JP2007512836A - Methods of treatment and diagnosis using targeting of cells expressing killer cell immunoglobulin-like receptor-like proteins - Google Patents

Abstract

Some cells belonging to a type of cancer cell such as KIRHy can express KIRHy mRNA. By targeting a KIRHy polypeptide, a nucleic acid encoding a KIRHy polypeptide, and an anti-KIRHy antibody, a method of killing or inhibiting the growth of a cancer cell that expresses a KIRHy protein is provided. The treatment and diagnosis methods for disorders involving KIRHy protein expressing cells such as acute myeloid leukemia (AML) are described.

Description

(1. Cross-reference of related applications)
This application is a US patent application Ser. No. 10 / 727,012, filed Dec. 2, 2003, entitled “Therapeutic and diagnostic methods targeting cells expressing killer cell immunoglobulin-like receptor-like proteins”, Acting This is a continuation-in-part of human number NUVO-02CP, which is filed in US patent application Ser. No. 10 / 414,539, filed Apr. 14, 2003, entitled “Killer Cell Immunoglobulin-like Receptor-like Protein” Therapeutic and Diagnostic Methods Targeting Cells ", Attorney Docket No. NUVO-02, a continuation-in-part application, filed PCT Application No. PCT / US01 / 02623, filed January 25, 200, This is a continuation-in-part application under the name “New Nucleic Acids and Polypeptides”, Attorney Docket No. 785CIP3 / PCT, which is PCT Application No. PCT / US This is a continuation-in-part of US patent application Ser. No. 09/631, filed January 25, 200, entitled “New Nucleic Acids and Polypeptides”, Attorney Docket No. 785CIP2-2C / PCT. No. 451, filed Aug. 3, 3000, name “Novel Nucleic Acids and Polypeptides”, Attorney Docket No. 785CIP2B, which is a continuation-in-part of US patent application Ser. No. 09 / 491,404 (currently Abandoned), filed January 25, 2000, partly continued application with name “New Contigs from Various Libraries”, Attorney Docket Number 785. The above and all other US patents and patent applications cited herein are hereby incorporated by reference in their entirety.

(2. Background)
(2.1 Technical field)
The present invention relates to compositions that target antibodies, polypeptides, polynucleotides, peptides, and low molecular weight products using cells that express a killer cell immunoglobulin-like receptor-like protein (abbreviated herein as KIRHy). And methods and their use in the treatment and diagnosis of various pathological conditions, including cancers such as acute myeloid leukemia (AML).

(2.2 Sequence Listing)
The sequences of the polynucleotides and polypeptides of the present invention are described in the sequence listing, prepared at 2:10:53 pm on April 6, 2004 using the Windows 2000 operating system on an IBM PC. Submit on a compact disc containing the file entitled “NUVO-02CP2 PCT.txt”. The entirety of which is incorporated herein by reference. A sequence listing, “NUVO-02CP2 PCT.txt” computer readable format (“CRF”) and three copies (“Copy 1”, “Copy 2”, and “Copy 3”) are also submitted. Applicants of this patent shall submit the original CRF and copy 1, copy 2, and copy 3 contents of the sequence listing as filed in accordance with Section 1.821 (c) and (e), respectively, of the 37 U.S. Patent Law Enforcement Rules. I swear here that they are the same.

(2.3 Background art)
Immunotherapy provides a way to utilize the immune system to treat a variety of pathological conditions, including cancer, autoimmune diseases, transplant rejection.

  For example, antibody therapy for cancer includes the use of antibodies or antibody fragments against tumor antigens that target cells that express the antigen. The antibody or antibody fragment can have a direct or indirect cytotoxic effect or can be bound or fused to a cytotoxic site. Direct effects include induction of apoptosis, growth factor receptor blocking, and formation of anti-idiotype antibodies. Indirect effects include antibody-dependent cell-mediated injury (ADCC) and complement-mediated cytotoxicity (CMCC). If an antibody or antibody fragment binds or fuses to a cytotoxic site, it opens a way to damage tumor antigen-expressing cells. (Green, et al., Cancer Treatment Reviews, 26: 269-286 (2000), which is incorporated by reference herein in its entirety).

  Antibody therapy targets cells that express specific antigens and can cross react with normal cells or tissues. Some cells, such as hematopoietic cells, easily replace precursors, but cross-reactivity with many tissues can have deleterious consequences. For this reason, considerable researchers are striving to find tumor-specific antigens. Tumor-specific antigens are found almost exclusively on tumors and are expressed in tumor cells at higher levels than normal tissues. Tumor-specific antigens are the targets of antibodies that target cells that express the antigen and are associated with cancer or other diseases. Antibodies having specificity for such tumor-specific antigens can bind to cytotoxic compounds, i.e., can be used alone in immunotherapy. Immunotoxins are targeted at cytotoxic compounds that cause cell death. For example, anti-CD22 antibodies that bind to deglycosylated lysine A can be used to treat B-cell lymphoma that has recurred after conventional therapy (Amlot, et al., Blood 82, which is incorporated herein by reference in its entirety). : 2624-2633 (1992)), promising results have been obtained in early clinical trials.

  The immune system serves to exclude organisms or cells that are recognized as non-self, including microorganisms, neoplasms, and transplants. Cell-mediated host responses to tumors include immune surveillance that destroys newly migrated tumor cells after recognizing tumor-related antigens (antigens related to tumors that cannot be recognized on normal cells). A concept that links cellular mechanisms and cellular immunity. Furthermore, through immunological processes such as antibody-dependent cell-mediated injury (ADCC) and complement-mediated lysis, where the response of body fluids to antigens associated with tumors triggers antibodies to bind to the surface of cells. Tumor cells can be destroyed.

  The avalanche phenomenon, including cytokin production, B cell proliferation, and subsequent antibody production, is triggered by antigen recognition of the immune system. Tumor cells often have a reduced ability to present antigen to effector cells, which delays the immune response to tumor-specific antigens. In some cases, the tumor specific antigen may not be recognized as non-self by the immune system, preventing an immune response to the tumor specific antigen. In such cases, stimulating or manipulating the immune system is an effective technique for the treatment of cancers that express one or more tumor-specific antigens.

  For example, rituximab (Rituxan®) is a surface molecule specific for CD20, a B cell found in more than 95% of non-Hodgkin's disease lymphoma B cells, both of which are incorporated herein by reference in their entirety. , Press, et al., Blood 69: 584-591 (1987); Malony, et al., Blood 90: 2188-2195 (1997)). Rituximab induces ADCC and prevents malignant B cells in vitro to inhibit cell proliferation (Maloney, et al., Blood 88: 637a (1996), which is incorporated herein by reference in its entirety). Rituximab is currently used as a treatment for the progression or recurrence of mild non-Hodgkin's disease lymphoma that was ineffective with conventional therapies.

  Active immunotherapy, in which a host is induced and initiates an immune response against tumor cells, can be achieved by using a therapeutic vaccine. Certain tumor-specific vaccines are purified from tumor cells, combined with hemocyanin (KLH) from mussel, mixed with adjuvant, and injected into patients with mild follicular lymphoma Idiotype proteins are used (Hsu, et al., Blood 89: 3129-3135 (1997), which is incorporated herein by reference in its entirety). Another type of vaccine uses antigen presenting cells (APCs) that present antigens to native T cells during immune response recognition and effector phases. Dendritic cells, a type of APC, can be used for cellular vaccines, in which dendritic cells are isolated from the patient, cultured with tumor antigens, and reinjected into the patient as a cellular vaccine (in its entirety herein). Hsu, et al., Nat. Med. 2: 52-58 (1996)), which is incorporated by reference in its entirety. An immune response can also be elicited by injection of naked DNA. When injected into mice, plasmid DNA expressing bicistronic mRNA encoding both low and high molecular weight chains of tumor idiotype proteins, such as from B cell lymphomas, can elicit a protective antitumor response. (Singh, et al., Vaccine 20: 1400-1411 (2002)).

  About 10,600 cases of acute myeloid leukemia (AML) are newly diagnosed every year in the United States. AML is also referred to by several disease names, including acute myelocytic leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute nonlymphocytic leukemia. AML is the most common leukemia in adults and accounts for about half of leukemia in children. Leukemia begins in the bone marrow but also spreads to the blood, lymph nodes, spleen, liver, central nervous system, and other organs. Usually it does not form a solid mass or tumor. Chemotherapy and blood stem cell transplantation are the main treatments, and many patients are cured. However, recurrence after treatment is a major problem and often results in death. Furthermore, the toxic effects of treatment outcomes and the existence of drug treatment resistance are major problems that must be overcome to improve the quality of life of cancer patients and reduce mortality. Graft-versus-host disease (GVHD) is a major problem in allogeneic blood stem cell transplantation caused by donors, whereas recurrence with potential AML cells uses autologous blood stem cells isolated from patients prior to chemotherapy. It is a problem in sex blood stem cell transplantation.

  The lack of tumor-related antigens that are tumor specific, highly immunogenic, and effective in a wide range of patients has hindered the use of immunotherapy as a treatment for AML and other cancers (Dalerba et al. al., Clin. Rev. Oncol. Hematol. 46: 33-57 (2003)). Therefore, there is a need in the art to find antigens that are clearly and specifically expressed on the surface of cancer cells that can be targets for various targeting techniques. For this reason, the applicant of the present patent finds useful molecular targets for therapeutic intervention in AML and provides here a method for diagnosing and treating AML.

(3. Summary of the Invention)
The present invention relates to target cells expressing a killer cell immunoglobulin-like receptor (KIR) -like protein (abbreviated herein as KIRHy1) in fragments or other modifications, peptides and low molecular weight forms of KIRHy poly Methods of treatment and diagnosis are provided by using a peptide, a nucleic acid encoding a KIRHy protein, and a target element such as an anti-KIRHy antibody. KIRHy protein is highly expressed in certain hematopoietic cancer cells compared to its expression in healthy cells. Therefore, targeting cells that express KIRHy can minimize hematopoietic cancer cells or inhibit growth while minimizing the effect on healthy tissue. Similarly, non-hematopoietic tumors (solid tumors) can be targeted if they have KIRHy antigen. For example, targeting a KIRHy-expressing cancer cell with an anti-KIRHy antibody can inhibit growth and / or destroy such cells. One embodiment of the present invention is a method of destroying KIRHy-expressing cells by binding a cell-disrupting material such as a radioisotope or other cytotoxic compound to an anti-KIRHy antibody.

  The present invention provides various targeting elements and compositions. One such embodiment is a composition comprising an anti-KIRHy antibody formulation. Examples of antibodies include a single anti-KIRHy antibody, any fragment of an antibody that retains one or more of the complementarity determining regions (CDRs) that recognize KIRHy, a portion or all of a CDR that recognizes KIRHy1 A single anti-KIRHy antibody, a combination of two or more anti-KIRHy antibodies, an anti-KIRHy antibody and a non-KIRHy antibody, including humanized anti-KIRHy antibodies, anti-KIRHy conjugates, and anti-KIRHy antibody fusion proteins Combinations, anti-KIRHy antibodies and therapeutic agents, anti-KIRHy antibodies and cell disrupting agents, bispecific anti-KIRHy antibodies, FabKIRHy antibodies or fragments thereof.

  Another target embodiment of the invention is a composition comprising a KIRHy antigen comprising, for example, a KIRHy polypeptide, or fragment thereof, and optionally a suitable adjuvant. Yet another target embodiment is a composition comprising a nucleic acid encoding KIRHy, or a fragment or variant thereof, optionally a recombinant vector.

  Another target embodiment of the invention is a composition comprising an antigen presenting cell transformed with a nucleic acid encoding KIRHy, or a fragment or variant thereof, optionally a recombinant vector.

  Yet another target embodiment of the invention is a formulation comprising a KIRHy polypeptide, or peptide fragment thereof. Another target embodiment of the invention is a non-KIRHy polypeptide or peptide that binds to a KIRHy polypeptide or polynucleotide of the invention.

  Another target embodiment of the invention is a formulation comprising a low molecular weight material that recognizes or binds to a KIRHy polypeptide or polynucleotide of the invention.

  The present invention further provides a method of targeting KIRHy-expressing cells comprising administering an effective amount of a targeting element or composition to the target KIRHy-expressing cells. An anti-KIRHy antibody preparation, a KIRHy antigen containing a KIRHy polypeptide or a fragment thereof, a nucleic acid encoding KIRHy or a fragment thereof or a variant thereof, optionally, a composition comprising a recombinant vector, or a nucleic acid encoding KIRHy Any of the targeting elements or compositions described herein can be used in the methods described above, including compositions comprising antigen-presenting cells or fragments thereof or variants thereof and optionally a recombinant vector.

  The present invention further provides a method of targeting KIRHy-expressing cells comprising administering an effective amount of a targeting element or composition to the target KIRHy-expressing cells. An anti-KIRHy antibody preparation, a KIRHy antigen containing a KIRHy polypeptide or a fragment thereof, a nucleic acid encoding KIRHy or a fragment thereof or a variant thereof, and optionally a composition comprising a recombinant vector, or a nucleic acid encoding KIRHy Antigen-presenting cells or fragments thereof or variants thereof, optionally further comprising a recombinant vector, KIRHy polypeptide or peptide fragment thereof, polypeptide, peptide or low molecular weight binding to the KIRHy polypeptide or polynucleotide of the invention Any of the targeting elements or compositions described herein can be used in the methods described above.

  The present invention also provides a method of inhibiting the growth of cancer cells, including hematopoietic cancer cells and KIRHy-expressing cancer cells, comprising administering an amount of a target element or composition effective to inhibit the growth of cancer cells. To do. Anti-KIRHy antibody preparation, KIRHy antigen containing KIRHy polypeptide or fragment thereof, KIRHy-encoding nucleic acid or fragment or variant thereof, optionally further comprising a recombinant vector, or transformed with KIRHy-encoding nucleic acid Antigen-presenting cells or fragments thereof or variants thereof, optionally further comprising a recombinant vector, KIRHy polypeptide or peptide fragment thereof, KIRHy polypeptide or polynucleotide of the invention, peptide or peptide Any of the targeting elements or compositions described herein, including molecular weights, can be used in the above methods.

  Furthermore, the present invention relates to a disorder associated with proliferation of KIRHy expressing cells in a subject in need of treatment, comprising administering an amount of a targeting element or target composition effective to treat a disease associated with KIRHy expressing cells. Provide a method of treating. An anti-KIRHy antibody preparation, a KIRHy antigen containing a KIRHy polypeptide or a fragment thereof, a nucleic acid encoding KIRHy or a fragment thereof or a variant thereof, and optionally a composition comprising a recombinant vector, or a nucleic acid encoding KIRHy Antigen-presenting cells or fragments thereof or variants thereof, optionally further comprising a recombinant vector, KIRHy polypeptide or peptide fragment thereof, polypeptide, peptide or low molecular weight binding to the KIRHy polypeptide or polynucleotide of the invention Any of the targeting elements or compositions described herein can be used in the methods described above.

  Examples of disorders associated with the proliferation of KIRHy-expressing cells include cancers such as acute myeloid leukemia (AML) and histiocytic lymphoma. Furthermore, other diseases related to the proliferation of KIRHy-expressing cells include Hodgkin's disease, non-Hodgkin B cell lymphoma, T cell lymphoma, malignant lymphoma, lymphosarcoma leukemia, chronic lymphocytic leukemia, multiple myeloma, (myeloid leukemia) (Also known as) chronic myelogenous leukemia, myelomonocytic leukemia, myelodysplastic syndrome, companion lymphoid tissue proliferative disorder; Epstein-Barr virus related symptoms such as mononucleosis, hyperproliferative disease; self Immune diseases; wound healing; diseases such as organ and tissue transplant rejection (including hyperacute, acute, chronic transplant rejection and xenograft rejection). Esophageal cancer, gastric cancer, colon cancer, colorectal cancer, polyp related to colorectal neoplasm, pancreatic cancer and gallbladder cancer, adrenocortical cancer, ACTH-producing tumor, bladder cancer, brain intrinsic cancer, neuroblastoma, star Glioma brain tumor, brain cancer including glioma, central nervous system invasion of metastatic tumor cells, head and neck cancer including Ewing tumor, oral cancer and pharyngeal cancer, kidney cancer including liver cell carcinoma, liver cancer, Lung cancer, including small cell lung cancer and non-small cell lung cancer, malignant ascites, malignant peritoneal water, malignant melanoma, tumorigenesis of human skin keratinocytes, epithelial cell carcinoma, squamous cell carcinoma, skin cancer including basal cell carcinoma, and Bone cancer, including sarcoma, mesothelioma, Kaposi sarcoma, fibrosarcoma and osteosarcoma, and bone cancer including sarcoma, uterine cancer, endometrial cancer, ovarian cancer, ovarian (germ cell) cancer, solid tumor in follicle Cancer of female reproductive organs, including vaginal cancer, vulva cancer, cervical cancer, Small cell and tube) breast cancer, penile cancer, prostate cancer, retinoblastoma, testicular cancer, thyroid cancer, nutritional cancer, and non-hematopoietic tumors with KIRHy1 antigen Wilms tumor or the like may also be targeted. The present invention further provides methods of modulating the immune system by inhibiting or stimulating growth factors and cytokines by administering a target element or composition of the present invention. The present invention passes through activation of immune cells (natural killer cells, T cells, B cells, and myeloid cells), through suppression of activation, or stimulates the growth of these cells by KIRHy peptide fragments or KIRHy antibodies Alternatively, a method of modulating the immune system by inhibiting is also provided.

  Thus, the present invention provides a method of treating immune related disorders by suppressing the immune system of a patient in need of treatment by administering a target element or composition of the present invention. Such immune related disorders include, but are not limited to, autoimmune diseases and organ transplant rejection.

  The present invention also provides a method of diagnosing a disorder associated with KIRHy-expressing cells, comprising measuring the expression pattern of KIRHy protein and / or related mRNA. According to another embodiment of the present invention, a method for diagnosing a disorder associated with a KIRHy-expressing cell is provided, comprising detecting the expression of KIRHy using an anti-KIRHy antibody. The expression level and expression pattern are then compared to a suitable standard efficacy for the desired diagnosis. Such diagnostic methods include compositions, kits and other methods for determining whether a patient is subject to KIRHy therapy that targets KIRHy.

  The present invention relates to a treatment used for the treatment and management of disorders relating to KIRHy-expressing cells by administering a KIRHy preparation of the above-mentioned KIRHy1 and therapeutic agents and adjuvants generally used for the treatment of the above-mentioned disorders. Also provided are methods for enhancing the effectiveness of agents and adjuvants.

(5. Detailed Description of the Invention)
The present invention uses target elements such as polypeptides, nucleic acids, antibodies, binding polypeptides, peptides, and low molecular weight substances, including fragments or other modifications of any of these elements, to express KIRHy The present invention relates to a method for targeting a cell that performs the process.

  The present invention provides a novel method for diagnosing and treating diseases and disorders associated with KIRHy. In this method, a preparation containing a target component such as KIRHy antigen, antigen-presenting cell, or the following target element, KIRHy polypeptide, nucleic acid encoding KIRHy, anti-KIRHy antibody, KIRHy polypeptide or polynucleotide that binds to polynucleotide Administration of an effective amount of a pharmaceutical composition comprising a peptide, peptide, and low molecular weight product is included. By targeting KIRHy on the cell membrane, it can be expected to inhibit the growth of these cells or destroy these cells. Effective amounts are those amounts necessary for these targeted formulations to target KIRHy on the cell surface, inhibit the growth of cells that express KIRHy or destroy cells and / or inhibit cell metastasis.

  Another embodiment of the present invention is in the treatment and management of disorders involving KIRHy by administering a targeted formulation that recognizes KIRHy, including therapeutic agents and adjuvants commonly used in the treatment of these disorders. It is to enhance the effect of the therapeutic agents and adjuvants used.

  Chemotherapeutic agents useful for the treatment of neoplastic diseases and antiproliferative and antiproliferative agents used for immunosuppression include alkylating agents such as nitrogen mustard, alkyl sulfates, nitrosoureas, triazenes; folic acid analogs , Pyrimidine analogs, and purine analogs; natural products such as vinca alkaloids, epipodophyllotoxins, antibiotics, and enzymes; platinum coordination complexes, substituted ureas, methylhydrazine derivatives, and adrenocortical inhibition And micelles such as drugs; and hormones and antagonists such as adrenocorticosteroids, progesterone, estrogen, androgen, and antiestrogens (Calebresi and Parks, pp, which is incorporated herein by reference in its entirety). 1240-1306 in, Ed . A. G. Goodman, L. S. Goodman, T. W. Rall, and F. Murad, The Pharmacological Basis of Therapeutics, Seventh Edition, MacMillan Publishing Company, New York, (1985)).

  Adjuvant therapies used to manage these disorders include, for example, the use of radiosensitizers, combinations of antigens and heterologous proteins such as globulins or β-galactosidase, or the addition of adjuvants during immunization. It is.

  Some therapeutic agents may require high doses to achieve levels that cause a target response, but are more likely to cause dose-dependent side effects. Thus, by combining the targeted therapies of the present invention with drugs commonly used in the treatment of disorders associated with KIRHy expression, the dose of such drugs can be significantly reduced and the long-term use of conventional therapeutics The potential for side effects associated with administration is also reduced. Thus, one of the benefits of the targeted therapy of the present invention is to reduce the side effects associated with conventional therapy for these disorders.

(5.1 Targeting KIRHy)
The function of the immune system is governed by a complex network of cell surface interactions and associated signaling processes. When a cell surface receptor is activated by a ligand, a signal is sent to the cell, which can be either an inhibitory signal or an activation signal, depending on the signaling pathway involved.

  The cytolytic activity of natural killer (NK) cells is controlled by the balance between the activation signal that initiates cell lysis and the inhibitory signal that blocks cell lysis. NK cells recognize and kill certain tumors, virus-infected cells, MHC class I non-compliant normal hematopoietic cells, and mediate acute rejection of bone marrow transplantation, the entirety of which is incorporated herein by reference. Salmon Divon et al., Bull. Math. Biol.65: 199-218 (2003)). When NK cells receive an excessive activation signal, the target cells are killed. When a target cell expresses a cell surface MHC class I antigen (ie, “self” MHC class I antigen), for which the NK cell has a specific receptor, the NK cell is suppressed and does not kill the target cell. There are two types of specific receptors for such NK cells, killer cell immunoglobulin (Ig) -like receptors (KIRs) and C-type lectin-like Ly49 receptor. KIRs send a negative signal when MHC ligands that down-regulate the cytotoxic activity of NK cells are involved. KIRs have an immunoreceptor tyrosine inhibitory motif (ITIM) in the cytoplasmic domain that is phosphorylated by tyrosine kinases. An ITIM motif common to many KIRs has the sequence I / L / VxYxxL / V, where “x” represents any amino acid (SEQ ID NO: 10), the entirety of which is incorporated herein by reference. Heid et al., Curr. Opin. Immunol. 15: 233-237 (2003)).

  Several soluble forms of these membrane receptors, such as FDF03 and CD54, have been reported and can be used as markers of pathological conditions (Borges and Cosman, Cytokine and, which are incorporated herein by reference in their entirety). Growth Factor Reviews 11: 209-217 (2000)). The variable region of Ig provides a site for specific binding site formation, while the stable region of Ig provides a more conservative counter-receptor binding module. Recently, CMRF-35 and PIGR-1 which are members of an immunoglobulin and have only one Ig variable region have been cloned (Shujian et al (1999), European Patent No. 1, which is incorporated herein by reference in its entirety). 087981A1).

  It is clear that inhibitory receptors are present in most hematopoietic cells, including dendritic cells, mononuclear cells, CD19 + B cells, CD3 + T cells (Borges and Cosman, the entirety of which is incorporated herein by reference). (2000) supra; De Maria et al., Proc. Natl. Acad. Sci. USA 94: 10285-88 (1997)). Immunoreceptors expressed by mast cells are also known to down-regulate cell activation signals (International Patent Application No. WO 98/48017).

  Receptors on NK cells and T cells mediate innate immunity and have been shown to play an important role in killing certain virus-infected and melanoma cells as well as bone marrow transplant rejection. ing. Immunoglobulin receptors are also involved in mediating autoimmune responses. Very recently it has also been shown that immunoglobulin receptors are required for dendritic cell growth and maturation (Fournier et al., J. Immunol. 165: 1197-1209, which is incorporated by reference herein in its entirety). (2000)). The addition of anti-Ig receptor monoclonal antibodies to T cells has been shown to induce cytolytic activity of T cells against target cells infected with HIV. NK cells whose downregulation of inhibitory receptors causes immune disorders such as rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), psoriasis, and inflammatory bowel disease (IBD), among others And clearly leads to the overall activation of T cells.

  It is clear that the activation and repression signals mediated by opposing kinases and phosphatases are very important in maintaining the balance of the immune system. An immune system with a dominant activation signal causes autoimmunity and inflammation. An immune system with a dominant suppressor signal is less able to combat infected or cancer cells. Isolating new activating and inhibitory receptors is highly desirable in studying biological signals transmitted through the receptors. In addition, the isolation of these molecules provides a means to control and treat conditions associated with autoimmunity, inflammation, and infection.

  For example, cell surface receptors with ITIM motifs, such as KIRHy, that are in an antagonistic relationship with antibodies or ligands, down-regulate cell function in conditions where the immune system is overactive and excessive infection or immune pathology is present. Can be used for On the other hand, antibodies that specifically antagonize KIRHy1 or soluble forms of KIRHy block the interaction between cell surface receptors and receptor ligands that activate specific immune responses in pathologies related to suppression of immune function Can be used for

  The KIRHy protein of the present invention, a homologue of the human NK inhibitory receptor precursor (gi2052982), is highly expressed in certain hematopoietic cancers, but not highly expressed in non-hematopoietic, healthy cells. . Therefore, targeting cells that express KIRHy can minimize the effect on healthy tissue while destroying cancer cells or inhibiting the growth of cancer cells. Similarly, non-hematopoietic tumors (ie solid tumors) can be targeted if they carry the KIRHy antigen. Targeting KIRHy can also be used to treat disorders associated with proliferation of KIRHy expressing cells. Examples of disorders related to the growth of KIRHy expressing cells include (also known as acute myeloid leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute granulocytic leukemia, and acute nonlymphocytic leukemia Cancers such as AML and histiocytic lymphoma. Further, non-Hodgkin B cell lymphoma, B cell leukemia, T cell lymphoma, acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), B cell large cell lymphoma, multiple myeloma, myelodysplastic syndrome Epstein-Barr virus-related symptoms such as symptomatic lymphoid tissue proliferative disorders, and mononucleosis; autoimmune disorders such as systemic lupus erythematosus; hyperproliferative disorders; organ and tissue transplant rejection; and certain allergies Reaction is included. Non-hematopoietic tumors such as breast cancer, colon cancer, prostate cancer, squamous cell carcinoma, or epithelial cell carcinoma that retain the KIRHy antigen can also be targeted.

  KIRHy polypeptides and polynucleotides encoding KIRHy polypeptides are described in co-pending U.S. Patent Application No. 09 / 631,451 and U.S. Patents corresponding to WO 01/55437 and WO 01/55437, respectively. No. 09 / 491,404. All of these and other US patents, patent applications, foreign patents, and international publications cited herein are hereby incorporated by reference in their entirety. US patent application Ser. No. 09 / 491,404, which is incorporated herein by reference in its entirety, is generally a collection or library of at least one novel nucleic acid sequence, particularly a contig, ie, an alignment of labels of expressed sequences. It is about. US patent application Ser. No. 09 / 631,451, which is hereby incorporated by reference in its entirety, includes (especially including the entire sequence of sequences covered), a recombinant molecule, a cloned gene, or a degenerate version thereof. Variants, particularly natural variants such as allelic variants, fragments or homologues or variants of natural polynucleotides or polypeptides, antisense polynucleotide molecules, polyclonal, monoclonal, single-stranded, bispecific Such as fragmented, human and humanized antibodies, antibodies that specifically recognize one or more antigenic determinants present on a polypeptide, and fusion hybrid tumors that produce monoclonal antibodies KIRHy polypeptide, a polynucleotide isolate encoding KIRHy polypeptide, including cells, These polynucleotides, polypeptides, and diagnostic and therapeutic applications, as well as screening assays antibodies are involved is disclosed.

