + and KIR2DL2 / 3 +, were brightly stained with DF200mAb (Figure 1). NK clones expressing one or the other (or even both) of these specific HLA class inhibitory receptors were used as effector cells against target cells expressing one or more HLA-C alleles. The cytotoxicity assays are carried out as follows. The cytolytic activity of the YTS-KIR2DL1 or YTS-Eco cell lines was evaluated by a standard 4 hour 51Cr release assay. Effector cells were tested in EBV positive or negative HLA-Cw4 cell lines and 721,221 cells transfected in HLA-Cw4. All the targets are used at 3000 cells per well in microtitre plate. The effector / objective relation is indicated in the figures. The cytolytic activity was performed with or without the indicated full length of the F (ab ') 2 fragments of mouse and human monoclonal antibodies. As expected, the KIR2DL1 + NK clones exhibit little if any of the target cells against cytolytic activity express HLA-Cw4 and the KIR2DL3 + NK clones exhibit little or no activity on the Cw3 positive targets. However, in the presence of DF200mAb (used to mask their KIR2DL receptors) the NK clones become unable to recognize their HLA-C ligands and exhibit a strong cytolytic activity in Cw3 or Cw4 targets. For example, the cell line C1R (cell line CW4 + EBV, ATCC No. CRL 1993) is not deleted by clones KIR2DL1"1" NK (CN5 / CN505), but the inhibition can be efficiently reversed by the use of either DF200 or a conventional anti KIR2DL1 mAb. On the other hand, NK clones expressing the phenotype KIR2DL2 / 3 + KIR2DL1"(CN12) efficiently eliminate C1R cells and this elimination is not affected by DF200mAb (Figure 2). Similar results are obtained with NK clones KIR2DL2- or KIR2DL3 -positive in Cw3 positive targets Similarly, the Cw4 + 221 EBV cell line is not deleted by NK cells transfected by KIR2DL1"1", but the inhibition can be efficiently reversed by the use of either DF200, a DF200 Fab fragment, or an anti KIR2DL1 mAb EB6 or conventional XA141. Also, the Cw3 + 221 EBV cell line is not removed by KIR2DL2"1" NK cells, but this inhibition may be reversed by the use of either DF200 or a DF200 Fab fragment. Finally, the last cell line Cw3 + 221 EBV is not removed by KIR2DL3"1" NK cells, but this inhibition can be reversed by the use of either a DF200 Fab fragment or a conventional KIR2DL3 mAb GL183 or Y249. The results are shown in Figure 3. F (ab ') 2 fragments were also tested for their ability to reconstitute the lysis of positive Cw targets. The F (ab ') 2 fragments of DF200 and EB6 Abs were both able to reverse the inhibition of lysis by NK cells transfected by KIR2DL-1 of cell line 221 transfected by Cw4 and cell line Cw4 + EBV TUBE. The results are shown in Figure 4.

