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Method for nk cell transduction

A technology of NK cells and cell membranes, applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, and can solve problems such as non-proliferating cells and cell cycle arrest

Active Publication Date: 2020-08-21
ミルテニイビオテックベーファーウントコーカーゲー
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, TBK1 is essential for mitosis and BX795 is generally known to cause cell cycle arrest, resulting in non-proliferating cells (Pillai et al., 2015; Bai et al., 2015)

Method used

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  • Method for nk cell transduction
  • Method for nk cell transduction
  • Method for nk cell transduction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0141] In one embodiment of the invention, a transgenic nucleic acid (eg, nucleic acid encoding a chimeric antigen receptor) is transferred into activated NK cells using a retroviral vector particle (eg, lentiviral vector particle) that Particles were pseudotyped with a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein capable of binding and fusing with hematopoietic cell membranes.

[0142] The pseudotyped retroviral vector particles, e.g., lentiviral vector particles, may comprise a chimeric envelope glycoprotein comprising the transmembrane and extracellular domain with or consisting of the cytoplasmic tail domain of the murine leukemia virus (MLV) envelope glycoprotein), or a modified BaEV envelope glycoprotein in which the cytoplasmic tail domain lacks a fusion-inhibiting R-peptide.

[0143] NK cells in a cell culture medium comprising NK cells are activated, for example, by adding IL-2 and / or IL-15 to the culture medium. After 2 to 5 days, the effect of...

Embodiment 1

[0187] Example 1: Surprisingly, lentiviral vectors pseudotyped with baboon envelope glycoprotein (BaEV) in NK cells Shows high transduction efficiency of primary NK cells upon activation.

[0188]For NK cell isolation from the buffy coat, peripheral blood mononuclear cell (PBMC) preparation was performed by standard density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare). NK Cell Isolation Kit-Human (MiltenyiBiotec) was used for NK cell untouched enrichment from PBMC. Isolated NK cells were divided into three groups, activated for different durations and transduced with a lentiviral vector pseudotyped with GFP-containing BaEV. All transductions were performed with 10 ng / ml vectofusin (Miltenyi Biotec) and spinoculation according to the manufacturer's protocol. The first group of NK cells were transduced immediately after isolation (no activation). The second group of NK cells was cultured in NKMACS medium (Miltenyi Biotec) containing 5% AB serum, 500U / ml ...

Embodiment 2

[0189] Example 2: The transduction of activated NK cells with BaEV is significantly higher than that with VSV-G transduction, is stable for a long time, and does not induce cell death

[0190] Isolation of primary NK cells as described in Example 1, resulting in pure CD3 - and CD56 + NK cells (Fig. 2A). According to the user manual, T cells were isolated from the same blood sample using anti-CD4 MicroBeads and anti-CD8 MicroBeads from Miltenyi to generate pure CD3-positive T cells (Fig. 2A). After isolation, NK cells were activated for 2 days in NK MACS medium containing 5% AB serum, 10 ng / ml IL-15 and 500 U / ml IL-2. After isolation, T cells were activated for 2 days in TexMACS medium from Mitenyi containing 200 U / mL IL-2 and T cell Transact human according to the user manual. Subsequently, activated primary T and NK cells, as well as the NK-92 cell line, were transduced with supernatants containing a lentiviral vector encoding GFP expression either pseudotyped with the...

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Abstract

The present invention discloses an in-vitro method for transferring biological material into activated NK cells with a pseudotyped retroviral vector particle or a virus-like particle thereof, comprising the steps a) activation of NK cells, and b) addition of said pseudotyped retroviral vector particle or virus-like particle thereof to said activated NK cells, wherein said pseudotyped retroviral vector particle or virus-like particle thereof comprises a modified baboon endogenous retrovirus (BaEV) envelope glycoprotein that is able of binding to and fusing with a hematopoietic cell membrane, thereby transferring biological material into said activated NK cells. Preferentially, the activating of NK cells is performed by the addition of an IL-1 family cytokine to the NK cells.

Description

technical field [0001] The present invention relates generally to the field of transfer of biological material into cells, and in particular to the transfer of biological material into activated natural killer (NK) cells. Background technique [0002] Natural killer cells (hereinafter also abbreviated as "NK cells") are a unique population of lymphocytes capable of detecting and destroying virus-infected cells and malignantly degenerated cells (also called tumor cells). In addition, NK cells produce and secrete cytokines upon exposure to tumor cells. These functional features make NK cells attractive drugs for the treatment of cancer. Clinical trials have highlighted the potential of NK cells for clinical use (Miller et al., 2005; Rubnitz et al., 2010; Childs & Berg 2013). [0003] The use of donor NK cells for patients ("allogeneic use") requires cell isolation methods to separate desired NK cell effects from unwanted, counter-indicating non-NK cell (eg, T cell) effects. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867
CPCC12N2740/15033C12N2740/15021C12N2740/15043C12N2740/15045C12N15/86C12N2740/10022C12N2740/16043C12N2740/16045A61K39/12A61K35/17C07K14/5443C07K14/545C07K14/55C07K2319/85C12N15/867C12N2740/13044
Inventor R·巴里M·格兰青W·伦格N·默克V·胡珀特
Owner ミルテニイビオテックベーファーウントコーカーゲー
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