  The KIRHy polypeptide of SEQ ID NO: 3 is a protein consisting of approximately 305 amino acids with a predicted molecular weight of 33 kD in the non-glycosylated state. The first methionine begins at SEQ ID NO: 2 at 114 and the putative stop codon begins at SEQ ID NO: 2 at 1028. The predicted 17 residue signal peptide is encoded by residues 1 through 17 of SEQ ID NO: 3 (ie, SEQ ID NO: 4). This signal peptide region was predicted using Neural Network SignalP V1.1 (Nielsen et al., Int. J. Neural Syst. 8: 581-599 (1997)), which is incorporated herein by reference in its entirety. did. Those skilled in the art will recognize that the actual rupture site may differ from what is predicted by the computer program. The predicted transmembrane domain is encoded by residues 159 to 186 of SEQ ID NO: 3 (ie, SEQ ID NO: 6). The transmembrane domain was predicted using the Kyte-Doolittle hydrophobicity prediction algorithm (J. Mol. Biol. 157: 105-131 (1982), which is incorporated herein by reference in its entirety). Those skilled in the art will recognize that the actual domain may differ from what is predicted by the computer program. Using the Pfam software program (Sonhammer et al., Nucl. Acids Res. 26: 320-322 (1998), which is incorporated herein by reference in its entirety), KIRHy from amino acid 33 to 110 (SEQ ID NO: 5) It was predicted to contain a single immunoglobulin (Ig) domain, the soluble portion of KIRHy is expressed by SEQ ID NO: 7.

  BLASTP algorithm (Altschul et al., J. Mol. Evol. 36: 290-300 (1993); Altschul et al., J. Mol. Biol. 21: both of which are incorporated herein by reference in their entirety. 403-410 (1990)), as a result of searching the protein database, SEQ ID NO: 3 is a human NK inhibitory receptor precursor (gi2052982) and a human CMRF35 homolog (“similar to CMRF35 leukocyte Ig-like receptor”), It was found to be homologous to gi20380183). FIG. 1 shows SEQ ID NO: 3 and human NK inhibitory receptor precursor (SEQ ID NO: 8) side by side. These two sequences show 94% similarity and 94% identity over the entire amino acid sequence of SEQ ID NO: 3. In the figure, A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, and M is methionine. , N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine. The gap is represented by a broken line. FIG. 2 shows SEQ ID NO: 3 and CMRF35 homologue (SEQ ID NO: 9) side by side, and these two sequences show 89% similarity and 89% identity over the entire amino acid sequence of SEQ ID NO: 3, A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is glycine, H is histidine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is Asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T is threonine, V is valine, W is tryptophan, and Y is tyrosine. The gap is represented by a broken line.

  The gene corresponding to SEQ ID NO: 3 is localized on chromosome 17 (see Example 3). The CMRF35 family is also located on chromosome 17 close to the psoriasis susceptibility locus (PSORS2), which can overlap the atopic dermatitis locus and the rheumatoid arthritis locus (both of which are present in their entirety). Clark et al., Tissue Antigens 57: 415-423 (2001); Speckman et al., Hum. Genet. Therefore, those belonging to the KIRHy1 and CMRF35 families can be involved in psoriasis, atopic dermatitis, rheumatoid arthritis, and autoimmune diseases in general.

  KIRHy is not only AML and histiocytic lymphoma, but also B cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma, undifferentiated large T cell lymphoma, multiple myeloma, T cell leukemia, chronic bone marrow Expressed in hematopoietic cancers, including sexual leukemia (CML), histiocytic lymphoma, plasmacytoma, non-Hodgkin lymphoma, and Hodgkin lymphoma, while most non-hematopoietic, healthy cells do not express KIRHy, Or even if expressed, the level is low (see FIG. 3 and Table 3). Therefore, targeting KIRHy is useful in the treatment of hematopoietic cancers such as AML.

  The KIRHy peptide itself can also be used to target toxins or radioisotopes to tumor cells in vivo. KIRHy may be a homophilic adhesion protein that binds to itself. In this case, the extracellular domain or domain fragment of KIRHy can bind to KIRHy expressed on tumor cells. This peptide fragment can then be used as a means of delivering cytotoxic agents to KIRHy-bearing tumor cells. In much the same way as antibodies, these fragments can specifically target cells that express the KIRHy antigen. Targeted delivery of cytotoxic agents to tumor cells results in tumor cell death and tumor growth inhibition. An example of the ability of an extracellular fragment to bind to and activate an intact receptor by homophilic binding has been shown for the CD84 receptor (Martin, et al., Incorporated herein by reference in its entirety. J. Immunol.167: 3668-3676 (2001)).

  Extracellular fragments of the KIRHy receptor can also be used to modulate immune cells that express the KIRHy receptor. The extracellular domain fragment of the receptor can bind to and activate the receptor itself expressed on the cell surface. For cells that retain the KIRHy receptor (such as NK cells, T cells, B cells, and myeloid cells), this stimulates the release of cytokines (such as interferon gamma) that activate or suppress the immune system. In addition, these fragments can also activate these cells and stimulate proliferation by binding to cells carrying the KIRHy receptor. Some fragments can bind to the intact KIRHy receptor and block activation signals and cytokine release by immune cells. Therefore, these fragments have an immunosuppressive effect. Fragments that activate and stimulate the immune system have anti-tumor properties. These fragments are capable of stimulating an immunological response that can lead to immune-mediated tumor cell death. The same fragment can stimulate the immune system to mount and enhance responses to external invaders such as viruses and bacteria. Fragments that suppress the immune response are useful in the treatment of inflammatory diseases such as lymphoid tissue proliferative disorders, autoimmune diseases, graft-versus-host disease, emphysema.

(5.2 Definition)
The term “fragment” of a nucleic acid means a sequence of nucleic acid residues that is at least 5 nucleotides, preferably at least 7 nucleotides, more preferably at least 11 nucleotides, and most preferably at least 17 nucleotides. Fragments are preferably less than 500 nucleotides, preferably less than 200 nucleotides, more preferably less than 100 nucleotides, more preferably less than 50 nucleotides, and most preferably less than 30 nucleotides. Fragments are preferably used in polymerase chain reaction (PCR), various hybridization methods or microarray methods to identify or amplify identical or related portions of mRNA or DNA molecules. Each nucleotide sequence of the present invention can be uniquely identified from the fragment or portion. It is preferred that the fragment comprises a sequence that is substantially identical to the portion of SEQ ID NO: 2. A polypeptide “fragment” is a length of at least 5 amino acids, preferably at least 7 amino acids, more preferably at least 9 amino acids, and most preferably at least 17 amino acids or more amino acid residues. The peptide is preferably less than 200 amino acids, more preferably less than 150 amino acids, and most preferably less than 100 amino acids. The peptide is preferably from 5 amino acids to 200 amino acids. In order to be active, each polypeptide must have a length sufficient to exhibit biological and / or immunological activity. The term “immunogenic” binds the ability of a natural, recombinant or synthetic KIRHy peptide, or any KIRHy peptide, to elicit a specific immune response in a suitable animal or cell and a specific antibody. It means ability.

  The term “KIRHy antigen” means a molecule that, when introduced into an animal, can stimulate a specific immune response against the KIRHy polypeptide of the invention or fragment thereof in the animal.

  The term “variant” (or “homolog”) refers to any polypeptide that differs from the native polypeptide by, for example, amino acid insertions, removals, and substitutions, as provided by recombinant DNA techniques. Indicators that determine which amino acid residues can be substituted, added, or removed without losing the activity in question include comparing sequences of peptides that are homologous to a specific polypeptide sequence, highly homologous regions ( It can be found by minimizing changes in the number of amino acid sequences in the conserved region) or replacing amino acids with consensus sequences.

  Alternatively, recombinant variants encoding the same or similar polypeptides can be synthesized or screened using “overlaps” in the genetic code. Various codon substitutions, such as silent changes that produce various restriction sites, can be introduced into plasmids or viral vectors or expression in specific prokaryotic or eukaryotic systems to optimize cloning. To alter properties such as ligand binding affinity, interstrand affinity, or degradation / turnover rate, mutations in the polynucleotide sequence can be converted to polypeptides to alter the properties of the polypeptide or any part of the polypeptide. It can be reflected in the domain of other peptides to be added.

The term “stringent” is used to describe a condition that is commonly recognized as strict in the art. Stringent conditions include highly stringent conditions (ie 0.5 M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS), hybridization of filter-bound DNA at 65 ° C. in 1 mM ETDA and 0.1 × SSC / 0. Washing at 68 ° C. with 1% SDS) and moderately stringent conditions (ie, washing at 42 ° C. with 0.2 × SSC / 0.1% SDS) are also included. Other exemplary hybridization conditions are described in the examples herein.

  In the example of deoxyoligonucleotide hybridization, exemplary hybridization conditions include washing with 6 × SSC / 0.05% sodium pyrophosphate at 37 ° C. (for 14 base oligonucleotides), 48 ° C. (for 17 base oligonucleotides). ), 55 ° C. (for 20 base oligonucleotides), and 60 ° C. (for 23 base oligonucleotides).

(5.3 Targeting with KIRHy antigen)
According to one embodiment of the invention, a composition comprising a KIRHy polypeptide that stimulates the immune system against KIRHy to target KIRHy-expressing cells is provided. The use of tumor antigens in compositions that provide cellular and human immunity for the purpose of anti-cancer treatment is well known in the art. For example, one type of tumor-specific antigen composition includes an idiot that is isolated from tumor cells, conjugated to hemocyanin (KLH) from shellfish, mixed with adjuvant, and injected into patients with mild follicular lymphoma. Type proteins are included (Hsu, et al., Blood 89: 3129-3135 (1997), which is incorporated herein by reference in its entirety). US Pat. No. 6,312,718, incorporated herein by reference in its entirety, describes a method of eliciting an immune response against malignant B cells, particularly lymphoma, chronic lymphocytic leukemia, and multiple myeloma Is described. The methods described in this patent include (1) an antigen related to the malignancy of at least one B cell, (2) IL-2 alone, or a combination with at least one cytokine or chemokine, and (3 ) Use a vaccine comprising liposomes with at least one lipid molecule. Methods for targeting KIRHy with a KIRHy antigen generally use KIRHy polypeptides, including fragments, analogs, and variants.

  As another example, dendritic cells, a type of antigen presenting cell, can be used in a cellular vaccine, which is isolated from a patient, cultured with tumor antigens, and reinjected as a cellular vaccine (Hsu, et al., Nat. Med. 2: 52-58 (1996), which is incorporated herein by reference in its entirety).

  Combinations of this antigen therapy with other types of therapeutic agents used in treatments such as chemotherapy or radiation therapy are also possible.

(5.4 Targeting with nucleic acids)
(5.4.1 Direct delivery of nucleic acids)
In some embodiments, a nucleic acid encoding KIRHy or a fragment, analog, or variant thereof in a recombinant vector is used. Such methods are known in the art. For example, an immune response can be elicited by injecting naked DNA. When injected into mice, plasmid DNA that expresses bicistronic mRNA encoding both light and heavy chains of tumor idiotype proteins from B cell lymphomas and the like can elicit a protective anti-tumor response (its Singh, et al., Vaccine 20: 1400-1411 (2002)), which is incorporated by reference herein in its entirety. KIRHy viral vectors are particularly useful for delivering nucleic acids encoding KIRHy of the present invention to cells. Examples of vectors include vectors from influenza, adenovirus, cowpox, herpes simplex virus, fowlpox, vesicular stomatitis virus, canarypox virus, poliovirus, adeno-associated virus, lentivirus, and Sindbis virus. Of course, non-viral vectors such as liposomes or even naked DNA are useful for delivering nucleic acids encoding KIRHy of the present invention to cells.

  Combinations of this type of treatment with other types of therapeutic agents used in treatments such as chemotherapy or radiation therapy are also possible.

(5.4.2 Nucleic acids expressed in cells)
In some embodiments, a vector comprising a nucleic acid encoding a KIRHy polypeptide (including fragments, analogs or variants thereof) is introduced into a cell such as a dendritic cell or macrophage. When expressed in antigen-presenting cells (APC), antibodies on the surface of KIRHy cells are presented to T cells, which induce an immune response against KIRHy. Such methods are also known in the art. Methods for introducing tumor antigens into APC and vectors useful therefor are described in US Pat. No. 6,300,090, which is hereby incorporated by reference in its entirety. A vector encoding KIRHy can be introduced into APCs in vivo. Alternatively, KIRHy or a nucleic acid encoding KIRHy can be introduced into APC ex vivo and then introduced into a patient to elicit an immune response against KIRHy. In another embodiment, cells presenting a KIRHy antigen are used to stimulate the development of anti-KIRHy cytotoxic T cells (CTLs) ex vivo, followed by introduction of the stimulated CTL into the patient ( US Pat. No. 6,306,388, which is incorporated herein by reference in its entirety.

  Combinations of this type of treatment with other types of therapeutic agents used in treatments such as chemotherapy or radiation therapy are also possible.

(5.4.3 antisense nucleic acid)
Another aspect of the invention pertains to isolated antisense nucleic acid molecules that can hybridize to or be complementary to KIRHy nucleotide sequences, or fragments, analogs, or variants thereof. An “antisense” nucleic acid has a nucleotide sequence that is complementary to a “sense” nucleic acid that encodes a protein (eg, complementary to the coding strand of a double-stranded cDNA or complementary to an mRNA sequence). In specific embodiments, an antisense nucleic acid that is complementary to at least 10 nucleotides, 25 nucleotides, 50 nucleotides, 100 nucleotides, 250 nucleotides, or 500 nucleotides of a KIRHy coding strand, or the entire KIRHy coding strand, or only a portion thereof. A molecule is provided. Also provided are nucleic acid molecules that encode fragments, homologues, derivatives, and analogs of KIRHy or antisense nucleic acids that are complementary to a KIRHy nucleic acid sequence.

  In one embodiment, the antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a KIRHy protein. The term “coding region” means a region of a nucleotide sequence that includes codons translated into amino acid residues. In other embodiments, the antisense nucleic acid molecule is antisense to a “acceptable region” of the coding strand of a nucleotide sequence encoding a KIRHy protein. The term “acceptable region” means 5 ′ and 3 ′ sequences adjacent to the coding region that are not translated into amino acids (ie, regions also referred to as 5 ′ and 3 ′ untranslated regions).

  Given the coding strand sequence encoding the KIRHy protein disclosed herein, the antisense nucleic acids of the invention can be designed according to the rules of Watson and Click or the Hoogstein base pairing rule. The antisense nucleic acid molecule can be complementary to the entire coding region of KIRHy mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or non-coding region of KIRHy mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of KIRHy mRNA. Antisense oligonucleotides can be, for example, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, 45 nucleotides, or 50 nucleotides in length. The antisense nucleic acids of the invention can be prepared by chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) are natural nucleotides, as well as duplexes formed to increase the biological stability of a molecule or between an antisense nucleic acid and a sense nucleic acid. It can be chemically synthesized using nucleotides that are variously modified to increase physical stability (eg, phosphorothioate derivatives and acridine substituted nucleotides can also be used).

  Examples of modified nucleotides that can be used for the preparation of antisense nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyl. Hydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcuocin, inosine, N6-isopentenyladenine, 1-methylguanine, 1- Methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino Methyl 2-thiouridine, β-D-mannosylcuocin, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutosine, pseudouracil, cuocin 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2 -Thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Alternatively, an antisense nucleic acid is one in which the nucleic acid is subcloned in the antisense orientation (ie, the RNA transcribed from the inserted nucleic acid is in the antisense orientation relative to the target nucleic acid in question, Can be biologically prepared using expression vectors (described further in the section).

  An antisense nucleic acid molecule of the invention is a cell that is generally administered to a patient or produced in situ to inhibit protein expression (eg, by inhibiting transcription and / or translation) and encodes a KIRHy protein. Hybridize with or bind to cellular mRNA and / or genomic DNA. Hybridization may be by conventional nucleotide complementarity to form a stable duplex or, for example, in the case of an antisense nucleic acid molecule that binds to a DNA duplex, a large duplex. It may be due to a specific interaction in the groove. An example of a route for administering an antisense nucleic acid molecule of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be denatured into target-selected cells and administered systemically. For example, for systemic administration, an antisense molecule is denatured to specifically bind to a receptor or antigen expressed on a selected cell surface (eg, an antisense nucleic acid molecule is combined with a cell surface receptor or antigen). By conjugation with a peptide or antibody that binds). Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. In order to deliver a sufficient amount of nucleic acid molecule, a vector structure in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter is preferred.

  In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. The α-anomeric nucleic acid molecule forms a specific double-stranded hybrid with the complementary RNA, in which the strands run squarely to each other, as opposed to the normal α-unit. See, for example, Gaultier, et al. , Nucl. Acids Res. 15: 6625-6641 (1987). Antisense nucleic acid molecules can be 2′-o-methylribonucleotides (see, eg, Inoue, et al., Nucl. Acids Res. 15: 6131-6148 (1987)) or chimeric RNA-DNA analogs (see, eg, Inoue, et al. al., FEBS Lett. 215: 327-330 (1987)), all of which are incorporated herein by reference in their entirety.

(5.4.4 Ribozyme component and PNA component)
In yet another embodiment, the antisense nucleic acid of the invention is a ribozyme. A ribozyme is a catalytic RNA having a ribonuclease activity that can cleave a single-stranded nucleic acid such as mRNA to form a complementary region to the ribozyme. Thus, ribozymes (eg, hammerhead structural ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591)) can be used for catalytic cleavage of mRNA transcripts, thereby inhibiting translation of mRNA. it can. A ribozyme having specificity for the nucleic acids of the invention can be designed based on the nucleotide sequence of the DNA disclosed herein (ie, SEQ ID NOs: 1-2). For example, Tetrahymena L-19 IVS RNA can be prepared to be complementary to the nucleotide sequence in the mRNA where the active site nucleotide sequence is cleaved. For example, Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, the mRNA of the present invention can be used to select catalytic RNA having specific ribonuclease activity from a population of RNA molecules. For example, Bartel et al. (1993) Science 261: 1411-1418.

  Alternatively, by targeting a nucleotide sequence that is complementary to a regulatory region (eg, promoter and / or enhancer) that forms a triple helix structure, transcription of the gene in the target cell is blocked and gene expression is inhibited. be able to. Helene. (1991) Anticancer Drug Des. 6: 569-84; Helene. (1992) Ann. N. Y. Acad. Sci. 660: 27-36; Maher (1992) Bioassays 14: 807-15.

  In various embodiments, the base moiety, sugar moiety, or phosphate ester backbone of the nucleic acids of the invention can be modified to improve, for example, stability, hybridization, or molecular solubility. For example, the deoxyribose phosphate backbone of nucleic acids can be modified to produce peptide nucleic acids (see Hyrup et al. (1996) Bioorg. Med. Chem. 4: 5-23). As used herein, the term “peptide nucleic acids” or “PNAs” refers to nucleic acid mimetics in which the deoxyribose phosphate backbone is replaced with a pseudo-peptide backbone and only four natural nucleobases remain. For example, it means a DNA mimetic. The neutral backbone of PNAs has been shown to allow specific hybridization with DNA and RNA under low ionic strength conditions. The synthesis of PNAs oligomers is described in Hyrup et al. (1996); Perry-O'Keefe et al. (1996) PNAS 93: 14670-675 can be performed according to standard solid phase peptide synthesis protocols.

  The PNAs of the present invention can be used for therapeutic and diagnostic applications. For example, PNAs can be used as antisense or anti-gene agents that cause sequence-specific denaturation of gene expression by inducing transcriptional or translational arrest or preventing replication. The PNAs of the present invention can be used as artificial restriction enzymes when used in combination with other enzymes such as S1 nuclease (Hyrup B. (1996) above); or as DNA sequences and hybridization probes or primers (see above). Hyrup et al. (1996); above-mentioned Perry-O'Keefe et al. (1996)), can also be used for analysis of single base pair mutations of genes by PCR clamping for PNA.

  In other embodiments, the PNAs of the present invention can be used to bind PNA-DNA chimeras by, for example, conjugating lipophilic groups or other useful groups to PNAs to enhance their stability or cellular uptake. It can be modified by forming or by using liposomes or other drug delivery techniques known in the art. For example, PNA-DNA chimeras can be prepared to combine the advantageous properties of PNA and DNA. Such chimera allows DNA to recognize enzymes that interact with DNA, such as liponuclease H and DNA polymerase, while the PNA moiety provides high binding affinity and specificity. PNA-DNA chimeras can be attached using linkers of suitable lengths selected based on base stacking, number of linkages between nucleobases, or orientation (Hyrup (1996) above). The synthesis of PNA-DNA chimeras is described in the literature of Hyrup (1996) and Finn et al. (1996) Nucl. Acids Res. 24: Can be performed as described in 3357-63. For example, a DNA strand can be synthesized on a solid support using standard phosphoramidite conjugation methods and nucleic acid analogs such as 5 ′-(4-methoxytrityl) that can bind PNA to the 5 ′ end of DNA. It can be denatured with amino-5′-deoxy-thymidine phosphoramidites (Mag et al. (1989) Nucl. Acids Res. 17: 5973-88). The PNA monomer is then sequentially coupled to prepare a chimeric molecule from the 5 'PNA segment and the 3' DNA segment (Finn et al. (1996) above). Alternatively, a chimeric molecule can be synthesized from a 5 'DNA segment and a 3' PNA segment. Petersen et al. (1975) Bioorg. Med. Chem. Lett. 5: 1119-11124.

  In other embodiments, the oligonucleotides are other adducts such as peptides (eg to target host cell receptors in vivo) or cell membranes (eg Letsinger et al., 1989, Proc. Natl. Acad. Sci. U. S. A. 86: 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84: 648-652: PCT Application WO 88/09810) or the blood-brain barrier (see For example, agents that facilitate transport across PCT application WO 89/10134) may be included. Furthermore, oligonucleotides can be triggered by cleaving agents (see, eg, Krol et al., 1988, BioTechniques 6: 958-976) or intercalating agents (see, eg, Zon, 1988, Pharm. Res. 5: 539-549). Can be modified by hybridization. For denaturation, oligonucleotides can be conjugated to other molecules, such as peptides, cross-linking agents, hybrids triggered by transport agents, hybrids triggered by cleavage agents, and the like.

(5.4.5 KIRHy nucleic acid)
An isolated polynucleotide of the present invention comprises a polynucleotide comprising any of the nucleotide sequences of SEQ ID NOs: 1-2; a fragment of SEQ ID NOs: 1-2; and a full-length protein encoding the sequence of SEQ ID NOs: 1-2. Including, but not limited to, a polynucleotide comprising a nucleotide sequence (eg, encoding SEQ ID NO: 3); and a nucleotide sequence encoding the mature protein sequence of any one of SEQ ID NOs: 1-2 . The polynucleotide of the present invention is, under stringent conditions, (a) the complement of any of the nucleotide sequences of SEQ ID NOs: 1-2; (b) any one of the polypeptides of SEQ ID NOs: 3-7, 11 (C) a polynucleotide that is an allelic variant of any of the polynucleotides listed above; (d) a polynucleotide that encodes a homologue of any of the proteins listed above; or ( e) A polynucleotide that forms a hybrid with a polynucleotide that encodes a polypeptide containing a specific domain or truncation of the polypeptide of SEQ ID NOs: 3 to 7, 11 is also included, but is not limited thereto. The domain of interest depends on the nature of the encoded polypeptide, eg, a receptor-like polypeptide domain, including a ligand-binding, extracellular, transmembrane, or cytoplasmic domain, or combinations thereof; variable A domain of an immunoglobulin-like protein, including an active immunoglobulin-like domain; a domain of an enzyme-like polypeptide, including a catalytic domain and a substrate-binding domain; and a domain of a ligand polypeptide, including a receptor-binding domain. It's okay.

  The polynucleotides of the present invention include natural DNA or all or part of synthetic DNA, such as cDNA, and genomic DNA, and RNA, such as mRNA. The polynucleotides of the present invention can comprise the entire coding region of a cDNA or present a portion of the coding region of a cDNA.

  The present invention also provides genes corresponding to the cDNA sequences disclosed herein. Corresponding genes can be isolated according to known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for gene identification and / or amplification in a suitable genomic library or other source of genomic material. In addition, 5 'and 3' sequences can be obtained using methods known in the art. For example, by using any of the polynucleotides of SEQ ID NO: 1-2 or a part thereof as a probe and screening an appropriate cDNA or genomic DNA library under suitable hybridization conditions, SEQ ID NO: 1-2 A full-length cDNA or genomic DNA corresponding to any of the polynucleotides can be obtained. Alternatively, the polynucleotides of SEQ ID NOs: 1-2 can be used as the basis for suitable primers that allow the identification and / or amplification of genes in an appropriate cDNA or genomic DNA library.

  The nucleic acid sequences of the present invention can be constructed from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases such as dbEST, gbpri, and UniGene. . EST sequences can provide sequence identification information, representative fragment or segment information, or new segment information for the entire gene length.

  The polynucleotides of the present invention also provide polynucleotides comprising a nucleotide sequence substantially equivalent to the polynucleotides listed above. A polynucleotide according to the invention is, for example, at least about 65%, at least about 70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least about 90%, 91%, 92%, 93%, or 94%, more typically at least about 95%, 96 %, 97%, 98%, or 99% sequence identity.

  Within the scope of the nucleic acid sequences of the invention are fragments of the polynucleotides of SEQ ID NOs: 1-2 whose fragments are more than 5 nucleotides, preferably more than 7 nucleotides, more preferably more than 9 nucleotides, most preferably more than 17 nucleotides. Nucleic acid sequence fragments that hybridize under stringent conditions with either or its complement are included. Fragments that are selective (ie, specifically hybridize to any one of the polynucleotides of the invention), eg, 15, 17, or 20 nucleotides or more are envisioned. A probe capable of specifically hybridizing with a polynucleotide can distinguish between a polynucleotide sequence of the invention and other polynucleotide sequences in the same gene family, or can distinguish between a human gene and another species of gene, and Preferably based on a unique nucleotide sequence.

  Sequences falling within the scope of the invention are not limited to these specific sequences, but also include allelic variants and species variants. Allelic variants and species variants are at least 90% homologous, preferably 95% homologous to the sequence shown in SEQ ID NOs: 1-2, representative fragments thereof, or SEQ ID NOs: 1-2. It can be determined mechanically by comparison with a nucleic acid sequence having a sequence from one type of another isolate. In addition, to accommodate for codon variation, the present invention also includes nucleic acid molecules that encode the same amino acid sequences as encoded by the specific ORFs disclosed herein. In other words, replacement of one codon with another codon encoding the same amino acid in the coding region of the ORF is clearly envisaged.

  Nucleic acids closest to the nucleic acids of the present invention, including SEQ ID NOs: 1-2, can be obtained by searching the database using an algorithm or program. To search for local sequence orientation, it is preferred to use BLAST, a basic local orientation search tool (Altshu, SF, J. Mol. Evol. 36: 290-300 (1993). And Altschul, SF, et al., J. Mol. Biol.21: 403-410 (1990)).

  Congeners (ie, orthologs) of the disclosed polynucleotide and protein species are also provided by the present invention. By preparing suitable probes or primers from the sequences provided herein and searching for suitable nucleic acid sources of the desired species, species homologs can be isolated or identified.

  The present invention relates to allelic variants of the disclosed polynucleotides and proteins, ie, natural forms separate from isolated polynucleotides that encode proteins identical, homologous, or related to the proteins encoded by the polynucleotides. Are also included.

  The nucleic acid sequence of the present invention also covers sequences encoding the aforementioned nucleic acid variants. These amino acid sequence variants can be prepared by introducing suitable nucleotide changes into the natural or variant polynucleotides by methods known in the art. There are two variables in the construction of amino acid sequence variants. The location of the mutation and the nature of the mutation. The nucleic acid encoding the amino acid sequence variant is preferably constructed by mutating the polynucleotide encoding the amino acid sequence to make it non-natural. These nucleic acid modifications can be made at different nucleic acid sites (variable sites) or highly conserved regions (stable regions) depending on the species. The site at such a position is generally continuously modified, for example, by first performing a conservative modification (eg, replacing a hydrophobic amino acid with another hydrophobic amino acid), followed by a larger modification (eg, hydrophobic The target amino acid is replaced with a charged amino acid) and then removed or inserted into the target site. In general, the removal of amino acid sequences extends from 1 to 30 residues, preferably 1 to 10 residues, usually adjacent. Amino acid insertions include amino and / or carboxyl terminal terminal fusions, ranging in length from 1 residue to 100 residues or more, as well as intrasequence insertions of one or more amino acid residues. In general, intrasequence insertions may range from 1 to 10 amino acid residues, preferably 1 to 5 residues. Examples of terminal insertions include heterologous signal sequences necessary for insertion or intracellular targeting in different host cells, and sequences such as FLAG® or polyhistidine sequences useful for purification of the expressed protein.