Example 4 Generation of new human mAbs Human monoclonal anti-KIR Abs are generated by immunized transgenic mice engineered to express a repertoire of human antibody with recombinant KIR protein. After different cell fusions, the mAbs are first selected for their ability to cross-react with the immobilized KIR2DL1 and KIR2DL2 protein. Several monoclonal antibodies, including 1-7F9, 1-4F1, 1-6F5 and 1-6F1, are found to react with KIR2DL1 and KIR2DL2 / 3. Positive monoclonal antibodies are separated by exclusion for their capacity for reconstitutive lysis by KIR2DL1 expressing NK positive transfectants EB6 of Cw4 positive target cells. NK cells expressing specific inhibitors of class I HLA are used as effector cells against target cells expressing one or more HLA-C alleles (Figures 5 and 6). Cytotoxicity assays are carried out as described above. The effector / target ratio is indicated in the Figures, and the antibodies were used at either 10 ug / ml or 30 ug / ml. As expected, KIR2DL1 + NK cells exhibit little if there is any cytolytic activity against the target cells expressing HLA-Cw4. However, in the presence of 1-7F9 mAb, the NK cells become unable to recognize their HLA-C ligands and exhibit strong cytolytic activity in the Cw4 targets. For example, the two cell lines tested (the transfected HLA-Cw4 cell lines 721.221 and CW4 + EBV) are not killed by KIR2DL1 + NK cells, but the inhibition can be efficiently reversed by the use of either Mab 1-7F9 or an anti KIR2DL1 mAb EB6 conventional. The Abs DF200 and panKIR (also referred to as NKVSFl) are compared with 1-7F9. Antibodies 1-4F1, 1-6F5 and 1-6F1 on the one hand are not able to reconstitute cell lysis by NK cells on positive Cw4 targets. Example 5 Analysis Biacore DF200 mAb / KIR2DL1 and interactions DF200 mAb / KIR2DL3 Production and purification of recombinant proteins The recombinant KIR2DL1 and KIR2DL3 proteins are produced in E. coli. The cDNA encoding the complete extracellular domain of KIR2DL1 and KIR2DL3 were amplified by PCR from vector 47.11 of clone pCDM8 (Biassoni et al, 1993) and vector 6 of clone RSVS (gpt) 183 (Wagtman et al, 1995) respectively , using the following primers: Sense: 5 '-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3' Anti-sense: 5 '-CGGGATCCCAGGTGTCTGGGGTTACC-3'. They are cloned into the expression vector pML1 in the structure with a sequence encoding the biotinylation signal (Saulquin et al, 2003).

The expression of the protein was carried out in the bacterial strain BL21 (DE3) (Invitrogen). The transfected bacteria are grown up to OD600 = 0. 6 to 37 ° C in a medium supplemented with ampicillin (100 μg / ml) and the expression is induced with 1 mM IPTG. Proteins are recovered from low inclusion bodies or denaturing conditions (8M urea). The refolding of the recombinant proteins is carried out in a 20 mM Tris buffer solution, pH 7.8, 150 mM NaCl containing L-arginine (400 iriM, Sigma) and β-mercaptoethanol (1 mM), at room temperature, by reducing the concentration of urea in a six-stage dialysis (4, 3, 2, 1, 0.5 and 0 M urea, respectively). Reduced and oxidized glutathione (5 mM and 0.5 mM respectively, Sigma) are added during the 0.5 and 0 M dialysis stages. Finally, the proteins are extensively dialyzed against 10 mM Tris buffer, pH 7. 5, NaCl 150 mM. The refolded, soluble proteins are concentrated and then purified on a Superdex 200 size exclusion column (Pharmacia, AKTA system). Surface resonance resonance measurements are made in a Biacore apparatus (Biacore). In all Biacore experiments, the HBS buffer solution supplemented with 0.05% P20 surfactant served as a running buffer. Immobilization of protein. The recombinant KIR2DL1 and KIR2DL3 proteins produced as described above are covalently immobilized to carboxyl groups in the dextran layer on a CM5 sensor chip (Biacore). The surface of the sensor chip is activated with EDC / NHS (N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, Biacore). The proteins, in coupling buffer solution (10 mM acetate, pH 4.5) were injected. Deactivation of the remaining activated groups was performed using 100 mM ethanolamine pH 8 (Biacore). Affinity measurements. For kinetic measurements, several concentrations of the soluble antibody (1 x 10 ~ 7 up to 4 x 10"10 M) were applied to the immobilized sample The measurements were made at a continuous flow rate of 20 ul / min. the surface of the sensor chip was regenerated by injection of 5 μl of 10 mM NaOH pH 11. The BIAlogue Kinetics evaluation program (BIAevaluation 3.1, Biacore) is used for data analysis The soluble analyte (40 μl at various concentrations) was injected at a flow rate of 20 μl / min in HBS buffer, in dextran layers containing 500 or 540 reflectance units (RU), and 1000 or 700 RU of KIR2DL1 and KIR2DL3, respectively. 6 independent experiments The results are shown in Table 1, below.

Table 1. BIAcore analysis of DF200 mAb linked to KIR2DL1 and KIR2DL3.

