End-to-end cell therapy automation
The present disclosure provides an automated method of producing genetically modified immune cells, including chimeric antigen receptor T (CAR T) cells, utilizing a fully-enclosed cell engineering system.
1. A method for automated production of a genetically modified T cell culture, the method comprising:
a) mixing a T cell culture with a magnetic selection reagent;
b) exposing the T cell culture to magnetic separation;
c) activating the T cell culture with an activation reagent selected from an antibody and a dendritic cell to produce an activated T cell culture;
d) transducing within a cell culture chamber the activated T cell culture with a viral vector encoding an ectodomain, a transmembrane domain and an endodomain, to produce a transduced T cell culture;
e) expanding within the cell culture chamber the transduced T cell culture;
f) centrifuging the expanded T cell culture; and
g) harvesting the T cell culture to produce a genetically modified T cell culture,
wherein (a) through (f) are performed within a fully enclosed, automated cell engineering system, and the cell culture chamber has a fixed area; and
wherein expansion of the transduced T cell culture in (c) produces at least 20% more genetically modified T cells than expansion utilizing manual cell culture with a flexible, gas permeable bag and transduction efficiency of the method is at least 20% higher than transduction efficiency utilizing manual cell culture with the flexible, gas permeable bag.
2. The method of claim 1 , wherein speed of the centrifuging is automatically adjusted.
3. The method of claim 1 , wherein the method further includes:
a) monitoring with one or more of a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and an optical density sensor; and
b) adjusting one or more of a temperature, a pH level, a glucose level, an oxygen level, a carbon dioxide level, and an optical density of the transduced T cell culture, based on the monitoring.
4. The method of claim 3 , wherein the method further includes adjusting parameters of the centrifuging step based on a pre-defined concentration of T cells of the expanded T cell culture.
5. The method of claim 1 , wherein the automated cell engineering system further includes one or more sampling ports to sample the transduced T cell culture.
6. The method of claim 5 , wherein cell density, glucose, and pH can be measured from a sample of the transduced T cell culture.
7. The method of claim 1 , wherein the magnetic selection reagent comprises antibody-conjugated beads.
8. The method of claim 1 , wherein the activating is within the cell culture chamber.
9. The method of claim 1 , wherein the T cell culture comprises at least one accessory cell.
10. The method of claim 9 , wherein the accessory cell is a monocyte, an antigen-presenting cell or a dendritic cell.
11. The method of claim 10 , wherein the accessory cell comprises antigens for a T cell receptor, selected from CD28, CD40, CD2, CD40L, ICOS and combinations thereof.
12. The method of claim 1 , wherein the transduced T cell culture is a chimeric antigen receptor T (CAR T) cell culture.
13. The method of claim 12 , wherein the ectodomain is a region that is exposed to extracellular fluid and includes three parts: a signaling peptide, an antigen recognition region, and a spacer.
14. The method of claim 12 , wherein the transmembrane domain is a hydrophobic α-helix that spans a membrane.
15. The method of claim 14 , wherein the transmembrane domain is a CD28 transmembrane domain or a CD3-ζ transmembrane domain.
16. The method of claim 12 , wherein the endodomain of the CAR is a CD3-ζ endodomain, which includes three immunoreceptor tyrosine-based activation motifs (ITAMs).
17. The method of claim 1 , wherein the automated cell engineering system includes at least one peristaltic pump.