  In a preferred method, a polynucleotide encoding a novel amino acid sequence is modified by site-directed mutagenesis. In this method, a polynucleotide encoding the desired amino acid variant as well as a sufficiently close nucleotide is prepared so that both side chains of the modified amino acid on either side of the modified site form a stable duplex. The oligonucleotide sequence to be modified is used. In general, site-directed mutagenesis techniques are well known to those skilled in the art and are described in Edelman et al. , DNA 2: 183 (1983). A convenient and efficient method for inducing site-specific mutations in polynucleotide sequences is described by Zoller and Smith, Nucleic Acids rev. 10: 6487-6500 (1982). PCR can also be used to prepare amino acid sequence variants of novel nucleic acids. When a small amount of template DNA is used as a starting material, a desired amino acid variant is produced with a primer slightly different in sequence from the corresponding region in the template DNA. PCR amplification produces large amounts of DNA fragments that differ in the site specified by the primer from the polynucleotide template encoding the polypeptide. This DNA fragment product replaces a corresponding region within the pyramide, resulting in a polynucleotide encoding the desired amino acid variant.

  Another technique for generating amino acid variants is described by Wells, et al. , Gene 34: 315 (1985), and other mutagenesis techniques well known in the art are described in, for example, Sambrook, et al. , Supra, and Current Protocols in Molecular Boilology, Ausubel, et al. The technology described in,. Due to the degeneracy inherent in the genetic code, other DNA sequences encoding substantially identical or functionally equivalent amino acid sequences can be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include DNA sequences that can form hybrids with suitable novel nucleic acid sequences under stringent conditions.

  A polynucleotide encoding a preferred polypeptide short body of the invention can be used to produce a polynucleotide encoding a chimeric or fusion protein comprising one or more of the domains of the invention and a heterologous protein sequence.

  The polynucleotides of the present invention further include the complement of any of the polynucleotides listed above. The polynucleotide may be DNA (genomic DNA, cDNA, amplified or synthesized) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art, for example, to determine hybridization conditions that can mechanically isolate a polynucleotide identical to the desired sequence. A method is also included.

  A polynucleotide sequence encoding any one of SEQ ID NO: 3 or a functional equivalent thereof according to the present invention and comprising a mature protein sequence is capable of expressing the nucleic acid or a functional equivalent thereof in a suitable host cell. Can be used to produce the desired recombinant DNA molecule. Also included are cDNA inserts of any of the clones identified herein.

  Polynucleotides according to the present invention can be produced by a variety of other nucleotide sequences using established recombinant DNA technology (see Sambrook, J. et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Can be combined with either. Nucleotide sequences useful for binding to polynucleotides include vector combinations well known in the art, such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like. Accordingly, the present invention also provides a vector comprising the polynucleotide of the present invention and a host cell comprising the polynucleotide. In general, a vector includes a replication source that functions in at least one organism, a convenient restriction endonuclease site, and a selectable host cell marker. Vectors according to the present invention include expression vectors, replication vectors, probe production vectors, and sequencing vectors. A host cell according to the present invention may be a prokaryotic cell or a eukaryotic cell, and may be a unicellular organism or part of a multicellular organism.

  The present invention also provides a recombinant structure comprising a nucleic acid having any of the nucleotide sequences of SEQ ID NOs: 1-2 or fragments thereof or any of the other polynucleotides of the present invention. In one embodiment, the recombinant structure of the invention comprises a plasmid or viral, into which a nucleic acid having any of the nucleotide sequences of SEQ ID NOs: 1-2 or a fragment thereof has been inserted in a forward or backward orientation. Includes vectors such as vectors. Where the vector includes one of the ORFs of the invention, the vector further includes regulatory sequences, including, for example, a promoter operably linked to the ORF. Many suitable vectors and promoters are known to those skilled in the art and are commercially available for constructing the recombinant structures of the present invention. For illustration, the following vectors are shown. Bacterial vectors: pBs, phagescript, PsiX174, PBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); : PWneo, pSV2cat, pOG44, PXTI, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

  In order to produce recombinant proteins, the isolated polynucleotides of the present invention can be prepared using the method of Kaufman et al. Nucleic Acids Res. 19: 4485-4490 (1991), and can be operably linked to an expression control sequence such as a pMT2 expression vector or a pED expression vector. Many suitable expression control sequences are known in the art. General methods for expressing recombinant proteins are also known. Kaufman, Methods in Enzymology 185: 537-566 (1990). As used herein, “operably linked” refers to a host cell into which an isolated polynucleotide and expression control sequence of the invention is transformed (transfected) by the polynucleotide / expression control sequence to be combined. Means that the protein is introduced into a vector or into a cell so that the protein is expressed.

  The promoter region can be selected from any desired gene using a CAT (chloramphenicol transferase) vector or other vector with a selectable marker. Two preferred vectors are pKK232-8 and pCM7. Specific presentable bacterial promoters include lacI, lacZ, T3, T7, gpt, λPR, and trc. Eukaryotic promoters include early CMV, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of suitable vectors and promoters is well possible for those skilled in the art. In general, recombinant expression vectors contain a replication source and a selectable marker such as E. coli that permit transformation of the host cell. E. coli ampicillin resistance gene and S. coli. cerevisiae TRP1 gene, as well as promoters from highly expressed genes that direct transcription of downstream structural sequences are included. Such promoters are obtained from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-elements, acid phosphatases, heat shock proteins, and others. The heterologous structural sequence is constructed from a translation initiation sequence and a translation termination sequence in an appropriate phase, more preferably from a leader sequence capable of directing secretion of the translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein comprising an amino terminal identification peptide that confers desired properties, eg, stabilization or purification of the expressed recombinant product. Useful expression vectors for use in bacteria are constructed by inserting a functional promoter into a structural DNA sequence encoding a suitable translation start signal and translation stop signal in the reading phase operable with the desired protein. The vector includes one or more phenotypic selectable markers as well as a source of replication that ensures maintenance of the vector and allows amplification in the host if desired. Suitable prokaryotic hosts for transformation include E. coli. Various species within E. coli, Bacillus subtilis, Salmonella typhimurium, and the genera Pseudomonas, Streptomyces, and Staphylococcus are included, but others can be used as a matter of choice.

  As a representative but non-limiting example, a useful expression vector for use in bacteria is a bacterial replication derived from a commercially available plasmid containing a selectable marker as well as the genetic elements of the well known cloning vector pBR322 (ATCC 37017). Sources can be included. Such commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, WI, UAS). These “backbone” parts of pKK223 are combined with a suitable promoter and the structural sequence to be expressed. After a suitable host strain has been transformed and the host strain has grown to a suitable cell density, the selected promoter is induced or repressed by suitable means (eg, temperature change or chemical induction) and the cells are further cultured. Is done. Usually, the cells are recovered by centrifugation, disrupted by physical or chemical means, and the crude extract is removed for subsequent purification.

  The polynucleotide of the present invention can also be used for mutagenesis. See, eg, Fan, et al., Incorporated herein by reference. , Nat. Biotech. 17: 870-872 (1999), the nucleic acid sequence encoding the polypeptide can be administered to the encoded polypeptide after local administration or injection of naked plasmid DNA, preferably after intramuscular injection of DNA. Can be used to form antibodies. The nucleic acid sequence is preferably inserted into a recombinant expression vector, but may be in the form of naked DNA.

(5.4.6 Gene therapy)
Mutations in the polynucleotides of the present invention can cause the encoded protein to lose normal function. Thus, the present invention provides gene therapy that reconstructs the normal activity of a polypeptide of the invention; or treats a pathological condition involving a polypeptide of the invention. Ex vivo, in situ by delivery of a functional gene encoding a polypeptide of the present invention to a suitable cell by use of a vector, more specifically a viral vector (eg, adenovirus, adeno-associated virus, or retrovirus). , Or in vivo, or ex vivo through the use of physical DNA delivery methods (eg, liposomes or chemical therapy). See, for example, Anderson, Nature, 392 (Suppl): 25-20 (1998). For a review of gene therapy techniques, see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990): and Miller, Nature, 357: 455-460 (1992). . All of which are incorporated herein by reference in their entirety. Introduction of a gene encoding any one of the nucleotides of the present invention or the polypeptide of the present invention is also achieved by extrachromosomal material (transient expression) or artificial chromosome (stable expression). In order to grow such cells or to produce the desired effect or activity on such cells, the cells can be cultured ex vivo in the presence of the protein of the invention. For treatment, the treated cells can then be introduced in vivo. Alternatively, in other human pathologies, it is envisaged that blocking the expression or inhibiting the activity of the polypeptides of the invention will be useful for the treatment of the pathology. It is envisioned that antisense or gene therapy is applied to negatively regulate the expression of the polypeptides of the invention.

  Another method of blocking protein expression includes introducing an antisense molecule into the nucleic acid of the invention, its complement, or its translated RNA sequence by methods known in the art. Furthermore, the polypeptides of the invention can be inhibited using targeted removal methods or by insertion of negative regulators such as tissue-specific silencers.

  The invention also provides a cell that has been genetically engineered in vivo to express a polynucleotide of the invention, wherein the polynucleotide is heterologous to the host cell that expresses the polynucleotide in the cell. Functionally linked to regulatory sequences. This method can be used to increase or decrease the expression of a polynucleotide of the invention.

  The knowledge of the DNA sequences provided by the present invention allows for the denaturation of cells that allow the expression, increase or decrease of expression of heterologous polypeptides. By replacing all or part of a native promoter with all or part of a heterologous promoter so that the cell expresses the protein at a high level, the cell is denatured to increase expression of the polypeptide (e.g., heterologous By recombination). The heterologous promoter is inserted so as to be operably linked to the sequence encoding the desired protein. See, for example, PCT application WO 94/12650, PCT application WO 92/20808, and PCT application WO 91/09955. All of which are incorporated herein by reference in their entirety. In addition to heterologous promoter DNA, heterologous marker DNA (eg, ada, dhfr, and multifunctional CAD genes encoding carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and / or intron DNA It can be inserted together with promoter DNA. Once bound to the sequence encoding the desired protein, the sequence encoding the desired protein is also amplified together in the cell if the marker DNA is amplified by standard sorting methods.

  In other embodiments of the invention, cells and tissues can be modified to express a heterologous gene comprising a polynucleotide of the invention under the control of an inducible regulatory element, wherein the regulatory element of the heterologous gene is Can be replaced by heterologous recombination. Using gene targeting to replace an existing regulatory region of a gene with a regulatory sequence isolated from another species or a novel regulatory sequence synthesized by genetic engineering methods, as described herein Can do. Such regulatory sequences can be included in promoters, enhancers, scaffold attachment regions, negative regulatory elements, transcription initiation sites, regulatory protein binding sites, or combinations of the above sequences. Alternatively, targeting can replace, remove, add, or add other modifications to sequences that affect the structure or stability of the RNA or protein produced. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences that enhance or denature the transport or secretion properties of proteins, and other that modify or improve the function or stability of a protein or RNA molecule. Contains an array.

  Targeting may be simple insertion of regulatory sequences, placing the gene under the control of new regulatory sequences, eg, inserting a new promoter or enhancer or both upstream of the gene. Alternatively, targeting may be simple removal of regulatory elements such as removal of tissue specific negative regulatory elements. Alternatively, targeting can be a replacement of an existing element, for example, a tissue specific enhancer can be replaced with an enhancer having a broader or different cell type specificity than the native element. In this case, the natural sequence is removed and a new sequence is added. In all of these, target identification uses one or more selectable marker genes that flank the target DNA and allow selection of cells that have exogenous DNA integrated into the cell's genome. By making it easier. Target identification is such that negative selectable markers are placed adjacent to the target sequence even when bound to exogenous DNA, and strictly homologous recombination between sequences within the host cell genome is negative selectable. It is also facilitated by using one or more marker genes that exhibit negative selection properties that do not result in stable integration of the marker. Markers useful for this include the herpes simplex virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene.

  Gene targeting and gene activation techniques that can be used in accordance with this aspect of the present invention are described in Chapel US Pat. No. 5,272,071, Sherwin et al. US Pat. No. 5,578,461, Selen et al. PCT / US92 / 09627 (International Publication No. WO 93/09222), Skultchi et al. International Application No. PCT / US90 / 06436 (International Publication No. 91/06667), all of which are described in their entirety. Is incorporated herein by reference.

(5.5 Anti-KIRHy antibody)
Alternatively, immune targeting includes the administration of components of the immune system, such as antibodies to the target, antibody fragments, or sensitized cells of the immune system. Methods for immunotargeting cancer cells using antibodies or antibody fragments are well known in the art. US Pat. No. 6,306,393 describes the use of anti-CD22 antibodies in immunotherapy of B cell malignancies and US Pat. No. 6,329,503 expresses a tortuous transmembrane antigen. Immunotargeting of cells has been described (both US patents are hereby incorporated by reference in their entirety).

  (Humanized antibody or human monoclonal antibody, or fragment thereof) so that the antibody binds to KIRHy expressed by cancer cells, mediates destruction of cancer cells and tumors and / or inhibits growth of cancer cells and tumors, Alternatively, other modified products thereof, including those optionally conjugated to cytotoxic agents) KIRHy antibodies can be introduced into the patient. The mechanisms by which such antibodies exert therapeutic effects include complement-mediated cell lysis, antibody-dependent cellular cytotoxicity (ADCC), alteration of the physiological function of KIRHy, inhibition of binding or signal transduction pathways, differentiation of tumor cells , Alteration of tumor angiogenesis factor profile, alteration of immunostimulatory or tumor suppressor cytokine and growth factor secretion, alteration of cell adhesion, and / or induction of apoptosis. Absent. A KIRHy antibody conjugated to a toxin agent or therapeutic agent such as a radioligand or cytosolic toxin can also be used as a therapy to deliver the toxin agent or therapeutic agent directly to tumor cells bearing KIRHy.

  KIRHy antibodies can be used to suppress the immune system in patients undergoing organ transplantation or in autoimmune diseases such as arthritis. Healthy immune cells become the target of these antibodies and die or are expelled from the system, suppressing the immune system.

  The KIRHy antibody can be used as an antibody therapy for solid tumors that exhibit this effect. Cancer immunotherapy using KIRHy antibodies provides a new method of treating cancers involving cells that specifically express KIRHy. As described above, KIRHy is highly expressed in AML and histiocytic lymphoma cell lines because KIRHy is a target for therapeutic antibodies or a diagnostic marker for some cell types or disorders (eg AML). It can be used as Immunotherapy using antibodies against other types of cancer has already been reported, including colon cancer (Arlen et al., Crit. Rev. Immunol. 18: 133-138 (1998)), multiple myeloma ( Ozaki et al., Blood 90: 3179-3186 (1997); B cell lymphoma (Funakoshi et al., J. Immunother. Emphasis Tumor Immunol. 19: 93-101 (1996)), leukemia (Zhong et al., Le. uk Res.20: 581-589 (1996)), colorectal cancer (Moun et al., Cancer Res. 54: 6160-6166 (1994); )), And breast cancer (Shepard et al., J. Clin. Immunol. 11: 117-127 (1991)). All of the above references are incorporated herein by reference in their entirety.

  KIRHy antibody therapy is useful for all stages of existing cancer, but is particularly suitable for advanced and metastatic cancers. A combination of antibody therapy and chemotherapy, radiation therapy, or surgical therapy is preferred for patients who have not been treated with chemotherapy, whereas treatment with antibody therapy has received one or more chemotherapy. It is shown to the patient who came. Furthermore, in combination with antibody therapy, the dose of chemotherapy can be reduced, especially for patients who are very tolerant to the toxicity of chemotherapeutic agents. Also, treatment of cancer patients with tumors resistant to chemotherapeutic agents with KIRHy antibody provides sensitivity and responsiveness to the chemotherapeutic agents used in combination.

  Prior to anti-KIRHy immunotargeting, the patient preferably exhibits an immunohistochemical assessment of tumor tissue, quantitative KIRHy imaging, quantitative RT-PCR, or other KIRHy1 presence and level of expression Other reliable methods that can be used can be evaluated for the presence and level of expression of KIRHy by cancer cells. For example, blood or biopsy samples can be evaluated by immunohistochemical methods to determine the presence or absence of KIRHy-expressing cells or to determine the extent of KIRHy expression on the surface of cells in the sample. Methods for immunohistochemical analysis of KIRHy fragments released into tumor tissue or serum are well known in the art.

  Anti-KIRHy antibodies useful for the treatment of cancer include anti-KIRHy antibodies that can cause a strong immune response against tumors and anti-KIRHy antibodies that can directly damage cells. In this regard, anti-KIRHy monoclonal antibodies can induce tumor cell lysis by either complement-mediated or ADCC mechanisms, including both effector cell Fc receptor sites or complement proteins. A complete Fc portion of an immunoglobulin molecule is required to interact. Furthermore, anti-KIRHy antibodies that have a direct biological effect on tumor growth are useful in the practice of the invention. The mechanisms by which such direct cytotoxic antibodies may function include inhibition of cell growth, modification of cell differentiation, modification of tumor angiogenesis factor profile, and induction of apoptosis. The mechanism by which a particular anti-KIRHy antibody exerts an anti-tumor effect can be determined using any number of in vivo assays designed to determine ADCC, ADMMC, complement-mediated cell lysis, etc., as is known in the art. Can be evaluated.

  The anti-tumor activity of a particular anti-KIRHy antibody or combination of anti-KIRHy antibodies can be assessed in vivo using a suitable animal model. An example is a cross-species AML cancer model in which human AML cells have been introduced into immune-deficient animals such as nude mice or SCID mice. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression, or metastasis, etc.

  Note that the use of mouse or other non-human monoclonal antibodies, human / mouse chimeric mAbs, can cause a moderate to strong immune response in some patients. In the most severe cases, the immune response can lead to widespread formation of immune complexes and cause renal failure. Thus, the monoclonal antibody used in the practice of the therapeutic methods of the present invention is a fully human monoclonal antibody or a fully humanized monoclonal antibody that binds to the target KIRHy antigen with high affinity but does not Does not show or shows low sex.

  The methods of the invention envisage administration of anti-KIRHy monoclonal antibodies (mAbs) alone and in combination, or “reaction mixtures” of different mAbs. Two or more monoclonal antibodies that bind to KIRHy show an improved effect compared to a single antibody. Alternatively, a combination of an anti-KIRHy antibody and an antibody that binds to a different antigen shows an improved effect compared to a single antibody. Such mAb reaction mixtures have several advantages, as well as including combinations of mAbs that use different effector mechanisms or direct cytotoxic mAbs and immune effector functions. Such combination mAbs can show a synergistic therapeutic effect. Further, the anti-KIRHy mAbs administered can be combined with other therapeutic agents including, but not limited to, various chemotherapeutic agents, antigen blocking agents, and immunodegenerative agents (eg, IL-2, GM-CSF). It is not something. Anti-KIRHy mAbs can be administered “naked”, ie, unbound, or can be conjugated to a therapeutic agent. In addition, bispecific antibodies can be used. Such an antibody has one antigen-binding domain specific for KIRHy and one antigen-binding domain specific for another antigen (for example, CD20). Finally, Fab KIRHy antibodies or fragments of those antibodies (including fragments bound to other protein sequences or toxins) can also be used as therapeutic agents.

Antibodies that specifically bind to KIRHy are useful in compositions and methods for immunotargeting cells that express KIRHy and for diagnosing diseases or disorders in which the cells are impaired by KIRHy expression. Such antibodies include monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional / bispecific antibodies, humanized antibodies, human antibodies, and complementarity determining regions (CDRs) that specifically recognize KIRHy. And / or complementarity determining region (CDR) grafted antibodies comprising a compound having an antigen binding sequence. Fab, Fab ', F (ab ') 2, and _{F v,} including a fragment of the antigen may also be useful.

  The term “specific” means that the variable region of an antibody exclusively recognizes and binds KIRHy (ie, regardless of sequence identity, homology, or similarity found within a family of polypeptides) KIRHy can be distinguished from similar polypeptides) but with other proteins (eg, S. aureus protein A or other antibodies in the ELISA method) by interaction between sequences other than the variable region of the antibody, particularly the stable region of the molecule. It is shown that the interaction is possible. Screening assays in antibodies that can assess the binding specificity of anti-KIRHy antibodies are well known in the art and are routinely performed (Chapter 6, Antibodies A, which is incorporated herein by reference in its entirety). Laboratory Manual, Eds. Harlow, et al., Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988)).

  KIRHy polypeptides can be used for immunization of animals to obtain polyclonal and monoclonal antibodies that specifically react with KIRHy. Such antibodies can be obtained using the whole protein or a fragment thereof as an immunogen. The peptide immunogen may further contain a cysteine residue at the carboxyl terminus and may be bound to a partial antigen such as hemocyanin (KLH) derived from mussel. Methods for synthesizing such peptides have already been reported (Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963), both of which are hereby incorporated by reference in their entirety; Krstenansky, et al., FEBS Lett. 211: 10 (1987)). Methods for preparing polyclonal and monoclonal antibodies with fusion cells capable of producing the desired antibody have also been previously disclosed (Cambell, Monoclonal Antibodies technology: Laboratory Technologies in Biochemistry, all of which are incorporated herein by reference in their entirety). Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth, et al., J. Immunol. 35: 1-21 (1990); 975), trioma technology, and human B cell fusion cell technology, see Kozbor, et al., Immunology Today 4: 72 (1983); Cole, et al., In Monoclonal Antibodies and Cancer Ther. pp. 77-96 (1985)).

  Any animal capable of producing antibodies can be immunized with a KIRHy peptide or polypeptide. Methods of immunization include subcutaneous and intraperitoneal injection of the polypeptide. The amount of KIRHy peptide or polypeptide used for immunization depends on the animal to be immunized, the antigenicity of the peptide, and the site of injection. A KIRHy peptide or polypeptide used as an immunogen can be denatured or administered with adjuvants to increase the antigenicity of the protein. Methods for increasing the antigenicity of a protein are well known in the art and include, but are not limited to, binding of an antigen to a heterologous protein (such as globulin or β-galactosidase) or inclusion of an adjuvant during immunization. It is not done.

  For monoclonal antibodies, spleen cells are removed from the immunized animal and fused with myeloma cells such as SP2 / O-Ag14 myeloma cells to form a fusion cell that produces monoclonal antibodies. Any one of a number of methods well known in the art can be used to identify fusion cells that produce antibodies with the desired properties. These methods include screening of fused cells by ELISA assay, Western blot analysis, or radioimmunoassay (Lutz, et al., Exp. Cell Res. 175: 109, incorporated herein by reference in its entirety). -124 (1988)). Fusion cells secreting the desired antibody are cloned and the class and subclass determined using methods known in the art (Cambell, AM Monoclonal Antibody, which is incorporated herein by reference in its entirety). Technology: Laboratory Technologies in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984). Techniques described for the production of single chain antibodies (US Pat. No. 4,946,778, incorporated herein by reference in its entirety) are applicable to the production of KIRHy single chain antibodies.

  For polyclonal antibodies, antiserum is removed from the immunized animal and antibodies having the desired specificity are screened using one of the means described above.

  Since antibodies derived from rodents are likely to induce a strong immune response against the antibodies when administered to humans, the effectiveness of such antibodies in the therapeutic methods of the present invention is limited. Methods for producing antibodies that do not cause a strong immune response to the administered antibody are well known in the art. For example, the anti-KIRHy antibody may be a non-human primate antibody. Methods for preparing such antibodies in baboons are described in PCT application WO 91/11465 and Losman et al. , Int. J. et al. Cancer 46: 310-314 (1990), which is incorporated herein by reference in its entirety. In one embodiment, the anti-KIRHy antibody is a humanized monoclonal antibody. Methods for producing humanized antibodies have already been reported. (US Pat. Nos. 5,997,867 and 5,985,279, Jones et al., Nature 321: 522 (1986); all of which are incorporated herein by reference in their entirety; Riechmann et al. , Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988); Carter et al., Proc. Nat'l Acad. Sci. Crit. Rev. Biotech.12: 437-465 (1992); and Singer, et al., J.Immun.150: 2844-2857 (199). )). In other embodiments, the anti-KIRHy antibody is a human monoclonal antibody. Humanized antibodies are produced by transgenic mice that have been transformed to produce human antibodies. Such mouse-derived fusion cells secrete large amounts of human monoclonal antibodies. Methods for obtaining human antibodies from transgenic mice are described in Green, et al. , Nature Genet. 7: 13-21 (1994); Lonberg, et al. , Nature 368: 856 (1994); and Taylor, et al. , Int. Immun. 6: 579 (1994), all of which are incorporated herein by reference in their entirety.

The present invention also includes the use of anti-KIRHy antibody fragments. Antibody fragments can be obtained by proteolysis of the antibody or E. coli. It can be regulated by the expression of DNA encoding the fragment in E. coli. Antibody fragments are obtained by digesting the entire antibody with pepsin or papain. For example, antibody fragments can be prepared by enzymatic cleavage of antibodies with pepsin into a 5S fragment termed F (ab ′) ₂ . This fragment can be further cleaved using a thiol reducing agent, optionally blocking the sulfhydryl groups resulting from the cleavage of disulfide bonds to give 3.5SFab ′ monovalent fragments. Alternatively, two monovalent Fab fragments and an Fc fragment can be directly prepared by enzymatic cleavage with pepsin. These methods have already been described (US Pat. Nos. 4,036,945 and 4,331,647, Nisonoff, et al., Arch Biochem, all of which are hereby incorporated by reference in their entirety. Biophys.89: 230 (1960); Porter, Biochem.J.73: 119 (1959); Edelman, et al., Meth.Enzymol.1: 422 (1967)). Other methods of cleaving antibodies, such as separating long chains to form monovalent short chain fragments, further cleaving fragments, using chemical or genetic methods, are also recognized by intact antibodies. It can be used as long as the fragment binds to the antigen. For example, _{F V} fragments comprise an association of _{V H} and _{V L} chains is likely to be the non-covalent (incorporated by reference in their entirety herein, Inbar et al., Proc. Nat'l Acad. Sci. USA 69: 2659 (1972)). Alternatively, the variable chains can be linked by intramolecular disulfide bonds or crosslinks with chemicals such as glutaraldehyde.

In one embodiment, the F _V fragment comprises a V _H chain and a V _L chain joined by a linker. These short antigens bound to a protein (sF _V ) are prepared by constructing a structural gene comprising DNA sequences encoding the V _H and V _L domains linked by an oligonucleotide. The structural gene is inserted into an expression vector and then E. coli. into a host cell such as E. coli. The recombined host cell synthesizes a single polypeptide chain that bridges the two V domains with a linker peptide. Methods of preparing sF _V is incorporated has been previously described (the whole, all of these reference herein, U.S. Patent No. 4,946,778, Whitlow, et al, Methods :. A Companion to Methods in Enzymology 2: 97 (1991); Bird, et al., Science 242: 423 (1988); Pack, et al., Bio / Technology 11: 1271 (1993)).

  Another form of antibody fragment is a peptide that encodes a single complementarity determining region (CDR). CDR peptides (“minimal recognition units”) are obtained by constructing a gene that encodes the CDRs of the antibody in question. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize variable regions from the RNA of antibody producing cells (all of which are incorporated herein by reference, Larick, et al. ., Methods: A Companion to Methods in Enzymology 2:... 106 (1991); Courtenay-Luck, pp 166-179 in, Monoclonal Antibodies Production, Engineering and Clinical Applications, Eds Ritter et al, Cambridge University Press (1995) Ward, et al., Pp 137-185 in, Monoclonal Antibodies Principles and Applications, Eds. Birch et al., Wiley-Liss, Inc. (1995)).

  The present invention further provides the above antibodies in a detectably labeled form. Detection of antibodies using radioisotopes, affinity labels (biotin, avidin, etc.), enzymatic labels (horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (FITC or rhodamine, etc.), paramagnetic atoms, etc. Can be labeled. Methods for performing such labeling have already been disclosed (Sternberger, et al., J. Histochem. Cytochem. 18: 315 (1970), all of which are hereby incorporated by reference in their entirety; Bayer, et al., Meth. Enzym.62: 308 (1979); Engval, et al., Immunol.109: 129 (1972); Goding, J. Immunol.Meth.13: 215 (1976)).

  Labeled antibodies can be used in in vitro, in vivo, and in situ assays to identify cells or tissues in which KIRHy is expressed. Furthermore, the labeled antibody can be used to confirm the presence or absence of KIRHy secreted in biological samples such as blood, urine, saliva samples.