KD: Dissociation constant Example 6 Biacore competitive binding analysis of murine and human anti-KIR antibodies Epitope mapping analysis was performed on KIR 2DL1 (900 RU), KIR 2DL3 (2000 RU) and KIR 2DS1 (1000 RU) ) immobilized with murine 2D anti-KIR antibodies DF200, Pan2D, gll83 and EB6, and human 2D anti-KIR antibodies 1-4F1, 1-6F1, 1-6F5 and 1-7F9 as previously described (Gauthier et al 1999 , Saunal and van Regenmortel 1995). All experiments are given at a flow rate of 5 μl / min in HBS buffer with 2 minute injection of the different antibodies at 15 μg / ml. For each antibody coupling, the competitive binding analysis was carried out in two stages. In the first step, the first monoclonal antibody (mAb) is injected into KIR 2D target protein followed by the second mAb (without removing the first mAb) and the second RU mAb value (RU2) is monitored. In the second step, the second mAb is first injected directly into the naked KIR 2D protein, and the RU mAb (RUI) value is monitored. The percentage inhibition of the second Ab bound to the KIR 2D protein by the first mab is calculated by: 100 * (1-RU2 / RU1). The results are shown in Tables 2, 3 and 4, where the antibodies designated "first antibody" are listed in the vertical column, and the "second antibody" is listed in the horizontal column. For each antibody combination tested, the values for the direct binding (RU) level of the antibodies to the microplate are listed in the table, where the direct link of the second antibody to the KIR2D chip is listed in the position above the field , and the value for the binding of the second antibody to the KIR2D chip when the first antibody is presented is listed in the position below the field. Listed to the right of each field is the percent inhibition of the bound second antibody. Table 2 shows the link to the KIR2DL1 chip, Table 3 shows the binding of the antibodies to the KIR2DL3 chip, and Table 4 shows the binding of the antibodies to the KIR2DS1 chip. The competitive binding of murine antibodies DF200, NKVSF1 and EB6, and human antibodies 1-4F1, 1-7F9 and 1-6F1 to KIR2DL1, KIR2DL2 / 3 and KIR2DS1 immobilized is evaluated. The epitope mapping (Figure 7) of the experiments with anti-KIR antibodies bound to KIR2DL1 shows that (a) antibody 1-7F9 is competitive with EB6 and 1-4F1, but not with NKVSF1 and DF200; (b) antibody 1-4 Fl is again competitive with EB6, DF200, NKVSF1 and 1-7 F9; (c) NKVSFl competes with DF200, 1-4F1 and EB6, but not with 1-7F9; and (d) DF200 competes with NKVSFl, 1-4F1 and EB6, but not with 1- 7F9. The epitope mapping (Figure 8) of experiments with anti-KIR antibodies linked to KIR2DL3 show that (a) 1-4F1 is competitive with NKVSF1, DF200, g83 and 1-7F9; (b) 1-7F9 is competitive with DF200, gll83 and 1-4F1, but not with NKVSFl; (c) NKVSFl competes with DF200, 1-4F1 and GL183, but not with 1-7F9; and (d) DF200 competes with NKVSFl, 1-4F1 and 1-7F9, but not with GL183. Epitope mapping (Figure 9) of experiments with anti-KIR antibodies bound to KIR2DS1 show that (a) 1-4F1 is competitive with NKVSF1, DF200 and 1-7F9; (b) 1-7F9 is competitive with 1-4F1 but is not competitive with DF200 and NKVSFl; (c) NKVSFl competes with DF200 and 1-4F1, but not with 1-7F9; and (d) DF200 competes with NKVSFl and 1-4F1, but not with 1-7F9. Example 7 Titration of anti-KIR mAb with NK cells of cinomolgus The anti-KIR antibody NKVSF1 was tested for its ability to bind to NK cells of cynomolgus monkeys. The binding of the antibody to monkey NK cells is shown in Figure 10. Purification of monkey PBMC and polyclonal NK cell volume generation. CPMC cynomolgus monkey PBMC were prepared from