Methods of producing modified natural killer cells and methods of use
Inventors
Childs, Richard W. • Allan, David S. J.
Assignees
United States, REPRESENTED BY SECRETARY Department And Of Health And Human Services AS • US Department of Health and Human Services
Publication Number
US-11293010-B2
Publication Date
2022-04-05
Expiration Date
2037-07-25
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Abstract
Disclosed herein are method of producing NK cells that include one or more heterologous nucleic acids. The methods include culturing a population of isolated NK cells in the presence of one or more cytokines to produce a population of activated NK cells. The population of activated NK cells are transduced with a viral vector comprising the one or more heterologous nucleic acids, for example by contacting the activated NK cells with viral particles including the viral vector. The resulting transduced NK cells are then cultured in the presence of one or more cytokines, and optionally in the presence of irradiated feeder cells, to produce a population of expanded transduced NK cells. Also disclosed are methods of treating a subject with a disorder (such as a tumor or hyperproliferative disorder) by administering to the subject NK cells produced by the methods described herein.
Core Innovation
The invention provides efficient viral vector-based methods for producing natural killer (NK) cells that include or express one or more heterologous nucleic acids, referred to as modified NK cells. The methods involve culturing isolated NK cells in the presence of cytokines such as IL-2, IL-15, and/or IL-21 to activate them, followed by transduction with a viral vector comprising the heterologous nucleic acids, and subsequent culture to expand the transduced NK cells, optionally in the presence of irradiated feeder cells.
The problem addressed is the difficulty in achieving efficient genetic modification of primary NK cells for therapeutic use. Although NK cells have intrinsic anti-tumor activity, malignant cells often resist NK cell lysis due to expression of MHC class I antigens. Previous clinical trials using adoptive transfer of autologous NK cells have had limited success. There is a need for simple and efficient gene transfer methods to introduce genes of interest into primary NK cells to enhance their anti-tumor and immunotherapeutic efficacy.
The disclosed methods overcome existing limitations by enabling stable and long-term robust expression of transgenes in primary human peripheral blood-derived NK cells using lentiviral vectors. The method is simpler and more cost-effective compared to prior protocols requiring multiple transductions and additional reagents. It supports high transduction efficiency without compromising NK cell viability or function. The protocol allows for large-scale expansion of genetically modified NK cells suitable for clinical applications.
Claims Coverage
The patent discloses two independent claim sets: one directed to methods of producing transduced NK cells, and another directed to methods of treating subjects by administering such NK cells.
Method of producing NK cells with heterologous nucleic acids
Culturing isolated NK cells in presence of 500 IU/ml IL-2 and absence of irradiated feeder cells for 2-3 days to activate; transducing the activated NK cells with a viral vector carrying one or more heterologous nucleic acids; and culturing transduced NK cells with 500 IU/ml IL-2 and irradiated feeder cells to expand the population.
Use of lentivirus vector for transduction
Utilization of lentiviral vectors for gene delivery, specifically lentivirus vectors comprising or consisting of nucleic acid sequences with at least 90% identity to any of SEQ ID NOs: 8-17.
Feeder cell ratio and type during expansion
Culturing transduced NK cells with irradiated feeder cells at a ratio ranging from 2:1 to 20:1, wherein feeder cells may be Epstein-Barr virus transformed lymphoblastoid cell lines or K562 cells.
Heterologous nucleic acid encoding therapeutic proteins or regulatory RNAs
Encoding high affinity CD16 (CD16-V158), CXCR4, CCR7, CXCR3, CD34, double negative TGFβ type II receptor, VLA-4, LFA-1; chimeric antigen receptors (CARs) targeting tumor antigens such as CD19, CD20, CD33, CD138, CS1, GD2, HER2, erbB2, CEA, EpCAM, NKG2D-L, or TRAIL-R1; truncated CD34 lacking intracellular domain; or shRNA/siRNA/antisense nucleic acids to modulate gene expression.
Method of treating subjects using modified NK cells
Obtaining NK cells from subject or donor; activating with IL-2 (500 IU/ml) without feeder cells for 2-3 days; transducing with viral vector; expanding with IL-2 and irradiated feeder cells; and administering the expanded modified NK cells to subjects with tumors, hematological malignancies, or viral infections.
The patent claims cover a method for efficiently producing genetically modified NK cells using lentiviral vectors with cytokine activation and feeder cell-based expansion, and methods for treating diseases by administering these modified NK cells expressing a variety of therapeutic transgenes.
Stated Advantages
The method provides efficient, robust, and highly reproducible stable gene transfer into primary human NK cells with long-term transgene expression.
The protocol is simpler and cheaper than previous methods, avoiding cumbersome steps like retronectin use, spinoculation, and multiple transductions that may harm NK cell viability.
The use of lentiviral vectors enhances safety due to improved vector design, including split genome packaging and self-inactivating promoters, reducing risks of insertional mutagenesis.
The modified NK cells retain normal phenotype and function, allowing their use in various gene therapy and immunotherapy applications.
Documented Applications
Treating subjects with tumors, including hematological malignancies such as leukemias, lymphomas, multiple myeloma, and solid tumors like breast, lung, pancreatic, and ovarian cancers.
Treating subjects with hyperproliferative disorders and viral infections including cytomegalovirus, adenovirus, respiratory syncytial virus, Epstein-Barr virus, and human immunodeficiency virus.
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