(5.5.1 Anti-KIRHy antibody conjugate)
The present invention contemplates the use of immunoconjugates as well as the use of “naked” anti-KIRHy antibodies. Immunoconjugates can be prepared by indirectly conjugating therapeutic agents such as cytotoxic agents to antibody components. Cytotoxic components include, for example, plant toxins such as abrin, ricin, modesin, biscumin, pokeweed antiviral protein, saporin, gelonin, momoridine, trichosanthin, barley toxin; Diptheria toxin, Pseudomonas endotoxin Exotoxins, bacterial toxins such as Staphylococcal enterotoxins; fungal toxins such as α-sarcin, restricotocin; cytotoxic ribonucleases such as extracellular pancreatic ribonuclease; deoxyribonuclease I (incorporated herein by reference in its entirety) Pastan, et al., Cell 47: 641 (1986); Goldenberg, Cancer Journal for Clinicians 44: 43 (1994)), calichiamycin, And radioactive isotopes such as ³² P, ⁶⁷ Cu, ⁷⁷ As, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹²¹ Sn, ¹³¹ I, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹⁴ Ir, ¹⁹⁹ Au, etc. Are incorporated herein by reference in their entirety, including Illidge and Block, Curr. Pharm. Design 6: 1399 (2000)). In humans, clinical trials with yttrium-90 conjugated to anti-CD20 antibodies for B-cell lymphoma are ongoing (Cancer Chemother Pharmacol 48 (Suppl 1), which is incorporated herein by reference in its entirety): S91-S95 (2001)).

  General techniques have already been described (US Pat. Nos. 6,306,393 and 5,057,313, Shih, et al., Int, all of which are hereby incorporated by reference in their entirety. J. Cancer 41: 832-839 (1988); Shih, et al., Int. J. Cancer 46: 1101-1106 (1990)). Common methods include the addition of an antibody component having an oxidized carbohydrate moiety and a plurality of drugs, toxins, chelators, boron additives, or other therapeutic agents that have at least one free amino group. Reaction with other carrier polymers. This reaction first forms a Schiff base (imine) bond, which is reduced to a secondary amine for stabilization and finally forms a conjugate.

  The carrier polymer is preferably aminodextran or a polypeptide of at least 50 amino acid residues, although other substantially equivalent polymer carriers can be used. The final immunoconjugate is preferably soluble in an aqueous solution, such as mammalian serum, for ease of administration and efficient targeting when used in therapy. Therefore, if there is a solubilizing group on the carrier polymer, dissolution of the final immunoconjugate in serum is facilitated. Specifically, aminodextran is preferable.

Typically, the process of preparing an immunoconjugate from an aminodextran carrier begins with the preparation of a dextran polymer, preferably a dextran polymer having an average molecular weight of about 10,000 to 100,000. Dextran reacts with an oxidizing agent, and the sugar ring part is oxidized to an aldehyde group by control. Oxidation is conveniently performed using a glycolytic agent such as NaIO ₄ according to conventional practice. Oxidized dextran is then reacted with a polyamine, preferably a diamine, more preferably a monohydroxydiamine or polyhydroxydiamine. Suitable amines include ethylenediamine, propylenediamine, or similar polymethylenediamine, diethylenetriamine or similar polyamine, 1,3-diamino-2-hydroxypropane, or other similar hydroxylated diamine or polyamine, etc. . An excess of amine relative to the aldehyde group of dextran is used to ensure conversion of the aldehyde group to a Schiff base group. In order to stably reduce the resulting Schiff salt intermediate, a reducing agent such as NaBH ₄ or NaBH ₃ CN is used. The resulting adduct can be purified by removing cross-linked dextran through a conventional sizing column or ultrafiltration membrane. Conventional methods of inducing dextran to introduce an amino group, such as reacting with cyanogen bromide and then reacting with an amine, can also be used.

  The aminodextran is then loaded in the activated form, preferably in the form of a carboxy-active derivative prepared using conventional means such as dicyclocarbodiimide (DCC) or a water-soluble variant thereof. Reacts with certain drugs, toxins, chelators, immunomodulators, boron additions, or derivatives of other therapeutic agents to form intermediate adducts. Alternatively, polypeptide toxins such as pokeweed antiviral protein or lysine A chain can be coupled to aminodextran by glutaraldehyde condensation, ie reaction of the activated carboxyl group of the protein with the amino group of aminodextran.

  Chelating agents for radioactive metals or magnetic resonance enhancing agents are well known in the art. Typical chelating agents are derivatives of ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Generally, these chelating agents have a group for bonding to a carrier on the side chain. Such groups include, for example, a benzylisothiocyanate group by which DTPA or EDTA can be attached to the amino group of the carrier. Alternatively, the carboxyl group or amino group on the chelator can be attached to the carrier by activation or pre-induction and subsequent attachment, all by well-known means.

  Boron additives such as carborane can be bound to antibody components by conventional methods. For example, as is well known in the art, carboranes can be prepared with a carboxyl group on the side chain. Binding of such carborane to a carrier, such as aminodextran, is achieved by activation of the carboxyl group of the carborane and condensation with an amino group on the carrier, producing an intermediate conjugate. Such intermediate conjugates are then coupled to antibody components, resulting in pharmacologically useful immunoconjugates as described below.

  A polypeptide carrier can be used in place of aminodextran, but the polypeptide carrier must have a chain of at least 50 amino acid residues, preferably 100-5000 amino acid residues. At least some of the amino acids must be lysine residues, glutamic acid residues or aspartic acid residues. The pendant amino group of lysine residues and the pendant carboxyl group of glutamate and aspartate are advantageous for conjugation with drugs, toxins, chelators, immunomodulators, boron additives, or other therapeutic agents. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and amino acids and others that confer to loaded carriers and immunoconjugates that provide the desired solubility characteristics. And a mixture of polymers of amino acids such as serine.

Conjugation of the intermediate conjugate with the antibody component involves the oxidation of the carbohydrate portion of the antibody component and the resulting aldehyde (and ketone) carbonyl group and drug, toxin, chelator, immunomodulator, boron additive, or other This is carried out by reacting with the amino group remaining on the carrier after loading with the therapeutic agent. Alternatively, the intermediate conjugate can bind to the oxidized antibody component via an amino group introduced into the intermediate conjugate after loading with the therapeutic agent. Oxidation is conveniently performed either chemically, for example using NaIO ₄ or other glycolytic agents, or enzymatically using, for example, neuraminidase and galactose oxidase. In the case of an aminodextran carrier, usually not all amino groups of aminodextran are used to load the therapeutic agent. The amino group of the remaining aminodextran condenses with the oxidized antibody component to form a Schiff base adduct, which is then reductively stabilized, usually with a borohydride reducing agent.

  Similar methods are used for the preparation of other immunoconjugates according to the invention. It is preferable that a free lysine residue for condensation with the oxidized carbohydrate moiety of the antibody component remains in the polypeptide carrier after loading. The carboxyl group on the polypeptide carrier can be converted to an amino group if necessary, for example by activation with DCC and reaction with excess diamine.

  Finally, the immunoconjugate is purified using conventional techniques such as sizing chromatography on Sephacryl S-300 or affinity chromatography using one or more KIRHy antigenic sites.

  Alternatively, immunoconjugates can be prepared by conjugating the antibody component directly with a therapeutic agent. The general procedure is similar to the indirect conjugation method, except that the therapeutic agent binds directly to the oxidized antibody component. It should be appreciated that other therapeutic agents can be substituted with the chelating agents described herein. Engineers in the field should be able to determine conjugation procedures without undue experimentation.

  As an additional explanation, the therapeutic agent can bind to the hinge region of the reduced antibody component by disulfide bond formation. For example, a tetanus toxin peptide can be constructed having one cysteine residue that is used to attach the peptide to the antibody component. Alternatively, such peptides can be synthesized using N-succinyl 3- (2-pyridyldithio) propionate (SPDP) (Yu, et al., Int. J., incorporated herein by reference in its entirety. Heterobifunctional crosslinkers such as Cancer 56: 244 (1994)) can be used to bind to antibody components. General techniques for such conjugation have already been described (Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press (1991), all of which are hereby incorporated by reference in their entirety; Upeslacis, et pp. 187-230 in, Monoclonal Antibodies Principes and Applications, Eds. Birch et al., Wiley-Liss, Inc. (1995); Price, pp. n. editions, Eds. Ritter et al., University University Press (1995)).

  As noted above, the carbohydrate portion of the Fc region of an antibody can be used to conjugate a therapeutic agent. However, if an antibody fragment is used as the antibody component of the immunoconjugate, there may be no Fc region. However, the carbohydrate moiety can be introduced into the light chain variable region of an antibody or antibody fragment (Leung, et al., J. Immunol. 154: 5919-5926, both of which are hereby incorporated by reference in their entirety. (1995); U.S. Pat. No. 5,443,953). The introduced carbohydrate moiety is used for binding with the therapeutic agent.

  In addition, those skilled in the art will appreciate that various conjugation methods can be modified. For example, the carbohydrate moiety can be used to bind to polyethylene glycol to increase the half-life of the intact antibody or antigen-binding fragment of the antibody in the blood, lymph, or other extracellular fluid. Furthermore, it is possible to construct a “bivalent immunoconjugate” by coupling a therapeutic agent to the carbohydrate moiety and a free sulfhydryl group. Such free sulfhydryl groups can be placed in the hinge region of the antibody component.

(5.5.2 Anti-KIRHy antibody fusion protein)
Where the therapeutic agent conjugated to the antibody is a protein, the present invention contemplates the use of a fusion protein comprising one or more anti-KIRHy antibody portions and an immunomodulator or toxin portion. Methods for preparing antibody fusion proteins have already been described (US Pat. No. 6,306,393, which is hereby incorporated by reference in its entirety). Antibody fusion proteins comprising an interleukin-2 component have also been previously disclosed (Boleti, et al., Ann. Oncol. 6: 945 (1995), all of which are incorporated herein by reference in their entirety; Nicolet, et al., Cancer Gene Ther. 2: 161 (1995); Becker, et al., Proc. Nat'l. Acad. Sci. USA 93: 7826 (1996); 2: 1951 (1996); Hu, et al., Cancer Res. 56: 4998 (1996)). Furthermore, Yang, et al., Which is incorporated herein by reference in its entirety. , Hum. Antibodies Hybridomas 6: 129 (1995) describes a fusion protein comprising an F (ab ′) ₂ fragment and the α component of tumor necrosis factor.

  Methods are also known in the art for preparing antibody-toxin fusion proteins having therein a recombinant molecule comprising one or more antibody components and a toxin or chemotherapeutic agent. For example, an antibody-Pseudomonas exotoxin A fusion protein has been described (Chaudary, et al., Nature 339: 394 (1989); all of which are incorporated herein by reference in their entirety; Brinkmann, et al. Nat'l.Acad.Sci.USA 88: 8616 (1991); Batra, et al., Proc.Natl.Acad.Sci. Immunol.150: 3054 (1993); Wels, et al., Int. J. Can.60: 137 (1995); Chem 271:.. 10560 (1996); Kuan, et al, Biochemistry 35:... 2872 (1996); Schimdt, et al, Int J. Can 65: 538 (1996)). Antibody-toxin fusion proteins containing diphtheria toxin components have been described (Kreitman, et al., Leukemia 7: 553 (1993); all of which are incorporated herein by reference in their entirety); Nichols, et al., J. Biol. Chem. 268: 5302 (1993); Thompson, et al., J. Biol.Chem. 270: 28037 (1995); Deonarain et al. (Tumor Targeting 1: 177 (1995)) describe an antibody-toxin fusion protein with a ribonuclease component, while Linardou et al. (Cell Biophys. 24-25: 243 (1994)) describe a deoxynuclease. An antibody-toxin fusion protein containing component I was prepared. All of the above references are incorporated herein by reference in their entirety. Gelonin and Staphylococcal enterotoxin-A are used as toxin components in antibody-toxin fusion proteins (both Wang, et al., Abstracts of the 209th ACS National Meeting, incorporated herein by reference in their entirety. Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005; Dohlsten, et al., Proc. Nat'l. Acad. Sci.

(5.5.3 Fab fragment and single chain KIRHy antibody)
In accordance with the present invention, techniques can be adapted to produce single chain antibodies specific for KIRHy (see, eg, US Pat. No. 4,946,778). In addition, _Fab expression libraries (eg, Huse, et al.) That allow rapid and efficient identification of monoclonal _Fab fragments with the desired specificity for a protein or derivative, fragment, analog or homologue thereof. _{., Science 246: 2 1275-1281 (} 1989) refer) of can adapt the technology for the construction. Antibody fragments containing idiotypes against protein antigens are: (i) preparation of F _{(ab ′) 2} fragments by pepsin digestion of antibody molecules; (ii) preparation of F _ab fragments by reduction of disulfide bridges of F (ab ′) ₂ fragments. Preparation; (iii) preparation of _Fab fragments by treatment of antibody molecules with papain and reducing agents; and (iv) can be prepared by techniques known in the art including, but not limited to, _Fv fragments. Absent.

(5.5.4 Bispecific KIRHy antibody)
Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In this case, one of the binding specificities is for the antigenic protein of the present invention. The second binding target is for any other antigen, preferably a cell surface protein or receptor or receptor subunit.

  Methods for preparing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, with the two heavy chains having different specificities (Milstein and Cuello, Nature). 305: 537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these fused cells can produce a mixture of 10 different antibody molecules, only one of which is the correct bispecific structure. It is. Purification of the correct molecule is usually done by affinity chromatography. Similar procedures are described in WO 93/08829 (Publication May 13, 1993) and Traunecker et al. , 1991 EMBO J. et al. , 10, 3655-3659.

  The variable region of an antibody having the desired binding specificity (antibody-antigen combining site) can be fused to an immunoglobulin stable region. This fusion preferably occurs in an immunoglobulin heavy chain stable region, including at least a portion of the hinge, CH2, and CH3 regions. It is preferred to have a first heavy chain stability region (CH1) containing at least the site necessary for light chain binding present in one of the fusions. The immunoglobulin heavy chain and, if desired, the immunoglobulin light chain, the coding DNA are inserted into separate expression vectors and introduced together into a suitable host organ. For details of bispecific antibody production, see, for example, Suresh et al. , Methods in Enzymology, 121: 210 (1986).

  According to another method described in WO 96/27011, the interface between antibody molecule pairs can be modified to maximize the proportion of heterodimers recovered from the recombinant cell culture. it can. Preferred interfaces include at least a portion of the CH3 region of the antibody stability region. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are associated with larger side chains (eg tyrosine or tryptophan). Large amino acid side chains are replaced with small amino acid side chains (eg, alanine or threonine) to form a compensatory “cavity” of the same or similar size as the large side chain on the interface of the second antibody molecule. . This is the mechanism by which the yield of heterodimers is increased over undesired end products such as homodimers.

Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg F (ab ′) ₂ bispecific antibodies). Techniques for preparing bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. , Science 229: 81 (1985) describe a method in which intact antibodies are cleaved with proteolytic enzymes to produce F (ab ′) ₂ fragments. These fragments are reduced in the presence of sodium arsenate, a dithiol complexing agent that stabilizes adjacent dithiols and prevents intermolecular disulfide formation. The resulting Fab ′ fragment is then converted to a thionitrobenzoic acid (TNB) derivative. One of the Fab′-TNB derivatives is reduced with mercaptoethylamine, converted to Fab′-thiol, and mixed with equimolar other Fab′-TNB derivatives to form bispecific antibodies. The generated bispecific antibody can be used as an enzyme selective immunizing agent.

In addition, Fab ′ fragments are can be recovered directly from E. coli and chemically coupled to form bispecific antibodies. Sharaby et al. , J. et al. Exp. Med. 175: 217-225 (1992) describes the generation of _two molecules of the fully specific bispecific antibody F (ab ′). Each Fab ′ fragment is separately E. coli. It is secreted from E. coli and is subject to in vitro direct chemical coupling, forming a bispecific antibody. The bispecific antibody thus formed can bind to cells overexpressing ErbB2 receptor and normal human T cells, and can also trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. .

Various techniques for generating and directly isolating bispecific antibody fragments in recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol. 148: 1547-1553 (1992)). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab ′ part of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Hollinger et al. , Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. This fragment has a heavy chain variable region (V _H ) joined to a light chain variable region (V _L ) by a linker that is too short to pair two domains on the same chain. Accordingly, the V _H region and the V _L regions of one fragment are not forced to pair with the complementary V _L and V _H regions of another fragment, thereby two antigen-binding sites are formed. Another method for preparing bispecific antibody fragments using single chain F _V (sF _v ) dimers has also been reported. Gruber et al. , J. et al. Immunol. 152: 5368 (1994).

  Antibodies with an antibody valency of 2 or more are envisioned. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147: 60 (1991)).

  A typical bispecific antibody can bind to two different antigenic sites, at least one of which is derived from a protein antigen of the invention. Alternatively, to focus cellular defense mechanisms on cells that express a particular antigen, the anti-antigenic arm of an immunoglobulin molecule is a leukocyte such as a T cell receptor molecule (eg, CD2, CD3, CD28, or B7). It can bind to the above trigger molecule or an arm that binds to Fc receptors for IgG (FcγR) such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Bispecific antibodies can also be used as direct cytotoxic agents against cells that express a particular antigen. These antibodies have an antigen-binding arm and an arm that binds to a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody binds to the protein antigen described herein and further binds to tissue factor (TF).

(5.5.5 heteroconjugated KIRHy antibody)
Heteroconjugate antibodies are also within the scope of the present invention. A heteroconjugated antibody consists of two covalently bound antibodies. Such antibodies, for example, target immune system cells to unwanted cells (US Pat. No. 4,676,980) or treat HIV infection (WO 91/00360; WO 92 / No. 23033; European Patent No. 03089). These antibodies are expected to be prepared in vitro using known methods of synthetic protein chemistry, including those using cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for construction include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in US Pat. No. 4,676,980.

(5.5.6 Effector functional engineering)
For example, it is desirable to modify the antibodies of the present invention with effector functions in order to enhance the effectiveness of the antibodies in cancer therapy. For example, cysteine residues can be introduced into the Fc region, thereby forming an interchain disulfide bond in this region. The homodimeric antibody thus generated can have improved uptake capacity and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Caron et al. , J. et al. Exp. Med. 176: 1191-1195 (1992) and Shopes, J. et al. Immunol. 148: 2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity are described in Wolff et al. , Cancer Research, 53: 2560-2565 (1993), can also be prepared using heterobifunctional crosslinkers. Alternatively, the antibody can be modified to have two Fc regions, thereby enhancing complement solubility and ADCC ability. Stevenson et al. , Anti-Cancer Drug Design, 3: 219-230 (1989).

(5.6 KIRHy polypeptide)
The isolated polypeptide of the present invention includes an amino acid sequence represented by any one of SEQ ID NOs: 3 to 7, 11 or an amino acid sequence encoded by any one of the nucleotide sequences of SEQ ID NOs: 1-2 Including, but not limited to, a polypeptide comprising a full-length protein or mature protein. The polypeptide of the present invention preferably has a biological activity or an immunological activity, and (a) a polynucleotide having any one of the nucleotide sequences represented by SEQ ID NOs: 1-2 or (b) a sequence A polynucleotide encoding any one of the amino acid sequences represented by the numbers 3 to 7, 11 or (c) hybridized with the complement of the polynucleotide of either (a) or (b) under strict hybridization conditions Also included are polypeptides encoded by polynucleotides that form The present invention relates to any of the polypeptide amino acid sequences set forth in SEQ ID NOs: 3-7, 11 or corresponding and “substantial equivalence” (eg, at least about 65%, at least about 70%, at least about 75% , At least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more usually at least about 90%, 91%, 92%, 93%, or 94%, even more commonly at least about 95%, 96%, 97%, 98%, or 99%, most commonly at least about 99% amino acid identity) Also provided are biologically or immunologically active variants of full-length or mature proteins of proteins that retain pharmacological activity. The polypeptide encoded by the allelic variant can have the same, increased, or decreased activity compared to the polypeptide having SEQ ID NOs: 3-7,11.

  Also included in the present invention are fragments of the proteins of the present invention that can exhibit biological activity. Protein fragments may be linear and may be obtained by known methods such as H.P. U. Saragovi, et al. , Bio / Technology 10: 773-778 (1992) and R.A. S. McDowell, et al. , J. et al. Amer. Chem. Soc. 114: 9245-9253 (1992). Both documents are incorporated herein by reference in their entirety. Such a fragment can be fused to a carrier molecule such as an immunoglobulin for a number of purposes, including increasing the valency of the protein binding site.

  The invention also provides both full-length forms of the disclosed proteins and mature forms (eg, lacking signal or precursor sequences). The protein coding sequence is identical to the sequence listed by translation of the disclosed polynucleotide sequence. A mature form of such a protein can be obtained by expression of the full-length polynucleotide in a suitable mammalian cell or other host cell. The mature protein sequence can also be determined from the full-length amino acid sequence. If the protein of the invention binds to the cell membrane, a soluble form of the protein is also provided. In the soluble form, some or all of the region that binds the protein to the cell membrane has been removed, and the protein is all secreted from the expressed cells.

  The protein composition of the present invention may further comprise an acceptable carrier, such as a hydrophilic carrier, for example a pharmaceutically acceptable carrier.

  The invention further provides an isolated polypeptide encoded by a nucleic acid fragment of the invention or a degenerate variant of the nucleic acid fragment of the invention. A “degenerate variant” means a nucleic acid fragment that differs in nucleic acid sequence from a nucleic acid fragment of the present invention (eg, ORF), but encodes the same polypeptide sequence due to the degeneracy of the genetic code. Preferred nucleic acid fragments of the invention are ORFs that encode proteins.

  Various techniques known in the art can be used to obtain any one of the isolated polypeptides or proteins of the invention. At the simplest level, amino acid sequences can be synthesized using commercially available peptide synthesizers. A protein sequence that is synthetically constructed with the same primary, secondary, or tertiary structure and / or conformational properties as the protein can have the same biological properties, including protein activity. This technique is particularly useful in the preparation of small peptide and large polypeptide fragments. Fragments are useful, for example, in generating antibodies against natural polypeptides. Thus, fragments can be used as biologically active or immunological substitutes for natural, purified proteins in immunological processes for therapeutic compound screening and antibody development.

  The polypeptides and proteins of the invention can alternatively be purified from cells that have been modified to express the desired polypeptide or protein. As used herein, a cell expresses a desired polypeptide or protein when genetically manipulated such that the cell does not normally produce or even produces low levels of the polypeptide or protein. Is said to have been modified. A person skilled in the art will be able to introduce a recombinant or synthetic sequence into a prokaryotic or eukaryotic cell for expression in order to generate a cell that produces one of the polypeptides or proteins of the invention. Can be easily adapted.

  The invention also relates to a method for producing a polypeptide comprising growing the host cells of the invention in a suitable culture medium and purifying the protein obtained from the cells or the culture medium in which the cells have grown. For example, the methods of the invention produce a polypeptide in which a host cell containing a suitable expression vector having a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide. Process for including. The polypeptide can be recovered by purifying a lysate prepared from the culture solution, conveniently from the culture medium, or from the host cells. Preferred embodiments include those in which the protein produced by such a process is a full-length or mature protein.

  In another method, the polypeptide or protein is purified from bacterial cells that naturally produce the polypeptide or protein. One skilled in the art can readily isolate polypeptides and proteins according to known methods to obtain one of the isolated polypeptides or proteins of the invention. Isolation methods include, but are not limited to, immunochromatography, HPLC, size exclusion chromatography, ion exchange chromatography, and immunoaffinity chromatography. See, for example, Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al. , In Molecular Cloning: A Laboratory Manual; Ausubel et al. , Current Protocols in Molecular Biology. Polypeptide fragments that retain biological / immunological activity include fragments containing more than about 100 amino acids, or more than about 200 amino acids, as well as fragments encoding particular protein domains.

  The purified polypeptide can be used in in vitro binding assays well known in the art to identify molecules that bind to the polypeptide. Molecules that bind to a polypeptide include, but are not limited to, for example, small molecules, combinatorial library molecules, antibodies, or other proteins. The molecules identified in the binding assay are then subjected to an antagonist test or antagonist activity test in an in vivo tissue culture or animal model, as is well known in the art. Briefly, molecules are titrated into multiple cell culture media or animals and tested to see if cells / animals die or that animal cell / cell life spans are extended.

  Furthermore, the peptides of the present invention or molecules capable of binding to peptides can be conjugated to toxins, such as lysine or cholera toxin, or other compounds that are toxic to cells. Toxin binding molecule complexes can be targeted to tumors or other cells by molecular binding specificity for SEQ ID NOs: 3-7,11.

  The protein of the present invention is transformed into a product of a transformed animal, eg, a transformed bovine, goat, pig or sheep, characterized in that the somatic cell or germ cell contains a nucleotide sequence encoding the protein of the present invention. It can also be expressed as a component of milk.

  Proteins provided herein also include proteins characterized in that the amino acid sequence is identical to that of the purified protein, but that contain natural or artificial modifications therein . For example, modifications in peptide or DNA sequences can be performed using techniques known to those skilled in the art. Modifications in the protein sequence of interest include modification, substitution, replacement, insertion or removal of selected amino acid residues within the coding sequence. For example, one or more cysteine residues can be removed or replaced with other amino acids to alter the conformation of the molecule. Such alteration, substitution, replacement, insertion, or removal techniques are well known to those skilled in the art (see, eg, US Pat. No. 4,518,584). It is preferred that the desired activity of the protein is retained after such modification, substitution, replacement, insertion, or removal. Protein regions important for protein function can be determined by various methods known in the art, including systematically replacing one or a stretch of amino acids with alanine and resulting alanine-containing Also included is an alanine scanning method to test the biological activity of the variant. This type of analysis determines the importance of the replaced amino acid in the biological activity. Regions of the protein that are important for protein function can be determined using the eMATRIX program.

  Other fragments and derivatives of the protein sequence, all or part of which retain the activity of the protein and which may be useful for screening and other immunological techniques, are readily accessible to those skilled in the art given the disclosure herein. Can be prepared. Such modifications are also included in the present invention.

Proteins can also be produced by operably linking an isolated polynucleotide of the present invention to one or more insect expression vectors and using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are described, for example, in Invitrogen, San Diego, Calif. , U. S. A. (The MaxBat ^™ kit), which can be purchased in kit form, and are generally used in Summers and Smith, Texas Agricultural Experiment Bulletin No. (incorporated herein by reference in their entirety). 1555 (1987), such methods are well known in the art. As used herein, an insect cell capable of expressing a polynucleotide of the invention is “transformed”.

  The protein of the present invention can be produced by culturing transformed host cells under culture conditions suitable for expression of the recombinant protein. The resulting expressed protein is then purified from the culture broth (ie, from the culture medium or cell extract) using known purification methods such as gel filtration and ion exchange chromatography. For protein purification, an affinity column containing reagents that bind to the protein; one or more columns on an affinity resin such as concanavalin A-agarose, heparin-toyopearl ™, or Cibachrom blue 3GA Sepharose ™ Also included are one or more column stages including hydrophobic interaction chromatography using a resin such as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

  Alternatively, the protein of the present invention may be expressed in a form that facilitates purification. For example, the protein of the present invention can be expressed as a fusion protein such as maltose-binding protein (MPB), glutathione-S-transferase (GST), or thioredoxin (TRX), or as a His tag. Kits for expression and purification of such fusion proteins can be purchased from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, NJ), and Invitrogen, respectively. Proteins can be purified using specific antibodies that tag antigenic sites and are subsequently directed to such antigenic sites. One such antigenic site (“FLAG®”) can be purchased from Kodak (New Haven, Conn.).

  Finally, the protein is further purified using, for example, one or more reverse phase high performance liquid chromatography (RP-HPLC) using silica gel as a medium with pendant methyl groups or other pendant aliphatic groups. be able to. Some or all of the current purification means can be used in various combinations to obtain a substantially homogeneous isolated recombinant protein. Such purified protein is substantially free of other mammalian proteins and is defined as an “isolated protein” according to the present invention.

  The polypeptides of the present invention include analogs (variants). The polypeptides of the present invention include KIRHy analogs. KIRHy analogs include fragments of KIRHy, as well as KIRHy polypeptides in which one or more amino acids have been removed, inserted, or substituted. Also, an analog of KIRHy can include a fusion of KIRHy polypeptide or a modification of KIRHy polypeptide, wherein the KIRHy polypeptide or analog is fused to other component or components, such as a targeting component or another therapeutic agent. The body is also included. Such analogs exhibit improved properties such as activity and / or stability. Examples of components that are fused to a KIRHy polypeptide or analog include, for example, targeting components that deliver the polypeptide to hematopoietic cells or cancer cells.