sodium citrate CPT (Becton Dickinson). Purification of NK cells was performed by negative elimination (macaque NK cell enrichment kit, Stem Cell Technology). Nk cells are cultured in irradiated human feeder cells, 300 U / rnl methylterucin 2 (Proleukin, Chiron Corporation) and Ing / ml Phytohemagglutin A (Invitrogen, Gibco) to obtain populations of polyclonal NK cells. Titration Pan2D mAb with cytotoxic NK cells. NK cells of cinomolgus (day 16 of volume NK) were incubated with different amounts of Pan2D mAb followed by anti-mouse IgG (H + L) antibodies from goat F (ab ') 2 fragments conjugated to PE. The percentage of positive cells is determined with an isotypic control (purified mouse IgGl). The samples are given in duplicate. Average fluorescence intensity = MFI. Table 2: Epitope Mapping KIR2DL1 <; -Second Ab- Table 3: Epitope Mapping KIR2DL3 < -Second Ab- » Table 4: Epitope Mapping KIR2DL1 < -Second Ab- First Ab DE200 Pai? D 1-4 Fl 1-7 F9 (below) DF200: 70% 660 87% 975 15% 80 825 Pan2D 100% 650 100% 920 45% * -8 500 1-7 F9 900 17% 1350 11% 660 96% 1090 1200 23 Example 8 Epitope Mapping of DF200- and pan2D- linked to KIR2DL1 Computer modeling of the extracellular domains of KIR2DL1, -2 and -3 (KIR2DL1-3), based on their published crystal structures (Maenaka et al. 1999), Fan et al (2001), Boyington et al. (2000)), predicts that amino acids R1311 are involved in the interaction between KIR2DL1 and the cross-reactive mouse monoclonal antibodies KIR2DL1-3 (mAb's) DF200 and pan2D . To verify this, fusion proteins consisting of the complete extracellular domain of KIR2DL1 (amino acids H1-H224), either wild type or mutated in point (for example R131W2), fused to human Fe (hFc) are prepared. The material and methods used to produce and evaluate the different KIR2DL1-hFc fusion proteins have been described (Winter and Long (2000)). In short, the cDNA vectors encoding KIR2DL1 (R131W) -hFc are generated, by PCR-based mutagenesis (Quickchange II, Promega) of CL42-Ig, a published cDNA vector APRA the production of wild-type KIR2DL1-hFc (Wagtmann et al. (nineteen ninety five)). KIR2DL1-hFc and KIR2DL1 (R131W) -hFc are procured in COS7 cells and isolated from the tissue culture medium, essentially as described (Wagtmann et al (1995)). To test its correct fold, KIR2DLl-hFc and KIR2DL1 (R131W) -hFc are incubated with LCL721.221 cells expressing either HLA-Cw3 (without KIR2DL1 ligand) or HLA-Cw4 (ligand KIR2DL1), and the interaction between KIR-Fc fusion proteins and cells analyzed by FACS, a standard technique for the study of protein interactions on the cell surface. An example of independent experiments is given in Figure 11, panel A. As predicted from the literature, none of the KIR2DL1-hFc fusion proteins linked to HLA-Cw3 express LCL721.221 cells. In contrast, both KIR2DLl-hFc and KIR2DL1 (R131W) -hFc bound to HLA-Cw4 express LCL721.221 cells, thereby confirming their correct folding. 1 Single-letter amino acid code Substitution of R by W at amino acid position 131 (from the N-terminus) in KIR2DL1 The binding of KIR2DL1 (R131W) -hFc and KIR2DLl-hFc to KIR-specific mAbs (DF200, pan2D, EB6 and GL183) was studied using ELISA, a standard technique for studying protein interactions. In short, KIR2DL1 (R131W) -hFc and KIR2DL1-hFc went to the 96 well plates by means of goat anti-human antibodies, after which the KIR specific mAbs are added at various concentrations (0-1 μg / ml in PBS). The interactions between KIR2DL1-hFc and mAb variants are visualized by spectrophotometry (450nm), using secondary antibodies coupled to the peroxidase specific for mouse antibodies to convert the TMB substrate.

