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Natural killer cell lines and methods of use

a technology of natural killer cells and cell lines, applied in the field of natural killer cells, can solve the problems of difficult immunotherapy work, difficult to work with and apply, and high cost of purging treatment, so as to promote cell growth, promote cytokine secretion, and reduce the cytotoxicity of cells

Inactive Publication Date: 2014-04-10
CONKWEST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent describes a new cell line called NK-92, which can be used to treat cancer and virus infections. The cells are modified to express a protein called interleukin-2, which promotes their growth. The cells can be further modified to respond to specific agents or to target cancer cells. The patent also describes a method for purging cancer cells from biological samples, such as blood or bone marrow, and a method for treating pathologies ex vivo in a mammal. The technical effects of the patent include the development of a new and effective cell line for cancer treatment and the ability to selectively destroy cancer cells while preserving other cells in the sample.

Problems solved by technology

This purging treatment is expensive and can cause serious side effects.
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.
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.

Method used

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  • Natural killer cell lines and methods of use
  • Natural killer cell lines and methods of use
  • Natural killer cell lines and methods of use

Examples

Experimental program
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example 1

NK-92 Cells

[0083]NK-92 cells (Gong et al. (1994)) were derived from cells obtained from a patient suffering from non-Hodgkin's lymphoma. PBMC from the patient were cultured in enriched alpha MEM supplemented with fetal calf serum (12.5%) and horse serum (12.5%) plus 10−6 M hydrocortisone and 1000 U / mL of recombinant human IL-2 (rhIL-2). Cells were cultured at 37° C. in humidified air containing 5% CO2. Subcultures were made after 4 weeks, and propagated indefinitely with twice-weekly changes in medium. In these later stages the hydrocortisone could be omitted without any effect on cell growth. This culture has been designated NK-92 and has been deposited with the American Type Culture Collection (ATCC; Rockville, Md.) under designation CLR-2407.

[0084]The cells have the morphology of large granular lymphocytes. The cells bear the CD56bright, CD2, CD7, CD11a, CD28, CD45, and CD54 surface markers. In contrast, they do not display the CD1, CD3, CD4, CD5, CD8, CD10, CD14, CD16, CD19, CD2...

example 2

Cytotoxic Activity of NK-92 against Different Leukemic Cell Lines

[0085]The cytotoxic activity of NK-92 against K562, Daudi, TF-1, AML-193, and SR-91 cells was determined (Gong et al. (1994)). K562 (erythroleukemia) and Daudi (Burkitt) lymphoma cell lines were obtained from ATCC. They were maintained in continuous suspension culture in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). TF-1 is a myelomonocytic cell line (Kitamura et al., J. Cell Physiol. 140:323-334 (1989)) that requires the presence of medium containing 2 ng / mL of human GM-CSF. AML-193 is a myeloid cell line that is maintained in the presence of 10% 5637-conditioned medium (Lange et al., Blood 70:192-199 (1987)). Both TF-1 and AML-193 cells were obtained from Dr. D. Hogge, Terry Fox Laboratory, University of British Columbia, Vancouver, BC. SR-91 is a cell line with features of early progenitor cells established by Gong et al. (1994) from a patient with acute lymphoblastic leukemia (ALL) (Klingemann et a...

example 3

Cytotoxicity of NK-92 Against Leukemia, Lymphoma, and Myeloma Target Cell Lines

[0087]K562 (Ph-chromosome positive [Ph+] erythroleukemia), HL60 (promyelocytic), U937 (myelomonocytic), KG1a (variant subline of the AML cell line KG1), DHL-10 (B-cell lymphoma), Daudi (Burkitt's lymphoma), Raji (B-cell lymphoma), Jurkat (T-cell lymphoma), U266 (IgE myeloma), NCI H929 (IgA myeloma), and RPMI 8226 (myeloma, light chain secreting) cell lines were obtained from ATCC. The lymphoma-derived cell lines Ly3 (B-lineage, diffuse large cell), Ly8 (immunoblastic), and Ly13.2 (T-lineage, diffuse large cell) were provided by Dr. H. Messner, Toronto, Ontario. Their characteristics have been described (Chang et al., Leuk. Lymphoma 19:165 (1995)). All lines were maintained in RPM1 1640 medium supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 U / mL penicillin, 25 mM HEPES (StemCell Technologies), and 5% heat-inactive FCS(RPMI / 5% FCS) at 37° C. in a humidified atmosp...

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Abstract

This invention relates to a natural killer cell line termed NK-92. The invention provides a vector for transfecting a mammalian cell which includes a nucleic acid sequence encoding a cytokine that promotes the growth of NK-92. Additionally, the invention provides an NK-92 cell, or an NK-92 cell modified by transfection with a vector conferring advantageous properties, which is unable to proliferate and which preserves effective cytotoxic activity. The invention further provides a modified NK-92 cell that is transfected with a vector encoding a cytokine that promotes the growth of NK-92 cells. The cell secretes the cytokine upon being cultured under conditions that promote cytokine secretion, and furthermore secretes the cytokine in vivo upon being introduced into a mammal. In a significant embodiment, the cytokine is interleukin 2. The present invention also provides methods of purging cancer cells from a biological sample, of treating a cancer ex vivo in a mammal, and of treating a cancer in vivo in a mammal employing a natural killer cell, such as NK-92 itself, an NK-92 cell which is unable to proliferate and which preserves effective cytotoxic activity, or natural killer cells transfected with a vector encoding a cytokine.

Description

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 k...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/0783A61K35/12C07K14/55C12N5/00
CPCC12N5/0646A61K38/00A61K2035/124C07K14/55C12N5/0093C12N2510/00A61K39/4644A61K2239/31A61K2239/48A61K39/4613A61K2239/38
Inventor KLINGEMANN, HANS
Owner CONKWEST
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