(5.6.1 Hematopoietic regulatory activity of KIRHy)
KIRHy can be involved in the regulation of hematopoiesis and thus can be involved in the treatment of myeloid or lymphoid cytotoxicity. Peripheral biological activity that helps colony-forming cells or factor-dependent cell lines indicates involvement in hematopoietic regulation. For example, assisting in the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines can treat various anemias, eg, to stimulate production of erythroid progenitor cells and / or erythroid cells Or useful in combination with radiation / chemotherapy. Helping the growth and proliferation of myeloid cells such as granulocytes and monocytes / macrophages (ie conventional CSF activity) is useful in combination with chemotherapy, for example to prevent or treat myelosuppression . Helping the growth and proliferation of megakaryocytes, which later become platelets, enables the prevention and treatment of various platelet disorders such as thrombocytopenia and generally replaces or assists with platelet transfusions. By assisting in the growth and proliferation of hematopoietic stem cells that mature to become any or all of the above hematopoietic cells (usually including but not limited to aplastic anemia and paroxysmal nocturnal hemoglobinuria, In various stem cell disorders (also treated by transplantation), after irradiation / chemotherapy, in vivo or ex vivo (ie simultaneously with bone marrow transplantation) or as normal cells or cells modified for gene therapy Pharmacological utility can be found in the regrowth of stem cell compartments in transplantation of peripheral progenitor cells (homologous or xenogeneic).

  The therapeutic composition of the present invention can be used as follows. A suitable assay for proliferation and differentiation of the various hematopoietic systems listed above.

  Embryonic stem cell differentiation assays (among others identifying proteins that affect hematopoietic embryonic differentiation) include those described in Johansson et al. Cell. Biol. 15: 141-151 (1995); Keller et al. , Mol. and Cell. Biol. 13: 473-486 (1993); McClanahan et al. , Blood 81: 2903-2915 (1993), but is not limited thereto.

  Stem cell viability and differentiation assays (among other things, identifying proteins that regulate lymphatic hematopoiesis) include methylcellulose colony formation assays, Freshney, M. et al. G. in Culture of Hematopoietic Cells, R.C. I. Freshney, et al. , Eds. Vol pp. 265-268, Wiley-Liss, Inc. , New York, N .; Y. 1994; Hirayama et al. , Proc. Natl. Acad. Sci. USA 89: 5907-5911, 1992; primitive hematopoietic colony forming cells with high proliferative ability, McNiece, I .; K. and Bridgedell, R.A. A. In Culture of Hematopoietic Cells, R.C. I. Freshney, et al. , Eds. Vol pp. 23-39, Wiley-Liss, Inc. , New York, N .; Y. 1994; Neben et al. , Exp. Hematol. 22: 353-359, 1994; Cobblestone-forming cell assay, Ploecher, R .; E. In Culture of Hematopoietic Cells, R.C. I. Freshney, et al. , Eds. Vol pp. 1-21, Wiley-Liss, Inc. , New York, N .; Y. 1994; long-term bone marrow culture in the presence of substrate cells, Sponcer, E. Dexter, M .; and Allen, T .; In Culture of Hematopoietic Cells, R.C. I. Freshney, et al. , Eds. Vol pp. 163-179, Wiley-Liss, Inc. , New York, N .; Y. 1994; long-term culture starting cell assay, Sutherland, H .; I. in Culture of Hematopoietic Cells, R.C. I. Freshney, et al. , Eds. Vol pp. 139-162, Wiley-Liss, Inc. , New York, N .; Y. 1994; but is not limited thereto.

(5.6.2 Immunostimulatory activity and immunosuppressive activity of KIRHy)
KIRHy can exhibit immunostimulatory and immunosuppressive activities, including those whose assays are described herein, but the assays are not limited to those described herein. The polynucleotide of the present invention can encode a polypeptide exhibiting such activity. Such proteins are useful for the treatment of various immunodeficiencies and immune disorders (including severe combined immunodeficiency (SCID)), for example (up and down) regulation of T lymphocyte / B lymphocyte growth and proliferation, As well as in activating cytolytic activity of NK cells and other cell populations. These immunodeficiencies may be due to genetic or viral (eg HIV) or even bacterial or fungal infections and may be due to autoimmune disorders. Specifically, infections caused by viruses, bacteria, fungi, or other infections can treat the proteins of the present invention, including HIV, hepatitis virus, herpes virus, mycobacterium, leish Various fungal infections such as Mania, Malaria infections, and candidiasis are included. Of course, in this regard, the proteins of the invention are also useful when additional to the immune system is generally required, ie in the treatment of cancer.

  Autoimmune disorders that can be treated using the proteins of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune lung inflammation, Guillain-Barre syndrome, autoimmune thyroiditis Insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease, and autoimmune inflammatory eye diseases. Those in which the protein of the invention (or an antagonist comprising the antibody) is useful in therapy include allergic reactions and symptoms (eg, hypersensitivity, serum disease, drug reaction, food allergy, insect venom allergy, mast cell hyperproliferation, Allergic rhinitis, hypersensitivity pneumonia, polymorphic exudative erythema, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, gender keratoconjunctivitis, giant papillary conjunctivitis, and contact allergy), asthma (especially allergic asthma) ), Or other respiratory disorders. Other conditions where immunosuppression is desirable (including, for example, organ transplantation) can also be treated using the proteins of the invention (including antagonists thereof). The therapeutic effect of the polypeptide of the present invention or an antagonist thereof can be determined by the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), the skin needle test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and mouse local lymph node assay (Kmber et al., J. Toxicol. Environ. Health 53: 563-79). Can be evaluated by in vivo animal model testing.

  The protein of the present invention can be used to modify the immune response in various ways. Down regulation is to inhibit or block an immune response that is already in progress, ie to participate in inhibiting the induction of an immune response. The function of activated T cells can be inhibited by suppressing T cell responses or introducing specific tolerance into T cells, or both. In general, immunosuppression of T cell responses is an active, non-antigen specific process that requires exposure of T cells to a continuous suppressor. Tolerance, including T cell unresponsiveness or anergy, is distinguished from immunosuppression in that tolerance is generally antigen-specific and persists after exposure to a tolerizing agent. In practice, tolerance can be determined by the absence of a T cell response upon re-exposure to a specific antigen in the absence of a tolerizing agent.

  Down-regulation, ie inhibition of one or more antigen functions (without limiting the antigen function of B lymphocytes (eg B7 etc.)), eg inhibition of high levels of lymphokine synthesis by activated T cells Useful in skin and organ transplantation and graft-versus-host disease (GVHD). For example, blocking T cell function leads to reduced tissue destruction in tissue transplantation. In general, in tissue transplantation, transplant rejection begins with T cells recognizing the graft as foreign, followed by an immune response that destroys the graft. Administration of the therapeutic composition of the present invention inhibits cytokine synthesis by immune cells such as T cells, and the therapeutic composition of the present invention can act as an immunosuppressant. Furthermore, because of the lack of costimulation, T cells are anergized and tolerance is introduced into the subject. The introduction of long-term tolerance with the B lymphocyte antigen blocking agent eliminates the need for repeated administration of the blocking agent. In order to provide sufficient immunosuppression or tolerance within a subject, it is also necessary to block the function of the B lymphocyte antigen combination.

  The effectiveness of a particular therapeutic composition in preventing organ transplant rejection or GVHD can be assessed using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart transplantation in rats and cross-species pancreatic islet cell transplantation in mice, see Lenschow et al. , Science 257: 789-792 (1992) and Turka et al. , Proc. Natl. Acad. Sci. USA, 89: 11102-11105 (1992), both have been used to test the in vivo immunosuppressive effects of CTLA4Ig fusion proteins. Furthermore, the mouse model in GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 844-847) can be used to measure the effect of the therapeutic composition of the present invention on GVHD progression.

  Blocking antigen function is also pharmacologically useful for the treatment of autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are active against their own tissues and promote the production of cytokines and antibodies related to the pathology of the autoimmune disease. By inhibiting the activation of autoreactive T cells, the symptoms of the disease can be reduced or completely cured. Administration of agents that block T cell stimulation can inhibit T cell activation and inhibit the production of autoantibodies or T cell-derived cytokines that are associated with the progression of the disease. Blocking agents also allow for the introduction of antigen-specific tolerance into autoreactive T cells and long-term release from the disease. The effectiveness of blocking agents in preventing or alleviating autoimmune diseases can be determined using a number of well-studied animal models for human autoimmune diseases. Examples include experimental autoimmune encephalitis in mice, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, autoimmune collagen arthritis in mice, diabetes mellitus in NOD and BB rats, experimental in mice Myasthenia gravis is included (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-846).

  Up-regulation of antigen function (eg, B lymphocyte antigen function) as a means of up-regulating the immune response is also useful for therapy. Up-regulation of the immune response is to promote an existing immune response or elicit an initial immune response. For example, promoting an immune response is useful in the case of viral infections, including systemic viral diseases such as influenza, common colds, and encephalitis.

  Alternatively, the antiviral immune response in an infected patient is the removal of T cells from the patient and expression of APCs injected with viral antigens that express the peptide of the invention or also express the stimulated form of the soluble peptide of the invention. Can be facilitated by co-stimulating T cells in vitro and reintroducing in vitro activated T cells into the patient. Another method of promoting an antiviral immune response is to remove infected cells from a patient and, as described herein, a nucleic acid encoding a protein of the invention, wherein the infected cells are present on their surface. Transforming to express all or part of the inventive protein and reintroducing the transformed cells into the patient. Infected cells can deliver costimulatory signals to T cells and activate T cells in vivo.

KIRHy polypeptides can provide the necessary stimulation signals to T cells and elicit T cell-mediated immune responses against transformed tumor cells. In addition, tumor cells lacking MHC class I or MHC class II molecules or unable to reexpress a sufficient amount of MHC class I or MHC class II molecules are treated with MHC class I or MHC class II proteins on the cell surface. Transformation with a nucleic acid encoding all or part of an MHC class I α chain protein and β ₂ microglobulin protein or MHC class II α chain protein and MHC class II β chain protein (eg, a cytoplasmic domain shortened portion) expressed in Can do. The expression of a suitable class I or class II MHC bound to a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3), thereby expressing T against transformed tumor cells. A cell-mediated immune response is elicited. Optionally, a gene encoding an antisense structure that blocks expression of MHC class II bound to a protein such as a non-mutant chain, and a peptide having the activity of a B lymphocyte antigen that promotes the presentation of tumor-bound antigen Can be transformed with DNA to induce tumor specific immunity. In this way, a T cell-mediated immune response sufficient to overcome tumor specific tolerance in the subject is elicited in the subject's human.

  In particular, the activity of the protein of the present invention can be measured by the following method.

  Suitable assays for cytotoxicity against thymocytes or spleen cells include the Current Protocols in Immunology, Ed by J. et al. E. Coligan, A.M. M.M. Kruisbeek, D.D. H. Margulies, E.M. M.M. Shevach, W.H. Strober, Pub. Green Publishing Associates and Wiley-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19); Chapter 7, Immunohistomeric Histology , Proc. Natl. Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al. , J. et al. bnmunol. 128: 1968-1974, 1982; Handa et al. , J. et al. bnmunol. 135: 1564-1572, 1985; Takai et al. , J Immunol. 137: 3494-3500, 1986; Takai et al. , J. et al. Immunol. 140: 508-512, 1988; Bowman et al. , J Virology 61: 1992-1998; Bertagnolli et al. Cellular Immunology 133: 327-341, 1991; Brown et al. , J Immunol. 153: 3079-3092, 1994. However, it is not limited to these.

  Assays for T cell-dependent immunoglobulin responses and isotype switching (in particular identifying proteins that denature T cell-dependent antibody responses and proteins that affect Th1 / Th2 profiles) are described in Maliszewski, J. et al. Immunol. 144: 3028-3033, 1990, including but not limited to, assays for B cell function are described in Mariszewski, J. et al. Immunol. 144: 3028-3033, 1990; and Assays for B cell function: in vitro antibody production, Mond, J. Biol. J. et al. and Brunswick, M.M. In Current Protocols in Immunology. J. et al. E. e. a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

  Mixed lymphocyte reaction (MLR) assays (which identify, among other things, proteins that primarily cause Th1 and CTL responses) are described in Current Protocols in Immunology, Ed by J. et al. E. Coligan, A.M. M.M. Kruisbeek, D.D. H. Margulies, E.M. M.M. Shevach, W.H. Strober, Pub. Green Publishing Associates and Wiley-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function 3.1-3.19); Chapter 7, Immunohistochemical Talc. , J. et al. Inmunol. 137: 3494-3500, 1986; Takai et al. , J. et al. bnmunol. 140: 508-512, 1988; Bertagnolli et al. , J. et al. Immunol. 149: 3778-3783, 1992, but is not limited thereto.

  Dendritic cell-dependent assays (in particular, identifying proteins expressed by dendritic cells that activate natural T cells) include Guery et al. , J Immunol. 134: 536-544, 1995; Inaba et al. , J. et al. Exp. Med. 173: 549-559, 1991; Mactonia et al. , J Immunol. 154: 5071-5079, 1995; Porgador et al. , J. et al. Exp. Med. 182: 255-260, 1995; Nair et al. , J Virology 67: 4062-4069, 1993; Huang et al. , Science 264: 961-965, 1994; Mactonia et al. , J. et al. Exp. Med. 169: 1255-1264, 1989; Bhardwaj et al. , J Clin. Invest. 94: 797-807, 1994; and Inaba et al. , J. et al. Exp. Med. 172: 631-640, 1990, but is not limited thereto.

  Lymphocyte viability / apoptosis assays (among others identifying proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include those described in Darzynkiewicz et al. , Cytometry 13: 795-808, 1992; Gorczyca et al. Leukemia 7: 659-670, 1993; Gorczyca et al. , Cancer Res. 53: 1945-1951, 1993; Itoh et al. , Cell 66: 233-243, 1991; Zacharchuk, J Immunol. 145: 4037-4045, 1990; Zamai et al. , Cytometry 14: 891-897, 1993; Gorczyca et al. , Int. J. et al. Oncology 1: 639-648, 1992, but is not limited thereto.

  For assays of proteins that affect T cell intervention and early stages of growth, see Antica et al. , Blood 84: 111-117, 1994; Fine et al. Cellular Immunology 155: 111-122, 1994; Galy et al. , Blood 85: 2770-2778, 1995; Told et al, Proc. Nat. Acad Sci. USA 88: 7548-7551, 1991, but is not limited thereto.

(5.6.3 Anti-inflammatory activity of KIRHy)
The composition of the present invention can also exhibit anti-inflammatory activity. Anti-inflammatory activity impairs the chemotaxis of cells involved in inflammatory progression by inhibiting or promoting cell-cell interactions (eg, cell adhesion) by stimulating cells involved in the inflammatory response. By inhibiting or promoting, by inhibiting or promoting cell extravasation, it is achieved by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Compositions having such activity can be used to treat inflammation, including chronic or acute symptoms, where inflammation involves endometritis (septic shock, sepsis, or systemic inflammatory response syndrome ( SIRS)), ischemic reperfusion injury, endotoxin-related lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytoquinone or chemoquinone-induced lung injury, inflammatory bowel disease, Crohn's disease, or TNF or IL Inflammation due to overproduction such as -1 is included, but is not limited thereto. The compositions of the present invention are also useful in the treatment of hypersensitivity or hypersensitivity to antigenic substances or materials. The composition of the present invention can be used for sepsis, acute pancreatitis, endotoxin shock, cytokine-induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage due to Mellitus type 1 diabetes, graft-versus-host disease, inflammation Prevention or prevention of symptoms such as inflammatory bowel disease, inflammatory inflammation related to respiratory diseases, other autoimmune or inflammatory diseases, acute or chronic myelogenous leukemia or premature labor following uterine infection, etc. Although it can be used for treatment, symptoms are not limited to these.

  The immunosuppressive effect of the compositions of the invention on rheumatoid arthritis is determined in an experimental animal model system. An experimental animal model system is rat-induced arthritis, the protocol of which is described in J. Am. Holoshitz, et al. , 1983, Science, 219: 56 or B.R. Waksman et al. , 1963, Int. Arch. Allergy Appl. Immunol. , 23: 129. Disease induction was accomplished by a single intradermal injection of a suspension of dead M. tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but in the case of rats, it was injected with the auxiliary mixture at the base of the tail. The peptide was administered as a phosphate buffer (PBS) at a dose of approximately 1-5 mg / kg. The control received PBS alone.

  The test of the effect of the test compound consists of intradermal injection of dead M. tuberculosis in CFA, administration of the test compound immediately after, and administration every other day up to the 24th day. On days 14, 15, 18, 20, 22, and 24 after M. tuberculosis CFA injection, J. Overall arthritis scores were taken as in Holskitz literature. Analysis of the data showed that the test compound had a significant impact on joint swelling, as a reduction in arthritic score was measured.

(5.7 peptide)
A KIRHy peptide, such as a fragment of the extracellular region, binds or interacts with an antibody on the cell surface of the invention expressed on tumor cells or diseased cells, thereby targeting the toxin or radioisotope against tumor cells in vivo. Can be used for Similar to antibodies, these fragments can specifically target cells that expressed this antigen. Targeted delivery of these cytotoxic agents to tumor cells results in cell death or tumor growth inhibition. An example of the ability of an extracellular fragment to bind to and activate a complete receptor by specific antibody binding is shown for the CD84 receptor (Martin et al., Which is incorporated herein by reference in its entirety). J. Immunol.167: 3668-3676 (2001)).

  The extracellular fragment of KIRHy can also be used to denature immune cells that express the protein of the invention. An extracellular region fragment of KIRHy can bind to and activate a receptor for it on the cell surface, thereby causing cytokines (eg, interferon gamma from NK cells, T cells, B cells, or spinal cord cells). Etc.) is stimulated to strengthen or suppress the immune system. Furthermore, binding of these fragments to cells with KIRHy of the present invention activates these cells and stimulates proliferation. Some fragments can bind to complete KIRHy and block activation signals and cytokine release from immune cells. Therefore, these fragments have an immunosuppressive effect. Fragments that activate and stimulate the immune system can have anti-tumor properties. These fragments stimulate an immunological response and are capable of immune-mediated tumor cell killing. The same fragment can stimulate the immune system to enhance the response to foreign invaders such as viruses and bacteria. Fragments that suppress the immune response are useful in the treatment of inflammatory diseases such as lymphoid tissue proliferative disorders, autoimmune diseases, graft-versus-host disease, and emphysema.

(5.8 Other binding peptides or small molecules)
Screening with a recombinantly expressed KIRHy protein of the present invention from a library of organic compounds or peptides can be used to identify therapeutic molecules that specifically bind to KIRHy and further inhibit KIRHy1 activity. It is useful in. Synthetic and natural products can be screened in a variety of ways that are considered routine work by those skilled in the art. A library of random peptides can be displayed for phage (phage display) or E. coli. Displayed for bacteria such as E. coli. Use these random peptide display libraries to display peptides that interact with known targets, which may be proteins or polypeptides, such as ligands or receptors, natural or synthetic macromolecules, organic or inorganic substances be able to. For example, a library of differences such as a random peptide or combinatorial peptide or non-peptide library can be screened to determine molecules that specifically bind to the KIRHy polypeptide. Many usable libraries are known in the art, namely chemically synthesized libraries, recombinant (ie phage display libraries), and in vitro translation libraries. Techniques for constructing and screening such random peptide display libraries are known in the art (Ladner et al., US Patent No. 5, which is incorporated herein by reference in its entirety). Ladner et al., US Patent No. 4,946,778; Ladner et al., US Patent No. 5,403,484; Ladner et al., US Patent No. 5,571,698), random peptide display libraries and kits for screening such libraries are described, for example, in Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego, CA), New England Biolabs, Inc. (Beverly, MA), and Pharmacia KLB Biotechnology Inc. (Piscataway, NJ). Random peptide display libraries can be screened using the KIRHy sequences disclosed herein to identify proteins that bind to KIRHy.

  Examples of chemical synthesis libraries include Fodor et al., All of which are incorporated herein by reference in their entirety. , Science 251: 767-773 (1991); Houghten et al. , Nature 354: 84-86 (1991); Lam et al. , Nature 354: 82-84 (1991); Medynski, Bio / Technology 12: 709-710 (1994); Gallop et al. , J. et al. Med. Chem. 37: 1233-1251 (1994); Ohlmyer et al. , Proc. Natl. Acad. Sci. USA 90: 10922-10926 (1993); Erb et al. , Proc. Natl. Acad. Sci. USA 91: 11142-11426 (1994); Houghten et al. , Biotechniques 13: 412 (1992); Jaywickreme et al. , Proc. Natl. Acad. Sci. USA 91: 1614-1618 (1994); Salmon et al. , Proc. Natl. Acad. Sci. USA 90: 11708-11712 (1993); International Publication No. WO 93/20242; Brenner and Lemer, Proc. Natl. Acad. Sci. USA 89: 5381-5383 (1992).

  Examples of phage display libraries include Scott and Smith, Science 249: 386-390 (1990); Devlin et al., All of which are hereby incorporated by reference in their entirety. , Science 249: 404-406 (1990); Christian et al. , J. et al. Mol. Biol. 227: 711-718 (1992); Lenstra, J. et al. Immunol Meth. 152: 149-157 (1992); Kay et al. Gene 128: 59-65 (1993); International Publication No. Those described in WO 94/18318 are included.

  In vitro translation libraries include International Publication No. 2, which is hereby incorporated by reference in its entirety. WO 91/05058, and Mattheakis et al. , Proc. Natl. Acad. Sci. USA 91: 9022-9026 (1994) are included, but are not limited thereto.

  As an example of a non-peptide library, a benzodiazepine library (see Bunin et al., Proc. Natl. Acad. Sci. USA 91: 4708-4712 (1994), which is incorporated herein by reference in its entirety). Can be adapted. Peptide libraries (Simon et al., Proc. Natl. Acad. Sci. USA 89: 9367-9371 (1992), which is incorporated herein by reference in its entirety) can also be used. Examples of other usable libraries in which the amide group in the peptide is fully methylated to construct a chemical transformation combinatorial library are described in (Oreshet et al., Which is hereby incorporated by reference in its entirety. al., (Proc. Natl. Acad. Sci. USA 91: 11138-11142 (1994)).

  Library screening can be done by any of a variety of known methods. See, for example, Palmley and Smith, Adv., Which is incorporated herein by reference in its entirety, which discloses screening of peptide libraries. Exp. Med. Biol. 251: 215-218 (1989); Scott and Smith, Science 249: 386-390 (1990); Fowlkes et al. Biotechniques 13: 422-427 (1992); Oldenburg et al. , Proc. Natl. Acad. Sci. USA 89: 5393-5397 (1992); Yu et al. Cell 76: 933-945 (1994); Staudt et al. , Science 241: 577-580 (1988); Bock et al. , Nature 355: 564-566 (1992); Tuerk et al. , Proc. Natl. Acad. Sci. USA 89: 6988-6992 (1992); Ellington et al. , Nature 355: 850-852 (1992); Rebar and Pabo, Science 263: 671-673 '(1993); and International Publication No. See WO 94/18318.

  In certain embodiments, contacting a library member with a KIRHy protein (or nucleic acid or derivative) immobilized on a solid phase and recovering the library member bound to the KIRHy protein (or nucleic acid or derivative) By this, screening can be carried out. An example of a screening method called “panning” technique is described in Parmley and Smith, Gene 73: 305-318 (1988); Fowlkes et al., All of which are incorporated herein by reference in their entirety. Biotechniques 13: 422-427 (1992); International Publication No. WO 94/18318 and the references cited above in this specification.

  In other embodiments, a two-hybrid system for selecting proteins that interact in yeast (Fields and Song, Nature 340: 245, both of which are hereby incorporated by reference in their entirety). Chien et al., Proc. Natl. Acad. Sci. USA 88: 9578-9582 (1991)) can be used to identify molecules that specifically bind to KIRHy or KIRHy derivatives.

  These “binding polypeptides” or small molecules that interact with KIRHy polypeptides can be used for tagging or targeting cells; isolating homologous polypeptides by affinity purification; Can be directly or indirectly bound to drugs, toxins, radionuclides, etc. These binding polypeptides or small molecules can also be used privately in expression libraries and neutralization activities to block the interaction between the ligand and the receptor, or binding of the virus to the receptor. . These binding polypeptides or small molecules are also used in diagnostic assays to determine the rotational level of the KIRHy polypeptide of the invention; detection or quantification of soluble KIRHy polypeptide as a marker of its underlying disease state or disease it can. These binding polypeptides or small molecules can also act as KIRHy “antagonists” that block KIRHy binding and signal transmission in vitro and in vivo. These binding polypeptides or small molecules are useful for inhibiting KIRHy activity, ie protein binding.

  Binding polypeptides can bind directly or indirectly to drugs, toxins, radionuclides, etc., and these conjugates can be used for in vivo diagnostic or therapeutic applications. A binding polypeptide can be fused to another polypeptide, eg, an immunoglobulin constant chain or a portion thereof, to increase the half-life of the binding polypeptide (eg, by branching or repeating units). ) Can be multivalent to increase the binding affinity for KIRHy. For example, the binding polypeptides of the invention can be used for the identification or treatment of tissues or organs that express the corresponding anti-complementary molecule (eg, receptor or antibody, respectively). More specifically, a binding polypeptide or biologically active fragment or portion thereof is linked to a cleavable molecule or cytotoxic molecule to express a cell, tissue, or anti-complementary molecule It can be delivered to animals with organs.

  Suitable detectable molecules can bind directly or indirectly to the binding polypeptide, and in addition, radionuclides, enzymes, substances, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles Others can be included. Suitable cytotoxic molecules can be conjugated directly or indirectly to the binding polypeptide, and can also be bacterial or plant toxins (eg, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin ), Etc.) or even (eg, binding directly to the binding polypeptide or indirectly to the binding polypeptide via a chelator moiety) such as iodine-131, rhenium-188, or yttrium-90 A therapeutic radionuclide can be included. A binding polypeptide can also bind to a cytotoxic agent such as adriamycin. To indirectly bind a detachable molecule or cytotoxic molecule, the detachable molecule or cytotoxic molecule can bind to a component of a complementary / anti-complementary pair, in which case the other component is Binds to a binding polypeptide. An example of a complement / anti-complementarity pair for this purpose is biotin / streptavidin.

  In other embodiments, the binding polypeptide-toxin fusion protein can be used for inhibition or excision of target cells or tissues (eg, for treating cancer cells or tissues). Alternatively, if the binding polypeptide has a multifunctional domain (ie other activation domain or ligand binding domain of the targeting domain), there is a problem with the detectable molecule, cytotoxic molecule, or complementary molecule A fusion protein containing only the targeting domain is preferred to direct any cell or tissue. If the fusion protein contains only the domain of the complementary molecule, the anti-complementary molecule can be bound to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins provide a genetic targeting means for cell / tissue specific delivery of genetic anti-complementary detectable / cytotoxic molecule conjugates.

(5.9 Diseases suitable for anti-KIRHy targeting)
In one embodiment, the present invention provides agents and methods useful for the treatment of diseases and conditions where cells are associated with diseases or disorders where KIRHy is expressed, such as AML. In addition, these diseases include cancer and hyperproliferative conditions such as hyperplasia, psoriasis, contact dermatitis, immunological disorders, wound healing, arthritis, and autoimmune diseases. Whether a cell is associated with a disease or condition that expresses KIRHy can be determined using the diagnostic methods described herein.

