CN1842541B - Compositions and methods for regulating NK cell activity - Google Patents

Abstract

The present invention relates to novel compositions and methods for modulating the immune response of a subject. The invention more particularly relates to specific antibodies that modulate NK cell activity in a mammalian subject and allow enhanced NK cell cytotoxicity. The present invention also relates to fragments and derivatives of such antibodies, as well as pharmaceutical compositions containing them, and their use, in particular for increasing NK cell activity or cytotoxicity in a subject in therapy.

Description

Compositions and methods for modulating NK cell activity

field of invention

The present invention relates to antibodies, antibody fragments and derivatives thereof that cross-react with two or more inhibitory receptors present on the surface of NK cells and enhance NK cell cytotoxicity in a mammalian subject or biological sample. The invention also relates to methods of making such antibodies, fragments, variants and derivatives; pharmaceutical compositions comprising said substances; the use of such molecules and compositions (particularly in therapy) in increasing NK cell activity or Use in Cytotoxicity. the

background

Natural killer (NK) cells are a subset of lymphocytes involved in unconventional immunity. NK cells can be obtained from, for example, blood samples, cytapheresis, collections, etc., by a variety of techniques well known in the art. the

Characteristics and biological properties of NK cells include: expression of surface antigens, including CD16, CD56, and/or CD57; lack of α/β or γ/δ TCR complexes on the cell surface; binding and killing by activation of specific cytolytic enzymes that do not express " The ability of cells to "self" MHC/HLA antigens; the ability to kill tumor cells or other diseased cells expressing NK-activating receptor-ligands; the ability to release cytokines that stimulate or suppress immune responses; and the ability to undergo multiple rounds of cell division, The ability to produce daughter cells with biological properties similar to those of the parent cell. In the context of the present invention, "active" NK cells refer to biologically active NK cells, more specifically NK cells having the ability to lyse target cells. For example, "active" NK cells are capable of killing cells that express NK-activating receptor-ligands and do not express "self" MHC/HLA antigens (KIR-incompatible cells). the

Based on their biological properties, various therapeutic and immunization strategies relying on the regulation of NK cells have been proposed in the art. However, NK cell activity is regulated by complex mechanisms involving stimulatory and inhibitory signals. Therefore, effective NK cell-mediated therapy may require stimulation of these cells and neutralization of inhibitory signals. the

NK cells are negatively regulated by major histocompatibility complex (MHC) class I-specific inhibitory receptors ( et al., 1986; et al., 1989). These specific receptors bind to polymorphic determinants of MHC class I molecules or HLA present on other cells and inhibit NK cell lysis. In humans, certain members of a family of receptors known as killer Ig-like receptors (KIRs) recognize HLA class I alleles.

KIRs are a large family of receptors present on certain subsets of lymphocytes, including NK cells. KIR nomenclature is based on the number of extracellular domains (KIR2D or KIR3D) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). In humans, the presence or absence of a given KIR is variable between NK cells within the NK population present in a single individual. There are also relatively high levels of polymorphisms of KIR molecules in the population, with certain KIR molecules being present in some (but not all) individuals. Certain KIR gene products cause stimulation of lymphocyte activity when bound by appropriate ligands. The proven stimulatory KIRs all have short cytoplasmic tails containing charged transmembrane residues that bind to linker molecules containing an immunostimulatory motif (ITAM). Other KIR gene products are repressive in nature. All confirmed inhibitory KIRs have long cytoplasmic tails and appear to interact with distinct subsets of HLA antigens according to KIR subtype. Inhibitory KIRs display in their cytoplasmic portion one or several inhibitory motifs that recruit phosphatases. Known inhibitory KIR receptors include KIR2DL and KIR3DL subfamily members. KIR receptors with two Ig domains (KIR2D) identify HLA-C allotypes: KIR2DL2 (originally designated p58.2) or the closely related gene product KIR2DL3 recognize HLA-C allotypes consisting of 2 groups of 3, 7 and 8), whereas KIR2DL1 (p58.1) recognizes epitopes shared by reciprocal group 1 HLA-C allotypes (Cw2, 4, 5 and 6). The presence of a Lys residue at position 80 of the HLA-C allele indicates KIR2DL1 recognition. The presence of an Asn residue at position 80 indicates KIR2DL2 and KIR2DL3 recognition. Importantly, most HLA-C alleles have Asn or Lys at position 80. One KIR with three Ig domains, KIR3DL1(p70), recognizes an epitope shared by HLA-Bw4 alleles. Finally, KIR3DL2 (p140), a homodimeric molecule with three Ig domains, recognizes HLA-A3 and HLA-A11. the

Although inhibitory KIRs and other class I inhibitory receptors (Moretta et al., 1997; Valiante et al., 1997a; Lanier, 1998) may be co-expressed by NK cells, expression is present in any given individual's NK repertoire Cells of a single KIR, and thus the corresponding NK cells, are blocked only by cells expressing the set of specific class I alleles. the

KIR-mismatched NK cell populations or clones (i.e. NK cell populations expressing KIRs that are incompatible with host HLA molecules) have been shown to be the most likely mediators of the graft-versus-leukemic effects seen in allografts (Ruggeri et al., 2002 ). One way to recreate this effect in a given individual would be to use agents that block the KIR/HLA interaction. the

A KIR2DL1-specific monoclonal antibody was shown to block the interaction of KIR2DL1 with the Cw4 (or analog) allele (Moretta et al., 1993). Anti-KIR2DL2/3 monoclonal antibodies have also been described to block the interaction of KIR2DL2/3 with HLACw3 (or analogue) alleles (Moretta et al., 1993). However, the use of such agents in a clinical setting would require the development of two therapeutic mAbs to treat all patients, regardless of whether any given patient expresses class 1 or class 2 HLA-C alleles. In addition, before deciding which therapeutic antibody to use, the HLA type expressed by each patient must be determined in advance, resulting in much more expensive treatment costs. the

Watzl et al., Tissue Antigens , 56, p. 240 (2000) made cross-reactive antibodies recognizing multiple KIR isoforms, but these antibodies did not demonstrate enhancement of NK cell activity. GM Spaggiara et al., Blood , 100, pp. 4098-4107 (2002) performed experiments with a number of monoclonal antibodies against various KIRs. One of these antibodies (NKVSF1) is said to recognize a common epitope of CD158a (KIR2DL1), CD158b (KIR2DL2) and p50.3 (KIR2DS4). There is no suggestion that NKVSF1 can enhance NK cell activity, nor that it could be used therapeutically. Therefore, there is currently no feasible and effective way to regulate NK cell activity in this field, and it is still necessary to use specific reagents to specifically intervene in HLA alleles.

Summary of the invention

The present invention now provides novel antibodies, compositions and methods to overcome existing difficulties in NK cell activation and provide additional advantageous features and benefits. In one exemplary aspect, the invention provides a single antibody that promotes human NK cell activation in substantially all humans. More specifically, the present invention provides novel specific antibodies that cross-react with and neutralize the inhibitory signal of various groups of inhibitory KIRs, resulting in the expression of NKs expressing such inhibitory KIR receptors. The cytotoxicity of NK cells in the cells was enhanced. The ability to cross-react with multiple KIR gene products allows the antibodies of the invention to be effectively used to increase NK cell activity in most human subjects without the burden or expense of pre-determining the subject's HLA type. the

In a first aspect, the invention provides antibodies, antibody fragments, and derivatives of any of these, wherein said antibodies, antibody fragments, or derivatives cross-react with at least two inhibitory KIR receptors on the surface of NK cells, neutralize NK Inhibitory signaling of cells and enhances NK cell activity. More preferably, the antibody binds to a common determinant of the human KIR2DL receptor. The antibodies of the invention bind even more specifically to at least the KIR2DL1, KIR2DL2 and KIR2DL3 receptors. For the purposes of the present invention, the term "KIR2DL2/3" refers to either or both of the KIR2DL2 and KIR2DL3 receptors. These two receptors share very high homology, are putative allelic forms of the same gene, and are considered interchangeable by the art. Therefore, KIR2DL2/3 are considered for the purposes of the present invention as a single inhibitory KIR molecule, and thus antibodies that cross-react only with KIR2DL2 and KIR2DL3 and not with other inhibitory KIR receptors are not within the scope of the present invention. the

The antibodies of the invention specifically inhibit the binding of MHC or HLA molecules to at least two inhibitory KIR receptors and promote NK cell activity. Both activities are inferred from the term "neutralizing the inhibitory activity of a KIR" as used herein. In the context of the present invention, the antibodies of the present invention "promote NK cell activity", "promote NK cell cytotoxicity", "promote NK cell", "enhance NK cell activity", "enhance NK cell cytotoxicity" or "enhance NK cell" Ability means that the antibody enables NK cells expressing an inhibitory KIR receptor on their surface to lyse cells expressing the corresponding ligand of that particular inhibitory KIR receptor (eg a particular HLA antigen) on their surface. In a specific aspect, the invention provides antibodies that specifically inhibit the binding of HLA-C molecules to KIR2DL1 and KIR2DL2/3 receptors. In another specific aspect, the invention provides antibodies that promote NK cell activity in vivo. the

Because at least one of KIR2DL1 or KID2DL2/3 is present in at least about 90% of the human population, the more preferred antibodies of the invention are capable of facilitating targeting of most HLA-C allotypes (Group 1 HLA-C allotypes and Group 2 HLA-C allotypes, respectively). NK cell activity of HLA-C allotype)-associated cells. Therefore, the composition of the present invention can be used to effectively activate or enhance NK cells in most human subjects (generally about 90% of human subjects or more). Thus, a single antibody composition according to the invention can be used to treat most human subjects with little need to determine allelic classes or use antibody mixtures. the

The present invention demonstrates for the first time that cross-reactive and neutralizing antibodies against inhibitory KIRs can be produced and that such antibodies allow efficient activation of NK cells in a broad range of populations. the

A particular object of the present invention is thus located on antibodies which specifically bind KIR2DL1 and KIR2DL2/3 human receptors and reverse the inhibition of NK cell cytotoxicity mediated by these KIRs. In one embodiment, the antibody competes with monoclonal antibody DF200 produced by hybridoma DF200. Optionally, the antibody that competes with antibody DF200 is not antibody DF200 itself. the

In another embodiment, the antibody competes with monoclonal antibody NKVSF1, optionally, wherein the antibody that competes with antibody NKVSF1 is not antibody NKVSF1. the

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

The antibody is preferably a chimeric antibody, a humanized antibody or a human antibody. the

When referring to a specific monoclonal antibody (e.g. DF200, NKVSF1, 1-7F9, EB6, GL183), the term "competes with" means that the antibody competes with the monoclonal antibody (e.g. DF200, NKVSF1, GL183) in a binding assay. 1-7F9, EB6, GL183) competition, the binding assay using recombinant KIR molecules or surface expressed KIR molecules. For example, an antibody "competes" with DF200 if it reduces the binding of DF200 to a KIR molecule in a binding assay. Antibodies that "compete" with DF200 may compete with DF200 for binding to the KIR2DL1 human receptor, the KIR2DL2/3 human receptor, or both the KIR2DL1 and KIR2DL2/3 human receptors. the

In preferred embodiments, the invention provides human antibodies that bind KIR2DL1 and KIR2DL2/3, reverse the inhibition of NK cell cytotoxicity mediated by these KIRs, and compete with DF200, 1-7F9 or NKVSF1 for binding to the KIR2DL1 human receptor, KIR2DL2 /3 human receptor or antibodies to both KIR2DL1 and KIR2DL2/3 human receptors. The antibody is optionally not NKVSF1. The antibody can optionally be a chimeric, human or humanized antibody. the

In another embodiment, the invention provides binding to KIR2DL1 and KIR2DL2/3 human receptors, reversing the inhibition of NK cell cytotoxicity mediated by these KIRs, and competing with EB6 for binding to KIR2DL1 human receptors, and GL183 for binding to KIR2DL2/3 human receptors. 3 human receptor, or an antibody that competes with EB6 for binding to the KIR2DL1 human receptor and with GL183 for binding to the KIR2DL2/3 human receptor. The antibody is optionally not NKVSF1; the antibody is optionally not DF200. The antibody can optionally be a chimeric, human or humanized antibody. the

In an advantageous aspect, the present invention provides an antibody that competes with DF200, which recognizes and binds to the epitope or "epitope site" on the KIR molecule that is substantially the same or the same as the monoclonal antibody DF200, or to which epitope or An "epitopic site" is immunospecific. The KIR molecule is preferably the KIR2DL1 human receptor or the KIR2DL2/3 human receptor. the

A particular object of the invention is directed to antibodies which bind to common determinants present on the KIR2DL1 and KIR2DL2/3 human receptors and reverse the inhibition of NK cell cytotoxicity mediated by these KIRs. These antibodies more specifically bind to substantially the same epitope on KIR as monoclonal antibody DF200 produced by hybridoma DF200 or antibody NKVSF1 produced by hybridoma NKVSF1, wherein said antibody is not NKVSF1. the

In preferred embodiments, the antibodies of the invention are monoclonal antibodies. The most preferred antibody of the present invention is monoclonal antibody DF200 produced by hybridoma DF200. the

The hybridoma producing antibody DF200 is deposited with the CNCM Depository Center, CollectionNationale de Cultures de Microorganismes, Institut Pasteur, 25, Rue du Docteur Roux, under the identification number "DF200", registration number CNCM I-3224 (registered on June 10, 2004) , F-75724 Paris Cedex 15, France. Antibody NKVSF1 is available from Serotec (Cergy Sainte-Christophe, France), catalog ref. MCA2243. NKVSF1 is also referred to herein as pan2D mAb. the

The invention also provides functional fragments and derivatives of the antibodies described herein that have substantially the same antigen specificity and activity (e.g., can cross-react with the parent antibody and enhance expression of inhibitory KIR receptors). NK cell cytotoxic activity), including (but not limited to) Fab fragments, Fab'2 fragments, immunoadhesins, miniature diabodies (diabodies), CDRs and ScFv. Additionally, antibodies of the invention may be humanized, human or chimeric. the

The invention also provides antibody derivatives comprising an antibody of the invention conjugated or covalently bound to a toxin, a radionuclide, a detectable moiety (eg, a fluorescent agent), or a solid support. the

The invention also provides pharmaceutical compositions comprising the above-disclosed antibodies, fragments thereof, or derivatives of any of them. Accordingly, the present invention also relates to the use of the antibodies disclosed herein in a method for the manufacture of a medicament. In preferred embodiments, the medicament or pharmaceutical composition is for the treatment of cancer or other proliferative diseases, infections, or for transplantation. the

In another embodiment, the invention provides compositions comprising antibodies that bind to at least two different human inhibitory KIR receptor gene products, wherein the antibodies are capable of neutralizing expression of the two different human inhibitory KIR receptors. Inhibition of KIR-mediated NK cell cytotoxicity on NK cells of at least one of the sex KIR receptors, wherein the antibody is incorporated into liposomes. The composition optionally includes additional substances selected from nucleic acid molecules for delivering genes for gene therapy; nucleic acid molecules for delivering antisense RNA, RNAi or siRNA for gene suppression in NK cells; or otherwise incorporated into the Toxins or drugs that target and kill NK cells in liposomes. the

