| Methods of treating tumors using natural killer cell lines -> Monitor Keywords |
|
|
 
Methods of treating tumors using natural killer cell linesRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus ContainingMethods of treating tumors using natural killer cell lines description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060110360, Methods of treating tumors using natural killer cell lines. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATION [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/403,910, filed on Oct. 27, 1999, which was based on, and claimed benefit of, U.S. Provisional Application Ser. No. 60/045,885, filed on Apr. 30, 1997. FIELD OF THE INVENTION [0002] This invention relates to natural killer cells and their use in the treatment of pathologies related to cancer or viral infections. Specifically, a particular cell line, NK-92, and modifications thereof, are disclosed. These cells are shown to be highly effective in the treatment of these pathologies. BACKGROUND OF THE INVENTION [0003] Certain cells of the immune system have cytotoxic activity against particular target cells. Cytotoxic T lymphocytes (CTLs) are specifically directed to their targets via antigen-derived peptides bound to MHC class I-specific markers. Natural killer (NK) cells, however, are not so restricted. NK cells, generally representing about 10-15% of circulating lymphocytes, bind and kill target cells, including virus-infected cells and many malignant cells, nonspecifically with regard to antigen and without prior immune sensitization (Herberman et al., Science 214:24 (1981)). Killing of target cells occurs by inducing cell lysis. MHC class restriction likewise is not involved. In these ways the activity of NK cells differs from antigen-specific and MHC class-specific T cells, such as cytotoxic T lymphocytes. Use of NK cells in the immunotherapy of tumors and malignancies is suggested by these properties, since many tumors are MHC class I deficient and therefore do not attract CTL activity. Adhesion molecules may also be involved in the targeting of NK cells; for example, it is observed that the Fc.gamma. receptor (CD16) is expressed on NK cells. NK cells are large granular lymphocytes which lack CD3, and in addition to CD16, also may express Leu19 (Lanier et al., J. Immunol. 136; 4480 (1986)). [0004] NK cells are activated when exposed to cytokines such as interleukin-2 (IL-2), IL-7, IL-12, and interferons (Alderson et al., J. Exp. Med. 172:577-587 (1990); Robertson et al., J. Exp. Med. 175:779-788 (1992)). The resulting cells are called lymphokine activated killer (LAK) cells. The spectrum of target cells is altered in activated NK cells compared to nonactivated cells, although the mechanism of killing may be identical or similar (Philips et al., J. Exp. Med. 164:814-825 (1986)). [0005] More generally, killing activity in the cells of the immune system may be induced by treating a population of cells, such as peripheral blood mononuclear cells (PBMCs), with lymphokines. Such preparations contain LAK cells. LAK cells may also be generated from autologous samples of peripheral blood lymphocytes. LAK cells have antitumor killing activity while having essentially no effect on normal cells. They appear to purge leukemia (Long et al., Transplantation 46:433 (1988); Xhou et al., Proc. Am. Assoc. Cancer Res. 34:469 (1993; abstract)), lymphoma (Schmidt-Wolf et al., J. Exp. Med. 174: 139 (1991); Gambacorti-Passerini et al., Br. J. Haematol. 18:197 (1991)) and neuroblastoma (Ades et al., Clin. Immunol. Immunopathol. 46:150 (1988)). NK cells, activated NK cells, and LAK cells are distinguishable by their cell surface markers and by the identity of the target cells that they kill. [0006] Activated and expanded (i.e., cultured to proliferate) NK cells and LAK cells have been used in both ex vivo therapy and in vivo treatment in patients with advanced cancer. Some success with ex vivo therapy has been observed in bone marrow related diseases, such as leukemia, breast cancer and certain types of lymphoma. In vivo treatment may be directed toward these and other forms of cancer, including malignant melanoma and kidney cancer (Rosenberg et al., N. Engl. J. Med. 316:889-897 (1987)). LAK cell treatment requires that the patient first receive IL-2, followed by leukophoresis and then an ex vivo incubation and culture of the harvested autologous blood cells in the presence of IL-2 for a few days. The LAK cells must be reinfused along with relatively high doses of IL-2 to complete the therapy. This purging treatment is expensive and can cause serious side effects. These include fluid retention, pulmonary edema, drop