  Patients respond to therapy targeting the KIRHy antigen of the present invention by comparing KIRHy1 mRNA and protein expression levels with diseased cells, tissues, or body fluids (blood, lymph, etc.) and corresponding normal samples Whether it is determined. Standard nucleic acid and protein detection and quantification techniques well known in the art are used for methods of detecting and quantifying the expression of KIRHy mRNA or protein, both of which are described herein in their entirety. Sambrook, et al., Incorporated by reference. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989) or Ausubel, et al. , Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1989). Methods for detecting and quantifying KIRHy mRNA include in situ hybridization using labeled KIRHy riboprobes (Gemou-Engesee, et al., Pediatrics 109: E24-E32 (2002), which is hereby incorporated by reference in its entirety). , Northern blots using KIRHy polynucleotide probes and related techniques (Kunzli, et al., Cancer 94: 228 (2002), RT-PCR analysis using KIRHy-specific primers, the entirety of which is incorporated herein by reference. (Angchaiskisiri, et al., Blood 99: 130 (2002), which is incorporated herein by reference in its entirety, Formation assay (Jardi, et al., J Viral Hepat. 8: 465-471 (2001), incorporated herein by reference in its entirety, transcription-mediated amplification (incorporated herein by reference in its entirety). Kimura, et al., J Clin. Microbiol. 40: 439-445 (2002), oligos, cDNAs, and monoclonal antibodies and other real-time PCR (Simpson, incorporated herein by reference in its entirety). , Et al., Molec.Vision, 6: 178-183 (2000), etc. Standard methods for detecting and quantifying KIRHy protein include Western blot analysis (Sambrook, 1989s). pr, Ausubel, 1989 supra), immunocytochemistry (Racila, et al., Proc. Natl. Acad. Sci. USA 95: 4589-4594 (1998), incorporated herein by reference in its entirety. A variety of immunoassays are included, including immunosorbent assays (ELISA), radioimmunoassays (RIA), and specific enzyme immunoassays (EIA) (Sambrook, 1989 supra, Ausubel, 1989 supra). Specific immunomagnetic beads (Racila, 1998 supra) or biotinylated KIRHy antibody (Soltys, et al., Incorporated herein by reference in its entirety). , J Immunol. 168: 1903 (2002) can also be used to analyze KIRHy expression in peripheral blood cells.

  Another related aspect of the invention is a method for assessing tumor aggressiveness by comparing the level of KIRHy protein or mRNA in tumor cells to the level of corresponding normal cells, the entirety of which is incorporated herein by reference. Orlandi, et al., Cancer Res., 62: 567 (2002) In one embodiment, the disease or disorder is cancer.

  Diseases treatable by the methods of the present invention preferably occur in mammals. Mammals include, for example, humans and other primates, as well as pets or pets such as dogs or cats, laboratory animals such as rats, mice, and squirrels, and livestock animals such as horses, pigs, sheep, and cows. It is.

  A tumor or neoplasm includes the growth of tissue cells in which the proliferation of cells is uncontrolled and progressive. Some of these growths are benign, while others are called “malignant” and can lead to the death of the organism. Malignant neoplasms or cancers are distinguished from benign growths in that they can invade surrounding tissues and metastasize besides exhibiting aggressive cell proliferation. Furthermore, malignant neoplasms are characterized in that they exhibit a significant loss of differentiation (substantial “dedifferentiation”) and loss of histology compared to themselves and the surrounding tissue. This property is also called “retirement”.

  Neoplasms treatable by the present invention include solid tumors / malignant or carcinomas, locally expanded tumors, and human soft tissue sarcomas. Carcinomas include malignant neoplasms derived from epithelial cells that penetrate (invade) the surrounding tissue and turn into metastatic cancer, including lymphatic metastases. Adenocarcinoma is a carcinoma derived from a tissue that is recognized as a gland-like tissue, that is, a gland-like structure. Another broad category, or cancer, includes sarcoma, a tumor whose cells are embedded in embryonic connective tissue-like fibrous material or homogeneous material. The present invention generally treats myeloid or lymphoid tissue cancers, including leukemias, lymphomas and other cancers that are not present as tumor masses but are dispersed in the vascular or lymphoreticular system. Is also possible.

  Suitable cancer or tumor types for treatment according to the present invention include AML. Other types of cancer that are effective by targeting KIRHy include chronic lymphocytic leukemia, chronic myelogenous leukemia, cutaneous T-cell lymphoma, ciliary cell leukemia, erythroleukemia, chronic myeloid (granulocyte) leukemia, Hodgkin's disease And non-Hodgkin lymphoma, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, polyps related to colorectal neoplasm, pancreatic cancer and gallbladder cancer, adrenocortical cancer, ACTH-producing tumor, bladder cancer including bladder cancer , Brain intrinsic cancer, neuroblastoma, astrocytic brain tumor, glioma, brain cancer including metastatic tumor cell invasion to central nervous system, central nervous system invasion of metastatic tumor cells, Ewing tumor , Head and neck cancer including oral cancer and pharyngeal cancer, kidney cancer including renal cell carcinoma, liver cancer, lung cancer including small cell lung cancer and non-small cell lung cancer, malignant malignant ascites, malignant peritoneal water, malignant melanoma, human skin horn Oncogenic cells, Skin cancer including cutaneous cell carcinoma, squamous cell carcinoma, basal cell carcinoma, and bone cancer including sarcoma, mesothelioma, Kaposi's sarcoma, fibrosarcoma and osteosarcoma, and bone cancer including sarcoma, uterine cancer, uterus Endometrial cancer, ovarian cancer, ovarian (germ cell) cancer, solid tumor in follicle, vaginal cancer, vulva cancer, cancer of female reproductive organs, including cervical cancer, breast cancer (penis), penile cancer, prostate Cancer, retinoblastoma, testicular cancer, thyroid cancer, trophoblast cancer, and Wilms tumor are included.

  The present invention is specifically described herein with reference to the treatment of certain experimentally defined cancers. In these illustrative treatments, standard current in vitro and in vivo models are used. These methods can be used to identify agents that appear to be effective in in vivo therapy. However, it should be understood that the method of the present invention is not limited to the treatment of these tumor types and extends to any cancer from any biological system. As shown in the Examples, KIRHy is highly expressed in hematopoietic cell related disorders such as AML and histiocytic lymphoma. Leukemia originates initially from unregulated B cell proliferation in the bone marrow, then spreads to peripheral blood, spleen, lymph nodes, and eventually to other tissues. Unregulated B cell proliferation is also caused by the progression of lymphomas that develop in the lymph nodes and then spread to the blood and bone marrow. KIRHy targeting includes myeloma, including but not limited to malignant B cells, leukemia, lymphoma, and multiple myeloma, Burkitt lymphoma, cutaneous B cell lymphoma, primary follicular cutaneous B cell lymphoma, B lineage acute Myeloblastic leukemia (ALL), B cell non-Hodgkin lymphoma (NHL), B cell chronic lymphocytic leukemia (CLL), acute myeloblastic leukemia, hairy cell leukemia (HCL), acute myeloid leukemia, acute Myelomonocytic leukemia, chronic myelogenous leukemia, lymphosarcoma cell leukemia, splenic marginal lymphoma, diffuse large B cell lymphoma, B cell large cell lymphoma, malignant lymphoma, prolymphocytic leukemia (PLL), lymph Plasmacytoid lymphoma, cerebral cortical cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, primary thyroid lymphoma, intravascular malignant lymphoma , Spleen lymphoma, Hodgkin's disease, and are useful in the treatment of graft within the vessel tropism large cell lymphoma. Example 4 includes acute monocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, undifferentiated large T cell lymphoma, B cell lymphoma, chronic myelogenous leukemia, diffuse large B cell lymphoma, follicle KIRHy expression has been shown in multiple lymphomas, histiocytic lymphomas, Hodgkin lymphomas, B cell large cell lymphomas, myeloma, non-Hodgkin lymphomas, and plasmacytoma cell lines and tissues, all with KIRHy antibodies Can be treated. Other diseases that can be treated by the methods of the present invention include multiple Castleman's disease, primary amyloidosis, Franklin's disease, Seligman's disease, primary exudative lymphoma, [related to EBV infection] post-transplantation lymphoid tissue increased disease (PTLD), ectopic neoplastic pemphigus, chronic lymphoid tissue proliferative disorder, companion lymphoid tissue proliferative syndrome (XLP), acquired angioedema, vascular immunoblastic lymphadenopathy with dysproteinemia, Hermann Syndrome, post-splenectomy syndrome, congenital erythropoiesis anemia type III, lymphoma-related phagocytic syndrome (LAHS), necrotizing ulcerative stomatitis, Kikuchi disease, lymphocytic granulomatosis, Rector syndrome, polycythemia vera ( PV), Gauche disease, Gougerot-Sjogren syndrome, Kaposi's sarcoma, cerebral lymphocyte plasmacytosis (Bind and Neel syndrome), companion Pas tissue proliferative disorder, mononucleosis (Epstein - Barr virus) pathogen-associated disorders such as, lymphoid plasma cell disorder, post-transplantation plasma cell disorder, and a good syndrome.

  The therapeutic compositions of the present invention include solid tumors / malignant, locally advanced tumors, adult and pediatric tumors including human soft tissue sarcomas; metastatic cancers including lymphatic metastases; multiple myeloma, acute and chronic Blood cell malignancies including leukemia and lymphoma; head and neck cancer including oral cancer, pharyngeal cancer, and thyroid cancer; lung cancer including small cell carcinoma and non-small cell carcinoma; breast cancer including small cell carcinoma and ductal carcinoma; Esophageal cancer, gastric cancer, colon cancer, colorectal cancer, polyp related to colorectal neoplasm, pancreatic cancer, gastrointestinal cancer including liver cancer; urinary cancer including bladder cancer and prostate cancer; ovarian cancer, (endometrial cancer Uterine cancer, female reproductive organ cancer including solid tumor in follicle; kidney cancer including renal cell carcinoma; brain intrinsic tumor, neuroblastoma, astrocytic brain tumor, glioma, metastatic tumor Brain with cell invasion to central nervous system Cancer; bone cancer including osteosarcoma; sarcoma including fibrosarcoma and osteosarcoma; malignant melanoma, tumor formation of human skin keratinocytes, epithelial cell carcinoma, squamous cell carcinoma, skin cancer including basal cell carcinoma; blood vessels Effective for surrounding cell tumors and Kaposi's sarcoma.

  Autoimmune diseases are associated with superactive B cell activity that produces autoantibodies. In addition, autoimmune diseases are characterized by unregulated protease activity (Wernike et al., Arthritis Rheum. 46: 64-74 (2002)) and abnormal cytoquinone activity (Rodenburg, the entirety of which is incorporated herein by reference). et al., Ann.Rheum.Dis.58: 648-652 (1999)). Inhibition of the growth or proliferation of autoantibody producing cells is pharmacologically effective in reducing the level of autoantibodies in autoimmune diseases. Inhibiting protease activity can reduce the degree of inflammation associated with tissue invasion and autoimmune diseases, including systemic lupus erythematosus, Hashimoto thyroiditis, Sjogren's syndrome, heart Erythematous erythema, Crohn's disease, graft-versus-host disease, Graves' disease, myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosis, malignant Anemia, Waldenstrom's macroglobulinemia, hyperviscosemia syndrome, macroglobulinemia, cold aggregating hemolytic anemia, unexplained monoclonal immunoglobulinemia, mottled skin atrophy and POEMS syndrome (polyneuropathy) Disorder, organometallic poisoning, endocrine disorder, M component, personality change), connective tissue disease, multiple sclerosis, cystic fibrosis, rheuma -Like arthritis, autoimmune pulmonary inflammation, psoriasis, Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, autoimmune inflammatory eye disease, Goodpasture's disease, Rasmussen encephalitis, herpetic dermatitis, thymoma Type 1 autoimmune multiglandular syndrome, primary and secondary membranous nephropathy, cancer-related retinopathy, type 1 autoimmune hepatitis, kidney-associated mixed cryoglobulinemia, cystic macular edema, uterus Includes, but is not limited to, endometriosis, immunoglobulin multiple neuropathy (including hyperimmunoglobulinemia), demyelinating disease, hemangiomatosis, and monoclonal immunoglobulinemia.

  KIRHy targeting is associated with allergic reactions and symptoms such as hypersensitivity, serum sickness, drug reactions, food allergies, insect venom allergy, mast cell hyperproliferation, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, Atopic dermatitis, allergic contact dermatitis, polymorphic exudative erythema, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, gender keratoconjunctivitis, giant papillary conjunctivitis, allergic gastrointestinal disease, inflammatory bowel It is also useful in the treatment of disease (IBD) and contact allergies such as asthma (especially allergic asthma) or other respiratory disorders.

  KIRHy targeting is also useful for the management or prevention of transplant rejection in patients in need of transplant, such as transplantation of stem cells, tissues, or organs. Accordingly, one aspect of the present invention is that after various diseases (which are usually treated with transplantation, including but not limited to aplastic anemia and paroxysmal nocturnal hemoglobinuria), and after radiation / chemotherapy, Pharmacological in regrowth of stem cell compartments in vivo or ex vivo (ie simultaneously with bone marrow transplant) or transplantation of peripheral progenitor cells (homogeneous or xenogeneic) as normal cells or cells modified for gene therapy Usefulness can be found.

  KIRHy targeting can also modify the immune response in a variety of ways. Down-regulation is a form that can inhibit or block an immune response that is already in progress, or can be involved in the prevention of immune response induction. Inhibition of one or more antigen functions, including down-regulation, ie, but not limited to, B lymphocyte antigen function, eg, denaturation or inhibition of high-level lymphokine synthesis by activated T cells, is tissue, skin And in organ transplantation and graft-versus-host disease (GVHD). For example, blockage of T cell function can reduce tissue destruction in tissue transplantation. In general, in tissue transplantation, rejection begins when T cells recognize the graft as foreign, followed by an immune response that destroys the graft. Administration of the therapeutic composition of the present invention inhibits cytokine synthesis by immune cells such as T cells, and the therapeutic composition of the present invention serves as an immunosuppressant. Furthermore, the lack of costimulation makes the T cells anergic and the subject is tolerated. The introduction of long-term tolerance with B lymphocyte antigen blocking agents eliminates the need for repeated administration of these blocking agents. It may also be necessary to block the function of the combination with the B lymphocyte antigen in order to achieve sufficient immunosuppression or tolerance in the subject.

  The effectiveness of a particular therapeutic composition in inhibiting organ transplant rejection or GVHD can be assessed using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart transplantation in rats and xenopancreatic islet cell transplantation in mice, both of which are incorporated herein by reference in their entirety. , Science 257: 789-792 (1992) and Turka et al. , Proc. Natl. Acad. Both have been used to test the immunosuppressive effects of CTLA4Ig fusion proteins in vivo, as described in Sci USA, 89: 11102-11105 (1992). In addition, a mouse model of GVHD (Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 844-847, the entirety of which is incorporated herein by reference) is used to treat GVHD according to the present invention. Can be used to measure the effect of the composition.

(5.10 administration)
The anti-KIRHy monoclonal antibody used to practice the methods of the invention can be prepared into a therapeutic composition comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with an anti-KIRHy antibody retains the anti-tumor function of the antibody and does not react with the subject's immune system. Examples of suitable carriers include, but are not limited to, any number of standard pharmacological carriers such as sterile phosphate buffered saline solution, bacteriostatic water, etc.

  The anti-KIRHy antibody formulation can be administered by any route capable of delivering the antibody to the tumor site. Effective routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, etc. A preferred route of administration is intravenous injection. Preferred formulations for intravenous injection are polyvinyl chloride or polyethylene dissolved in preserved bacteriostatic water, sterile non-preserved water and / or containing 0.9% sterile saline from USP for injection. Contains anti-KIRHy mAbs diluted in bags. The anti-KIRHy 1 mAbs product is lyophilized and stored as a sterile powder, preferably under vacuum, and redissolved in bacteriostatic water, eg, containing benzyl alcohol preservative, or sterile water prior to injection.

Treatment generally involves repeated administration of an anti-KIRHy antibody formulation by administration by an acceptable route such as intravenous injection, usually at a dose of about 0.1 mg / kg body weight to about 10 mg / kg body weight, but 0.01 mg Examples of doses ranging from / kg to approximately 100 mg / kg are also envisioned. Doses in the range of 10-500 mg mAb per week are effective and well tolerated. Rituximab (Rituxan®), a chimeric CD20 antibody used for B-cell lymphoma, non-Hodgkin's lymphoma, and recurrent analgesic lymphoma, is usually administered iv at 375 mg / m ² once a week for 4-8 doses. The It may be necessary to repeat this twice, but it must not be repeated more than three times. An effective dose range for Rituxan® is 50-500 mg / m ² (Maloney, et al., Blood 84: 2457-2466 (1994), both of which are hereby incorporated by reference in their entirety); Davis, et al., J Clin. Oncol. 18: 3135-3143 (2000)). Clinical experience with trastuzumab (Herceptin®), a humanized monoclonal antibody used to treat HER2 (human epidermal growth factor 2) -positive metastatic breast cancer, which is incorporated herein by reference in its entirety , Et al., Mol Cell Biol. 9: 1165 (1989)), with an anti-KIRHy mAb formulation as the first loading dose, approximately 4 mg / kg patient body weight intravenously and then weekly approximately 2 mg / kg intravenously. (Slamon, et al., N. Engl. J. Med. 344: 783 (2001)), which is hereby incorporated by reference in its entirety. It is preferable to administer the loading dose over 90 minutes or longer, the first dose being well tolerated In such cases, the regular maintenance dose is administered over 30 minutes or longer, although as will be appreciated by those skilled in the art, various factors will affect the desired mode of administration in specific cases. These factors include, for example, the binding affinity and half-life of the mAb or the mAb used, the degree of KIRHy overexpression in the patient, the degree of circulating excreted KIRHy antigen, the concentration level of the desired steady state antibody, the therapeutic level The frequency includes the effects of chemotherapeutic agents used in combination with the treatment methods of the present invention.

Treatment also includes anti-KIRHy antibodies conjugated to radioisotopes. Studies using radiolabeled anti-carcinoembryonic antigen (anti-CEA) monoclonal antibodies give dose guidance from tumor regression when injected 2-3 times at 30-80 mCi / m ² (both (Behr, et al. Clin, Cancer Res. 5 (10 Suppl.): 3232s-3242s (1999), Juweid, et al., J. Nucl. Med. 39, incorporated herein by reference in its entirety. 34-42 (1998)).

Alternatively, dendritic cells transformed with mRNA encoding KIRHy can be used as a vaccine to stimulate a T cell mediated anti-tumor response. Studies using dendritic cell transformed with mRNA of prostate specific ^antigen, 1x10 ^{7 ~5x10} 7 cells cycle intravenous combination with 2-6 weeks done 10 ⁷ intradermal injection of the cells Has been shown to be a suitable dose (both are incorporated herein by reference in their entirety, (Heiser, et al., J. Clin. Invest. 109: 409-417). ... (2002); Hadzantonis and O'Neill, Cancer Mother Radiopharm 1: 11-22 (1999)) can also be envisaged other doses such as between from 1x10 ⁵ 1x10 ⁹ pieces during or 1x10 ⁶ cells 1x10 ⁸ .

  Naked DNA vaccines using a plasmid encoding KIRHy can elicit an immunological anti-tumor response. Injecting and administering naked DNA directly intradermally or intramuscularly is not necessarily related to the limitations encountered when using viral vectors, such as the progression of adverse immune responses and the risk of mutagenesis by insertion ( (Hengge, et al., J. Invest. Dermatol. 116: 979 (2001)), which is incorporated herein by reference in its entirety Direct injection of exogenous cDNA into muscle tissue provides a strong immune response and protection Studies have shown that it induces immunity (Ilan, Curr Opin. Mol. Ther. 1: 116-120 (1999)), which is incorporated herein by reference in its entirety. Gene guns, electroporation) and chemical introduction methods (cationic lipids or polymers) DNA has been improved to increase the efficiency of gene transport and target cell specificity (incorporated herein by reference in its entirety, (Nishikawa and Huang, Hum. Gene Aer. 12: 861-870 (2001)). Plasmid DNA can also be administered to the lungs by aerosol delivery (Densmore, et al., Mol. Ther. 1: 180-188 (2000)) Direct injection of naked or lipid-coated plasmid DNA The gene therapy by is contemplated for use in the prevention, treatment, and cure of diseases such as cancer, acquired immune deficiency syndrome, cystic fibrosis, cerebrovascular disease, and hypertension (both in their entirety herein). (Prazeres, et al., Trend) Biotechnol.17: 169-174 (1999)); Wehl, et al., Neurosurgery 44: 239-252 (1999)) Dose escalation studies with HIV-1 DNA vaccines have been conducted at It has been shown that administration is preferred (incorporated herein by reference in its entirety (Weber, et al., Eur. J. Clin. Microbiol. Infect. Dis. 20: 800 (2001)). When naked DNA was injected into the mouse cerebrum, a reporter protein was expressed, expression was dose dependent, with maximum expression at the time of 150 μg DNA injection (incorporated herein by reference in its entirety, Schwartz, et al. , Gene Ther. 3: 405-411 (1996)). Gene expression in mice after intramuscular injection of nanospheres containing 1 μg of β-galactosidase plasmid was higher and longer than when injected with the same amount of naked DNA or a complex of DNA with lipofectamine (Truong, et al., Hunt. Gene Ther. 9: 1709-1717 (1998), which is incorporated herein by reference in its entirety). In the study of introducing a plasmid-mediated gene into skeletal muscle as a means of providing a therapeutic source of insulin, four plasmid constructs, including the mouse furin cDNA transgene and the rat proinsulin cDNA, are present in male Balb / c mice. Injected into the calf muscle, the preferred dose for most constructs was 100 μg plasmid DNA (Kon, et al. J Gene Med. 1: 186-194 (incorporated herein by reference in its entirety). 1999)). Other doses of 1-1000 μg / dose or 10-500 μg / dose are also envisioned.

  In order to provide reference data for determining the most effective mode of administration and related factors, it is desirable for patients to be evaluated for the concentration of circulating excreted KIRHy antigen in the serum. Such an assessment can also be used for monitoring throughout the treatment period and is useful in assessing the success of treatment in conjunction with assessment of other parameters.

(5.10.1 KIRHy targeting composition)
Compositions for targeting KIRHy expressing cells are within the scope of the present invention. Pharmacological compositions comprising antibodies are described in detail, for example, in US Pat. No. 6,171,586, which is hereby incorporated by reference in its entirety. Such compositions are pharmaceutically acceptable as a mixture, as described herein, in a therapeutically or prophylactically effective amount of an antibody, or fragment, variant, derivative, or fusion thereof. Contains drugs. Usually, the KIRHy targeting agent is purified enough to be administered to an animal.

  Pharmacological compositions modify, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, absorption or penetration of the composition Formulation materials can be included for, maintaining, or storing. Suitable formulation materials include amino acids (such as glycine, glutamic acid, aspartic acid, arginine, or lysine), antibacterial substances, antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite), buffering agents (borate, heavy Carbonate, Tris-HCl, citrate, phosphate, other organic acids, etc.), extenders (such as mannitol or glycine), chelating agents [ethylenediaminetetraacetic acid (EDTA), etc.], complexing agents (caffeine, Polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, polysaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (serum albumin, gelatin, or immunity) Globulin), coloring agents , Fragrances and diluents, suspending agents, hydrophilic polymers (polyvinylpyrrolidone, etc.), low molecular weight polypeptides, salt-forming counterions (sodium, etc.), preservatives (benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phene Sil alcohol, methyl paraben, propyl paraben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvent (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohol (such as mannitol or sorbitol), emulsion, surfactant Or wetting agents (plalonics, PEG, sorbitanate, polysorbate 20, polysorbate such as polysorbate 80, triton, tromethamine, lectin, cholesterol, tyloxapol, etc.), stability enhancer (sucrose or sorbi ), Tonicity enhancing agents (alkali metal chlorides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.), delivery agents, diluents, excipients and / or adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, Ed. A. R. Gennaro, Mack Publishing Company, (1990), which is incorporated herein by reference in its entirety).

  The optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the KIRHy targeting agent.

  The primary medium or carrier in the pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection, physiological saline or artificial cerebrospinal fluid, possibly supplemented with other materials common to compositions for parenteral administration. Natural balanced saline or saline mixed with serum albumin is another exemplary medium. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which further contains sorbitol or suitable Alternatives can be included. In one embodiment of the present invention, the KIRHy targeting agent composition may be selected from any formulation (Remington's Pharmaceutical Sciences, supra) that exhibits a desired degree of purity in the form of a lyophilized cake or aqueous solution. ) And can be prepared for storage. Further, the binder product can be formulated as a lyophilizate using appropriate excipients such as sucrose.

  The pharmaceutical composition can be selected for parenteral release. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract, such as orally. The manufacture of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are present in an acceptable concentration at the site of administration. For example, the buffer is used to maintain the composition at physiological pH or at a slightly lower pH, particularly in the pH range of about 5 to about 8. When parenteral administration is achieved, the therapeutic composition for use in the present invention includes a KIRHy targeting agent in a pharmaceutically acceptable vehicle, is pyrogen free, and is parenterally acceptable aqueous. It can be in the form of a solution. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the KIRHy targeting agent is formulated as a suitably stored sterile, isotonic solution. Yet another manufacture is an injectable microsphere, biodegradable particle, polymerizable compound (polyacetic acid, polyglycolic acid), bead, or that provides controlled or sustained release of the product that can then be delivered via a depot injection Formulation of the desired molecule with agents such as liposomes can be included. Hyaluronic acid can also be used, and this can show the effect of driving the duration of the cycle. Other suitable means for introduction of the desired molecule include implantable drug delivery devices.

  In another aspect, a pharmaceutical formulation suitable for parenteral administration is in an aqueous solution, preferably a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or physiologically balanced saline. Can be prescribed. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty acid oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Non-fatty polycation amino polymers can also be used for delivery. For convenience, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound and address the production of concentrated solutions.

  In another embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the KIRHy targeting agent can be formulated as a dry powder for inhalation. Polypeptide or nucleic acid molecule inhalation solutions can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized. Pulmonary administration is further described in International Application No. PCT / US94 / 001875, which is incorporated herein in its entirety, and it describes pulmonary delivery of chemically modified proteins.

  It is also anticipated that a given formulation can be administered orally. In one embodiment of the invention, the KIRHy targeting agent administered in this form may be formulated with or without a carrier conventionally used in compounding solid dosage forms such as tablets and capsules. For example, a capsule can be intended to release the active portion of the formulation at that time in the gastrointestinal tract when bioavailability is maximized and systemic disintegration is previously minimized. Another agent can be included to facilitate absorption of the binder molecule. Diluents, flavors, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

  Pharmaceutical compositions for oral administration can also be formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by the patient.

  Pharmaceutical products for oral use can be obtained through combining the active compound with solid excipients and processing the product mixture of granules to obtain tablets or dragee cores. If desired, suitable adjuvants can be added. Suitable excipients include sugars including lactose, sucrose, mannitol, and sorbitol; starch obtained from corn, wheat, rice, potato or other plants; methylcellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose Cellulose such as; gum including gum arabic and tragacanth; and hydrocarbons or protein fillers such as proteins such as gelatin and collagen. If desired, disintegrating or stabilizing agents such as cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof such as sodium alginate may be added.

  Dragee cores can be prepared with suitable coatings, such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or concentrated sugar solutions that can also contain titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Can be used in conjunction. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie, dosage.

  Pharmaceutical products that can be used orally also include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules may contain active ingredients mixed with a filler or binder such as lactose or start; a lubricant such as talc or magnesium stearate, and, optionally, a stabilizer. In soft capsules, the KIRHy targeting agent can be dissolved or suspended in a suitable liquid, such as fatty acid oil, or liquid polyethylene glycol, with or without stabilizers.

  Another pharmaceutical composition may comprise an effective amount of a KIRHy targeting agent in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or other suitable medium, solutions can be prepared in unit dosage form. Suitable excipients include, but are not limited to, an inert diluent such as calcium carbonate, carbonate or sodium bicarbonate, lactose or calcium phosphate; a binder such as starch, gelatin, or acacia; or magnesium stearate, stearin Examples include lubricants such as acids or talc.

  Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations that include a KIRHy targeting agent in a sustained or controlled delivery formulation. Techniques for formulating various other sustained or controlled delivery means such as liposomal carriers, biodestructive microparticles or porous beads and depot injections are also well known to those skilled in the art. See, for example, PCT / US93 / 00829, which is incorporated herein in its entirety, which describes the controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Another example of a sustained release formulation includes a semipermeable polymer matrix in the form of a shaped article, such as a film or microcapsule. The sustained-release matrix is a polyester, hydrogel, polylactide (US Pat. No. 3,773,919; European Patent No. 58,481), a copolymer of L-glutamic acid and gamma-ethyl-L-glutamate ( Sidman et al, Biopolymers 22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al, J. Biomed Mater Res, 15: 167-277 (1981)) and (Langer et al. Chem Tech, 12: 98-105 (1982)), ethylene vinyl acetate (Langer et al, supra) or poly-D (-)-3-hydroxybutyric acid (European Patent No. EP 133,988, all here To the whole Embedded). Sustained release compositions also include liposomes that can be produced by any of several methods known in the art. For example, Epstein et al. Proc Natl Acad Sci (USA), 82: 3688-3692 (1985); see European Patent Nos. EP36,676, EP88,046 and EP143,949, all of which are hereby incorporated by reference in their entirety. It is incorporated.