The present invention also provides a method for regulating human NK cell activity in vitro, ex vivo or in vivo, comprising combining human NK cells with a therapeutically effective amount of an antibody of the present invention, a fragment of such an antibody, any derivative thereof, or at least comprising any of the above-mentioned A pharmaceutical composition contact. A preferred method comprises administering a therapeutically effective amount of a pharmaceutical composition of the present invention and is directed at increasing the cytotoxic activity of human NK cells (most preferably ex vivo or in vivo) in a subject suffering from cancer, an infectious disease or an immune disease. ). the

In another aspect, the invention provides a hybridoma comprising: (a) a B cell from a mammalian host (typically a non-human mammalian host) immunized with an antigen comprising an epitope present on an inhibitory KIR polypeptide, said B cells are fused with (b) an immortalized cell line (e.g., a myeloma cell), wherein the hybridoma produces a monoclonal antibody that binds to at least two different human inhibitory KIR receptors and is capable of at least substantially neutralizing and inhibition of KIR-mediated NK cell cytotoxicity in a population of NK cells expressing said at least two different human inhibitory KIR receptors. Optionally, the hybridoma does not produce monoclonal antibody NKVSF1. The antibody preferably binds KIR2DL1 and KIR2DL2/3 receptors. The antibody preferably binds to a common determinant present on KIR2DL1 and KIR2DL2/3. The hybridoma preferably produces an HLA-c allele molecule that inhibits the binding of an HLA-c allele molecule with a Lys residue at position 80 to the human KIR2DL1 receptor, and an HLA-C allele molecule with an Asn residue at position 80 that binds to human KIR2DL2/3 Receptor-bound antibodies. The hybridoma preferably produces an antibody that binds to substantially the same epitope on KIR2DL1 or KIR2DL2/3 or both KIR2DL1 and KIR2DL2/3 as monoclonal antibody DF200 produced by hybridoma DF200. An example of such a hybridoma is DF200. the

The present invention also provides methods for producing antibodies that cross-react with multiple KIR2DL gene products and neutralize the inhibitory activity of such KIRs, the methods comprising the steps of:

(a) immunizing a non-human mammal with an immunogen containing a KIR2DL polypeptide;

(b) preparing antibodies from said immunized mammal, wherein said antibodies bind said KIR2DL polypeptide,

(c) selecting an antibody in (b) that cross-reacts with at least two different KIR2DL gene products, and

(d) Selecting an antibody that enhances NK cells in (c). In one embodiment, the non-human mammal is a transgenic animal designed to express a human antibody repertoire (e.g., a non-human mammal containing human immunoglobulin loci and lacking native immunoglobulin genes, such as Xenomouse™ (Abgenix-Fremont , CA, USA), or a non-human mammal containing a minilocus of a human Ig-encoding gene, such as HuMab-mouse ^™ (Medarex-Princeton, NJ, USA)). The method optionally further comprises selecting antibodies that bind primate (preferably macaque) NK cells or KIR polypeptides. The invention optionally further comprises a method of evaluating antibodies, wherein antibodies produced according to the methods described above are administered to a primate, preferably a macaque, preferably the monkey is observed for the presence or absence of signs of antibody toxicity.

The inventors also provide methods of producing antibodies that bind to at least two different human inhibitory KIR receptor gene products, wherein the antibodies are capable of neutralizing the expression of the at least two different human inhibitory KIR receptor gene products. Inhibition of NK cell cytotoxicity mediated by KIR on the NK cell population of the product, the method comprises the steps of:

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

b) preparing an antibody from said immunized animal, wherein said antibody binds said KIR polypeptide,

c) selecting an antibody in (b) that cross-reacts with at least two different human inhibitory KIR receptor gene products, and

Selecting (c) an antibody capable of neutralizing inhibition of NK cell cytotoxicity mediated by KIR on NK cell populations expressing said at least two different human inhibitory KIR receptor gene products, wherein steps (c) and The order of (d) can be reversed arbitrarily, and any step can be repeated 1 or more times. The inhibitory KIR polypeptide used for immunization is preferably a KIR2DL polypeptide, and the antibody selected from step (c) cross-reacts with at least KIR2DL1 and KIR2DL2/3. The antibody preferably recognizes a common determinant present on at least two different KIR receptor gene products; most preferably the KIRs are KIR2DL1 and KIR2DL2/3. The method optionally further comprises selecting antibodies that bind to primate (preferably macaque) NK cells or KIR polypeptides. The invention optionally further comprises a method of evaluating antibodies, wherein antibodies produced according to the methods described above are administered to a primate, preferably a macaque, preferably the monkey is observed for the presence or absence of signs of antibody toxicity. the

The antibody selected from step c) or d) of the above method is optionally not NKVSF1. The antibody prepared in step (b) of the above method is preferably a monoclonal antibody. The antibody selected from step (c) of the above method preferably inhibits the binding of HLA-C allele molecules having a Lys residue at position 80 to the human KIR2DL1 receptor, and HLA-C allele molecules having an Asn residue at position 80 Binding of gene molecules to human KIR2DL2/3 receptors. The antibody selected from step (d) of the method above preferably causes an increase in NK cell cytotoxicity, such as any significant increase in NK cell toxicity, or an increase of at least 5%, 10%, 20%, 30% or more, such as targeting NK cells Toxicity is at least about 50% enhanced (e.g., NK cell cytotoxicity is at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 65-100%) ). The antibody preferably binds substantially the same epitope on KIR2DL1 and/or KIR2DL2/3 as monoclonal antibody DF200. Optionally, the method also or additionally comprises the steps of making fragments of selected monoclonal antibodies, making derivatives of selected monoclonal antibodies (e.g., by conjugation with radionuclides, cytotoxic agents, reporter molecules, etc.), or making derivatives of antibody fragments As an additional step, the antibody fragments are produced from or contain sequences corresponding to the sequences of such monoclonal antibodies. the

The present invention additionally provides methods for producing antibodies that bind to at least two different human inhibitory KIR receptor gene products, wherein the antibodies are capable of neutralizing the expression of the at least two different human inhibitory KIR receptor genes Inhibition of NK cell cytotoxicity mediated by KIR on the NK cell population of the product, the method comprises the steps of:

(a) selecting from a library or library a monoclonal antibody or antibody fragment that cross-reacts with at least two different human inhibitory KIR2DL receptor gene products, and

(b) selecting an antibody of (a) capable of neutralizing inhibition of KIR-mediated NK cell cytotoxicity on a population of NK cells expressing said at least two different human inhibitory KIR2DL receptor gene products. The antibody preferably binds to a common determinant present on KIR2DL1 and KIR2DL2/3. The antibody selected from step (b) is optionally not NKVSF1. The antibody selected from step (b) preferably inhibits the binding of HLA-c allele molecules having a Lys residue at position 80 to the human KIR2DL1 receptor, and HLA-C allele molecules having an Asn residue at position 80 Binding to human KIR2DL2/3 receptors. The antibody selected from step (b) preferably causes an enhancement of NK cytotoxicity, such as any significant enhancement of NK cytotoxicity, or an enhancement of at least 5%, 10%, 20%, 30% or more, such as an enhancement of target NK cytotoxicity of at least About 50% (eg, NK cell cytotoxicity is enhanced by at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (eg, about 65-100%)). The antibody preferably binds substantially the same epitope on KIR2DL1 and/or KIR2DL2/3 as monoclonal antibody DF200. The method optionally comprises the additional step of making a fragment of the selected monoclonal antibody, making a derivative of the selected monoclonal antibody, or making a derivative of the selected monoclonal antibody fragment. the

In addition, the present invention provides methods for producing antibodies that bind to at least two different human inhibitory KIR receptor gene products, wherein the antibodies are capable of neutralizing expression of the at least two different human inhibitory KIR receptors. Inhibition of NK cell cytotoxicity mediated by KIR in the NK cell population of the gene product, the method comprises the steps of:

a) cultivating the hybridoma of the present invention under conditions that allow the production of the monoclonal antibody; and

b) isolating said monoclonal antibody from the hybridoma. The method optionally comprises the additional step of making the monoclonal antibody fragment, making a derivative of the monoclonal antibody, or making a derivative of such a monoclonal antibody fragment. The antibodies preferably bind to common determinants present on KIR2DL1 and KIR2DL2/3. the

The present invention also provides methods for producing antibodies that bind to at least two different human inhibitory KIR receptor gene products, wherein the antibodies are capable of neutralizing the expression of the at least two different human inhibitory KIR receptor genes The inhibition of NK cell cytotoxicity mediated by KIR in the NK cell population of the product, the method comprises the steps of:

a) isolating nucleic acid encoding said monoclonal antibody from the hybridoma of the present invention;

b) optionally modifying the nucleic acid such that a modified nucleic acid comprising a sequence encoding a modified or derived antibody comprising a functional sequence corresponding to said monoclonal antibody or substantially similar (e.g., at least about 65% identical to such sequence) is obtained , at least about 75%, at least about 85%, at least about 90%, at least about 95% (eg, about 70-99%) amino acid sequence identical), wherein the antibody is selected from a humanized antibody, a chimeric antibody, a single chain antibody , immunoreactive fragments of antibodies or fusion proteins containing such immunoreactive fragments;

c) inserting said nucleic acid or modified nucleic acid (or related nucleic acid encoding the same amino acid sequence) into an expression vector, wherein when the expression vector exists in a host cell grown under suitable conditions, the encoded antibody or antibody fragment can be obtained Express;

d) transfecting a host cell with said expression vector, wherein said host cell does not otherwise produce immunoglobulin protein;

e) culturing said transfected host cell under conditions that result in expression of said antibody or antibody fragment; and

f) isolating antibodies or antibody fragments produced by the transfected host cells. The antibodies preferably bind to common determinants present on KIR2DL1 and KIR2DL2/3. the

It is to be understood that the present invention also provides compositions comprising antibodies, wherein said antibodies bind at least two different human inhibitory KIR receptor gene products, wherein said antibodies are capable of neutralizing genes expressing at least two different human inhibitory KIR receptors. Inhibition of KIR-mediated NK cell cytotoxicity in NK cells, one of the KIR receptors, the antibody is present in an amount effective to detectably enhance NK cell cytotoxicity in a patient or in a biological sample containing NK cells; also containing a druggable carrier or excipient. Antibodies preferably bind to common determinants present on KIR2DL1 and KIR2DL2/3. The composition may optionally contain another therapeutic agent selected from, for example, immunomodulators, hormonal agents, chemotherapeutic agents, anti-angiogenic agents, apoptotic agents, agents that bind to and inhibit inhibitory KIR receptors Another antibody, anti-infective, targeting agent or adjunct compound. Favorable immunomodulators may be selected from IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10. IL-11, IL-12, IL-13, IL-15, IL-21, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, or IFN-γ. Examples of such chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, doxorubicin, actinomycin D, mitomycin, carmine, daunomycin, doxorubicin, Tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), cytarabine, cyclophosphamide, thiote Pie, methotrexate, camptothecin, actinomycin D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatin, other vinca alkaloids and their derivatives or prodrugs. Examples of hormonal agents include leuprolide, goserelin, triptorelin, buserelin, tamoxifen, toremifene, flutamide, nilutamide, cyproterone Lutamide Anastrozole, exemestane, letrozole, fadrozole medroxy, chlormadinone, megestrol, other LHRH agonists, other antiestrogens, other antiandrogens, other aromatase inhibitors , and other progestogens. Said another antibody that binds to and inhibits an inhibitory KIR receptor is preferably an antibody or a derivative or fragment thereof that binds an epitope of an inhibitory KIR receptor, wherein said epitope is different from that present in at least two different human inhibitory KIR receptors. Epitopes bound by antibodies that bind to common determinants on gene products of sex KIR receptors. the

The present invention further provides a method of detectably enhancing NK cell activity in a patient in need thereof, comprising the step of administering to the patient a composition of the present invention. A patient in need of enhanced NK cell activity can be any patient with a disease or disorder in which such enhancement is likely to facilitate, enhance and/or induce a therapeutic effect (or at least in patients with the disease or disorder, or with substantially similar characteristics) Facilitating, enhancing and/or inducing such effects in a significant portion of , as can be determined, for example, by clinical trials). Patients in need of such treatment may suffer from, for example, cancer, other proliferative, infectious or immune diseases. The method preferably comprises the additional step of administering to the patient a suitable additional therapeutic agent selected from the group consisting of immunomodulators, hormonal agents, chemotherapeutic agents, anti-angiogenic agents, apoptotic agents, binding and inhibiting inhibitory KIR receptors Another antibody, anti-infective agent, targeting agent, or adjuvant compound of the present invention, wherein the additional therapeutic agent is administered to the patient together with the antibody in a single dosage form, or in separate dosage forms. The dose of the antibody (or antibody fragment/derivative) and the dose of the additional therapeutic agent taken together are sufficient to detectably induce, promote and/or enhance (including enhancing NK cell activity) a therapeutic response in the patient. When administered separately, the antibody, fragment or derivative and the additional therapeutic agent are administered as needed under conditions that produce a detectable benefit of the combination therapy in the patient (eg, with respect to timing, amount of agent, etc.). the

Further encompassed by the invention are antibodies of the invention capable of specifically binding non-human primate (preferably monkey) NK cells and/or monkey KIR receptors. Also encompassed are methods of evaluating the toxicity, dosage and/or activity or efficacy of an antibody of the invention as a drug candidate. In one aspect, the invention comprises a method of determining a dose of an antibody that is toxic to an animal or a target tissue, comprising administering an antibody of the invention to a non-human primate recipient having NK cells, assessing the effect of the agent on the animal or, preferably, on the target tissue Any toxicity or harmful or adverse effects. In another aspect, the invention is a method of identifying an antibody that is toxic to an animal or a target tissue comprising administering an antibody of the invention to a non-human primate recipient having NK cells, assessing the effect of the agent on the animal or preferably on the target tissue Any toxicity or harmful or adverse effects. In another aspect, the present invention is a method for identifying an effective antibody for treating an infected person, a disease or a tumor, comprising administering the antibody of the present invention to an infection, disease or cancer model of a non-human primate, identifying and improving infection, disease or cancer, or antibodies to its symptoms. The antibodies of the invention are preferably antibodies that (a) cross-react with at least two inhibitory human KIR receptors on the surface of human NK cells, and (b) cross-react with NK cells or KIR receptors of non-human primates. the

Other content included in the present invention is a method for detecting NK cells with inhibitory KIR on the cell surface in a biological sample or a living organism, the method comprising the steps of:

a) contacting said biological sample or living organism with an antibody of the invention, wherein said antibody is conjugated or covalently linked to a detectable moiety; and

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

The present invention also provides a method for purifying NK cells with inhibitory KIR on their cell surface from a sample, comprising the steps of:

a) contacting the sample with an antibody of the invention, wherein the antibody is conjugated to a solid support (e.g., a bead, matrix, etc.) or covalently linked; and

b) Elution of bound NK cells from the antibody conjugated or covalently linked to the solid support. the