in blood pressure, and high fever. In some cases in which these side effects occur, intensive care unit management is required. [0007] Purging techniques have been applied in other circumstances as well. Cytotoxic drugs or monoclonal antibodies combined with complement, and toxins, may be administered in order to bring about remission. In such cases bone marrow or blood stem cells, purged to reduce the number of residual leukemic cells present, have been infused back into the patient after the drug treatment (Uckun et al., Blood 79:1094 (1992)). Gene marking studies have shown that unpurged bone marrow may contribute to relapse in patients presumed to be in remission (Brenner et al., Lancet 341:85 (1993)). This suggests that some form of purging of autologous marrow or blood prior to transplantation is necessary (Klingemann et al., Biol. Blood Marrow Transplant. 2:68-69 (1996)). [0008] Recently, preclinical studies have also demonstrated promising antitumor activity in vivo with a lethally irradiated, MHC-unrestricted, cytotoxic T-cell leukemic clone (TALL-104) (Cesano et al., Cancer Immunol. Immunother. 40:139-151 (1995); Cesano et al., Blood 87:393-403 (1996)). These cells were derived from leukemia T cell lines obtained from patients having acute T lymphoblastic leukemias (ALL). They bear the CD3 cell surface marker, but not the CD56 marker, in distinction to NK cells which have the converse immunophenotype (CD3.sup.- CD56.sup.+). Adoptive transfer of these cells was able to eliminate human leukemic cell lines in xenografted sever combined immunodeficient (SCID) mice and to induce remissions of spontaneous lymphomas in dogs without producing T-cell leukemia in the animal models (Cesano et al. (1995); Cesano et al. (1996); Cesano et al., J. Clin. Invest. 94:1076-1084 (1994); Cesano et al., Cancer Res. 56:3021-3029 (1996)). [0009] In spite of the advantageous properties of NK cells in killing tumor cells and virus-infected cells, they remain difficult to work with and to apply in immunotherapy. It is difficult to expand NK cells ex vivo that maintain their tumor-targeting, tumoricidal, and viricidal capabilities in vivo. This remains a major obstacle to their clinical use in adoptive cell immunotherapy (Melder et al., Cancer Research 48:3461-3469 (1988); Stephen et al., Leuk. Lymphoma 377-399 (1992); Rosenberg et al., New Engl. J. Med. 316:889-897 (1987)). Studies of the mechanisms whereby NK cells exert their tumoricidal and viricidal effects are also limited by difficulties in enriching the NK cell fractions without compromising their biological functions and in obtaining pure NK cells that are not contaminated by T cells or other immune effector cells. In an attempt to overcome these problems, many investigators have turned to the use of established NK-like cell lines to explore the mechanisms whereby target cells are susceptible to cytotoxic cells (Hercend et al., Nature 301:158-160 (1983): Yodoi et al., J. Immunol. 134:1623-1630 (1985); Fernandez et al., Blood 67:925-930 (1986); Robertson et al., Exp. Hematol. 24:406-415 (1996); Gong et al., Leukemia 8:652-658 (1994)). NK cell lines described in earlier work carry T lymphocyte-associated surface markers, in spite of the fact that they were developed from precursor cells depleted of T cells (Rosenberg, et al. (1987); Hercend, et al., (1983)). [0010] There thus remains a need for a method of treating a pathology related to cancer or a viral infection with a natural killer cell line that maintains viability and therapeutic effectiveness against a variety of tumor classes. This need encompasses therapeutic methods in which samples from a mammal are treated ex vivo with natural killer cells, as well as methods of treatment of these pathologies with natural killer cells in vivo in a mammal. There is also a need for a natural killer cell line that maintains its own propensity for viability and cytolytic activity by secreting a cytokine which fosters these properties. There also remains a need for such natural killer cell lines which are modified to be more effective, convenient, and/or useful in treatment of cancer and viral infection. It is the objective of this invention to provide NK cells and methods that address these needs. SUMMARY OF THE INVENTION [0011] The cell line described by Gong et al. (1994), termed NK-92, proliferates in the presence of IL-2 and has high cytolytic activity against a variety of cancers. The present invention employs the NK-92 cell line, as well as modified NK-92 cell lines, to provide cancer treatment and virus treatment systems. The invention also provides the vectors that transfect NK-92, as well as the modified