Methods of generating natural killer cells

A natural killing, cell technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of difficult to maintain tumor targeting and killing ability, difficult to apply immunotherapy, etc.

Active Publication Date: 2013-05-08
ANTHROGENESIS LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although NK cells have favorable properties in killing tumor cells and virus-infected cells, they are still difficult to use with and in immunotherapy, mainly due to the difficulty in maintaining their tumor targeting during culture and expansion. and tumor killing ability

Method used

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  • Methods of generating natural killer cells
  • Methods of generating natural killer cells
  • Methods of generating natural killer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0268] 7.1. Example 1: Recovery of hematopoietic stem cells from human placental perfusate and umbilical cord blood

[0269] Human placental perfusate (HPP) and umbilical cord blood (UCB) cells are usually purified using Ficoll or ammonium chloride to obtain total nucleated cells (TNC). TNCs were then used in a positive selection procedure using anti-CD34 beads and RoboSep according to the manufacturer's (StemCell Technologies, Inc.) protocol to isolate CD34 + cell. In this experiment, CD34 was isolated with a purity greater than 90% + cell. As another option, the Human Progenitor Cell Enrichment Kit (StemCell Technologies, Inc.) was developed by using the Human Progenitor Cell Enrichment Mixture and the following human cell surface antigens: CD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin A monoclonal antibodies were used in a negative selection procedure to exclude lineage-committed cells. Using negative selection, 90% of CD34 was recovere...

Embodiment 2

[0272] 7.2. Example 2: Feeder-free Expansion and Differentiation of Hematopoietic Stem Cells to Natural Killer Cells

[0273] CD34 + Cells were cultured in the following media formulations for up to 48 days, and cell aliquots were taken for cell counts, assessment of cell viability, identification of natural killer cell differentiation, and functional assessment.

[0274] NK1 medium : GBGM (Glycostem Basal Growth Medium, Glycostem Cat. No.: CCT-SCB500, Clear cell technology) supplemented with pen / strep (Cat. No.: 15140, Gibco), 20 ng / niL SCF (Cat. No.: 255-SC, R&D Systems), 10ng / mL Flt-3 Ligand (Cat. No.: 308-FK, R&D system), 20ng / mL TPO (Cat. No.: 288-TP, R&D system), 20ng / mL IL-7 (Cat. No.: 207 -IL, R&D Systems), 200IU / mL IL-2 (catalogue number: 202-IL, R&D Systems), and 10ng / mL IL-15 (catalogue number: 247-IL, R&D Systems).

[0275] NK2 medium : DMEM (Cat. No.: MT-10-013-CV, Fisher): Ham's F12 medium (Cat. No.: BW12-615F, Fisher) (1:2) supplemented with 2 mM L-gluta...

Embodiment 3

[0295] 7.3. Embodiment 3: the culture of NK cell in CNK culture medium

[0296] Enhance NK cell expansion and cytotoxicity

[0297] On day 27 (D27), CD34 cultured in NK2A medium + Cells were further cultured in one of the following media:

[0298] • A two-stage medium comprising CNK medium and maintenance medium. The CNK medium was IMDM (Invitrogen) supplemented with 10% FCS (Hyclone), 200 IU / mL IL-2 (R&D Systems), 35 μg / mL transferrin (Sigma-Aldrich), 5 μg / mL insulin (Sigma-Aldrich ), 2×10-5M ethanolamine (Sigma-Aldrich), 1 μg / mL oleic acid (Sigma-Aldrich), 1 μg / mL linoleic acid (Sigma-Aldrich), 0.2 μg / mL palmitic acid (Sigma-Aldrich), 2.5 μg / mL BSA (Sigma-Aldrich) and 0.1 μg / mL phytohemagglutinin (PHA-P, Sigma-Aldrich). CD56 cultured in NK2A medium + CD3 - NK cells were resuspended at 2.5×l05 viable cells / mL in CNK medium in cell culture-treated 24-well plates or square flasks. Allogeneic PBMC and K562 cells (chronic myelogenous leukemia cell line) treated with mi...

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Abstract

Provided herein are methods of producing natural killer cells using a two-step expansion and differentiation method. Also provided herein are methods of suppressing tumor cell proliferation, of treating individuals having cancer or a viral infection, comprising administering the NK cells produced by the method to an individual having the cancer or viral infection.

Description

[0001] 1. Related applications [0002] The present application claims the rights and interests of U.S. Provisional Patent Application Serial No. 61 / 363981 filed on July 13, 2010 and U.S. Provisional Patent Application Serial No. 61 / 497897 filed on June 16, 2011, the disclosure content of the above patent applications They are hereby incorporated by reference in their entirety. 2. Technical field [0003] Provided herein are methods of producing natural killer cell populations, e.g., natural killer cells derived from placenta, e.g., derived from placental perfusate (e.g., human placental perfusate), such as placenta-derived intermediate natural killer cells, or derived from In other tissues, for example, cord blood or peripheral blood. Also provided herein are expanded natural killer cell populations produced by the methods provided herein. Also provided herein are methods of inhibiting tumor cell proliferation using placental perfusate and natural killer cells derived from ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/12
CPCA61K2035/124C12N2500/25C12N2500/33C12N2501/998C12N5/0646C12N2500/44C12N2501/91C12N2501/26C12N2500/46C12N2501/125C12N2501/2302C12N2501/2315C12N2501/145C12N2500/38C12N2501/2307C12N2500/05C12N2501/59C12N2500/36C12N2501/2303A61K35/17C12N2500/30C12N2506/11A61P35/00A61P35/02A61P35/04Y02A50/30A61P31/12C12N2500/84
Inventor 罗伯特·J·哈黎里穆罕默德·A·黑德兰斯蒂芬·亚什科康琳埃里克·劳阿贾伊·帕尔芭娃妮·斯托特瓦内萨·沃斯基纳里安-贝尔斯安德鲁·查特林张小葵
Owner ANTHROGENESIS LLC
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