  The pharmaceutical composition to be used for in vivo administration must generally be sterile. This can be achieved by filtration through sterile filtration membranes. If the composition is lyophilized, sterilization using this method can be performed either prior to or subsequent to lyophilization and reorganization. Compositions for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are generally replaced in a sterile evaluation port, such as a venous solution bag container, or via a vial having a puncture stopper with a hypodermic injection needle.

  Once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid or a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form that requires reorganization prior to administration (eg, lyophilized).

  In a particular embodiment, the present invention is directed to a kit for generating a single dose dosage unit. Both kits can include both a first container having a dry KIRHy targeting agent and a second container having an aqueous formulation. Further included within the scope of the present invention are kits containing single and multi-chamber prefilled syringes (eg, liquid syringes and lio syringes).

(5.10.2 dose)
The effective amount of the pharmaceutical composition to be used is therapeutically dependent, for example, on the therapeutic content and the subject. Those skilled in the art will appreciate that the appropriate dosage level for treatment is in part determined by the molecule delivered, the indication that the KIRHy targeting agent will be used, the route of administration, and the patient size (weight, body surface or Identify changes in organ size) and symptoms (age and general health). Thus, medical personnel can titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages can range from about 0.1 mg / kg to about 100 mg / kg, or more, depending on the factors specified above. In other embodiments, the dosage is from 0.1 mg / kg to about 100 mg / kg; or from 0.01 mg / kg to 1 g / kg; or from 1 mg / kg to about 100 mg / kg, or from 5 mg / kg. It is in the range up to about 100 mg / kg. In other embodiments, dosages range from 10 mCi to 100 mCi per dose for radioimmunotherapy, up to about 1 × 10 ⁷ −5 × 10 ⁷ cells per injection or infusion, or from about 1 × 10 ^5. Up to 1 × 10 ⁹ cells, or about 1 × 10 ⁶ to 1 × 10 ⁸ cells, or from 30 μg to 300 μg per dose, or 1-1000 μg / dose or 10-500 μg / dose, depending on the factors listed above It can be naked DNA.

  For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models such as mice, rats, rabbits, dogs, or pigs. The animal model can also be used to determine the appropriate concentration range or route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

  The exact dosage will be determined in terms of factors associated with the subject in need of treatment. Dosage amount and administration are adjusted to provide sufficient levels of the active compound or to maintain the desired effect. Factors that may be considered include the severity of the disease state, the subject's general health, the subject's age, weight and sex, time and frequency of administration, drug combination, response sensitivity, and response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or every two weeks, depending on the half-life and clearance of the particular formulation.

  The frequency of administration will depend on the pharmacological parameters of the KIRHy targeting agent in the formulation used. In particular, the composition is administered until a dosage is reached that achieves the desired effect. Thus, the composition may be administered as a single dose or as multiple doses (at the same or different concentrations / doses), over time, or as a continuous infusion. Further purification of appropriate doses is routinely done. Appropriate dosages can be ascertained through use of appropriate dose-response data.

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredient is included in an effective amount to achieve its intended purpose. More particularly, a therapeutically effective amount means an amount useful to prevent or alleviate the occurrence of an existing indication of a subject to be treated. Determination of an effective amount is well within the tolerance of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from those appropriate in in vitro assays. For example, a dose can be formulated in animal models to achieve a periodic concentration range that can be used to more accurately determine useful doses in humans. For example, to achieve a periodic concentration range that includes an IC ₅₀ as measured in cell culture (ie, half the biological activity of the protein—the concentration of the test compound that achieves maximum inhibition) Can be prescribed to the model. Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose corresponds to the amount of compound that results in alleviation of symptoms or prolonged survival in a patient. The toxicity and therapeutic efficiency of such compounds can be determined, for example, in cell culture or to determine LD ₅₀ (dose that is lethal for 50% of the population) and ED ₅₀ (dose that is therapeutically effective for 50% of the population). It can be determined by standard pharmaceutical means in laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD ₅₀ and ED ₅₀ . Compounds that exhibit high therapeutic indices are preferred. Data obtained from these cell culture assays and animal studies can be used to formulate multiple dosages for use in humans. The dosage of such compounds lies preferably within a range of periodic concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's symptoms. See, for example, Fingl et al. , 1975, “The Pharmacological Basis of Therapeutics”, Ch. 1p. See 1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. The dosage required to achieve MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

  Dosage intervals can also be determined using the MEC value. Compounds should be administered using a treatment regimen that maintains plasma levels above the MEC for between 10-90%, preferably between 30-90%, and most preferably between 50-90% It is. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

  An exemplary dosage regime for a polypeptide or other composition of the invention is in the range of about 0.01 μg to 100 mg / kg of patient weight daily, and a preferred dose is about 0.1 μg of patient weight daily. Up to 25 mg / kg and varies in adults and children. Administration can be delivered once daily, or equivalent doses can be delivered at long or short intervals.

  The amount of composition administered will, of course, depend on the subject to be treated, the age and weight of the subject, the severity of the affliction, the means of administration, and the judgment of the prescribing physician.

(5.13 Route of administration)
The route of administration of the pharmaceutical composition may be, for example, orally, through an injection by intravenous, intraperitoneal, intracerebral (parenchymal), intracerebral, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes. Intramembrane, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, urethral, vaginal, or rectal means, by sustained release system, by implantation device, or through inhalation Consistent with known methods. If desired, the composition can be administered by bolus injection, continuously by infusion, or by implantation device.

  Alternatively or additionally, the composition may be administered via implantation of a membrane, sponge or another suitable material in which the KIRHy targeting agent is absorbed or encapsulated. When using an implantation device, the device can be implanted into any suitable tissue or organ and delivery of the KIRHy targeting agent can be via diffusion, time-release bolus, or continuous administration.

  In some cases, it may be desirable to use the medical composition by ex-vitro means. In such examples, cells, tissues or organs removed from the patient are exposed to the pharmaceutical composition after the cells, tissues and / or organs are subsequently transplanted back into the patient.

  In other cases, the KIRHy targeting agent can be delivered by transplanting certain cells that have been genetically engineered to express and secrete the polypeptide. Such cells can be animal or human cells and can be from the same species, xenogeneic, or xenogeneic. For convenience, the cells can be immortalized. To reduce the chance of an immunological response, cells can be encapsulated to avoid permeation of surrounding tissue. The encapsulating material, in particular, allows the release of the protein product, but prevents the destruction of cells by the patient's immune system or by other critical factors from surrounding tissues, a semi-compatible polymer It is an inclusion.

(5.11 Combination therapy)
The KIRHy targeting agent of the present invention can be utilized in combination with other therapeutic agents and requires a small daily dose, a low total dose or a reduced frequency of administration to achieve the same therapeutic effect with low toxicity. As such, the effects of these other therapeutic agents may be enhanced. For cancer, these other therapeutic agents include, for example, radiation therapy, chemotherapeutic agents, and other growth factors. For transplant rejection or autoimmune diseases, these other therapeutic agents include, for example, immunosuppressants such as cyclosporine, azathioprine corticosteroids, tacrolimus or mycophenolate mofuethyl.

  In one embodiment, anti-KIRHy antibody is used as a radiosensitizer. In such embodiments, the anti-KIRHy antibody is conjugated with a radiosensitizer. As used herein, the term “radiosensitizer” is defined as a molecule, preferably a low molecular weight molecule, which is administered to an animal in a therapeutically effective amount and is sensitive to cells that should be radiosensitive to electromagnetic radiation. And / or facilitate the treatment of diseases that are treatable with electromagnetic radiation. Diseases that can be treated with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancerous cells.

As used herein, the terms “electromagnetic radiation” and “irradiation” include, but are not limited to, radiation exhibiting wavelengths from 10 ⁻²⁰ to 100 meters. Preferred embodiments of the present invention include gamma irradiation (10 ⁻²⁰ to 10 ⁻¹³ m), X-ray irradiation (10 ⁻¹² to 10 ⁻⁹ m), ultraviolet light (10 nm to 400 nm), visible light (from 400 nm Use electromagnetic irradiation of up to 700 nm), infrared irradiation (from 700 nm to 1.0 mm), and microwave irradiation (from 1 mm to 30 cm).

  Radiosensitizers are known to increase the sensitivity of cancerous cells to the toxic effects of electromagnetic radiation. Many cancer treatment protocols currently use radiosensitizers that are activated by electromagnetic radiation of X-rays. Examples of X-ray activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU1069, SR4233, EO9, RB6145, nicotinamine, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same .

  Cancer photodynamic therapy (PDT) uses visible light as a radioactive activator of susceptibility agents. Examples of photodynamic radiation sensitizers include, but are not limited to: hematoporphyrin derivatives, photofurin (r), benzoporferrin derivatives, NPe6, tin etioporphyrin (SnET2), phenoborbide -A, bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine, and therapeutically effective analogues and derivatives thereof.

  Chemotherapy includes, for example, nitrogen mustards such as mechlorethamine, cyclophosphoamide, ifosfamide, melphalan and chlorambucil; nitrosourea such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); triethylenemelamine (TEM), triethylene, thiophosphoramide (thiopeta), ethyleneimine / methylmelamine such as hexamethyl melanin (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as decarbazine (DTIC); methotrexate and Antimetabolites including folic acid analogs such as trimethrexate; 5-fluorouracil, fluorodeoxycytidine, gemcitabine, cytosine arabinoside (AraC Rabin), 5-azacytidine, pyrimidine analogs such as 2,2′-difluorodeoxycytidine; 6-mercaptopurine, 6-thioguanine, azathioprine, 2′-deoxycoformycin (pentostatin), erythrohydroxynonyladenine ( EHNA), purine analogs such as fludarabine phosphate, and 2-chlorodeoxyadenosine (Cladribine, 2-CdA); vinca alkaloids including paclitaxel, vinblastine (VLB), vincristine and vinorelbine, taxotera, estramustine and Natural products including anti-mitotics such as estramustine phosphate; pipepodophyllotoxins such as etoposide and teniposide; actimomycin D, tauno Antibiotics such as isine (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, priomycin (mythramycin), mitomycin C and actinomycin; enzymes such as L-asparaginase; interferon-alpha, IL-2, G -Biological response modifiers such as CSF and GM-CSF; miscellaneous agents including platinum conjugated complexes such as cisplatin and carboplatin; anthracenediones such as mitoxantrone, such as hydroxyurea Adrenocortical inhibitors such as substituted ureas, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, mitotane (o, p'-DDD) and aminoglutethimide; prednisone and equivalents Hormones and antagonists, including various corticosteroid antagonists; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalent; tamoxifen Alkylates, including androgens including testosterone propionate and furoximesterone / equivalents; antiandrogens such as flutamide, gonadotropin releasing hormone analogs and leuprolide; and nonsteroidal antiandrogens such as flutamide Anti-neoplastic agents, including agents, can be used.

  Combination therapy with growth factors is M-CSF, GM-CSF, TNF, IL-1, 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-14, IL-15, IL-16, IL-17, IL-18, IFN, TNF0, TNF1, TNF2, G -Include cytokines, lymphokines, growth factors, or other hematopoietic factors such as CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Other compositions may include known angiopoietins such as vascular endothelial growth factor (VEGF). As growth factors, angiogenin, bone morphogenesis protein-1, bone morphogenesis protein-2, bone morphogenesis protein-3, bone morphogenesis protein-4, bone morphogenesis protein-5, bone morphogenesis protein-6, Bone morphogenetic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic protein-13, bone Morphogenic protein-14, bone morphogenetic protein-15, bone morphogenic protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor, cytokine induction Neutrophil chemotaxis factor 1, cytokine-induced neutrophil chemotaxis factor 2, endothelial cell growth Child, endothelin 1, epidermal growth factor, epithelial neutrophil attractant, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7, fibroblast growth factor 8. Fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, acidic fibroblast growth factor, basic fibroblast growth factor, derived from glial cell line Neutrophil factor receptor 2, growth-related protein, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor Binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor, nerve growth factor, nerve growth factor receptor Neutrophin-3, Neutrophin-4, Placental growth factor, Placental growth factor 2, Platelet-derived epidermal growth factor, Platelet-derived epidermal growth factor A chain, Platelet-derived epidermal growth factor AA, Platelet-derived epidermal growth factor AB, Platelet-derived epithelium Growth factor B chain, platelet-derived epidermal growth factor BB, platelet-derived epidermal growth factor receptor, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor, transforming growth factor 1, transforming growth factor 1 .2, transforming growth factor 2, transforming growth factor 3, transforming growth factor 5, latent transforming growth factor 1, transforming growth factor binding protein I, transforming growth factor binding protein II, trans Forming growth factor binding protein III, type I tumor necrosis factor receptor, type II tumor necrosis factor receptor, urokinase type plasminogen activator receptor, vascular endothelial growth factor, and chimeric protein and biologically or immunologically active fragments thereof Can be mentioned.

(5.12 KIRHy diagnostic use)
(5.12.1 Assay to determine KIRHy expression status)
Measuring the status of the KIRHy expression pattern in an individual (individual) can be used to diagnose cancer and provide prognostic information in defining appropriate treatment options. Similarly, the expression status of KIRHy may provide information useful for detecting susceptibility to a particular disease stage, progression, and / or tumor aggressiveness. The present invention provides methods and assays for measuring KIRHy expression status and diagnosing cancers that express KIRHy.

  In one aspect, the present invention, when available, in a test cell or tissue or fluid sample is associated with an apparent increase or decrease in KIRHy mRNA, or expression level in a corresponding normal cell or tissue. An assay useful for measuring the presence of cancer in an individual (individual), including detecting protein expression, is provided. In one embodiment, the presence of KIRHy mRNA is assessed in a lymphoma tissue sample. The presence of obvious KIRHy expression may be useful to indicate whether the lymphoma is sensitive to the KIRHy target using the targeting composition of the present invention. In a related embodiment, KIRHy expression status can be measured at the protein level rather than at the nucleic acid level. For example, such a method or assay measures the level of KIRHy expressed by cells in a test tissue sample, and the level so measured is the level of KIRHy expressed in the corresponding normal sample. Comparing with In one embodiment, the presence of KIRHy is assessed using, for example, an immunohistochemical method. KIRHy antibodies capable of detecting KIRHy expression can be used in a variety of assay formats well known in the art for this purpose.

  Peripheral blood can be conveniently analyzed for the presence of cancer cells, including lymphomas and leukemias, using RT-PCR to detect HIRHy expression. The presence of RT-PCR amplifiable KIRHy mRNA provides an indication of the presence of one of these types of cancer. A sensitive assay for detecting and characterizing cancer cells in the blood can be used (Racila et al., Proc. Natl. Acad. Sci. USA 95: 4589-4594 (1998), generally referred to herein). Incorporated). This assay combines multi-parameter flow cytometry and immunohistochemical analysis with immunomagnetic enrichment and is reportedly capable of detecting one epithelial cell in 1 ml of peripheral blood. Very sensitive to detection of cancer cells.

  A related aspect of the invention is directed to detecting susceptibility to developing cancer in an individual. In one embodiment, the method of detecting susceptibility to cancer comprises detecting KIRHy mRNA or KIRHy in a tissue sample, and its presence is proportional to the degree of morbidity in the presence of KIRHy mRNA expression. Shows susceptibility to cancer.

  Yet another related aspect of the invention is directed to a method of assessment of tumor aggressiveness (Orlandi, et al., Cancer Res. 62: 567 (2002), incorporated herein by reference in its entirety). In one embodiment, the method of assessing tumor aggressiveness detects the level of KIRHy mRNA or KIRHy protein expressed by cells in a sample of tumor and collects the level so measured from the same individual. Comparing the level of KIRHy mRNA or KIRHy protein expressed in the corresponding matched normal tissue or normal tissue control sample, wherein the degree of KIRHy mRNA or KIRHy protein in the tumor sample relative to the normal sample is Indicates the degree of aggression.

  Methods for detecting and quantifying the expression of KIRHy mRNA or protein are described herein and use standard nucleic acid and protein detection and quantification techniques well known in the art. Standard method for detection and quantification of KIRHy mRNA uses in situ hybridization using a labeled KIRHy1 riboprobe (Gemoo-Engeeth, et al., Pediatrics, 109: E24-E32 (2002)), using a KIRHy polynucleotide probe Northern blots and related techniques (Kunzli, et al., Cancer 94: 228 (2002)), RT-PCR analysis using primers specific for KIRHy (Angchaiskiri, et al., Blood 99: 130 (2002)) And, for example, branched DNA (Jardi, et al., J. Viral Hepat. 8: 465-471 (2001)), SISBA, TMA (Kim ra, et al., J. Clin. Microbiol. 40: 439-445 (2002)) and other amplified detection methods such as various types of microassay products such as oligos, cDNAs and monoclonal antibodies. . In certain embodiments, real-time RT-PCR can be used to detect and quantify KIRHy mRNA expression (Simpson, et al., Molec. Vision 6: 178-183 (2000)). Standard methods for protein detection and quantification can be used for this purpose. In certain embodiments, polyclonal or monoclonal antibodies that are specifically reactive with wild-type KIRHy can be used in immunohistochemical assays of biopsy tissues (Ristimaki, et al., Cancer Res. 62: 632 ( 2002), incorporated herein by reference in its entirety.

(5.12.2 Diagnostic Assays and Kits)
The invention further provides for the presence or expression of KIRHy, or homologues thereof, in a test sample using nucleic acid probes and antibodies of the invention that are conveniently conjugated or otherwise conjugated with a suitable label. A method of identifying is provided.

  In general, a method for detecting a KIRHy polynucleotide comprises contacting a sample with a polynucleotide and a compound that binds or forms a complex for a period of time sufficient to form a complex, and detects the complex, resulting in: Where a complex is detected, it can include detecting a polynucleotide of the invention in a sample. Such a method involves contacting a sample under stringent hybridization conditions with a nucleic acid primer that anneals to the polynucleotide of the invention under such conditions and amplifying the annealed polynucleotide so that the polynucleotide is When amplified, the polynucleotides of the invention can also include being detected in a sample.

  In general, a method for detecting a polypeptide of the invention comprises contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period of time sufficient to form a complex, and detects the complex; If the complex is detected as a result, it can include the polypeptide of the invention being detected in the sample.

  Specifically, such methods involve incubating a test sample with one or more antibodies or one or more nucleic acid probes of the present invention and nucleic acid probes or antibodies against components in the test sample. Analysis of the binding.

  The conditions under which the nucleic acid probe or antibody is incubated with the test sample will vary. Incubation conditions depend on the format used in the assay, the detection method used, and the type and characteristics of the nucleic acid probe or antibody used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can be well adapted to use the nucleic acid probes or antibodies of the present invention. Examples of such assays are described in Chard, T .; , An Introduction to Radioimmunoassay and Related Technologies, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. et al. R. et al. , Techniques in Immunocytochemistry, Academic Press, Orlando, FL, Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijsssen, P .; , Practice and Theory of Immunoassays: Laboratory Technologies in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam. Test samples of the present invention include cells, cellular protein or membrane extracts, or biological fluids such as saliva, blood, serum, plasma, or urine. The test sample used in the method described above will vary based on the assay format, the nature of the detection method, and the tissue, cell or extract used as the sample to be analyzed. Methods for producing cellular protein extracts or membrane extracts are well known in the art and can be well adapted to obtain a sample that is compatible with the system utilized.

  In another embodiment of the invention, a kit is provided that contains the reagents necessary to perform the assay of the invention. In particular, the present invention detects during closure inhibition (a) a first container containing one of the probes or antibodies of the present invention; and (b) the following: detection of the presence of a wash reagent, bound probe or antibody Compartment kits are provided that receive one or more containers, including one or more other containers that include one or more of the reagents capable of.

  Specifically, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or plastic or paper strips. Such containers are from one section to another so that the sample and reagents are not cross-mixed and the agent or solution in each container is added in quantitative form from one section to another. Allow for sufficient movement of reagents. Such containers include containers that allow test samples, containers that contain antibodies used in the assay, containers that contain wash reagents (such as phosphate buffered saline, Tris buffer), and detect bound antibodies or probes. A container containing the reagents used to do so. Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or alternatively, enzymatic or antibody binding reagents capable of reacting with the labeled antibody when the primary antibody is labeled. Those skilled in the art will appreciate that the disclosed probes and antibodies of the present invention can be fully incorporated into one of the established kit formats well known in the art.

(5.12.3 Medical images)
A KIRHy antibody that recognizes KIRHy and fragments thereof is useful for medical images of sites that express KIRHy. Such methods include chemical attachment of labels or imaging agents such as ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, ²¹² Bi, or pharmaceutically acceptable, pharmaceutically acceptable. Administration of labeled antibodies and fragments to a subject with a possible carrier and imaging the labeled antibodies and fragments in vivo at the target site. Radiolabeled anti-KIRHy antibodies or fragments thereof may be particularly useful in in vivo images of KIRHy expressing cancers such as lymphomas or leukemias. Such antibodies can provide a sensitive method for detecting metastasis of KIRHy expressing cancer.

  In light of the present disclosure, one of ordinary skill in the art appreciates that many other embodiments and variations can be made within the scope of the present invention. Accordingly, it is intended that the broad aspects of the invention not be limited to the disclosure of the following examples.

Example 1
(Isolation of SEQ ID NO: 1 from cDNA library of human cells)
A novel nucleic acid of SEQ ID NO: 1 was obtained from various human cDNA libraries using standard PCR and sequenced by hybridization sequencing analysis and Sanger sequencing techniques. Library inserts were amplified by PCR using primers specific for the vector sequence adjacent to the insert. These sequences were spotted on a nylon membrane and searched with oligonucleotide primers to obtain sequence signatures. Clones were collected within groups of similar or identical sequences and a single representative clone was selected from each group for gel sequencing. The 5 ′ sequence of the amplified insert was estimated using reverse M13 sequencing primers in a typical Sanger sequencing protocol. The PCR product was purified and subjected to fluorescent dye terminator cycle sequencing. Single gel passage sequencing was performed using a 377 Applied Biosystems (ABI) sequencer. These inserts were novel sequences not previously obtained from this library and not previously reported in a suitable database. This sequence is designated as SEQ ID NO: 1 in the attached sequence listing.

(Example 2)
(Group of SEQ ID NO: 2)
The novel nucleic acid (SEQ ID NO: 2) of the present invention was obtained from various cDNA libraries by the method described in Example 1 above, and in some cases from one or more suitable databases. . The final sequence was combined using an EST sequence as a seed. The recursive algorithm is then used to derive seeds from the extended population by pulling further sequences from different databases belonging to this population (ie, Hyseq databases containing EST sequences, dbEST, gb pri, and UniGene). Spread out. The algorithm was terminated when there were no more sequences from the database that could expand the population. Inclusion of component sequences into the population was based on BLASTN hits against the expanded population with a BLAST score greater than 300 and a percent identity greater than 95%.

  (Table 1)

（実施例３）
（ＫＩＲＨｙ１ポリヌクレオチドの組織発現解析およびクロモソーム局在）
ハイブリッド形成によるスクリーニングから確立されたＨｙｓｅｑ独占データベースにより、ＳＥＱ ＩＤ ＮＯ：２が、以下のヒト組織／細胞ｃＤＮＡに発現していることがわかった。

(Example 3)
(Tissue expression analysis and chromosome localization of KIRHy polynucleotide)
The Hyseq exclusive database established from screening by hybridization revealed that SEQ ID NO: 2 is expressed in the following human tissue / cell cDNA.

  (Table 2)

ＳＥＱ ＩＤ ＮＯ：２に相当する遺伝子は、ヒトゲノム配列でのＢＬＡＳＴ解析によって、ヒトクロモソーム１７にマップされた。

The gene corresponding to SEQ ID NO: 2 was mapped to human chromosome 17 by BLAST analysis on the human genome sequence.

Example 4
(KIRHy mRNA is highly expressed in B-cell lineages and tissues)
FIG. 3 shows the relative expression of mRNA from B-cell lineage, healthy tissue, and tumor tissue from B cell lymphoma, follicular lymphoma, and myeloma.

  Total mRNA derived from tissues and cell lines was analyzed using quantitative real-time PCR (TaqMan) (Simpson, et al., Molec. Vision, 6: 178-183, incorporated herein by reference). 2000)) to determine the relative expression of KIRHy mRNA. Cell lineage (obtained from ATCC, Manassas, Va.) Total mRNA was isolated using standard protocols. Cell lines include acute myeloid leukemia (AML193), acute myeloid leukemia (AM565), acute myeloid leukemia (KG1), aplastic giant T cell lymphoma (L5664), B cell lymphoma (RA1), chronic myelogenous leukemia (K562), diffuse giant B cell lymphoma (L22601), diffuse lymphoma grade II / III (L5856), histiocytic lymphoma (U937), Hodgkins lymphoma (HD5664), giant B cell lymphoma (DB), non-Hodgkin Derived from Slymphoma (RL) and plasmacytoma.

  MRNA derived from tumor tissue was prepared from malignant B cells isolated from the tumor. Tumor samples were B cell lymphoma (H02-85T, H02-86T, H02-87T, H02-88T, H02-89T), follicular lymphoma (H02-74T, H02-75T, H02-76T, H02-77T, H02). -78T), and myeloma (H02-79T, H02-80T, H02-81T, H02-82T, H02-83T, H02-84T) from different patients. The DNA sequence encoding elongation factor 1 was used as a positive control and normalization factor in all samples. All assays were performed in duplicate and the values obtained were averaged. The y-axis shows the relative expression of KIRHy mRNA, with the lowest expression set equal to 1 and the remaining values expressed as relative values to 1.

  FIG. 3 shows that there is relatively little expression of the KIRHy gene in healthy tissues, except for tissues produced or infiltrated by the B lymphoma, ie, lymph nodes and small intestine. The results show that KIRHy is most highly upregulated in AML and diffuse lymphoma, which may be used as a therapeutic target for these types of diseases or as a diagnostic target Suggests that.

(Example 5)
(Production of KIRHy1-specific antibodies)
Cells expressing KIRHy are identified using antibodies against KIRHy. Polyclonal antibodies are produced by DNA vaccination or by injection of peptide antigens into rabbits or other hosts. Animals such as rabbits are immunized with peptides from the extracellular region of KIRHy conjugated to a carrier protein such as BSA (bovine serum albumin) or KLH (keyhole limpet hemocyanin). The rabbit is first immunized with a conjugated peptide in complete Freund's adjuvant, followed by a booster shot every two weeks by injection of the conjugated peptide in non-complete Freund's adjuvant. Anti-KIRHy antibody was affinity purified from rabbit serum using KIRHy peptide conjugated to Affi-Gel 10 (Bio-Rad) in phosphate buffered saline containing 0.1% sodium azide. save.

  One such polyclonal antibody is the amino acid sequence Glu-Glu-Pro-Thr-Glu-Tyr-Ser-Thr-Ile-Ser-Arg-, corresponding to amino acid residues 294 to 305 of SEQ ID NO: 3. Produced using KLH conjugated to immunogenic KIRHy with Pro (SEQ ID NO: 11). The anti-KIRHy peptide polyclonal antibody is referred to herein as 10458a. In order to determine that 10458a is KIRHy1-specific, a vector encoding V5 / His-tagged KIRHy (pIntron-KIRHy, Nucleo Inc.) was introduced into mammalian COS-7 cells. Western blot analysis of protein extracts of untransfected cells and KIRHy-1 containing cells, 10458a as the first antibody, horseradish peroxidase-labeled anti-rabbit antibody (Donkey anti-rabbit IgG) as the second It was performed using as an antibody. An approximately 48 kD band was detected in KIRHy-containing cells and not in control cells, indicating that 10458a was specific for KIRHy.

  Monoclonal antibodies are produced by injecting mice with KIRHy peptide, with or without adjuvant. Subsequently, the mice are boosted every two weeks until an appropriate immune response is identified (typically 1-6 months), at which point the spleen is removed. The spleen is subdivided to release spleen cells and fused with rodent myeloma cells (in the presence of polyethylene glycol). The resulting cells (hybridomas) are grown in culture and screened for antibody production by clone selection. The antibody is secreted into the culture supernatant and facilitates the screening process, such as screening by enzyme immunoassay (ELISA). Alternatively, a humanized monoclonal antibody is genetically engineered to a chimeric rodent / human monoclonal antibody in which a rodent-specific antibody region is replaced by a human counterpart and is produced in a mammalian cell. Or by immunizing the transgenic mouse, as described above, with a transgenic “knockout” mouse in which the naive antibody gene has been replaced with a human antibody gene.