Examples of independent experiments are given in Figure 11, panel B. Although the GL183 mAb specific for KIR2DL2-3 is not capable of binding any of the KIR2DL1-hFc fusion proteins, the EB6 mAb specific for KIR2DL1, DF200 and pan2D bind KIR2DL1-hFc variants in a dose-dependent manner. The single point mutation (R131W) affects the binding of DF200 and pan2D with a reduction in the binding compared to the wild type of ~ 10% at higher concentrations of mAb (1 μg / ml), confirms that R131 is part of the DF200 binding site and pan2D in the extra-cellular domain 2 of KIR2DL1. REFERENCES Moretta, A., Bottino, C., Pende, D., Tripodi, G., Tambussi, G., Viale, O., Orengo, A., Barbaresi, M., Merli, A., Ciccone, E., and et al. (1990) . Identification of four subsets of human CD3-CD16 + natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific allogeneic recognition. J Exp Med 172, 1589-1598. Moretta, A., Vítale, M., Bottino, C, Orengo, A.M., Morelli, L., Augugliaro, R., Barbaresi, M., Ciccone, E., and Moretta, L. (1993). P58 molecules as putative receptors for major stocompatibility complex (MHC) class I molecules in human natural killer (NK) cells. Anti-p58 antibodies reconstitute lysis of MHC class I-protected cells in NK clones displaying different specif icities. J Exp Med 178, 597-604. Pende, D., Parolini, S., Pessino, A., Sivori, S., Augugliaro, R., Morelli, L., Marcenaro, E., Accame, L., Malaspina, A., Biassoni, R., et al. (1999) . Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells. J Exp Med 190, 1505-1516. Ruggeri, L., Capanni, M., Urbani, E., Perruccio, K., Shlomchik, W. D., Tosti, A., Posati, S., Rogaia, D., Frassoni, F., Aversa, F., et al. (2002). Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 295, 2097-2100. Wagtmann N, Biassoni R, Cantoni C, Verdiani S, Malnati MS, Vítale M, Bottino C, Moretta L, Moretta A, Long EO. Molecular clones of the p58 NK cell receptor reveal immunoglobulin-related molecules with diversity in both the extra- and intracellular dsmains. Ipttiunity. 1995 May; 2 (5): 439-49. Biassoni R, Verdiani S, Cambiaggi A, Romeo PH, Ferrini S, Moretta L. Human CD3- CD 16+ natural killer cells express the h.GATA-3 T cell transcription factor and an unrearranged 2.3-kb TcR delta transcript. Eur J Immunol. 1993 May; 23 (5): 1083-7. Saulquin X, Gastinel LN, Vivier E. Crystal structure of the natural human killer cell activating receptor ER2DS2 (CD158J) J Exp Med. 2003 Apr 7; 197 (7): 933-8. Gauthier, L., Lemmers, B., Guelpa-Fonlupt, V., Fougereau, M., and Schiff, C. pl-SLC physico-chemical interactions of the human preB cell receiver: implications for VH repertoire selection and cell signaling at the preB cell stage. Journal of Immunology, 162., 41-50. ( 1999) .