In another aspect, the present invention provides an antibody, an antibody fragment, or any derivative thereof, said antibody, antibody fragment, or any derivative thereof comprising the light chain variable region of antibody DF200 or antibody Pan2D or one or more light chain variable regions. Chain variable region CDR, as shown in Figure 12. In another aspect, the present invention provides an antibody, an antibody fragment, or any derivative thereof, which contains a sequence of the light chain variable region of DF200 or Pan2D or one of these antibodies or The gold part or substantially all of one or more light chain variable region CDRs of the two are highly similar sequences. the

In another aspect, the present invention provides an antibody, antibody fragment, or any derivative thereof, which comprises the heavy chain variable region or one or more light chain variable regions of antibody DF200. Region CDR, as shown in Figure 13. In another aspect, the present invention provides an antibody, an antibody fragment, or any derivative thereof, which contains a sequence that is highly similar to all or substantially all of the heavy chain variable region sequence of DF200 . the

These and other advantageous aspects and features of the present invention will be further described elsewhere herein. the

Brief description of the drawings

Figure 1 depicts the monoclonal antibody DF200, which binds to common determinants of multiple human KIR2DL receptors. the

Figure 2 depicts the monoclonal antibody DF200, which neutralizes the KIR2DL-mediated inhibition of cytotoxicity of KIR2DL1-positive NK cells on Cw4-positive target cells. the

Figure 3 depicts the monoclonal antibody DF200, DF200 Fab fragment, and conventional antibodies specific for KIR2DL1 or KIR2DL2/3, which neutralize the KIR2DL-mediated inhibition of cytotoxicity of KIR2DL1-positive NK cells on Cw4-positive target cells, and on Cw3-positive NK cells. Inhibition of cytotoxicity of KIR2DL2/3-positive NK cells mediated by KIR2DL on positive target cells. the

Figure 4 depicts cell lysis by NK clones of HLA Cw4 positive target cells in the presence of DF200 and EB6 antibody F(ab')2 fragments. the

Figures 5 and 6 depict monoclonal antibody DF200, NKVSF1 (pan2D), human antibodies 1-7F9, 1-4F1, 1-6F5 and 1-6F1, and conventional antibodies specific for KIR2DL1 or KIR2DL2/3, which neutralize in Inhibition of KIR2DL1-positive NK cell cytotoxicity mediated by KIR2DL on Cw4-positive target cells (Cw4-transfected cells in Figure 5 and EBV cells in Figure 6). the

)分析获得，其中重叠的圆圈指结合KIR2DL1的重叠。结果显示1-7F9在KIR2DL1 上与EB6和1-4F1竞争，但是不与NKVSF1和DF200竞争。抗体1-4F1依次与EB6、DF200、NKVSF1和1-7F9竞争。抗体NKVSF1在KIR2DL1上与DF200、1-4F1和EB6竞争，但是不与1-7F9竞争。DF200在KIR2DL1上与NKVSF1、1-4F1和EB6竞争，但是不与1-7F9竞争。  Figure 7 depicts epitope mapping showing the results of a competition binding experiment with an anti-KIR antibody against KIR2DL1 by surface plasmon resonance (

) analysis, where overlapping circles refer to overlapping of binding KIR2DL1. The results show that 1-7F9 competes with EB6 and 1-4F1, but not NKVSF1 and DF200, on KIR2DL1. Antibody 1-4F1 competed sequentially with EB6, DF200, NKVSF1 and 1-7F9. Antibody NKVSF1 competed with DF200, 1-4F1 and EB6, but not 1-7F9, on KIR2DL1. DF200 competes with NKVSF1, 1-4F1 and EB6, but not 1-7F9, on KIR2DL1.

分析获得，其中重叠的圆圈指结合KIR2DL3的重叠。结果显示1-4F1在KIR2DL3上与NKVSF1、DF200、g1183和1-7F9竞争。1-7F9在KIR2DL3上与DF200、g1183和1-4F1竞争，但是不与NKVSF1竞争。NKVSF1在KIR2DL3上与DF200、1-4F1和GL183竞争，但是不与1-7F9竞争。DF200在KIR2DL3上与NKVSF1、1-4F1和1-7F9竞争，但是不与GL183竞争。  Figure 8 depicts epitope mapping showing the results of a competition binding experiment passed with an anti-KIR antibody against KIR2DL3

Analysis obtained, where overlapping circles refer to overlaps binding KIR2DL3. The results show that 1-4F1 competes with NKVSF1, DF200, g1183 and 1-7F9 on KIR2DL3. 1-7F9 competed with DF200, g1183 and 1-4F1 on KIR2DL3, but not NKVSF1. NKVSF1 competes with DF200, 1-4F1 and GL183 on KIR2DL3, but not 1-7F9. DF200 competed with NKVSF1, 1-4F1 and 1-7F9 on KIR2DL3, but not GL183.

Figure 9 depicts epitope mapping showing the results of a competition binding experiment passed with an anti-KIR antibody against KIR2DS1 Analysis obtained, where overlapping circles refer to overlaps binding KIR2DS1. The results show that antibody 1-4F1 competes with NKVSF1, DF200 and 1-7F9 on KIR2DS1. Antibody 1-7F9 competed with 1-4F1 but not DF200 and NKVSF1 on KIR2DS1. NKVSF1 competes with DF200 and 1-4F1, but not 1-7F9, on KIR2DS1. DF200 competes with NKVSF1 and 1-4F1, but not 1-7F9, on KIR2DS1.

Figure 10 depicts NKVSF1(pan2D) mAb titration demonstrating NKVSF1(pan2D) mAb binding to macaque NK cells. Cynomolgus NK cells (total NK for 16 days) were incubated with varying amounts of Pan2D mAb followed by PE-conjugated goat F(ab')2 fragment anti-mouse IgG(H+L) antibody. The percentage of positive cells was determined with an isotype control (purified mouse IgGl). Samples were assayed in duplicate. Mean fluorescence intensity = MFI. the

Figure 12 provides an alignment of the light chain variable region of antibody DF200 and Pan2D mAb and the CDR amino acid sequence of the light chain variable region. the

Figure 13 provides the heavy chain variable region of antibody DF200. the

Detailed description of the invention

Antibody

The present invention provides novel antibodies and fragments or derivatives thereof that bind to a common determinant of human inhibitory KIR receptors, preferably a determinant present on at least two different KIR2DL gene products , and caused enhancement of NK cells expressing at least one of those KIR receptors. The present invention discloses for the first time that such cross-reactive and neutralizing antibodies can be produced represents an unexpected result and opens the way to entirely new and effective NK-based treatments, especially in human subjects. In preferred embodiments, the antibody is not monoclonal antibody NKVSF1. the

In the context of the present invention, "common determinant" refers to a determinant or epitope shared by several human inhibitory KIR receptor gene products. Common determinants are preferably shared by at least two members of the KIR2DL receptor group. This determinant is more preferably shared by at least KIR2DL1 and KIR2DL2/3. In addition to recognizing multiple KIR2DL gene products, certain antibodies of the invention may also recognize determinants present on other inhibitory KIRs, such as gene products of the KIR3DL receptor group. Determinants or epitopes may represent peptide fragments or conformational epitopes shared by the members. In a more specific embodiment, an antibody of the invention specifically binds substantially the same epitope as recognized by monoclonal antibody DF200. This determinant is present on KIR2DL1 and KIR2DL2/3. the

In the context of the present invention, the term "antibody that binds" a common determinant refers to an antibody that binds said determinant with specificity and/or avidity. the

Unless otherwise stated or clearly contradicted by context, the term "antibody" as used herein refers to polyclonal and monoclonal antibodies, as well as fragments and derivatives of said polyclonal and monoclonal antibodies. Depending on the type of heavy chain constant region, full-length antibodies are generally assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Several of these are further divided into subclasses or isotypes, such as IgGl, IgG2, IgG3, IgG4, etc. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called "α," "δ," "ε," "γ," and "μ," respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. IgG and/or IgM are the preferred classes of antibodies employed in the present invention because they are the most common antibodies under physiological conditions and because they are the easiest to produce in a laboratory setting. The antibodies of the invention are preferably monoclonal antibodies. Since one of the goals of the present invention is to block the interaction of inhibitory KIRs with their corresponding HLA ligands in vivo without depleting NK cells, an isotype corresponding to an Fc receptor mediating low effector function, such as IgG4, is generally preferred. the

Antibodies of the invention can be produced by a variety of techniques known in the art. Antibodies of the invention are generally produced by immunizing 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 contain the full-length sequence of a human inhibitory KIR polypeptide or a fragment or derivative thereof, generally an immunogenic fragment, that is, the portion of the polypeptide containing an epitope exposed on the surface of a cell expressing an inhibitory KIR receptor. Such fragments generally contain at least about 7 contiguous amino acids of the mature polypeptide sequence, and even more preferably at least about 10 contiguous amino acids thereof. Fragments are generally substantially derived from the extracellular domain of the receptor. More preferred is a human KIR2DL polypeptide comprising at least one (more preferably two) extracellular Ig domains of a full-length KIRDL polypeptide and capable of mimicking at least one conformational epitope present in the KIR2DL receptor. In another embodiment, the polypeptide contains at least about 8 contiguous amino acids of the extracellular Ig domain of amino acids 1-224 of the KIR2DL1 polypeptide (the amino acid code is according to the PROW website http://www.ncbi.nlm describing the KIR gene family .nih.gov/prow/guide/1326018082.htm ).

In the most preferred embodiment, the immunogen comprises wild-type human KIR2DL polypeptide in a lipid membrane (typically on the cell surface). In a specific embodiment, the immunogen comprises whole NK cells, in particular whole human NK cells, optionally processed or lysed. the

Stimulation of antibodies with antigens can be performed in any manner known in the art to stimulate antibody production in mice (see, for example, E. Harlow and D. Lane, " Antibodies: A Laboratory Manual ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)). A step for immunizing a non-human mammal. The immunogen is suspended or dissolved in a buffer, optionally with an adjuvant (eg, complete Freund's adjuvant). Methods for determining the amount of immunogen, buffer type and adjuvant amount are well known to those skilled in the art and do not limit the present invention in any way. These parameters may vary for different immunogens but are easy to elucidate.

Similarly, the location and frequency of immunization sufficient to stimulate antibody production is also well known in the art. In a typical immunization protocol, the non-human animal is injected intraperitoneally with the antigen on the first day and about a week later. This is followed by antigen recall injection on about day 20, optionally with an adjuvant such as Freund's incomplete adjuvant. Memory injections are given intravenously and can be repeated over consecutive days. Thereafter, intravenous or intraperitoneal booster injections are given on day 40, generally without adjuvant. This regimen results in the production of antigen-specific antibody-producing B cells after about 40 days. Other protocols can also be utilized so long as they result in the production of B cells expressing antibodies to the antigen used for immunization. the

For the preparation of polyclonal antibodies, sera are obtained from immunized non-human animals and the antibodies present therein are isolated by well known techniques. Antibodies reactive with inhibitory KIR receptors can be obtained by affinity purifying serum using any of the above immunogens attached to a solid support. the

In an alternative embodiment, non-immunized non-human mammalian lymphocytes are isolated and cultured in vitro. It is then exposed to the immunogen in cell culture. Lymphocytes were then harvested and the fusion steps described below were performed. the

For monoclonal antibodies, the next step is the isolation of splenocytes from the immunized non-human mammal and the subsequent fusion of those splenocytes with immortalized cells to form antibody-producing hybridomas. Isolation of splenocytes from non-human mammals is well known in the art and generally involves removing the spleen from an anesthetized non-human mammal, dicing it into small pieces, and squeezing the splenocytes out of the splenic capsule and passing them through the nylon mesh of the cell strainer Wells are dipped into an appropriate buffer to generate a single cell suspension. Cells were washed, centrifuged and resuspended in buffer to lyse any red blood cells. The solution was centrifuged again and the lymphocytes retained in the pellet were finally resuspended in fresh buffer. the

Once isolated and present as a single-cell suspension, lymphocytes can be fused with immortalized cell lines. These are typically mouse myeloma cell lines, although many other immortalized cell lines for producing 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. U.S.A., X63 Ag8653, and those available from SP-2 cells obtained from American Type Culture Collection, Rockville, Maryland U.S.A. Fusion is achieved using polyethylene glycol or the like. The resulting hybridomas are then cultured on selective media containing one or more substances that inhibit the growth of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which Prevents the growth of HGPRT-deficient cells. the

Hybridomas are typically cultured on macrophage feeder layers. Macrophages are preferably derived from littermates of non-human mammals used for isolation of spleen cells, and are generally primed with Freund's incomplete adjuvant or the like several days before hybridoma inoculation. Fusion methods are described in Goding, "Monoclonal Antibodies: Principles and Practice", pp. 59-103 (Academic Press, 1986), the disclosure of which is incorporated herein by reference. the

Cells are allowed to grow on selective medium for a time sufficient to form colonies and produce antibodies. Usually between about 7 and about 14 days. The hybridoma clones were then assayed for production of antibodies that cross-react with various inhibitory KIR receptor gene products. The assay is generally a colorimetric ELISA-type assay, although any assay that can be adapted to the wells in which the hybridomas are grown can be used. Other assays include immunoprecipitation and radioimmunoassays. Check wells positive for desired antibody production for one or more different clones. If more than one clone is present, the cells can be cloned again and cultured to ensure that the clone producing the desired antibody is produced only by a single cell. Positive wells with a single apparent clone are generally subcloned and reassayed to ensure that only one monoclonal antibody is detected and produced. the

Antibodies can also be produced by selection of combinatorial libraries of immunoglobulins, as disclosed in Ward et al., Nature , 341 (1989) at page 544.