NK-92 cells. For purposes of this invention and unless indicated otherwise, the term "NK-92" is intended to refer to the original NK-92 cell lines as well as the modified NK-92 cell lines disclosed herein. [0012] One aspect of the invention provides a vector for transfecting NK-92 cells, wherein the vector includes a nucleic acid sequence encoding a protein that is either a cytokine which promotes the growth of the NK-92 cells, a cellular component responsive to an agent, a cancer cell receptor molecule, or any combination of these proteins. When transfected with the vector, the NK-92 cells constitutively express the protein. In an important embodiment, the protein is the cytokine interleukin 2. In especially important embodiments of this aspect of the invention, the vectors are MFG-hIL-2 and pCEP4LTRhIL-2. In additional significant embodiments, the protein is a cellular component responsive to an agent, such that when the vector transfects NK-92 cells and the agent is taken up by the cells, the cells are inactivated. In still more significant embodiments the agent is either acyclovir or gancyclovir. [0013] A further embodiment of the invention provides a cell population containing NK-92 cells that have been modified by a physical treatment or by transfection with a vector. [0014] In significant embodiments of this population, the physical treatment renders them non-proliferative yet does not significantly diminish the cytotoxicity of the cells, and in particularly significant embodiments, the treatment is irradiation. In additional important embodiments the cells have been transfected by a vector that encodes a cytokine promoting the growth of the cells. The cells secrete the cytokine both upon being cultured under conditions that promote cytokine secretion or in vivo upon being introduced into a mammal. In particularly important embodiments of this aspect of the invention, the cytokine is interleukin 2. In still further important embodiments, the NK-92 cells are the cells NK-92MI, modified by transfection with the vector MFG-hIL-2 encoding, and the cells NK-92CI modified by transfection with the vector pCEP4-LTRhIL-2 encoding interleukin-2. The NK-92MI and NK-92CI cell lines have been in the American Type Culture Collection under the designations CRL-2408 and CRL-2409, respectively. In additional important embodiments, the NK-92 cells are transfected by a vector including a sequence that encodes a cellular component responsive to an agent such that, when the NK-92 cell so transfected takes up the agent, the cell is inactivated. In particularly important embodiments thereof, the agent is acyclovir or gancyclovir. In yet additional embodiments, the cell population is transfected with a vector encoding a cancer cell receptor molecule. [0015] The present invention also provides a method of purging cells related to a pathology from a biological sample including the steps of (i) obtaining a biological sample from a mammal that is suspected of containing cells related to the pathology, and (ii) contacting the sample with a medium comprising NK-92 or modified NK-92 natural killer cells, wherein the modified NK-92 cells have been modified by a physical treatment or by transfection with a vector. In significant embodiments of this method, the pathology is a cancer, or is an infection by a pathogenic virus such as human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), or herpes virus. In additional important embodiments, the modified NK-92 cells have undergone a physical treatment that renders them non-proliferative, yet which does not significantly diminish their cytotoxicity, or have been transfected with a vector, or they have been treated by any combination of these modifications. In significant embodiments of this method, the vector encodes a cytokine that promotes the growth of the cells, a protein that is responsive to an agent, a cancer cell receptor molecule, or a combination of these coding sequences. In a further embodiment, the medium also includes a cytokine that promotes the growth of the cells. The sample, once purged of cancer cells, may be further treated, including, for example, being returned to the mammal from which it was obtained. In important embodiments of the method, the biological sample is blood or bone marrow, the mammal is a human, and/or the natural killer cell is immobilized on a support. [0016] The invention additionally provides a method of treating a pathology ex vivo in a mammal including the steps of (i) obtaining a biological sample suspected of containing cells related to the pathology from the mammal; (ii) contacting the biological sample with a median including