(Example 6)
(Diagnostic method using KIRHy1-specific antibody to detect KIRHy expression)
Leukemia and myeloma cell line expression was detected by Western blot analysis using anti-KIRHy peptide polyclonal antibody 10458a (see Example 5 for Western details). All AML samples and histiocytic cell lines were positive for KIRHy expression (see Table 3).

  (Table 3)

結果は、ＫＩＲＨｙ１が、急性骨髄性白血病（ＡＭＬ）および組織球性リンホーマにて高く発現されていることを示している。さらに、これらの結果は、ＫＩＲＨｙ１ ｍＲＮＡの相対的発現と一致し（実施例４を参照のこと）、このことは、ＫＩＲＨｙ１標的化が、これらの疾病に対する治療的処置または診断アッセイとして有用であることを示唆している。

The results indicate that KIRHy is highly expressed in acute myeloid leukemia (AML) and histiocytic lymphoma. Furthermore, these results are consistent with the relative expression of KIRHy mRNA (see Example 4), indicating that KIRHy targeting is useful as a therapeutic treatment or diagnostic assay for these diseases. It suggests.

  The expression of KIRHy in tissue samples (normal or acute myeloid leukemia (AML) bone marrow) was detected using an anti-KIRHy peptide polyclonal antibody, 10458a (see Example 5). Samples were fixed in tissue in 10% formalin, embedded in paraffin, and cut with standard techniques to allow immunohistochemistry (IHC) analysis (LifeSpan Biosciences, Inc. Seatle, The sections were stained with 10458a followed by incubation with a second horseradish peroxidase (HRP) -conjugated antibody and visualized by the product of the HRP enzyme reaction. As can be seen in Table 4, the data show that KIRHy is highly expressed (5 out of 5 patient samples) on the cell surface of AML bone marrow tissue. No expression of KIRHy was observed (5 out of 5 patient samples). Is, KIRHy1 expression was seen in AML tissues (see Example 4) which are shown to be consistent with the relative expression of KIRHy1 mRNA.

  (Table 4)

さらに、抗体ブロッキングアッセイを、ＩＣＨによって実施し、解析した。正常およびＡＭＬ骨髄組織試料を、以上で言及したように、ＩＨＣのために調製したが、しかし１０４５８ａと試料をインキュベートする前に、１０４５８ａを、過剰なＳＥＱ ＩＤ ＮＯ：１１で前処理し、ついで上述したように処理した。試験した全ての試料（５中５）において、ＳＥＱ ＩＤ ＮＯ：１１が、ＡＭＬ骨髄試料中で、１０４５８ａの反応性を阻害した（表５を参照のこと）。

In addition, antibody blocking assays were performed and analyzed by ICH. Normal and AML bone marrow tissue samples were prepared for IHC as mentioned above, but prior to incubating the sample with 10458a, 10458a was pretreated with excess SEQ ID NO: 11 and then described above. Processed as you did. In all samples tested (5 out of 5), SEQ ID NO: 11 inhibited 10458a reactivity in AML bone marrow samples (see Table 5).

  (Table 5)

血液試料中の細胞表面上のＫＩＲＨｙ１の発現を、フローサイトメトリーによって検出する。末梢血単核球（ＰＢＭＣ）を、標準の技術を用いて血液試料から単離する。細胞を、氷冷ＰＢＳで洗浄し、氷上で、ＫＩＲＨｙ１−特異的ポリクローナル抗体と、３０分間インキュベートする。細胞を穏やかにペレット化し、ＰＢＳで洗浄し、蛍光抗−ウサギ抗体と共に３０分間、氷上でインキュベートする。インキュベーションの後、細胞を穏やかにペレット化し、氷冷ＰＢＳで洗浄し、０．１％アジ化ナトリウムを含むＰＢＳ中に再懸濁させ、解析まで氷上で保存する。試料を、ＦＡＣＳｃａｌｉｂｕｒフローサイトメーター（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）およびＣＥＬＬＱｕｅｓｔソフトウェア（（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）を用いて解析する。器具の設定は、ＦＡＣＳ−Ｂｒｉｔｅ検定ビーズ（Ｂｅｃｔｏｎ Ｄｉｃｋｉｎｓｏｎ）を用いて決定する。

The expression of KIRHy on the cell surface in the blood sample is detected by flow cytometry. Peripheral blood mononuclear cells (PBMC) are isolated from blood samples using standard techniques. Cells are washed with ice-cold PBS and incubated with KIRHy1-specific polyclonal antibody for 30 minutes on ice. Cells are gently pelleted, washed with PBS, and incubated with fluorescent anti-rabbit antibody for 30 minutes on ice. Following incubation, the cells are gently pelleted, washed with ice-cold PBS, resuspended in PBS containing 0.1% sodium azide and stored on ice until analysis. Samples are analyzed using a FACScalibur flow cytometer (Becton Dickinson) and CELLQuest software ((Becton Dickinson). Instrument settings are determined using FACS-Brite assay beads (Becton Dickinson).

A tumor expressing KIRHy is imaged using a KIRHy1-specific antibody conjugated to a radionuclide such as ¹²³ I and injected into the patient to target the tumor, followed by X-ray or magnetic Resonance imaging.

(Example 7)
(In vitro antibody-dependent cytotoxicity assay)
The ability of KIRHy1-specific antibodies to induce antibody-dependent cell-mediated cytotoxicity (ADCC) is determined in vitro. ADCC is performed using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI) (Hornick et al., Blood 89: 4437-4447, (1997)) and effector and target cells. Peripheral blood mononuclear cells (PBMC) or neutrophil multinucleated leukocytes (PMN) are two examples of effector cells that can be used in this assay. PBMCs are isolated from healthy human donors by Ficoll-Paque gradient centrifugation and PMN is purified by centrifugation through discontinuous Percoll gradients (70% and 62%) followed by hypotonic lysis to remove residual red blood cells To do. AML cells (for example) are used as target cells.

AML cells are suspended in RPMI 1640 medium containing 2% fetal bovine serum and plated in 96-well V-bottom microtiter plates at 2 × 10 ⁴ cells / well. KIRHy1-specific antibody is added in triplicate to individual wells at 1 μg / ml and effector cells are added at various effector: target cell ratios (12.5: 1 to 50: 1). Plates are incubated for 4 hours at 37 ° C. The supernatant is then collected, lactate dehydrogenase release is measured, and percent specific lysis is calculated using the manufacturer's protocol.

(Example 8)
(Toxin-conjugated KIRHy1-specific antibody)
Antibodies against KIRHy are conjugated to toxins to assess the effect of such conjugates in animal models of cancer. Chemotherapeutic agents such as calicheamicin and carboplatin, or toxic peptides such as ricin toxin are used in this approach. Antibody-toxin conjugates are used to target cytotoxic agents specifically to antigen bearing cells. Antibody-toxins bind to these antigen-containing cells, internalize by receptor-mediated endocytosis, and subsequently destroy the targeted cells. In this case, the antibody-toxin conjugate targets KIRHy-expressing cells such as AML cells and delivers cytotoxic agents to the tumor resulting in the death of the tumor cells.

  One example of a toxin that can be conjugated to an antibody is carboplatin. The mechanism by which this toxin is conjugated to the antibody is described in Ota et al. , Asia-Oceania J .; Obstet. Gymaecol. 19: 449-457 (1993). Cytotoxicity of carboplatin-conjugated KIRHy1-specific antibodies, for example, incubating KIRHy1-expressing target cells (such as AML cell line) with various concentrations of conjugated antibody, medium alone, carboplatin alone, or antibody alone In vitro. Antibody-toxin conjugates specifically target and kill cells with KIRH1y antigen, while cells without antigen, or cells treated with medium alone, carboplatin alone, or antibody alone are cytotoxic. Not shown.

  The anti-tumor efficacy of carboplatin-conjugated KIRHy1-specific antibodies is demonstrated in an in vivo rodent tumor model. 5-6 week old, athymic nude mice are implanted with tumors subcutaneously or via intravenous injection. Mice are treated with KIRHy1-carboplatin conjugates or with non-specific antibody-carboplatin conjugates. Tumor xenografts in mice containing KIRHy antigen are targeted and bound by KIRHy1-carboplatin conjugates. This results in tumor cell killing as evidenced by tumor necrosis, tumor shrinkage and increased survival of treated mice.

  Other toxins are conjugated to KIRHy1-specific antibodies using methods known in the art. An example of a toxin-conjugated antibody in human clinical trials is CMA-676, which is an antibody against the CD33 antigen in AML that is conjugated to calikiamycin toxin (Larson, Semin. Hematol. 38 (Suppl 6): 24-31 (2001). )).

Example 9
(Radioimmunotherapy using KIRHy1-specific antibody)
Animal models are used to assess the effect of antibodies against KIRHy as a vector in radionuclide transmission in radioimmunotherapy to treat AML and histiocytic lymphoma. Human tumors are grown in 5-6 week old athymic mice by subcutaneous injection of carcinoma cell lines or tumor cells. Tumor-bearing animals are injected intravenously with a radiolabeled anti-KIRHy antibody (eg, labeled with 30-40 μCi of ¹³¹ I) (Behr, et al., Int. J. Cancer 77: 787-795 (1988). ). Tumor size is measured before injection and on a regular basis after injection (ie weekly) and compared to tumors in mice that received no treatment. Anti-tumor efficacy is calculated by correlating acid average tumor volume with the degree of induced growth delay. To confirm tumor and organ tissue, animals are sacrificed by cervical dislocation and necropsied. Organs are fixed in 10% formalin, embedded in paraffin and minced. Sections are stained with hematoxylin-eosin.

(Example 10)
(Immunotherapy using KIRHy1-specific antibody)
An animal model is used to evaluate the effect of KIRHy1-specific antibodies as targets for antibody-based immunotherapy with monoclonal antibodies. Human AML cells are injected into the tail vein of 5-6 week old nude mice devoid of natural killer cells. To assess the ability of KIRHy1-specific antibodies in preventing tumor growth, mice were given KIRHy1-specific antibodies by intraperitoneal injection either 1 or 15 days after tumor implantation, followed by Give 20 μg or 100 μg daily, once or twice a week, respectively (Ozaki, et al., Blood 90: 3179-3186 (1997)). The level of human IgG (from the immune response caused by human tumor cells) is measured in rodent serum by ELISA.

The effect of KIRHy1-specific antibodies on the proliferation of AML cells is tested in vitro using the ³ H-thymidine incorporation assay (Ozaki et al., Supra). Cells are cultured in 96-well plates at 1 × 10 ⁵ cells / ml in 100 μl / well and incubated with various amounts of KIRHy antibody or control IgG (˜100 μg / ml) for 24 hours. Cells are incubated with 0.5 μCi ³ H-thymidine (New England Nuclear, Boston, Mass.) For 18 hours and harvested on glass filters using an automated cell harvester (Packard, Meriden, Conn.). Incorporated radioactivity is measured by a liquid scintillation counter.

The cytotoxicity of KIRHy monoclonal antibody is tested by the effect of complement in AML using a ⁵¹ Cr-release assay (Ozaki et al., Supra). AML cells are labeled with 0.1 mCi ⁵¹ Cr-sodium chromate for 1 hour at 37 ° C. ⁵¹ Cr-labeled cells are incubated for 30 minutes on ice with various concentrations of KIRHy monoclonal antibody or control IgG. Unbound antibody is removed by washing with media. Cells are dispersed in 96-well plates and incubated for 2 hours at 37 ° C. with a serial dilution series of rabbit complement. The supernatant is collected from each cell and the amount of ⁵¹ Cr released is measured using a gamma counter. Spontaneous release of ⁵¹ Cr is measured by incubating the cells in medium alone, while maximal ⁵¹ Cr release is measured by treating the cells with 1% NP-40 to break the plasma membrane. Percent cytotoxicity is measured by dividing the difference between experimental and spontaneous ⁵¹ Cr release by the difference between maximum and spontaneous ⁵¹ Cr release.

Antibody-dependent cell-mediated cytotoxicity (ADCC) for the KIRHy monoclonal antibody is measured using a standard 4 hour ⁵¹ Cr-release assay (Ozaki et al., Supra). Spleen mononuclear cells from SCID mice are used as effector cells and cultured (for example) with or without recombinant interleukin-2 for 6 days. ⁵¹ Cr-labeled target AML cells (1 × 10 ⁴ cells) are plated in 96-well plates with various concentrations of anti-KIRHy monoclonal antibody or control IgG. Effector cells are added to the wells at various effector to target ratios (12.5: 1 to 50: 1). After 4 hours, the culture supernatant is removed and counted in a gamma counter. The percentage of cell lysis is determined as above.

(Example 11)
(KIRHy1-specific antibody as an immunosuppressant)
An animal model is used to assess the effect of KIRHy1-specific antibodies to inhibit signaling through the KIRHy receptor and to suppress immune diseases such as rheumatism or other inflammatory conditions, or rejection of organ transplants. Immunosuppression is tested by injecting mice with horse red blood cells (HRBCs) and assaying the level of HRBC-specific antibodies (Yang, et al., Int. Immunopharm. 2: 389-397 (2002)). . The animals are divided into 5 groups, 3 of which are injected with anti-KIRHy antibody for 10 days and 2 groups are not treated. Two experimental groups and one control group are injected with either Eagle Balance Salt Solution (RBSS) containing 5-10 × 10 ⁷ HRBCs, or EBSS alone. Anti-KIRHy antibody treatment continues in one group while the other group does not receive antibody treatment. After 6 days, all animals are retroorbital puncture bleeding followed by cervical dislocation and spleen removal. Spleen cell supernatants are prepared and serum is removed by centrifugation for analysis.

Immunosuppression is measured by the number of B cells producing HRBC-specific antibodies. Ig isotypes (eg, IgM, IgG1, IgG2, etc.) are determined using the IspDetect ^™ Isotyping kit (Stratagene, La Jolla, Calif.). Once the Ig isotype is known, rodent antibodies against HRBCs are measured using an ELISA procedure. A 96-well plate is coated with HRBCs and incubated with anti-HRBC antibody-containing serum isolated from the animals. Plates are incubated with alkaline phosphatase-labeled secondary antibody and color development is measured on a microplate reader (SPECTRAmax 250, Molecular Devices) at 405 nm using p-nitrophenyl phosphate as a substrate.

Lymphocyte proliferation is measured in response to the T and B cell activities concanavalin A and lipopolysaccharide, respectively (Jiang, et al., J. Immunol. 154: 3138-3146 (1995)). Mice were randomly divided into two groups, one group receiving anti-KIRHy antibody treatment for 7 days, one group being the control. At the end of the treatment, the animals are sacrificed by cervical dislocation, the spleen is removed and the spleen cell supernatant is prepared as described above. For ex vivo studies, the same number of spleen cells is used, whereas for in vivo studies, anti-KIRHy antibody is added into the culture at the start of the experiment. Cell proliferation is also assayed using the above (Ozaki, et al., Blood 90: 3179 (1997)) ³ H-thymidine incorporation assay.

(Example 12)
(Cytokine secretion in response to KIRHy peptide fragment)
The assay assesses the activity of fragments of KIRHy proteins, such as IgG domains, to stimulate cytokine secretion and stimulate immune responses in NK cells, B cells, T cells and bone marrow cells. Such an immune response can be used to stimulate and suppress the immune system to recognize and / or mediate tumor cell killing or growth inhibition. Similarly, this immune stimulation can be used to target bacterial or viral infections. Alternatively, fragments of KIRH1 that inhibit activation through the KIRHy receptor may be used to prevent immune stimulation in natural killer (NK), B, T and bone marrow cells.

  A fusion protein comprising a fragment of KIRHy, such as an Ig domain (KIRHy1-Ig), is generated by inserting a CD33 leader peptide followed by a KIRHy domain fused to the Fc region of human IgG1 into a mammalian expression vector; For example, stably transfected into NS-1 cells. The fusion protein is secreted into the culture supernatant, which is a cytokine, such as an interferon-γ (IFN-γ) secretion assay (Martin, et al., J. Immunol. 167: 3668-3676 (2001)). Recover for use in the assay.

  PBMCs are activated with suboptimal concentrations of soluble CD3 and various concentrations of purified, soluble anti-KIRHy monoclonal antibody or control IgG. For the KIRHy1-Ig cytokine assay, anti-human Fc Ig at 5 or 20 μg / ml is bound to a 96-well plate and incubated at 4 ° C. overnight. Excess antibody is removed and either KIRHy1-Ig or control Ig is added at 20-50 μg / ml and incubated for 4 hours at room temperature. The plate is washed to remove excess fusion protein before adding cells and anti-CD-3 to various concentrations. Supernatants are harvested after 48 hours of culture and IFN-γ levels are measured by sandwich ELISA using primary and biotinylated secondary anti-human IFN-γ antibodies as recommended by the manufacturer. To do.

(Example 13)
(Tumor imaging using KIRHy1-specific antibody)
KIRHy1-specific antibodies are used to image KIRHy1-expressing cells in vivo. Six week old athymic nude mice are radiated from a cesium source at 400 rads. Three days later, radiated mice are implanted with 4 × 10 ⁷ RA1 cells and 4 × 10 ⁶ human fetal lung fibroblast feeder cells subcutaneously in the thigh. When the tumor reaches approximately 1 cm in diameter, mice are injected intravenously with inoculum containing 100 μCi / 10 μg of ¹³¹ I-labeled KIRHy1-specific antibody. At 1, 3 and 5 days after injection, mice are anesthetized by subcutaneous injection of 0.8 mg sodium pentobarbital. Next, the Spectrum with a pinhole collimator set to record 5,000-10,000 counts using a Nuclear MAX Plus image analysis software package (MEDX Inc. Wood Dale, IL). 91 images (Raytheon Media Systems), Melrose Park, IL) and imaged in the prone position (Hornick, et al., Blood 89: 4437-4447 (1997)).

(Example 14)
(In vivo tumor model)
Tumor suppressive activity of KIRHy targeting molecules was demonstrated by injecting subcutaneously either 10 ⁶ cells, control (M12 pcDNA), KIRHy1 expressing clones, or low expressing clones into 4-10 nude, athymic male mice. Test (Spenger et al., Cancer Research 59: 2370-2375 (1999), all of which are incorporated herein by reference). The lowest level KIRHy clone is used as a comparative benchmark. Mice are monitored for 8 weeks for weight gain / loss and tumor formation. Tumor volume is calculated using the formula (l × w ² ) / 2, where l = tumor length and w = tumor width (Id.).

  Statistical analysis using the Kruskal-Wallit method for tumor formation comparison and the Mann-Whitney U test to compare tumor dose is performed to determine statistical significance between groups.

After 8 weeks, mice are sacrificed, tumors are removed and digested with 0.1% collagenase (type I) and 50 μg / ml DNase (Worthington Biochemical Corp., Freehold, NJ). The dispersed cells are plated for 24 hours at 37 ° C. in ITS medium / 5% FBS at %% CO ₂ and allowed to adhere. After 24 hours, the culture medium is changed to a serum-free medium. Cells will divide, media and RNA will be collected and Western immunoblot and Northern blot will be performed to detect KIRHy.

(Example 15)
(In vitro assay of cell proliferation and migration)
The effect of a KIRHy1-specific antibody or therapeutic peptide on the proliferation of AML cells was compared with the ³ H-thymidine incorporation assay (Ozaki et al., Blood 90: 3179-3186, all of which are incorporated herein by reference). (1997)). Tumor cells in 100 [mu] l / well at 1 × 10 ⁵ cells / ml, were cultured in 96-well plates with various amounts of the antibody or control IgG (~100μg / ml), incubated for 24 hours. Cells are incubated for 18 hours with 0.5 μCi ³ H-thymidine (New England Nuclear, Boston, Mass.) And harvested on glass filters using an automated cell harvest phase (Packard, Meriden, Conn.). Incorporated radioactivity is measured using a liquid scintillation counter.

Cell migration is performed in 24-well, 6.5-mm internal diameter Transwell culture plates (Corning Coastar, Cambridge, Mass.). Briefly, 10 ⁵ cells / 75 μl are placed on fibronectin (5 μM) -coated polycarbonate membrane (8-μm pore size) and the two chambers of the transwell are separated (all of which are hereby incorporated by reference). Incorporated, Tai et al., Blood 99: 1419-1427 (2002) Medium with or without anti-KIRHy antibody is added to the lower chamber of the Transwell culture plate and induced into the lower chamber after 8-16 hours. Cells are counted by hematocytometer using a Coulter counter ZBII (Beckman Coulter).

(Example 16)
(Clinical study on AML)
For examples of AML clinical trials using immunotherapy, Larson, Semin. Hematol. 38 (Suppl 6): 24-31 (2001). For examples of clinical trials using small molecules, see Fiedler et al. , Blood 102: 2763-2767 (2003), O'Farrel et al. , Clin. Cancer Res. 9: 5465-5476 (2003), Ohno et al. , J .; Clin. Oncol. 8: 1907-1912 (1990).

  At the beginning of the study, patients identified as AML with sufficient liver and kidney function are confirmed to be KIRHy1-positive. Patients are treated with physiological doses of anti-KIRHy targeted drugs and tested for side effects, toxicity, and end response criteria. Complete blood counts indicating peripheral blood counts are measured daily for the first 4 days, every 2 days for the first 2 weeks, and twice a week for the remainder of the study. Bone marrow tests are performed after the first week and every two weeks for the remainder of the study. Safety assessments include evaluation of side effects and fatal signs, hematology tests, biochemical tests, urinalysis, and physical examination. Toxicity is graded according to the National Toxicity Criteria of the National Cancer Institute. Response criteria include complete remission using conventional criteria, complete remission without complete platelet recovery, but no longer dependent on platelet transfusion. Standards include 5% blasts, peripheral blood free blasts, hemoglobin greater than or equal to 9 g / dL, neutrophil absolute count equal to or greater than 1500 / μl, transfusion non-ionic, and 100,000 / μl It consists of bone marrow with a platelet count equal to or greater than

FIG. 1 is a diagram in which the BLASTP amino acid sequence (ie, SEQ ID NO: 3) of the protein encoded by SEQ ID NO: 2 and human natural killer (NK) inhibitory receptor precursor (SEQ ID NO: 8) are arranged. Over the entire amino acid sequence of number 3, these two sequences are shown to have 94% similarity and 94% identity. FIG. 2 is a diagram in which the BLASTP amino acid sequence of KIRHy1 (ie, SEQ ID NO: 3) and the human CMRF35-like protein (similar to CMRF35 leukocyte-like receptor) (SEQ ID NO: 9) are aligned, as encoded by SEQ ID NO: 2. It shows that these two sequences have 89% similarity and 89% identity across the 149 amino acids of SEQ ID NO: 3. FIG. 3 shows healthy tissues, acute monocytic leukemia (AML193), acute myeloid leukemia (AML565), acute myeloid leukemia (KG1), anaplastic large cell T cell lymphoma (L5664), B cell lymphoma (RA1). ), Chronic myelogenous leukemia (K562), diffuse large B-cell lymphoma (L22601), grade II / III follicular lymphoma (L5856), histiocytic lymphoma (U937), Hodgkin lymphoma (HD5664), large cells Cell lines derived from type B cell lymphoma (DB), non-Hodgkin lymphoma (RL), and plasmacytoma (RPMI), and B cell lymphoma (H02-85T, H02-86T, H02-87T, H02-88T, H02- 89T), follicular lymphoma (H02-74T, H02-75T, H02-76T, H02-77T, H 2-78T) and expression of KIRHy mRNA (determined by RT-PCR) in tumor tissue from myeloma (H02-79T, H02-80T, H02-81T, H02-82T, H02-83T, H02-84T) FIG.

Claims (35)

An isolated polynucleotide encoding the polypeptide of SEQ ID NO: 11. The polynucleotide of claim 1 which is a DNA sequence. A vector comprising the polynucleotide of claim 1. An expression vector comprising the polynucleotide according to claim 1. A host cell genetically engineered to contain the polynucleotide of claim 1. A host cell genetically engineered to contain the polynucleotide of claim 1 operably linked to a regulatory sequence that regulates expression of the polynucleotide in the host cell. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 11. An antibody that specifically binds to SEQ ID NO: 11. 9. The antibody of claim 8, wherein the antibody is a monoclonal antibody or an antibody fragment thereof. The antibody according to claim 9, wherein the antibody is a polyclonal antibody or an antibody fragment thereof. The antibody of claim 9, wherein the antibody is 12434a. A pharmaceutical composition comprising an anti-KIRHy antibody specific for cells causing acute myeloid leukemia (AML), wherein the antibody specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof . 13. The pharmaceutical composition according to claim 12, wherein the antibody is a monoclonal anti-KIRHy antibody or an antigen-binding fragment thereof. The pharmaceutical composition according to claim 12, wherein the antibody is labeled with a radioisotope. 13. The pharmaceutical composition according to claim 12, wherein the antibody is labeled with a toxin. 13. The pharmaceutical composition of claim 12, wherein the antibody is administered in an amount effective to kill or inhibit the growth of cells that cause AML. A method of targeting a KIRHy protein in a cell that causes AML comprising the step of administering to the cell a composition in an amount effective to target the KIRHy expressing cell, wherein The method wherein the composition is an anti-KIRHy antibody that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof. A method of killing or inhibiting the growth of a KIRHy-expressing cell that causes AML, wherein the composition is applied to the cell in an amount effective to kill the cancer cell or inhibit its growth. Administering, wherein the composition is an anti-KIRHy antibody that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof. A method of killing or inhibiting the growth of a KIRHy-expressing cell that causes AML, wherein the compound is administered to the cell in an amount effective to kill the cancer cell or inhibit its growth Wherein the compound comprises a KIRHy antigen. A method of killing or inhibiting the growth of a KIRHy-expressing cell that causes AML, wherein the composition is applied to the cell in an amount effective to kill the cancer cell or inhibit its growth. Administering, wherein the composition comprises a nucleic acid encoding KIRHy or an immunogenic fragment thereof in a recombinant vector. A method of killing or inhibiting the growth of a KIRHy-expressing cell that causes AML, wherein the composition is applied to the cell in an amount effective to kill the cancer cell or inhibit its growth. Administering, wherein the composition comprises an antigen presenting cell comprising a nucleic acid encoding KIRHy1 or an immunogenic fragment thereof in a recombinant vector. A method of killing or inhibiting the growth of a KIRHy-expressing cell that causes AML, wherein the composition is applied to the cell in an amount effective to kill the cancer cell or inhibit its growth. Administering, wherein the composition comprises a small molecule that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof. A method of killing or inhibiting the growth of a KIRHy-expressing cell that causes AML, wherein the composition is applied to the cell in an amount effective to kill the cancer cell or inhibit its growth. Administering, wherein the composition comprises a non-KIRHy polypeptide that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3, or an immunogenic fragment thereof. 24. The method according to any one of claims 17-23, wherein the cell is contacted with a second therapeutic agent. 19. The method of claim 17 or 18, wherein the anti-KIRHy antibody composition is administered in an amount effective to achieve a dosage range of about 0.1 mg / kg body weight to about 10 mg / kg body weight. Method. 24. The method of any one of claims 17-23, wherein the pharmaceutical composition is administered in a sterile preparation with a pharmaceutically acceptable carrier. A method of diagnosing a cancer selected from the group consisting of AML, comprising:
a) detecting or measuring the expression of KIRHy protein on the cell; and b) comparing the expression to normal tissue;
Including the method. 28. The method of claim 27, wherein the expression is the expression of KIRHy mRNA. 28. The method of claim 27, wherein the expression is detected or measured using an anti-KIRHy antibody. Use of an anti-KIRHy antibody in the preparation of a medicament for killing or inhibiting the proliferation of KIRHy-expressing cells that cause AML, wherein the antibody comprises a polypeptide having the amino acid sequence of SEQ ID NO: 3 or an immunogen thereof Use that specifically binds to a sex fragment. Use of a KIRHy antigen in the preparation of a medicament for killing or inhibiting the growth of KIRHy expressing cells that cause AML. Use of a nucleic acid encoding KIRHy or an immunogenic fragment thereof in a recombinant vector in the preparation of a medicament for killing or inhibiting the growth of KIRHy-expressing cells that cause AML. Use of an antigen presenting cell comprising a nucleic acid encoding KIRHy or an immunogenic fragment thereof in a recombinant vector in the preparation of a medicament for killing or inhibiting the growth of KIRHy expressing cells that cause AML. Use of a small molecule that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof in the preparation of a medicament for killing or inhibiting the growth of KIRHy-expressing cells that cause AML . Non-KIRHy polypeptide that specifically binds to a polypeptide having the amino acid sequence of SEQ ID NO: 3 or an immunogenic fragment thereof in the preparation of a medicament for killing or inhibiting the growth of KIRHy-expressing cells that cause AML Use of.

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