Back to results
US 12,655,391 B2Granted
Modified K562 feeder cell line expressing factors that enhance the activation and proliferation of natural killer cells and a method for its production
AssigneeGene Solutions Joint Stock Company
InventorsLe Son Tran, Thi Bao Tram Tran, Thi Van Anh Bui
Life of this patent
Estimating the term…
Abstract
A method for generating or expanding a population of natural killer cells (NK cells) via using modified K562 feeder cells having expression of factors capable of increasing activation and proliferation of natural killer cells, wherein the factors capable of increasing activation and proliferation of NK cells include: (A) a co-stimulatory molecule comprises CD80 molecules, and 4-1BBL molecules; and (B) a cytokine, specifically membrane-bound IL-21 (mbIL-21).
Claims2
A method for generating or expanding a population of natural killer (NK) cells, comprising:
- (i) isolating and purifying NK cells from a cord blood sample by gradient centrifugation;
- (ii) preparing a modified K562 feeder cell line expressing factors that increase NK cell activation and proliferation;
- wherein the factors that increase NK-cell activation and proliferation comprise:
- a co-stimulatory molecule combination which is CD80 and 4-1BB ligand (4-1BBL);
- and
- a cytokine which is membrane-bound interleukin-21 (mbIL-21);
- wherein step (ii) comprises:
- (A) producing lentiviral vectors encoding the co-stimulatory molecule combination and the cytokine;
- and
- (B) transducing K562 cells with the lentiviral vectors to obtain the modified K562 feeder cell line;
- (iii) generating or expanding a population of NK cells by culturing the NK cells isolated in step (i) in the presence of irradiated cells of the modified K562 cell line from step (ii) for 14 days, wherein the culturing comprises:
- co-culturing the NK cells with irradiated cells of the modified K562 feeder cell line at a feeder-to-NK cell ratio of 5:1 at day 0 in a serum-free lymphocyte culture medium supplemented with 10% fetal bovine serum (FBS) and 200 IU/ml interleukin-2 (IL-2);
- enumerating the NK cells and adding irradiated cells of the modified K562 feeder cell line to the NK cells at day 7 to maintain a feeder-to-NK cell ratio of 5:1 in a fresh serum-free lymphocyte culture medium supplemented with 10% FBS and 200 IU/ml IL-2;
- after that performing lentiviral chimeric antigen receptor (CAR) transduction of the NK cells at day 7, and culturing the post transduction NK cells in the fresh serum-free lymphocyte culture medium supplemented with 10% FBS and 200 IU/ml IL-2:
- replacing the culture medium with the fresh serum-free lymphocyte culture medium supplemented with 10% FBS and 200 IU/ml IL-2 at day 8;
- enumerating the NK cells and adding irradiated cells of the modified K562 feeder cell line to the NK cells at day 10 to establish a feeder-to-NK cell ratio of 1:1 in the fresh serum-free lymphocyte culture medium supplemented with 10% FBS, and 200 IU/ml IL-2;
- and
- collecting the population of NK cells at day 14;
- wherein the population of NK cells is functionally competent to exhibit high cytotoxic activity.
16 more elements
A population of natural killer (NK) cells exhibiting enhanced cytotoxic activity relative to NK cells expanded without a modified K562 feeder cell line expressing CD80, 4-1BB ligand (4-1BBL), and membrane-bound interleukin-21 (mbIL-21), and without defined temporal modulation of feeder-to-NK cell ratios;
- wherein the population of NK cells is generated or expanded by a method comprising:
- (i) isolating and purifying NK cells from a cord blood sample by gradient centrifugation;
- (ii) preparing a modified K562 feeder cell line expressing factors that increase NK cell activation and proliferation;
- wherein the factors that increase NK-cell activation and proliferation comprise:
- a co-stimulatory molecule combination which is CD80 and 4-1BB ligand (4-1BBL);
- and
- a cytokine which is membrane-bound interleukin-21 (mbIL-21);
- wherein step (ii) comprises:
- (A) producing lentiviral vectors encoding the co-stimulatory molecule combination and the cytokine;
- and
- (B) transducing K562 cells with the lentiviral vectors to obtain the modified K562 feeder cell line;
- (iii) generating or expanding a population of NK cells by culturing the NK cells isolated in step (i) in the presence of irradiated cells of the modified K562 cell line from step (ii) for 14 days, wherein the culturing comprises:
- co-culturing the NK cells with irradiated cells of the modified K562 feeder cell line at a feeder-to-NK cell ratio of 5:1 at day 0 in a serum-free lymphocyte culture medium supplemented with 10% fetal bovine serum (FBS) and 200 IU/ml interleukin-2 (IL-2);
- enumerating the NK cells and adding irradiated cells of the modified K562 feeder cell line to the NK cells at day 7 to maintain a feeder-to-NK cell ratio of 5:1 in a fresh serum-free lymphocyte culture medium supplemented with 10% FBS and 200 IU/ml IL-2;
- after that performing lentiviral chimeric antigen receptor (CAR) transduction of the NK cells at day 7, and culturing the post transduction NK cells in the fresh serum-free lymphocyte culture medium supplemented with 10% FBS and 200 IU/ml IL-2;
- replacing the culture medium with the fresh serum-free lymphocyte culture medium supplemented with 10% FBS and 200 IU/ml IL-2 at day 8;
- enumerating the NK cells and adding irradiated cells of the modified K562 feeder cell line to the NK cells at day 10 to establish a feeder-to-NK cell ratio of 1:1 in the fresh serum-free lymphocyte culture medium supplemented with 10% FBS, and 200 IU/ml IL-2;
- and
- collecting the population of NK cells at day 14;
- wherein the population of NK cells is functionally competent to exhibit high cytotoxic activity.
17 more elements
Dependencies are inferred from the claim text.