Saunal, H. and Van Regenmortel, M. H. V., Mapping of viral conformation epitopes using biosensor measurements. Journal of Immunology, 183: 33-41 (1995). Boyington JC; Motvka SA; Schuck P; Brooks AG; Sun PD.

Nature, Vol. 405 (6786) pp. 537-543 (2000) Fan OR Long Long; Wilev DC. Nature immunology, Vol. 2 (5) pp. 452-460 (2001) Maenaka K; Juji T; Stuart Pl; Jones EY. Structure with Folding and Design, Vol. 7 (4) pp. 391-398 (1999) Wagtmann N; Raiagopalan S; Winter CC; Peruzzi M; Long EO Immunity, Vol. 3 (6) pp. 801-809 (1995) Winter CC; Long EO Natural Killer Cells Protocols (edited by Campbell KS and Colonna M). Human Press, pp. 219-238 (2000) All references, including publications, patent applications and patents, cited herein, are hereby incorporated by reference to the same extent as if each reference individually and specifically indicated to be incorporated for reference and to be established in its entirety in the present. All titles and subtitles are used at present only for convenience and should not be construed as limiting the invention in any way. The terms "a" and "an" and "the" and similar references as used in the context of the description of the invention, are constructed to cover both singular and plural, unless otherwise indicated herein or clearly contradict the context. The mention of the ranges of values herein is intended only to serve as a stenographic method to refer individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if recited individually in the present. Unless stated otherwise, all exact exemplary values provided with respect to a particular factor or measurement may be considered to also provide a corresponding approximate measurement, modified by "around", where appropriate. All methods described herein may be performed in any appropriate order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended solely to better illuminate the invention and has no limitation on the scope of the invention unless otherwise indicated. another way. No language in the specification should be constructed as indicating any essential element for the practice of the invention, unless it is established very explicitly. The citation and incorporation of patent documents herein is for convenience only and does not reflect any view of validity, patentability, and / or enforcement of such patent documents. The description herein of any aspect or mode of the invention using terms such as "comprises", "having", "including", or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention which "consists of", "consists essentially of", or "substantially comprises" whose particular element or elements, unless otherwise stated or clearly contradicted by the context (e.g. composition described herein as comprising a particular element should be understood as also describing a composition consisting of such an element, unless otherwise stated or clearly contradicted by context). The invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law. It is noted that with this date, the best method known to the applicant to carry out the practice of said invention, is that which is clear from the present description of the invention.

Claims (19)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property.

1. A method for producing an antibody which cross-reacts with multiple KIR2DL gene products and which neutralizes the inhibitory activity of KIRs, characterized in that it comprises the steps of: (a) immunizing a non-human mammal with an immunogen comprising a polypeptide KIR2DL; (b) preparing the antibodies of the immunized mammal, wherein the antibodies bind to the KIR2DL polypeptide, (c) selecting antibodies from (b) that cross-react with at least two different KIR2DL gene products, and (d) selecting antibodies from (c) that potentiate NK cells. (e) selecting an antibody that binds an NK cell or primate KIR polypeptide.

2. The method according to claim 1, characterized in that the primate in step (e) is a cynomolgus monkey.

3. A method for evaluating the toxicity of an antibody, characterized in that the antibody produced according to the method of claim 1 or 2 is administered to a primate.

4. The method according to claim 3, characterized in that the primate is a cynomolgus monkey.

5. An antibody, antibody fragment, or antibody derivative, characterized in that it comprises the light chain variable region sequence of DF-200 as set forth in Fig. 12.

6. An antibody, antibody fragment, or antibody derivative, characterized in that it comprises the light chain variable region sequence of Pan2D as set forth in Fig. 12.

7. An antibody, antibody fragment, or antibody derivative, characterized in that it comprises one or more of the regions light variables CDRs of DF-200 as set forth in Fig. 12.

8. An antibody, antibody fragment, or antibody derivative, characterized in that it comprises one or more light variable regions of Pan2D CDRs as set forth in Fig. 12.

An antibody, antibody fragment, or antibody derivative, characterized in that it comprises the heavy chain variable region sequence of DF-200 coo is set forth in Fig. 13.

10. An antibody, antibody fragment, or antibody derivative, characterized in that it comprises one or more of the heavy variable regions CDRs of DF-200 as set forth in FIG. 13.

11. An antibody characterized in that it binds exclusively to KIR2DL1 within a region defined by the 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).

12. An antibody characterized by binding to KIR2DL1 and KIR 2DL2 / 3 without interacting with the residues of ainino acids outside of the region defined by the 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).

13. An antibody characterized by binding to KIR2DL1 and which does not bind to a mutant of KIR2DL1 in which R131 is Ala. 1 .

An antibody characterized in that it binds to KIR2DL1 and which does not bind to a mutant of KIR2DL1 in which R157 is Ala.

15. An antibody characterized in that it binds to KIR2DL1 and which does not bind to a mutant of KIR2DL1 in which R158 is Ala.

16. An antibody characterized by binding to KIR2DL1 residues (131, 157, 158).

17. An antibody characterized by binding to KIR2DS3 (R131W), but not to wild type KIR2DS3.

18. An antibody characterized in that it binds both KIR2DL1 and KIR2DL2 / 3 as well as KIR2DS4.

19. An antibody characterized by binding to both KIR2DL1 and KIR2DL2 / 3, but not to KIR2DS4.