Antibodies of the invention may also neutralize KIR-mediated inhibition of NK cell cytotoxicity; specifically inhibition mediated by the KIR2DL receptor and more specifically at least KIR2DL1 and KIR2DL2/3 inhibition. These antibodies are thus "neutralizing" or "inhibitory" antibodies in the sense that they at least partially or detectably block inhibitory activity mediated by KIR receptors when they interact with MHC class I molecules. signal path. More importantly, the inhibitory activity involves several types of inhibitory KIR receptors, preferably several KIR2DL receptor gene products, more preferably at least KIR2DL1 and KIR2DL2/3, so that these antibodies can be efficiently used in a variety of subjects. Inhibition of KIR-mediated inhibition of NK cell cytotoxicity can be assessed by a variety of assays or tests (eg, binding or cellular assays). the

Once antibodies that cross-react with various inhibitory KIR receptors are identified, they can be tested for their ability to neutralize the inhibitory effects of those KIR receptors in intact NK cells. In certain variants, neutralizing activity can be demonstrated by the ability of the antibodies to reproduce lysis of HLA-C positive target cells by KIR2DL positive NK clones. In another specific embodiment, the neutralizing activity of the antibody is additionally preferably defined by the antibody's ability to inhibit the binding of HLA-C molecules to the KIR2DL1 and KIR2DL3 (or closely related KIR2DL2) receptors as the antibody alters:

- an HLA-C molecule selected from the group consisting of Cw1, Cw3, Cw7 and Cw8 (or an HLA-C molecule with an Asn residue at position 80) that binds KIR2DL2/3; and

- the ability of an HLA-C molecule selected from Cw2, Cw4, Cw5 and Cw6 (or an HLA-C molecule with a Lys residue at position 80) to bind KIR2DL1. the

In another variation, the inhibitory activity of the antibodies of the invention can be assessed in a cell-based cytotoxicity assay, as disclosed in the Examples provided herein. the

In another variant, the inhibitory activity of the antibodies of the invention can be assessed in a cytokine release assay in which NK cells are combined with the test antibody and target cells expressing an HLA-C allele recognized by NK population KIR molecules Incubate NK cells to stimulate NK cells to produce cytokines (eg, produce IFN-γ and/or GM-CSF). In an exemplary protocol, after approximately 4 days in culture, IFN-γ production from PBMCs is assessed by cell surface and intracytoplasmic staining and flow cytometry analysis. Briefly, Brefeldin A (Sigma Aldrich) can be added at a final concentration of approximately 5 μg/ml in the last approximately 4 hours of incubation. Cells can then be incubated with anti-CD3 and anti-CD56 mAbs before permeabilization (IntraPrep ^™ ; Beckman Coulter) and stained with PE-anti-IFN-γ or PE-IgG1 (Pharmingen). GM-CSF and IFN-γ production by polyclonal activated NK cells can be measured in supernatants using ELISA (GM-CSF: DuoSetElisa, R&D Systems, Minneapolis, MN; IFN-γ: OptE1A set, Pharmingen).

The antibodies of the present invention can partially or completely neutralize KIR-mediated inhibition of NK cell cytotoxicity. The term "neutralizing KIR-mediated inhibition of NK cell cytotoxicity" as used herein refers to when a population of NK cells expressing a given KIR is contacted with a target cell expressing the relevant MHC class I molecule recognized by the KIR expressed on NK cells , at least about 20%, preferably at least about 30%, increase in specific lysis obtained at the same rate for NK cells or NK cell lines not blocked by their KIRs, compared to the level of specific lysis obtained without the antibody, A capacity of at least about 40%, at least about 50% or more (eg, about 25-100%) (as measured by the cytotoxic classical chromium release test). For example, preferred antibodies of the invention are capable of inducing lysis of a matched or HLA-compatible or autologous target cell population (ie, a cell population that would not be efficiently lysed by NK cells in the absence of the antibody). Therefore, the antibody of the present invention can also be defined as promoting the activity of NK cells in vivo. the

Additionally, the term "neutralizes KIR-mediated inhibition" means that the level of cytotoxicity obtained with antibodies in the chromium assay should be obtained with known blocking anti-MHC class I molecules (e.g. W6/32 anti-MHC class I antibody) At least about 20%, preferably at least about 30%, at least about 40%, at least about 50% (e.g., about 25-100%) or more of the cytotoxicity of , wherein the chromium assay uses a protein that expresses one or several inhibitory KIRs NK cell clones or transfectants and target cells expressing only one HLA allele recognized by one of the NK cell KIRs. the

In specific embodiments, the antibody binds substantially the same epitope as monoclonal antibody DF200 (produced by hybridoma DF200). Such antibodies are referred to herein as "DF200-like antibodies". In another preferred embodiment, the antibody is a monoclonal antibody. A more preferred "DF200-like antibody" of the present invention is an antibody other than the monoclonal antibody NKVSF1. Most preferred is monoclonal antibody DF200 (produced by hybridoma DF200). the

The term "binding to substantially the same epitope or determinant" as the antibody of interest means that the antibody "competes" with said antibody of interest. The term "binds to substantially the same epitope or determinant" as monoclonal antibody DF200 means that the antibody "competes" with DF200. Generally, an antibody that "binds to substantially the same epitope or determinant" as a monoclonal antibody of interest (e.g., DF200, NKVSF1, 17F9) means that the antibody competes with the antibody of interest for any one of a plurality of KIR molecules (preferably selected from KIR2DL1 and KIR2DL2/ 3 KIR molecules). In other embodiments, antibodies that bind to substantially the same epitope or determinant on the KIR2DL1 molecule as the antibody of interest "compete" with the antibody of interest for binding to KIR2DL1. Antibodies that bind to substantially the same epitope or determinant on the KIR2DL2/3 molecule as the antibody of interest "compete" with the antibody of interest for binding to KIR2DL2/3. the

The term "binding to substantially the same epitope or determinant" as the antibody of interest means that the antibody "competes" with said antibody of interest for any and all KIR molecules for specific binding by the antibody of interest. The term "binding to substantially the same epitope or determinant" as monoclonal antibody DF200 means that the antibody "competes" with DF200 for any and all KIR molecules for which DF200 specifically binds. For example, an antibody that binds essentially the same epitope or determinant as monoclonal antibody DF200 or NKVSF1 competes with DF200 or NKVSF1 for binding to KIR2DL1, KIR2DL2/3, KIR2DS1 and KIR2DS2, respectively. the

The identity of one or more antibodies that recognize substantially or essentially the same expression as the monoclonal antibodies described herein can be readily determined using any of a variety of immunological screening assays that can assess antibody competition. bit. Many such assays are routinely performed and are well known in the art (see, eg, US Patent No. 5,660,827, issued August 26, 1997, expressly incorporated herein by reference). It should be understood that determining the binding epitope of an antibody described herein is not actually required in any way to identify an antibody that binds the same or substantially the same epitope as a monoclonal antibody described herein. the

For example, when the test antibodies to be examined are obtained from animals of different origin, or even of different Ig isotypes, simple competition assays can be employed in which a control (e.g. DF200) and test antibody are mixed (or pre-adsorbed), and applied to samples containing KIR2DL1 and KIR2DL2/3 (each known to be bound by DF200). The use of ELISA, radioimmunoassay, Western blot-based protocols and BIACORE assays (listed eg in the Examples section) are suitable for such simple competition studies. the

In certain embodiments, the control antibody (eg, DF200) should be premixed with varying amounts of the test antibody (eg, about 1:10 or about 1:100) for a period of time prior to application to the inhibitory KIR antigen sample. In other embodiments, the control and varying amounts of test antibody can be simply mixed during exposure to the KIR antigen sample. As long as it is possible to distinguish bound from free antibody (e.g., using separation or washing techniques to DF200), it will be possible to determine whether the test antibody reduces the binding of DF200 to these two different KIR2DL antigens, thus indicating that the test antibody recognizes substantially the same epitope as DF200. Binding of a (labeled) control antibody can serve as a control high in the absence of a completely unrelated antibody. Control low values can be obtained by incubating labeled (DF200) antibody with unlabeled fully isotype antibody (DF200) (where competition will occur and reduce binding of labeled antibody). A marked decrease in the reactivity of the labeled antibody in the presence of the test antibody in the test assay is indicative of a test antibody that recognizes substantially the same epitope (ie, an antibody that "cross-reacts" with the labeled (DF200) antibody). At any ratio of DF200:test antibody between about 1:10 and about 1:100, the binding of DF200 to each of the KIR2DL1 and KIR2DL2/3 antigens is reduced by at least about 50%, such as at least about 60%, or more Preferably at least about 70% (eg, about 65-100%) of the antibodies tested are considered to be antibodies that bind substantially the same epitope or determinant as DF200. Such test antibodies preferably reduce the binding of DF200 to each of the KIR2DL antigens by at least about 90% (eg, about 95%). the

Competition can be assessed, for example, by flow cytometry assays. In such an assay, cells with a given KIR can first be incubated with, for example, DF200, and then with a fluorochrome or biotinylated test antibody. If the binding obtained upon pre-incubation with a saturating amount of DF200 is about 80%, preferably about 50%, about 40% or lower (e.g. about 30%) of the binding obtained when the antibody is not pre-incubated with DF200 (measured by fluorescence ), the antibody is said to compete with DF200. Alternatively, if the binding obtained with DF200 labeled (with a fluorescent dye or biotin) on cells pre-incubated with a saturating amount of the test antibody is about 80%, preferably about 50%, about 40% or less (eg, about 30%), the antibody is said to compete with DF200. the

Simple competition assays can also be advantageously employed, in which test antibodies are pre-adsorbed at saturating concentrations and applied to surfaces to which KIR2DL1 and KIR2DL2/3 are also immobilized. The surface in simple competition assays is preferably a BIACORE chip (or other medium suitable for surface plasmon resonance analysis). A control antibody (eg, DF200) is then contacted to the surface at a concentration saturating for KIR2DL1 and KIR2DL2/3, and KIR2DL1 and KIR2DL2/3 surface binding of the control antibody is measured. The binding of the control antibody was compared to the binding of the control antibody to surfaces containing KIR2DL1 and KIR2DL2/3 in the absence of the test antibody. In the test experiment, the significantly reduced binding of the control antibody to surfaces containing KIR2DL1 and KIR2DL2/3 in the presence of the test antibody indicates that the test antibody recognizes substantially the same epitope as the control antibody, so the test antibody "cross-reacts" with the control antibody ". A test antibody that reduces the binding of a control (e.g. DF200) antibody to each of the KIR2DL1 and KIR2DL2/3 antigens by at least about 30%, or more preferably about 40%, can be considered to bind substantially the same epitope or determinant as the control (e.g. DF200). cluster antibodies. Such test antibodies preferably reduce binding of a control antibody (eg, DF200) to each of the KIR2DL antigens by at least about 50% (eg, at least about 60%, at least about 70%, or more). It will be appreciated that the order of the control and test antibodies may be reversed: ie, in a competition assay, the control antibody may be bound to the surface first, followed by contacting the test antibody with the surface. Antibodies with high avidity for KIR2DL1 and KIR2DL2/3 antigens preferably bind first to surfaces containing KIR2DL1 and KIR2DL2/3, as binding by a second antibody (assuming the antibodies are cross-reactive) is expected to decrease by a greater amount. Further examples of such assays are provided in the Examples and for example Saunal and Regenmortel, (1995) J. Immunol. Methods 183: 33-41, where Saunal and Regenmortel, (1995) J. Immunol. Methods 183: 33-41 The disclosure of is hereby incorporated by reference. the

Although DF200 is used as an example for exemplary purposes, it should be understood that the immunological screening assays described above can also be used to identify antibodies that compete with NKVSF1, 1-7F9, EB6, GL183 and other antibodies of the invention. the

Following immunization and antibody production of the vertebrate or cell, specific selection steps may be performed to isolate antibodies of the invention. In this regard, in particular embodiments, the invention also relates to methods of producing such antibodies, comprising:

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

(b) preparing an antibody from said immunized animal, wherein said antibody binds said KIR polypeptide,

(c) the antibody of (b) that cross-reacts with at least two different inhibitory KIR gene products, and

(d) selecting the antibody of (c) capable of neutralizing inhibition of KIR-mediated NK cell cytotoxicity on a population of NK cells expressing said at least two different human inhibitory KIR receptor gene products. the

Selection of antibodies that cross-react with at least two different inhibitory KIR gene products can be achieved by screening antibodies against two or more different inhibitory KIR antigens, such as described above. the

In a more preferred embodiment, the antibody prepared in step (b) is a monoclonal antibody. Thus, the term "preparing antibodies from said immunized animal" as used herein includes obtaining B cells from the immunized animal and using those B cells to produce antibody-expressing hybridomas, as well as obtaining antibodies directly from the serum of the immunized animal. In another preferred embodiment, the antibody selected in step (c) is an antibody that cross-reacts with at least KIR2DL1 and KIR2DL2/3. the

In another preferred embodiment, as measured in a standard chromium release assay, step (d ) causes at least about 10% specific lysis, preferably at least about 40% specific lysis, at least about 50% specific lysis, or more preferably at least about 70% specific lysis mediated by NK cells to target cells lysed (eg, about 60-100% specific lysed), the NK cells display at least one KIR recognized by the antibody, and the target cells express the relevant HLA class I molecules. Additionally, when the antibody selected in step (d) is used in the chromium assay, the level of cytotoxicity obtained with that antibody should be that of cells obtained with a blocking anti-MHC class I mAb (e.g. W6/32 anti-MHC class I antibody) At least about 20%, preferably at least about 30%, or more of toxicity in the chromium assay employing NK cell clones expressing one or several inhibitory KIRs and expressing only one HLA recognized by one of the KIRs on the NK clone target cells for alleles. the

The order of steps (c) and (d) of the method described immediately above may be varied. Optionally, the method also or may additionally comprise the additional step of making a monoclonal antibody fragment or a derivative of a monoclonal antibody or such fragment, eg as otherwise described herein. the

In a preferred embodiment, the non-human animal for producing antibodies applicable according to the present invention is a mammal, such as a rodent (e.g. mouse, rat, etc.), cow, pig, horse, rabbit, goat, sheep wait. Non-human mammals can also be genetically modified or engineered to produce "human" antibodies, such as Xenomouse ^™ (Abgenix) or HuMAb-Mouse ^™ (Medarex).

