Mononuclear cell derived NK cells
Cord blood or peripheral blood NK cells are prepared from whole blood mononuclear cells without the need to isolate CD34+ hematopoietic stem cells or NK cells, and without the need for a feeder layer. Advantageously, the methods presented herein use an enrichment process that uses antiCD16 agonist antibodies, antiCD3 antibodies, and N-803. Moreover, contemplated processes are suitable for adaptation into a fully automated production process (GMP in a box).
1. A method of producing NK cells, comprising:
isolating from whole blood or cord blood a mixture of mononuclear cells,
contacting, in a container, the mixture of the mononuclear cells with an anti-CD16 antibody, an anti-CD3 antibody, and N-803 to activate NK cells;
sequentially feeding, in the same container, the activated NK cells by adding to the container a medium containing N-803 to promote preferential growth of NK cells in the mixture until the activated NK cells constitute at least 90% of all live cells or until the activated NK cells are enriched to an at least 80-fold expansion.
2. The method of claim 1 wherein the mixture of mononuclear cells is not further processed to enrich NK cells.
3. The method of claim 1 wherein the mixture of the mononuclear cells contains about 100-500×10 6 cells.
4. The method of claim 1 wherein the step of sequentially feeding is performed until a total cell number of about 0.5-5.0×10 9 cells is reached.
5. The method of claim 1 wherein the step of sequentially feeding the activated NK cells is performed until NK cells are enriched to an at least 100-fold expansion.
6. A method of expanding NK cells in a mixture of mononuclear cells isolated from whole blood or cord blood, comprising:
providing a mixture of the mononuclear cells that contains NK cells;
contacting, in a container, the mixture of the mononuclear cells with an anti-CD16 antibody, an anti-CD3 antibody, and N-803 to activate NK cells;
feeding, in the same container, the activated NK cells by adding to the container a medium containing N-803 to promote preferential growth of NK cells in the mixture until the activated NK cells constitute at least 90% of all live cells or until the activated NK cells are enriched to an at least 80-fold expansion.
7. The method of claim 6 wherein the anti-CD16 antibody is present at a concentration of between 0.05-1.0 mcg/ml.
8. The method of claim 6 wherein the N-803 is present at a concentration of between 0.1-1.0 nM.
9. The method of claim 6 wherein the anti-CD3 antibody is present at a concentration of between 0.1-1.0 ng/ml.
10. The method of claim 6 wherein the step of feeding the activated NK cells is performed until NK cells are enriched to an at least 100-fold expansion.
11. A method of expanding NK cells, comprising:
incubating a mixture of mononuclear cells isolated from whole blood or cord blood in an activation medium containing N-803, an anti-CD3 antibody, and an anti-CD16 antibody for a time sufficient to activate NK cells;
wherein the mixture of mononuclear cells is contained in a cell culture container while incubating the mixture;
measuring growth of the cells while the cells are in the container;
automatically feeding the cells in the same cell culture container by adding a medium containing N-803 to promote preferential growth of NK cells in the mixture, wherein the feeding is controlled by a predetermined schedule and/or a result from the step of measuring growth of the cells, and wherein the cells are fed until the NK cells are enriched to an at least 80-fold expansion;
terminating feeding the cells wherein the terminating is controlled by a predetermined schedule and/or a result from the step of measuring growth of the cells.
12. The method of claim 11 wherein the container has a volume of between about 200 ml and about 2,500 ml.
13. The method of claim 11 wherein the step of measuring growth of the cells is performed by counting.
14. The method of claim 11 wherein the activation medium contains N-803 at a concentration of between 0.1-1.0 nM and the anti-CD16 antibody at a concentration of between 0.05-1.0 mcg/ml.
15. The method of claim 11 wherein the cells are fed until a total cell number of about 0.5-5.0×10 9 cells is reached.
16. The method of claim 11 wherein the cells are fed until NK cells are enriched to an at least 100-fold expansion.