natural killer cells, either NK-92 cells or modified NK-92 cells that have been modified by a physical treatment or by transfection with a vector, thereby selectively destroying the cells related to the pathology in the sample and producing a purged sample, and (iii) returning the purged sample to the mammal. The pathology may be a cancer, such as a leukemia, a lymphoma, or a multiple myeloma. Alternatively, the pathology may be infection by a pathogenic virus such as HIV, EBV, CMV, or herpes. In this method the natural killer cells may be NK-92 itself or modified NK-92 cells. Examples of such modified NK-92 cells include those that have been modified by a physical treatment that renders them non-proliferative yet does not significantly diminish their cytotoxicity, and modification by transfection with a vector. The vector encodes a cytokine that promotes the growth of the cells, or a protein that is responsive to an agent, or a cancer cell receptor molecule, or the vector may include any combination of these modifications In important embodiments of this method, the biological sample is blood or bone marrow, the mammal is a human, and/or the natural killer cell is immobilized on a support. In additional significant embodiments, the medium further includes a cytokine that promotes the growth of the cells, and/or the cancer is a leukemia, a lymphoma or a multiple myeloma. [0017] The present invention further provides a method of treating a pathology in vivo in a mammal including the step of administering to the mammal a medium comprising natural killer cells, either NK-92 cells or NK-92 cells that have been modified by a physical treatment that renders them non-proliferative yet does not significantly diminish their cytotoxicity, by treatment that inhibits expression of HLA antigens on the NK-92 cell surface, or by transfection with a vector. The vector encodes a cytokine that promotes the growth of the cells, or a protein that is responsive to an agent, or a cancer cell receptor molecule, or they have been treated by any combination of these modifications. In important embodiments, the pathology is a cancer, such as a leukemia, a lymphoma, or a multiple myeloma. Alternatively, in important embodiments the pathology is infection by a pathogenic virus such as HIV, EBV, CMV, or herpes. Advantageous embodiments of this method include administering the cells intravenously to a human and administering a cytokine that promotes the growth of the cells to the mammal in conjunction with administering the medium comprising the natural killer cell. The present methods are especially adapted for the treatment of leukemia, lymphoma or multiple myeloma. [0018] In yet an additional embodiment of the in vivo method of treating cancer, the NK-92 is modified by transfection with a vector comprising an element responsive to an agent such that when the agent is taken up by the cell, the cell is inactivated. According to this method, an amount of the magent effective to inactivate the cell can be administered to a mammal after a time sufficient for the natural killer cell to treat the cancer has elapsed or at a time desirable to effectively end the treatment. A significant aspect of this embodiment is one in which the agent is acyclovir or gancyclovir. Such transfected cells can, in effect, be "turned off" as desired by administering the agent. BRIEF DESCRIPTION OF THE DRAWING [0019] FIG. 1. Cytotoxic activity of NK-92 against different leukemic target cell lines tested in a 4 hour .sup.51Cr release assay. The results represent the mean.+-.the standard deviation (SD) for three replicate experiments. Continue reading about Methods of treating tumors using natural killer cell lines... Full patent description for Methods of treating tumors using natural killer cell lines Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods of treating tumors using natural killer cell lines patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Methods of treating tumors using natural killer cell lines or other areas of interest. ### Previous Patent Application: Cellular delivery of natriuretic peptides Next Patent Application: Methods of making viral particles having a modified cell binding activity and uses thereof Industry Class: Drug, bio-affecting and body treating compositions ### FreshPatents.com Support Thank you for viewing the Methods of treating tumors using natural killer cell lines patent info. IP-related news and info Results in 0.31579 seconds Other interesting Feshpatents.com categories: Accenture , Agouron Pharmaceuticals , Amgen , AT&T , Bausch & Lomb , Callaway Golf 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