In another variant, the invention provides a method of obtaining an antibody comprising:

(a) selecting from a library or a library a monoclonal antibody, monoclonal antibody fragment, or derivative of any thereof that is resistant to at least two different human inhibitors Sexual KIR2DL receptor gene product cross-reactivity, and

(b) selecting an antibody, fragment or derivative in (a) which is capable of neutralizing the expression of KIR on a population of NK cells expressing said at least two different human inhibitory KIR2DL receptor gene products Inhibition of NK cell-mediated cytotoxicity. the

The library may be any (recombinant) library of antibodies or fragments thereof, optionally displayed by any suitable structure (eg phage, bacteria, synthetic complex, etc.). Selection of inhibitory antibodies can be performed as disclosed above and further illustrated in the Examples. the

According to another embodiment, the invention provides hybridomas comprising B cells of a non-human host, wherein said B cells produce antibodies that bind to determinants present on at least two different human inhibitory KIR receptor gene products, wherein Said antibodies are capable of neutralizing the inhibitory activity of the receptor. More preferably, the hybridoma of this aspect of the invention is not a hybridoma that produces the monoclonal antibody NKVSF1. The hybridomas of this aspect of the invention can be produced by fusing immunized non-human mammalian spleen cells with an immortalized cell line as described above. Hybridomas produced by the fusion can be screened for the presence of such cross-reactive antibodies as otherwise described herein. The hybridomas preferably produce antibodies that recognize at least determinants present on two different KIR2DL gene products, which antibodies elicit enhancement of NK cells expressing at least one of those KIR receptors. More preferably, the hybridoma produces an antibody that binds substantially the same epitope or determinant as DF200 and that enhances NK cell activity. Most preferably the hybridoma is hybridoma DF200 which produces monoclonal antibody DF200. the

Hybridomas confirmed to produce the monoclonal antibodies of the present invention can be cultured in a suitable medium (eg, DMEM or RPMI-1640) in large quantities. Alternatively, hybridoma cells can be cultured in vivo as ascites tumors in animals. the

After sufficient culture to produce the desired monoclonal antibody, the growth medium (or ascitic fluid) containing the monoclonal antibody is separated from the cells, and the monoclonal antibody present therein is purified. Purification is typically achieved by gel electrophoresis, dialysis, chromatography using protein A or protein G-Sepharose, or anti-mouse Ig attached to a solid support such as agarose or Sepharose beads (all described e.g. in the Antibody Purification Handbook , Amersham Biosciences, Publication No. 18-1037-46, AC Edition, wherein "Antibody Purification Handbook" is hereby incorporated by reference). Bound antibodies are generally eluted from protein A/protein G columns by direct neutralization of antibody-containing fractions using low pH buffers (glycine or acetate buffers at pH 3.0 or lower). Fractions were pooled, dialyzed and concentrated as necessary. the

According to an alternative embodiment, DNA encoding an antibody binding to at least two different human inhibitory KIR receptors present in at least two different human inhibitory KIR receptors is isolated from the hybridoma of the present invention and placed into a suitable expression vector for transfection of a suitable host. Determinants on body gene products. The host is then used to recombinantly produce the antibody or variant thereof (eg, a humanized form of the monoclonal antibody, an active fragment of the antibody, or a chimeric antibody containing the antigen recognition site of the antibody). The DNA used in this embodiment preferably encodes antibodies that recognize determinants present on at least two different KIR2DL gene products, which antibodies cause enhancement of NK cells expressing at least one of those KIR receptors. More preferably, the DNA encodes an antibody that binds substantially the same epitope as DF200 and enhances NK cell activity. Most preferably the DNA encodes the monoclonal antibody DF200. the

DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional protocols (eg, using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Once isolated, the DNA can be placed into expression vectors which are then transfected into host cells (e.g. Escherichia coli (E. coli) cells that do not otherwise produce immunoglobulins, simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells), resulting in monoclonal antibody synthesis in recombinant host cells. Recombinant expression of antibody-encoding DNA in bacteria is well known in the art (see e.g. Skerra et al., Curr. Opinion in Immunol. , 5, pp. 256 (1993); Pluckthun, Immunol. Revs. , 130, pp. 151 (1992) .

Monoclonal antibody fragments and derivatives

Antibody fragments and derivatives of the present invention (unless otherwise stated or clearly contradicted by context, the term "antibody" as used in this application includes antibody fragments and derivatives), preferably DF-200-like antibodies, can be produced by techniques known in the art. An "immunoreactive fragment" contains a portion of an intact antibody, usually the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab') ₂ , and Fv fragments; miniature diabodies; any antibody fragment that is an uninterrupted sequence of consecutive amino acid residues in its primary structure. sequence of polypeptides (referred to herein as "single-chain antibody fragments" or "single-chain polypeptides"), including but not limited to (1) single-chain Fv (scFv) molecules (2) single-chain Fv (scFv) molecules containing only one light chain variable domain chain polypeptides, or fragments thereof containing the three CDRs of the light chain variable domain, without the associated heavy chain portion, and (3) single-chain polypeptides containing only one heavy chain variable domain, or containing the heavy chain variable structure The three CDRs of a domain, fragments without the associated light chain portion; and multispecific antibodies formed from antibody fragments. For example, Fab or F(ab')2 fragments can be produced by protease digestion of isolated antibodies according to conventional techniques. It will be appreciated that immunoreactive fragments may be modified using known methods, eg to delay clearance in vivo and to obtain more desirable pharmacokinetic profiles. Fragments may be modified with polyethylene glycol (PEG). Coupling of PEG to Fab' fragments is 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) and site-specific conjugation methods, the disclosures of Leong et al. and Delgado et al. are incorporated herein by reference.

In particular aspects, the invention provides antibodies, antibody fragments and antibody derivatives comprising the DF200 light chain variable region sequence set forth in FIG. 12 . In another specific aspect, the invention provides antibodies, antibody fragments and antibody derivatives comprising the Pan2D light chain variable region sequence set forth in FIG. 12 . In another aspect, the invention provides antibodies, antibody fragments and derivatives thereof comprising one or more of the light chain variable region CDRs of DF200 listed in FIG. 12 . In another aspect, the invention provides antibodies, antibody fragments and derivatives thereof comprising one or more of the light chain variable region CDRs of Pan2D listed in FIG. 12 . Functional variants/analogues of such sequences can be generated by making appropriate substitutions, additions, and/or deletions in these disclosed amino acid sequences using standard techniques (and sequence comparisons are also possible). Thus, for example, CDR residues conserved between Pan2D and DF-200 may be suitable targets for modification, since such residues may not contribute to the distinct characteristics of these antibodies in competition with other antibodies disclosed herein (although Pan2D compete with DF-200), and thus may not have contributed to the specificity of these antibodies for their respective particular epitopes. On the other hand, where a residue occurs in the sequence of one of these antibodies, but not the other, that position may be suitable for deletion, substitution and/or insertion. the

In particular aspects, the invention provides antibodies, antibody fragments and antibody derivatives comprising the DF200 heavy chain variable region sequence set forth in FIG. 13 . In another aspect, the invention provides antibodies, antibody fragments and derivatives thereof comprising one or more of the heavy chain variable region CDRs of DF200 listed in FIG. 13 . Functional variants/analogues of such sequences can be generated by making appropriate substitutions, additions, and/or deletions in these disclosed amino acid sequences using standard techniques, with the benefit of sequence comparison. On the other hand, those positions where the residue is present in the sequence of one of these antibodies but not in the other sequence may be suitable for deletion, substitution and/or insertion. the

In addition, hybridoma DNA producing antibodies of the invention (preferably DF-200-like antibodies) can be modified to encode fragments of the invention. The modified DNA is then inserted into an expression vector and used to transform or transfect appropriate cells, which then express the desired fragment. the

In alternative embodiments, homologous non-human sequences can be replaced by, for example, human heavy and light chain constant domain coding sequences (e.g. Morrison et al., Proc. Natl. Acad. Sci. USA , 81 ) prior to insertion into the expression vector. , 6851 (1984)), or hybridoma DNA modified to produce antibodies of the invention (preferably DF-200-like antibodies) by covalently linking all or part of a non-immunoglobulin polypeptide coding sequence to an immunoglobulin coding sequence. In this format, "chimeric" or "hybrid" antibodies are produced that have the binding specificity of the original antibody. Typically, the constant domains of the antibodies of the invention are replaced with such non-immunoglobulin polypeptides.

Thus, according to another embodiment, the antibodies (preferably DF-200-like antibodies) of the invention are humanized. "Humanized" forms of antibodies according to the invention are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g. Fv, Fab, Fab', F(ab') ₂ , or other antigen-binding subsequence). In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the complementarity determining regions (CDRs) of the recipient are replaced by residues from the CDRs of the original antibody (donor antibody). Substitution of residues while retaining the desired specificity, affinity, and potency of the original antibody. In some instances, Fv framework residues of the human immunoglobulin can be substituted with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or in imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all at least one (usually two) variable domains in which all or substantially all CDR regions correspond to those of the original antibody, and all or substantially all The FR regions are those of the human immunoglobulin consensus sequence. The humanized antibody preferably will also comprise at least a portion of an immunoglobulin constant region (Fc) (typically, a human immunoglobulin constant region). For further details see Jones et al., Nature , 321, 522 (1986); Reichmann et al., Nature , 332, 323 (1988); and Presta, Curr. Op. Struct. Biol. , 2, 593 (1992 ).

Methods for humanizing the antibodies of the invention are well known in the art. Typically, a humanized antibody according to the invention has one or more amino acid residues introduced into it from the original antibody. These murine or other non-human amino acid residues are often referred to as "imported" residues and are typically taken from an "imported" variable domain. Can basically according to the method of Winter and co-workers (Jones et al., Nature , 321, 522 pages (1986); Riechmann et al., Nature , 332, 323 pages (1988); Verhoeyen et al., Science , 239, 1534 pages (1988) ) to perform humanization. Thus, such "humanized" antibodies are chimeric antibodies in which substantially less than an intact human variable domain is substituted by the corresponding sequence of the original antibody (Cabilly et al., US Patent No. 4,816,567). In practice, humanized antibodies according to the invention are generally human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous positions in the original antibody.

The choice of human variable domains (light and heavy chains) used to make humanized antibodies is very important to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequences of the antibodies of the invention are screened against the complete library of known human variable domain sequences. The human sequence closest to the mouse sequence was then accepted as the human framework (FR) of the humanized antibody (Sims et al., J. Immunol. , 151, p. 2296 (1993); Chothia and Lesk, J.Mol.Biol. , 196, pp. 901 (1987)). Another method uses a specific framework from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA , 89, 4285 (1992); Presta et al., J. Immunol. , 51, 1993 et al )).

(Abgenix，Fremont，CA)作为免疫小鼠。XenoMouse是根据本发明的鼠科宿主，其免疫球蛋白基因被功能性人类免疫球蛋白基因取代。因此，由该小鼠或由该小鼠B细胞制作的杂交瘤产生的抗体已经经过人源化。在美国专利No.6,162,963中描述了XenoMouse，其中美国专利No.6,162,963在此全文引用作为参考。使用HuMAb-Mouse^TM (Medarex)可以完成类似方法。  It is more important to humanize antibodies while maintaining high affinity for various inhibitory KIR receptors and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by analyzing the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows the analysis of the likely role of the residues in the functioning of the candidate immunoglobulin, ie the analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and imported sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the CDR residues are directly and primarily involved in affecting antigen binding. Another way to make "humanized" monoclonal antibodies is to use

(Abgenix, Fremont, CA) as immunized mice. The XenoMouse is a murine host according to the invention whose immunoglobulin genes are replaced by functional human immunoglobulin genes. Thus, antibodies produced by the mouse or hybridomas produced by the mouse B cells have been humanized. XenoMouse is described in US Patent No. 6,162,963, which is incorporated herein by reference in its entirety. A similar approach can be accomplished using HuMAb-Mouse ^™ (Medarex).

Human antibodies can also be produced according to a variety of other techniques, such as using other transgenic animals designed to express human antibody repertoires for immunization (Jakobovitz et al., Nature 362 (1993) 255), or using phage display methods to select antibody repertoires. Such techniques are well known to the skilled artisan and can be performed starting with the monoclonal antibodies disclosed in this application. the

Antibodies of the invention (preferably DF-200-like antibodies) can also be derivatized as "chimeric" antibodies (immunoglobulins), as long as they exhibit the desired biological activity, wherein heavy and/or A portion of the light chain is identical or homologous to the corresponding sequence of the original antibody, while the remainder of the chain is identical or homologous to the corresponding sequence of antibodies derived from other species or belonging to other antibody classes or subclasses, and fragments of such antibodies ( Cabilly et al., supra; Morrison et al., Proc. Natl. Acad. Sci. USA , 81, p. 6851, (1984)).

Other derivatives within the scope of the invention include functionalized antibodies, i.e. with toxins (e.g. ricin, diphtheria toxin, abrin and Pseudomonas exotoxin); detectable moieties (e.g. fluorescent moieties, radioisotopes or imaging agents); or antibodies conjugated or covalently linked to a solid support (eg, agarose beads, etc.). Methods of conjugating or covalently linking these other agents to antibodies are well known in the art. the

Conjugation to the toxin is beneficial for the targeted killing of NK cells displaying one of the cross-reactive KIR receptors on their cell surface. Once the antibody of the present invention binds to the cell surface of such cells, it will be internalized and the toxin will be released into the cell to selectively kill the cell. Such uses are alternative embodiments of the present invention. the

Conjugation to a detectable moiety is useful when the antibodies of the invention are used for diagnostic purposes. Such purposes include, but are not limited to, assaying biological samples for the presence of NK cells with cross-reactive KIRs on their cell surfaces, and detecting the presence of NK cells with cross-reactive KIRs in living organisms. Such assay and detection methods are also alternative embodiments of the invention. the

Antibodies of the invention conjugated to a solid support are useful as a tool for affinity purification of NK cells with cross-reactive KIRs on their cell surface from sources such as biological fluids. As with the resulting purified NK cell population, the purification method is another alternative embodiment of the invention. the

In an alternative embodiment, antibodies (including NKVSF1 ) can be incorporated into liposomes ("immunoliposomes"), either alone or with another substance for targeted delivery to an animal, wherein the antibody is associated with Binding of a common determinant present on at least two different human inhibitory KIR receptor gene products capable of neutralizing the presence of KIR on NK cells expressing at least one of said two different human inhibitory KIR receptors of the invention Inhibition of NK cell-mediated cytotoxicity. Such other substances include nucleic acids for delivering genes for gene therapy, or antisense RNA, RNAi or siRNA for gene suppression in NK cells; or toxins or drugs for targeted killing of NK cells. the

In silico prediction of 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)) Certain regions involved in the interaction between KIR2DL1 and KIR2DL1-3 cross-reactive mouse monoclonal antibodies DF200 and NKVSF1 or KIR2DL1, 2 and 3 were identified. Thus, in one embodiment, the present invention provides a sequence consisting of 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) within the region defined by antibodies that specifically bind to KIR2DL1. In another embodiment, the invention provides binding to KIR2DL1 and KIR 2DL2/3 without binding to 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) interacting antibodies with amino acid residues outside the region defined.

In another embodiment, the invention provides an antibody that binds to KIR2DL1 and does not bind to a KIR2DL1 mutant in which R131 is Ala. the

In another embodiment, the invention provides an antibody that binds to KIR2DL1 and does not bind to a KIR2DL1 mutant in which R157 is Ala. the

In another embodiment, the invention provides an antibody that binds to KIR2DL1 and does not bind to a KIR2DL1 mutant in which R158 is Ala. the

In another embodiment, the invention provides antibodies that bind to residues (131, 157, 158) of KIR2DL1. the

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

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

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

Determining whether an antibody binds within one of the above-defined epitope regions can be performed by methods well known to those skilled in the art. As an example of such mapping/characterization methods, the epitope region of an anti-KIR antibody can be determined by epitope "foot-printing" using chemical modifications of exposed amino/carboxyl groups in the KIR2DL1 or KIR2DL2/3 proteins. A specific example of this type of footprinting technique is the use of HXMS (Hydrogen-Deuterium Exchange with Mass Spectrometry Detection), where hydrogen/deuterium exchange, incorporation and backexchange of receptor and ligand protein amide protons occurs, where the involved The protein-bound backbone amido group is protected from exchange back and thus will remain deuterated. Regions of interest can be identified at this point by pepsin proteolysis, fast microbore HPLC separation and/or electrospray ionization mass spectrometry. See eg Ehring H, Analytical Biochemistry, Vol. 267(2) pp. 252-259 (1999) and/or Engen, JR and Smith, DL (2001) Anal. Chem. 73, 256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), which generally compares the location of signals in two-dimensional NMR spectra for free antigen and antigen complexed with an antigen-binding peptide (eg, antibody). Generally, the antigen is labeled with ¹⁵ N selective isotope, so only the signal corresponding to the antigen can be seen on the NMR spectrum, but no signal from the antigen-binding peptide. Antigen signals originating from amino acids involved in the interaction with the antigen-binding peptide will generally be shifted in the complex profile compared to the free antigen profile so that the amino acids involved in binding can be identified. See, eg, 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 Jun ;9(3):516-24.

Epitope mapping/characterization can also be performed using mass spectrometry methods. See eg Downward, J Mass Spectrom. 2000 Apr;35(4):493-503 and Kiselar and Downard, Anal Chem. 1999 May 1;71(9):1792-801. the

Protease digestion can also be useful in epitope mapping and identification. Antigenic determination can be confirmed by protease digestion (e.g., with trypsin at a ratio of approximately 1:50 to KIR2DL1 or KIR2DL2/3 at 37°C and pH 7-8 o/n) followed by mass spectrometry (MS) analysis for peptide identification Cluster-associated regions/sequences. Peptides that are protected from trypsin cleavage by the anti-KIR conjugate can then be identified by comparing samples subjected to trypsinization with samples incubated with antibodies and then subjected to eg trypsinization (thus showing a footprint for the conjugate). Other enzymes like chymotrypsin, pepsin, etc. can also or additionally be used in similar epitope characterization methods. Furthermore, enzymatic digestion may provide a rapid method for analyzing the presence of potential epitopic sequences within the KIR2DL1 region in the context of non-surface exposed and thus most likely not immunogenic/antigenically relevant anti-KIR polypeptides. For a discussion of similar techniques see, eg, Manca, Ann Ist Super Sanita. 1991;27(1):15-9. the

Cross-reactivity with macaques

The antibody NKVSF1 was found to also bind to macaque NK cells, see Example 7. The present invention thus provides antibodies and fragments and derivatives thereof, wherein said antibodies, fragments or derivatives cross-react with at least two inhibitory human KIR receptors on the surface of human NK cells and bind to macaque NK cells. In one embodiment, the antibody is not antibody NKVSF1. The present invention also provides a method for testing the toxicity of antibodies, fragments and derivatives thereof, wherein said antibodies, fragments or derivatives cross-react with at least two inhibitory human KIR receptors on the surface of human NK cells, wherein the method comprises testing the Antibody. the

Composition and application

The present invention also provides pharmaceutical compositions comprising antibodies, fragments and derivatives thereof, in a suitable carrier in an amount effective to detectably enhance NK cell cytotoxicity in a patient or a biological sample containing NK cells, wherein the antibody, fragment or The derivatives cross-react with at least two inhibitory KIR receptors on the surface of NK cells, neutralize their inhibitory signals and enhance the activity of those cells. The composition additionally contains a pharmaceutically acceptable carrier. Such compositions are also referred to as "antibody compositions of the invention". In one embodiment, an antibody composition of the invention comprises an antibody disclosed in the antibody embodiments above. Antibody NKVSF1 is included among the antibodies that may be present in the antibody composition of the present invention. the

The term "biological sample" as used herein includes, but is not limited to, biological fluids (e.g., serum, lymph, blood), cell samples or tissue samples (e.g., bone marrow). the

Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbitol acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, Polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin. the

Compositions of the invention may be employed in methods of enhancing NK cell activity in a patient or biological sample. The method includes the step of contacting the composition with a patient or a biological sample. Such methods would be beneficial for diagnostic and therapeutic purposes. the

For use in conjunction with a biological sample, depending on the nature of the sample (liquid or solid), the antibody composition can be administered by simply mixing with the sample or by applying it directly to the sample. The biological sample can be brought into direct contact with the antibody in any suitable device (plate, bag, bottle, etc.). For use in conjunction with a patient, the composition must be formulated for administration to the patient. the

The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. The composition is preferably administered orally, intraperitoneally or intravenously. the

Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspensions. Suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable non-toxic diluent or solvent, for example a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. Solutions or suspensions of these oils Liquids may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants (such as Tweens, spans, and other emulsifying agents or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms) may also be used for formulation purposes. 

Compositions of the present invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. A lubricating agent, such as magnesium stearate, is 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. Certain sweetening, flavoring or coloring agents can also be added, if desired. the

Additionally, the compositions of the present invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which 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 glycols. the

Topical administration of the compositions of the present invention may also be used, especially when the target of treatment comprises areas or organs readily accessible by topical application (including eye, skin, or lower intestinal disorders). Suitable topical formulations are readily prepared for each of these areas or organs. the

Topical application to the lower intestinal tract can be achieved by rectal suppository formulation (see above) or by suitable enema formulation. Topically transdermal patches may also be used. the

For topical application, the composition may be formulated in a suitable ointment containing the active components suspended or dissolved in one or more carriers. Carriers for topical administration of a compound of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polyoxypropylene compound, emulsifying wax and water. Alternatively, the composition can be formulated in a suitable lotion or cream containing the active ingredients 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 esters wax, cetyl alcohol, 2-octyldodecanol, benzene methanol and water. the

For ophthalmic use, the composition may be formulated as a micronized suspension in, or preferably a solution in, pH-adjusted isotonic sterile saline, with or without such as benzylalkonium chloride preservative. Additionally, for ophthalmic use, the composition can be formulated in an ointment, such as petrolatum. the

Compositions of the invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be dissolved in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons and/or other conventional solubilizing or dispersing agents. prepared as a solution. the

Several monoclonal antibodies have been shown to be effective in clinical settings, such as Rituxan (Rituximab), Herceptin (Trastuzumab) or Xolair (Omalizumab), and similar administration regimens (i.e. formulation and/or dosage and/or method of administration) can be used for the antibodies of the invention ). The regimen and dosage for administering the antibodies in the pharmaceutical compositions of the invention can be determined according to known methods for these products (eg, using manufacturer's instructions). For example, the antibodies present in the pharmaceutical compositions of the invention can be stocked at a concentration of 10 mg/mL in 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product was formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and sterile water for injection. Adjust the pH to 6.5. A suitable exemplary dosage range for the antibody in the pharmaceutical composition of the invention may be between about 10 mg/m ² and 500 mg/m ² . However, it should be understood that these protocols are exemplary and that the optimal protocol can be adjusted taking into account the avidity and tolerability of the specific antibody in the pharmaceutical composition, which is a clinical trial. must be determined. The amount and regimen of injecting the antibody in the pharmaceutical composition of the present invention to infiltrate NK cells for 24 hours, 48 hours, 72 hours or one week or one month will be determined considering antibody affinity and its pharmacokinetic parameters.

According to another embodiment, the antibody compositions of the invention may additionally contain another therapeutic agent, including agents normally used for the specific therapeutic purpose for which the antibody is administered. The additional therapeutic agent is usually present in the composition in the amount that the agent would normally use in monotherapy for the treatment of the particular disease or condition. Such therapeutic agents include, but are not limited to, therapeutic agents for the treatment of cancer, therapeutic agents for the treatment of infectious diseases, therapeutic agents for other immunotherapies, cytokines (such as IL-2 or IL-15), Other antibodies and fragments of other antibodies. the

For example, many therapeutic agents for cancer treatment are available. The antibody compositions and methods of the invention can be combined with any other method commonly used to treat a particular disease, particularly a tumor, cancer disease, or other disease or disorder manifested by a patient. As long as a particular method of treatment is known to be inherently harmless to the patient and does not substantially counteract the activity of the antibody in the compositions of the invention, it is envisaged to combine it with the present invention. the

With regard to solid tumor treatment, the pharmaceutical composition of the present invention can be used in combination with classical methods (eg, surgery, radiotherapy, chemotherapy, etc.). The present invention thus provides combination therapy wherein the pharmaceutical composition of the present invention is used simultaneously, before or after surgery or radiation therapy; or in addition to conventional chemotherapy, radiation therapy or anti-angiogenic agents, or targeted immunotoxins or coaguligand Simultaneously, before or after administration to the patient. the

Where one or more agents are used in combination with an antibody-containing composition of the invention in a treatment regimen, it is not required that the result of the combination be additive to the effects observed when each treatment is performed individually. Any increased anticancer effect over that of a single treatment would be advantageous, although at least an additive effect will generally be required. Likewise, there is no specific requirement for combination treatments to exhibit synergy, although it is certainly possible and advantageous. the

To practice combination anticancer therapy, the antibody composition of the present invention is simply administered to an animal in combination with another anticancer agent in such a manner as to effectively elicit its combined anticancer effect in the animal. The agents are thus provided in an effective amount and for a period of time to cause their combined presence within the tumor vasculature and their combined effects in the tumor environment. To achieve this goal, the antibody composition and anticancer agent of the present invention can be administered to animals simultaneously in a single combined composition or as two different compositions using different routes of administration. the

In addition, the antibody composition of the present invention may be administered before or after anticancer agent treatment at intervals ranging, for example, from minutes to weeks and months. One will ensure that the anticancer agent and the antibody in the antibody composition of the invention exert a beneficial combined effect on cancer. the

Most anti-cancer agents will be administered prior to the inhibitory KIR antibody compositions of the invention in anti-angiogenic therapy. However, when immunoconjugates of antibodies are used in the antibody composition of the present invention, multiple anticancer agents may be administered simultaneously or subsequently. the

In some cases, it may even be necessary to extend the treatment time course significantly, with several days (2, 3, 4, 5, 6 or 7) between the respective administration of the anticancer agent or anticancer treatment and the administration of the antibody composition of the invention , weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even months (1, 2, 3, 4, 5, 6, 7 or 8). This would be advantageous where anti-cancer therapy is aimed at substantially destroying the tumor (eg surgery or chemotherapy), administration of the antibody composition of the invention is aimed at preventing micrometastasis or tumor regrowth. the

It is also envisioned that more than one administration of an inhibitory KIR antibody-based composition or anticancer agent of the invention will be utilized. These agents may be administered alternately on alternate days or weeks; or a cycle of treatment with an inhibitory KIR antibody composition of the invention followed by a cycle of treatment with an anticancer agent. Regardless, in order to achieve tumor suppression using combination therapy, all that is required, regardless of the number of administrations, is to deliver a combined amount of both agents effective to exert an antitumor effect. the

With regard to surgery, any surgical intervention can be performed in combination with the present invention. With regard to radiation therapy, any mechanism that induces local damage to DNA within cancer cells is considered, such as gamma radiation, X-rays, UV radiation, microwaves, or even electron emission, among others. Targeted delivery of radioisotopes to cancer cells is also contemplated, which can be used in conjunction with targeting antibodies or other targeting means. the

In other aspects, immunomodulatory compounds or regimens may be administered in combination with or as part of the antibody compositions of the invention. Preferred examples of immunomodulatory compounds include cytokines. A variety of cytokines can be employed in such combinatorial approaches. Examples of cytokines useful in combinations contemplated by the present invention include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-1 -9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, TGF-β, GM-CSF, M-CSF, G-CSF, TNF-α, TNF-β , LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-α, IFN-β, IFN-γ. Cytokines used in combination therapies or compositions of the invention are administered according to standard protocols consistent with the clinical indication (eg, patient condition) and relative toxicity of the cytokines. the

In certain embodiments, therapeutic compositions of the invention comprising a cross-reactivity inhibiting KIR antibody may be administered in combination with, or may additionally contain, a chemotherapeutic or hormonal therapeutic agent. A variety of hormonal therapy and chemotherapeutic agents can be used in the combination treatment methods disclosed herein. Exemplary chemotherapeutic agents considered include, but are not limited to, alkylating agents, antimetabolites, cytotoxic antibodies, vinca alkaloids such as doxorubicin, actinomycin D, mitomycin, carmine Daumycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16), 5-fluorouracil (5FU), Cytarabine, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatin and its derivatives substances and prodrugs. the

Hormonal agents include, but are not limited to, for example, LHRH antagonists such as leuprolide, 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 progestins such as medroxy, progesterone and megestrol. the

As will be appreciated by those of ordinary skill in the art, appropriate dosages of chemotherapeutic agents will approximate those already employed in clinical therapy, where the chemotherapeutic agent is administered alone or in combination with other chemotherapeutic drugs. By way of example only, cisplatin and other DNA alkylating agents such as cisplatin may be used. Cisplatin has been widely used in the treatment of cancer, with an effective dose of 20mg/ ^m2 used in clinical applications for 5 days every three weeks, a total of three courses of treatment. Cisplatin is not absorbed orally and must therefore be delivered by intravenous, subcutaneous, intratumoral, or intraperitoneal injection.

Other useful chemotherapeutic agents include compounds that interfere with DNA replication, mitosis and chromosome segregation, and agents that disrupt the synthesis and fidelity of polynucleotide precursors. A number of exemplary chemotherapeutic agents for use in combination therapy are listed in Table C of US Patent No. 6,524,583, the disclosure of which reagents and descriptions is expressly incorporated herein by reference. Each listed reagent is exemplary and not limiting. The skilled artisan is guided by "Remington's Pharmaceutical Sciences", Fifteenth Edition, Chapter 33, specifically pages 624-652. Variations in dosage may occur depending on the condition being treated. The administering physician will be able to determine the appropriate dosage for the individual subject. the

The cross-reactivity inhibiting KIR antibody compositions of the invention may be used in combination with one or more other anti-angiogenic therapies, or additionally contain anti-angiogenic agents. Examples of such agents include neutralizing antibodies, respectively, against VEGF or VEGF receptors, antisense RNA, siRNA, RNAi, RNA aptamers, and ribozymes (US Patent No. 6,524,583, the disclosure of which is incorporated herein by reference). Variants of VEGF having antagonistic properties may also be employed, as described in WO 98/16551, which is incorporated herein by reference. Other exemplary anti-angiogenic agents useful for combination therapy are listed in Table D of U.S. Patent No. 6,524,583, the disclosure of which agents and indications is expressly incorporated herein by reference. the

The inhibitory KIR antibody compositions of the invention may also advantageously be used in combination with methods of inducing apoptosis, or contain an apoptotic agent. For example, many oncogenes that inhibit apoptosis or programmed cell death have been identified. Exemplary oncogenes in this category include, but are not limited to, bcr-ab1, bcl-2 (different from bcl-1, cyclin D1; GenBank Accession Nos. M14745, X06487; U.S. Patent Nos. 5,650,491; and 5,539,094; per are incorporated herein by reference) and family members including Bcl-x1, Mcl-1, Bak, A1 and A20. Overexpression of bcl-2 was first discovered in T-cell lymphomas. The oncogene bcl-2 works by binding to and inactivating the protein Bax in the apoptotic pathway. Inhibition of bcl-2 function prevents the inactivation of Bax and allows the apoptotic pathway to proceed. Inhibition of such oncogenes using, for example, antisense nucleotide sequences, RNAi, siRNA, or small chemical compounds is contemplated for use in the present invention to increase apoptosis (U.S. Patent Nos. 5,650,491; 5,539,094; and 5,583,034; each incorporated herein as refer to). the

The inhibitory KIR antibody compositions of the invention may also comprise or be used in combination with molecules (e.g., antibodies, ligands, or conjugates thereof) containing targeting moieties directed against target cells ( For example, target tumor cells) specific markers ("targeting agents"). In general, targeting agents for use in these additional aspects of the invention will preferably recognize accessible tumor antigens that are preferentially or specifically expressed at the tumor site. A targeting agent will generally bind to a surface expressed, surface accessible or surface localized component of the tumor cell. The targeting agent will also preferably exhibit high affinity properties; and will not exert significant side effects in vivo on normal vital tissues such as heart, kidney, brain, liver, bone marrow, colon, breast , prostate, thyroid, gallbladder, lungs, adrenal glands, muscles, nerve fibers, pancreas, one or more tissues in the skin, or other life-sustaining organs or tissues in the body. The term "not exerting significant side effects" as used herein refers to the fact that a targeting agent, when administered in vivo, will produce only negligible or clinically manageable side effects, such as those commonly encountered during chemotherapy. the

类和大麻素类；双膦酸化合物例如唑来膦酸和帕米膦酸；以及造血生长因子例如促红细胞生成素和G-CSF，例如非格司亭、来格司亭和达比波亭(darbepoietin)。  In tumor therapy, the antibody composition of the present invention may additionally contain auxiliary compounds or be used in combination therewith. As examples, ancillary compounds may include antiemetics such as serotonin antagonists and therapeutic agents such as phenothiazines, substituted benzamides, antihistamines, butyrophenones, corticosteroids, benzodiazepines

bisphosphonates such as zoledronic acid and pamidronic acid; and hematopoietic growth factors such as erythropoietin and G-CSF such as filgrastim, legrastim, and darbibotine (darbepoietin).

In another embodiment, two or more antibodies of the invention with different cross-reactivity, including NKVSF1, can be combined in a single composition to neutralize as many inhibitory KIR gene products as possible. effect. Compositions containing combinations of cross-reactive inhibitory KIR antibodies or fragments or derivatives thereof of the invention will allow broader utility due to the low percentage of the population that may lack each inhibitory KIR gene product recognized by a single cross-reactive antibody That may exist. Similarly, compositions of the invention may additionally contain one or more antibodies that recognize a single inhibitory KIR isoform. Such combinations would again provide broader utility in therapeutic settings. the

The present invention also provides a method for enhancing NK cell activity in a patient in need thereof, comprising the step of administering to said patient a composition according to the present invention. The method is more specifically directed to enhancing NK cell activity in a patient suffering from a disease in which it is beneficial to increase NK cell activity in a patient that involves, affects, or is caused by cells that are susceptible to lysis by NK cells , or the disease is caused by or is characterized by insufficient NK cell activity (eg, cancer, another proliferative disease, an infectious disease, or an immune disorder). The methods of the invention are more particularly useful in the treatment of a variety of cancers and other proliferative diseases including, but not limited to, bladder, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid and Cancer of the skin (including squamous cell carcinoma); hematopoietic neoplasms of the lymphoid lineage, including leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma Lymphoma, pilocytic lymphoma, and Burketts lymphoma; hematopoietic neoplasms of the myeloid lineage, including acute and chronic myelogenous leukemia and promyelocytic leukemia; neoplasms of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other neoplasms, including melanoma Tumors, seminomas, teratomas, neuroblastomas, and gliomas; central and peripheral nervous system tumors, including astrocytomas, neuroblastomas, gliomas, and schwannomas Tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other neoplasms, including melanoma, xeroderma pigmentosa, keratoacanthoma, seminoma, follicular carcinoma of the thyroid, and teratoma tumor. the

Preferred diseases that may be treated in accordance with the present invention include hematopoietic tumors of the lymphoid lineage, such as T-cell and B-cell tumors (including but not limited to T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including small cell and encephalic-like cell types large granular lymphocytic leukemia (LGL), preferably T-cell type; Sezary syndrome (SS); adult T-cell leukemia-lymphoma (ATLL); a/d T-NHL hepatosplenocyte lymphoma; Lymphoma (pleomorphic and immunoblastic subtypes); angioimmunoblastic T-cell lymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; Intestinal T-cell lymphoma; T-lymphoblastic; and lymphoma/leukemia (T-Lbly/T-ALL). 

Other proliferative diseases may also be treated according to the present invention, including, for example, hyperplasia, fibrosis (particularly pulmonary, but including other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis, and intravascular smooth muscle Hyperplasia, such as stenosis or restenosis after angioplasty. The cross-reactivity inhibiting KIR antibody of the present invention can be used to treat or prevent infectious diseases, preferably including any infection caused by viruses, bacteria, protozoa, molds or fungi. Such viral infectious organisms include, but are not limited to, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella virus, adenovirus, herpes simplex type I (HSV-1), 2 Herpes simplex type (HSV-2), rinderpest virus, rhinovirus, Ocovirus, rotavirus, respiratory syncytial virus, papillomavirus (papillomavirus), cytomegalovirus, echinovirus, arbovirus, huntvirus, coxsackievirus, mumps virus, measles virus, rubella virus, polio virus, and human immunodeficiency virus type 1 or 2 (HIV-1, HIV-2). the

Bacterial infections that may be treated in accordance with the present invention include, but are not limited to, infections caused by Staphylococci; Streptococci, including S. pyogenes; Enterococci; Bacillus, including Bacillus anthracis and Lactobacillus; Listeria; Corynebacterium diphtheriae; Gardnerella, including G. vaginalis; Nocardia; Streptomyces; Thermoactinomyces vulgaris ); Treponema; Campylobacter; Pseudomonas, including Raeruginosa; Legionella; Neisseria, including N. gonorrhoeae and N. meningitides; Flavobacteria, including F. meningosepticum and F. odoraturn; Brucella; Bordetella, including B. pertussis and Bordetella bronchitis B. bronchiseptica; Escherichia, including Escherichia coli (E.coli), Klebsiella; Enterobacter, Serratia, including S. marcescens and liquefied sand S. liquefaciens; Edwardsiella; Proteus, including P. mirabilis and P. vulgaris; Streptobacter; Rickettsia, including R. fickettsfi, Chlamydia, Including C. psittaci and C. trachomatis; Mycobacteria, including M. tuberculosis, M. intracellulare, M. folluiturn, leprosy M.laprae, M.avium, M.bovis, M.africanum, M.kansasii, intracellular Mycobacterium (M. intracellulare) and Mycobacterium leprae (M. lepraemurium); and Nocardia. the

Protozoan infections that may be treated in accordance with the present invention include, but are not limited to, infections caused by leishmania, kokzidioa, and trypanosoma. A complete list of infectious diseases can be found at the National Center for Infectious Diseases (NCID) (http://www.cdc.gov/ncidod/diseases/ ) website of the Centers for Disease Control (CDC), which is incorporated herein by reference. All of the above diseases are candidates for treatment using the cross-reactivity inhibiting KIR antibodies of the invention.

Such methods of treating various infectious diseases may employ the antibodies of the invention alone or in combination with other treatments and/or therapeutic agents known to be used in the treatment of such diseases, including antiviral agents, antifungal agents, Antibacterial, antibiotic, antiparasitic and antiprotozoal agents. When the methods involve additional treatment with additional therapeutic agents, these agents can be administered together with the antibodies of the invention in a single dose or in separate, multiple doses. When administered in separate dosage forms, the additional agent may be administered before, concurrently with, or subsequent to administration of the antibody of the invention. the

Other aspects and advantages of the present invention will be disclosed in the following experimental part, which should be considered as illustrative rather than limiting the scope of the application. the

Example 1

Purification of PBLs and generation of polyclonal or clonal NK cell lines.

PBLs were obtained from healthy donors via a Ficoll Hypaque gradient with cells depleted of adherent plastic. To obtain enriched NK cells, PBL were incubated with anti-CD3, anti-CD4 and anti-HLA-DR mAbs (4°C, 30 min), followed by goat anti-mouse magnetic beads (Dynal) (4°C, 30 min) and using Immunomagnetic selection by methods known in the art (Pende et al., 1999). CD3 ⁻ , CD4 ⁻ , DR ⁻ cells were cultured on irradiated feeder cells with 100 U/ml interleukin 2 (Proleukin, Chiron Corporation) and 1.5 ng/ml lectin A (Gibco BRL) to obtain polyclonal NK cell populations. NK cells were cloned by limiting dilution, and NK cell clones were characterized for cell surface receptors by flow cytometry.

The mAbs used were JT3A (IgG2a, anti-CD3), EB6 and GL183 (IgG1, against KIR2DL1 and KIR2DL3, respectively), XA-141 IgM (anti-KIR2DL1 with the same specificity as EB6), anti-CD4 (HP2.6), and Anti-DR (D1.12, IgG2a). Commercially available mAbs of the same specificity (Beckman Coulter Inc., Fullerton, CA) can be used in place of JT3A, HP 2.6 and DR1.12 produced by the applicant. EB6 and GL183 are commercially available (Beckman Coulter Inc., Fullerton, CA. XA-141 is not commercially available, but lysis can be reproduced using EB6 as a control as described in (Moretta et al., 1993)). the

Cells were stained with the appropriate antibody (4°C, 30 minutes) followed by PE or FITC-conjugated polyclonal anti-mouse antibody (Southern Biotechnology Associates Inc). Samples were analyzed by cytofluorometric analysis on a FACSAN instrument (Becton Dickinson, Mountain View, CA). the

The following clones were used in this study. CP11, CN5 and CN505 were KIR2DL1 positive clones stained by EB6 (IgG1 anti-KIR2DL1) or XA-141 (IgM anti-KIR2DL1 with the same specificity compared to EB6 antibody). CN12 and CP502 are KIR2DL3 positive clones, stained by GL183 antibody (IgG1 anti-KIR2DL3). the

^The cytolytic activity of NK clones was assessed by a standard 4-hour Cr release assay in which effector NK cells were tested on Cw3 or Cw4 positive cell lines ^known for their sensitivity to NK cell lysis . All target cells were used at 5000 cells per well in microtiter plates, effector cell: target cell ratios are shown in the graph (typically 4 effector cells per target cell). Cytolytic assays were performed with or without 1/2 dilutions of the indicated monoclonal antibody supernatants. The procedure was essentially the same as described in (Moretta et al., 1993).

Example 2

Generation of new mAbs

mAbs were produced by immunizing 5-week-old Balb C mice with activated polyclonal or monoclonal NK cell lines as described (Moretta et al., 1990). Following fusion of different cells, mAbs were first selected for their ability to cross-react with EB6 and GL183 positive NK cell lines and clones. Positive monoclonal antibodies were further screened for their ability to reproduce Cw4 or Cw3 positive target cell lysis by EB6 positive or GL183 positive NK clones, respectively. the

Perform cell staining as follows. Anti-mouse IgG (H+L) or PE-conjugated goat F with a panel of antibodies (1 μg/ml or 50 μl supernatant, 4°C, 30 minutes) followed by PE-conjugated goat F(ab')2 fragment Cells were stained with (ab')2 fragment anti-human IgG (Fcγ) antibody (Beckman Coulter). Cytofluorometric analysis was performed on an Epics XL.MCL instrument (Beckman Coulter). the

One of the monoclonal antibodies (DF200mAb) reacted with multiple KIR family members including KIR2DL1, KIR2DL2/3. Both KIR2DL1+ and KIR2DL2/3+ NK cells were brightly stained by DF200mAb (Figure 1). the

NK clones expressing one or the other (or even both) of these HLA class I-specific inhibitory receptors are used as effector cells against target cells expressing one or more HLA-C alleles. Cytotoxicity assays were performed as follows. The cytolytic activity of the YTS-KIR2DL1 or YTS-Eco cell lines was assessed by a standard 4- ^hour51Cr release assay. Effector cells were tested on HLA-Cw4 positive or negative EBV cell lines and HLA-Cw4 transfected 721.221 cells. All target cells were used at 3000 cells per well in microtiter plates. Effector cell/target cell ratios are shown in the graph. Cytolytic assays were performed with or without full-length or F(ab')2 fragments of the indicated monoclonal mouse or human antibodies. KIR2DL1 ⁺ NK clones displayed little if any cytolytic activity on HLA-Cw4 expressing target cells as expected, and KIR2DL3 ⁺ NK clones displayed little or no activity on Cw3 positive target cells. However, in the presence of DF200mAb (used to mask its KIR2DL receptor), NK cells became unable to recognize their HLA-C ligands and displayed strong cytolytic activity against Cw3 or Cw4 target cells.

For example the C1R cell line (CW4 ⁺ EBV cell line, ATCC n°CRL 1993) was not killed by the KIR2DL1 ⁺ NK clone (CN5/CN505), but this inhibition was effectively reversed with DF200 or conventional anti-KIR2DL1 mAb. On the other hand, NK clone (CN12) expressing KIR2DL2/3 ⁺ KIR2DL1 ^- phenotype efficiently killed C1R cells, and this killing was not affected by DF200mAb (Fig. 2). Similar results were obtained with KIR2DL2 or KIR2DL3 positive NK clones on Cw3 positive target cells.

Similarly, the Cw4+221 EBV cell line was not killed by KIR2DL1 ⁺ transfected NK cells, but this inhibition could be effectively reversed using DF200, DF200Fab fragments, or conventional anti-KIR2DL1 mAbs EB6 or XA141. Likewise, the Cw3+221 EBV cell line was not killed by KIR2DL2 ⁺ NK cells, but this inhibition could be reversed using DF200 or the DF200Fab fragment. Finally, the latter Cw3+ 221 EBV cell line was not killed by KIR2DL3 ⁺ NK cells, but this inhibition could be reversed using DF200 Fab fragments or conventional KIR2DL3 mAbs GL183 or Y249. The results are shown in FIG. 3 .

F(ab')2 fragments were tested for their ability to reproduce lysis of Cw4 positive target cells. Both DF200 and the F(ab')2 fragment of the EB6 antibody reversed the inhibition of KIR2DL1-transfected NK cells lysing Cw4-transfected 221 cell line and Cw4+ TUBO EBV cell line. The results are shown in Figure 4. the

Example 4

Generation of novel human mAbs

Human anti-KIR monoclonal antibodies were generated by immunizing transgenic mice engineered to express a human antibody repertoire with recombinant KIR proteins. Following fusion of different cells, mAbs were first selected for their ability to cross-react with immobilized KIR2DL1 and KIR2DL2 proteins. Several monoclonal antibodies (including 1-7F9, 1-4F1, 1-6F5, and 1-6F1) react with KIR2DL1 and KIR2DL2/3. the

Positive monoclonal antibodies were further screened for their ability to reproduce lysis of Cw4-positive target cells by EB6-positive NK transfectants expressing KIR2DL1. NK cells expressing HLA class I-specific inhibitory antibodies were used as effector cells against target cells expressing one or more HLA-C alleles (Figures 5 and 6). Cytotoxicity assays were performed as described above. Effector cell/target cell ratios are shown in the graph, antibodies were used at 10 ug/ml or 30 ug/ml. the

KIR2DL1 ⁺ NK cells exhibit little if any cytolytic activity against HLA-Cw4 expressing target cells as expected. However, in the presence of 1-7F9 mAb, NK cells became unable to recognize their HLA-C ligands and displayed strong cytolytic activity against Cw4 target cells. For example, the two cell lines tested (HLA-Cw4-transfected 721.221 and the CW4 ⁺ EBV cell line) were not killed by KIR2DL1 ⁺ NK cells, but this inhibition was effectively reversed using mAb 1-7F9 or the conventional anti-KIR2DL1 mAb EB6. Antibodies DF200 and panKIR (also known as NKVSF1 ) were compared to 1-7F9. On the other hand, antibodies 1-4F1, 1-6F5 and 1-6F1 were unable to reproduce the cytolysis of Cw4 positive target cells by NK cells.

Example 5

Biacore analysis of the interaction between DF200 mAb/KIR2DL1 and DF200mAb/KIR2DL3

Production and purification of recombinant proteins

Production of KIR2DL1 and KIR2DL3 recombinant proteins in E. coli. The cDNAs encoding the complete extracellular domains of KIR2DL1 and KIR2DL3 were amplified using PCR from the pCDM8 clone 47.11 vector (Biassoni et al., 1993) and the RSVS(gpt)183 clone 6 vector (Wagtman et al., 1995), respectively, using the following primers:

Sense: 5'-GGAATTCCAGGAGGAATTTAAAATGCATGAGGGAGTCCACAG-3'

Antisense: 5'-CGGGATCCCAGGTGTCTGGGGTTACC-3'

This was cloned in-frame with the sequence encoding the biotinylation signal into the pML1 expression vector (Saulquin et al., 2003). the

Protein expression was performed in BL21(DE3) strain (Invitrogen). The transfected bacteria were grown to OD ₆₀₀ =0.6 in a medium supplemented with ampicillin (100 μg/ml) at 37° C., and the expression was induced with 1 mM IPTG.

Proteins were recovered from inclusion bodies under denaturing conditions (8M urea). By reducing the concentration of urea in six steps of dialysis (4, 3, 2, 1, 0.5 and 0 M urea, respectively), in 20 mM Tris containing L-arginine (400 mM, Sigma) and β-mercaptoethanol (1 mM) , pH 7.8, NaCl 150mM buffer at room temperature for refolding of recombinant proteins. Reduced and oxidized glutathione (5 mM and 0.5 mM, respectively, Sigma) were added during the 0.5 and 0 M urea dialysis steps. Finally, the protein was extensively dialyzed against 10 mM Tris, pH 7.5, NaCl 150 mM buffer. Soluble refolded proteins were concentrated and then purified on a Superdex 200 size exclusion column (Pharmacia; AKTA Systems). the

Surface plasmon resonance measurements were performed on a Biacore instrument (Biacore). In all Biacore experiments, HBS buffer supplemented with 0.05% surfactant P20 was used as the running buffer. the

Protein immobilization. the

The recombinant KIR2DL1 and KIR2DL3 proteins produced as described above were covalently immobilized onto the carboxyl groups of the CM5 (Biacore) dextran layer of the Sensor Chip. The sensor chip surface was activated with EDC/NHS (N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, Biacore). Proteins were injected in coupling buffer (10 mM acetic acid, pH 4.5). Deactivation of residual activated groups was performed using 100 mM ethanolamine pH 8 (Biacore). the

Affinity Measurements. the

For kinetic measurements, various concentrations of soluble antibody (1×10 ⁻⁷ to 4×10 ⁻¹⁰ M) were applied to immobilized samples. Measurements were performed at a continuous flow rate of 20 μl/min. For each cycle, 5 μl of 10 mM NaOH pH 11 was injected to regenerate the sensor chip surface. Data were analyzed using the BIAlogueKinetics Evaluation program (BIAevaluation 3.1, Biacore). Soluble analyte (various concentrations, 40 μl) in HBS buffer was injected at a flow rate of 20 μl/min on a dextran layer containing 500 or 540 reflectance units (RU) and 1000 or 700 RU, respectively, of KIR2DL1 and KIR2DL3 . Data are representative of six independent experiments. The results are shown in Table 1, see below.

Table 1. BIAcore analysis of DF200 mAb binding to immobilized KIR2DL1 and KIR2DL3. the

protein K _D (10 ^-9 M) KIR2DL1 10.9+/-3.8 KIR2DL3 2.0+/-1.9

K _D : dissociation constant.

Example 6

Biacore Competitive Binding Analysis of Mouse and Human Anti-KIR Antibodies

Mouse anti-KIR2D antibodies DF200, Pan2D, g1183 and EB6 and human anti-KIR2D Antibodies 1-4F1, 1-6F1, 1-6F5 and 1-7F9 were subjected to epitope mapping analysis. the

All experiments were done with two minute injections of 15 μg/ml of the different antibodies in HBS buffer at a flow rate of 5 μl/min. Competitive binding assays were performed for each pair of antibodies in two steps. In the first step, the first monoclonal antibody (mAb) is injected to the KIR 2D target protein, followed by the second mAb (the first mAb is not removed), and the RU value of the second mAb (RU2 ). In the second step, a second mAb was first injected directly onto the naked KIR2D protein, and the RU value of the mAb (RU1) was monitored. The percent inhibition of KIR 2D protein binding by the second mAb by the first mAb was calculated by 100*(1-RU2/RU1). the

The results are shown in Tables 2, 3 and 4, where the antibody designated "first antibody" is listed in the vertical column and the "second antibody" is listed in the horizontal column. For each antibody combination tested, the values (RU) of the level of direct binding of the antibody to the chip are listed in the table, where the direct binding of the second antibody to the KIR2D chip is listed in the upper part of the grid, the second when the first antibody is present Values for antibody binding to the KIR2D chip are listed in the lower part of the grid. Listed to the right of each cell is the percent inhibition of second antibody binding. Table 2 shows the binding on the KIR2DL1 chip, Table 3 shows the binding of the antibody to the KIR2DL3 chip, and Table 4 shows the binding of the antibody 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 immobilized KIR2DL1, KIR2DL2/3 and KIR2DS1 was assessed. Epitope mapping from anti-KIR antibody binding KIR2DL1 experiments (Figure 7) shows that (a) antibody 1-7F9 competes with EB6 and 1-4F1, but not with NKVSF1 and DF200; (b) antibody 1-4F1 in turn competes with EB6 , DF200, NKVSF1 and 1-7F9; (c) NKVSF1 competes with DF200, 1-4F1 and EB6, but not with 1-7F9; and (d) DF200 competes with NKVSF1, 1-4F1 and EB6, but not with 1-7F9 competition. Epitope mapping from anti-KIR antibody binding KIR2DL3 experiments (Figure 8) showing (a) 1-4F1 competes with NKVSF1, DF200, g1183 and 1-7F9; (b) 1-7F9 competes with DF200, g1183 and 1-4F1 , but not NKVSF1; (c) NKVSF1 competes with DF200, 1-4F1 and GL183, but not 1-7F9; and (d) DF200 competes with NKVSF1, 1-4F1 and 1-7F9, but not GL183 compete. Epitope mapping from anti-KIR antibody binding KIR2DS1 experiments (Figure 9) showing (a) 1-4F1 competes with NKVSF1, DF200 and 1-7F9; (b) 1-7F9 competes with 1-4F1 but not DF200 and NKVSF1 Competes; (c) NKVSF1 competes with DF200 and 1-4F1, but not 1-7F9; and (d) DF200 competes with NKVSF1 and 1-4F1, but not 1-7F9. the

Example 7

Titration of anti-KIR mAb with macaque NK cells

The ability of anti-KIR antibody NKVSF1 to bind macaque NK cells was tested. Binding of antibodies to monkey NK cells is shown in FIG. 10 . the

Purification of monkey PBMCs and generation of total polyclonal NK cells.

Rhesus monkey PBMCs were prepared from sodium citrate CPT tubes (Becton Dickinson). NK cell purification was performed using negative depletion (Macaque NK Cell Enrichment Kit, Stem Cell Technology). NK cells were cultured on irradiated human feeder cells, 300 U/ml interleukin 2 (Proleukin, Chiron Corporation) and 1 ng/ml lectin A (Invitrogen, Gibco) to obtain polyclonal NK cell populations. the

Pan2D mAb was titrated with macaque NK cells.

Cynomolgus NK cells (total NK 16 days) were incubated with varying amounts of Pan2D mAb followed by PE-conjugated goat F(ab')2 fragment anti-mouse IgG(H+L) antibody. An isotype control (purified mouse IgGl) was used to determine the percentage of positive cells. Samples were processed in duplicate. Mean fluorescence intensity = MFI. the

Example 8

Epitope mapping of DF200 and pan2D binding to KIR2DL1

In silico prediction of KIR2DL1, 2 and 3 (KIR2DL1-3) extracellular domains based on their published crystal structures ( Maenaka et al., (1999); Fan et al., (2001); Boyington et al., (2000)) Amino acid R131 ¹ is involved in the interaction between KIR2DL1 and the KIR2DL1-3 cross-reactive mouse monoclonal antibody (mAb) DF200 and pan2D. To test this prediction, a fusion protein consisting of the entire extracellular domain (amino acids H1-H224) of wild-type or point mutant (eg ^R131W2 ) KIR2DL1 fused to a human Fc (hFc) was prepared. Materials and methods for producing and evaluating various KIR2DL1-hFc fusion proteins are described in (Winter and Long (2000)). Briefly, cDNA encoding KIR2DL1(R131W)-hFc was generated based on PCR mutagenesis (Quickchange II, Promega) of the published cDNA vector CL42-Ig (Wagtmann et al., (1995)) for the production of wild-type KIR2DL1-hFc. carrier. KIR2DL1-hFc and KIR2DL1(R131W)-hFc were produced in COS7 cells and isolated from tissue culture medium essentially as described (Wagtmann et al., (1995)). To test for its correct folding, KIR2DL1-hFc and KIR2DL1(R131W)-hFc were incubated with LCL721.221 cells expressing HLA-Cw3 (no KIR2DL1 ligand) or HLA-Cw4 (KIR2DL1 ligand), by studying cell surface protein interaction Interaction between KIR-Fc fusion protein and cells is analyzed by standard technique FACS. Examples of independent experiments are given in Figure 11A column. As predicted from the literature, no KIR2DL1-hFc fusion protein bound to HLA-Cw3 expressing LCL721.221 cells. In contrast, both KIR2DL1-hFc and KIR2DL1(R131W)-hFc bound to LCL721.221 cells expressing HLA-Cw4, confirming its correct folding.

Binding of KIR2DL1(R131W)-hFc and KIR2DL1-hFc to KIR-specific mAbs (DF200, pan2D, EB6 and GL183) was studied using ELISA, a standard technique for studying protein interactions. Briefly, KIR2DL1(R131W)-hFc and KIR2DL1-hFc were attached to 96-well plates via goat anti-human antibody, after which KIR-specific mAbs were added at various concentrations (0-1 μg/ml in PBS). Antibody specific for mouse

¹ single-letter amino acid code

² R was substituted for W at amino acid 131 in KIR2DL1 (counting from the N-terminus). Secondary antibody-converted TMB substrates coupled with different peroxidases were visualized spectrophotometrically (450 nm) with KIR2DL1-hFc variants and Interactions between mAbs. Examples of independent experiments are given in Figure 11B column. While KIR2DL2-3-specific mAb GL183 was unable to bind any KIR2DL1-hFc fusion protein, KIR2DL1-specific mAbs EB6, DF200 and pan2D bound KIR2DL1-hFc variants in a dose-dependent manner. A single point mutation (R131W) affects the binding of DF200 and pan2D, and the highest concentration of mAb (1 μg/ml) reduces the binding by about 10% compared with the wild type, confirming that R131 is the extracellular domain 2 of DF200 and pan2D in KIR2DL1 Part of the binding site in .

references

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All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference, and are set forth in their entirety herein. the

All headings and subheadings are used herein for convenience only and should not be construed as limiting the invention in any way. the

Unless otherwise indicated herein or otherwise clearly contradicted by context, the invention encompasses any combination of the above-described elements in all possible variations thereof. the

Unless otherwise indicated herein or otherwise clearly contradicted by context, the terms "a," "an," and "the," and similar references, as used in the text describing the invention, are to be construed to encompass both the singular and the plural. the

Unless otherwise indicated herein, ranges of values recited herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is referred to in the specification as if it were individually recited herein. Same. Unless otherwise stated, all exact values provided herein represent corresponding approximations (for example, all exact exemplary values provided for a particular factor or measurement can be considered to also provide corresponding approximate measurements, where appropriate indicated by "approximately "express). the

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. the

The use of any and all examples, or exemplary terms (eg, "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No term in this specification should be construed as indicating that any ingredient is essential to the practice of the invention unless likewise expressly stated. the

Citation and introduction of patent documents herein are for convenience only and do not reflect any view on the validity, patentability and/or mandatory nature of such patent documents. the

Unless otherwise stated or clearly contradicted by context, descriptions of elements in any aspect or embodiment of the invention using terms such as "comprising", "having", "comprising" or "containing" are intended to describe the invention as "comprising" of particular elements. Composed of", "essentially composed of" specific ingredients

Similar aspects or embodiments that "consist of" or "consist essentially of" a particular ingredient provide support (eg, unless otherwise stated or clearly contradicted by context, a composition described herein as comprising a particular ingredient should be understood to also describe composition). the

This invention includes all modifications and equivalents of the aspects and claims set forth herein to the maximum extent permitted by applicable law. the

Claims (9)

1. antibody, described antibody contain 3 variable region of heavy chain CDR of the DF-200 that lists among 3 variable region of light chain CDR of the DF-200 that lists among Figure 12 and Figure 13.

2. according to claim 1 antibody, described antibody contain the weight chain variabl area sequence of the DF-200 that lists among the light chain variable region sequence of the DF-200 that lists among Figure 12 and Figure 13.

3. according to claim 2 antibody, it is by being preserved in the antibody DF200 that the hybridoma DF200 at CNCM preservation center is produced on June 10th, 2004 with number of registration CNCM I-3224.

4. according to claim 1 and 2 antibody, it is Double function miniature antibody or the multi-specificity antibody that formed by antibody fragment.

5. the antibody fragment of each antibody according to claim 1-3, it is Fab, Fab ', Fab '-SH, F (ab ') ₂Or single chain antibody fragments.

6. each antibody according to claim 1-3, but it is further with toxin, radionuclide test section, solid support or PEG puts together or covalent attachment.

7. produce the method for antibody, the inhibition of described antibody and the cross reaction of multiple KIR2DL gene product and this class KIR that neutralizes is active, and described method comprises the following steps:

(a) containing the immunogen immune non-human mammal of KIR2DL polypeptide,

(b) from described Mammals Dispersal risk through immunity, wherein said antibody is combined with described KIR2DL polypeptide,

(c) antibody in the selection (b), described antibody and KIR2DL1 and KIR2DL2/3 cross reaction, and

(d) antibody in the selection (c), described antibody strengthens the NK cell,

(e) select antibody, described antibody is combined with primate NK cell or KIR polypeptide.

8. according to claim 7 method, wherein the primate in the step (e) is macaque.

9. according to claim 7 or 8 method, wherein said antibody be according to claim 1-3 in each antibody.

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