Methods for transduction and cell processing

Patent metadata

Field	Value
Publication number	US-11802295-B2
Application number	US-201916541083-A
Country	US
Kind code	B2
Filing date	Aug 14, 2019
Priority date	Nov 5, 2014
Publication date	Oct 31, 2023
Grant date	Oct 31, 2023

Abstract

Provided are methods, systems, and kits for cell processing, e.g., for therapeutic use, such as for adoptive cell therapy. The provided methods include transduction methods, in which cells and virus are incubated under conditions that result in transduction of the cells with a viral vector. The incubation in some embodiments is carried out in an internal cavity of a generally rigid centrifugal chamber, such as a cylindrical chamber made of hard plastic, the cavity of which may have a variable volume. The methods include other processing steps, including those carried out in such a chamber, including washing, selection, isolation, culture, and formulation. In particular, the disclosure relates to method providing advantages over available processing methods, such as available methods for large-scale processing. Such advantages include, for example, reduced cost, streamlining, increased efficacy, increased safety, and increased reproducibility among different subjects and conditions.

First claim

The invention claimed is:

1. A transduction method comprising incubating, in an internal cavity of a centrifugal chamber, an input composition comprising cells and viral particles containing a recombinant viral vector, wherein:
the centrifugal chamber comprises one or more openings comprising at least one opening that is capable of permitting intake of liquid into the internal cavity and expression of liquid from the internal cavity and at least one opening that is capable of permitting intake of gas into the internal cavity and expression of gas from the internal cavity;
the internal cavity has variable volume, and the method comprises, prior to or during the incubation, effecting intake of a gas into the internal cavity to increase the volume of the internal cavity, the internal cavity comprising the input composition and the gas during at least a portion of the incubation;
the centrifugal chamber is rotating around an axis of rotation during at least a portion of the incubation; and
the method generates an output composition comprising a plurality of the cells transduced with the recombinant viral vector.

2. The method of claim 1 , wherein the centrifugal chamber comprises a movable member capable of moving within the centrifugal chamber to vary the volume of the internal cavity, and the effecting the intake of the gas effects movement of the movable member.

3. The method of claim 2 , wherein the movable member is capable of moving axially within the centrifugal chamber to vary the volume of the internal cavity.

4. The method of claim 2 , wherein the movable member is a piston.

5. The method of claim 1 , wherein the rotating comprises rotation at a relative centrifugal force at a surface layer of the cells that is from about 500 g to about 2500 g.

6. The method of claim 1 , wherein the rotating is for a time that is from about 5 minutes to about 60 minutes.

7. The method of claim 1 , wherein the maximum liquid volume of the input composition present in the internal cavity at any one time during the incubation is no more than about 5 milliliters per square inch of the maximum internal surface area of the internal cavity during the incubation.

8. The method of claim 1 , wherein the number of cells in the input composition is no more than two times the number of the cells sufficient to form a monolayer on the inner surface of the internal cavity during the rotating.

9. The method of claim 1 , wherein:
the rotating comprises rotation at a relative centrifugal force at a surface layer of the cells that is from about 500 g to about 2500 g; and
the number of cells in the input composition is no more than two times the number of the cells sufficient to form a monolayer on the inner surface of the internal cavity during the rotating.

10. The method of claim 1 , wherein the input composition comprises at least or at least about 1 infectious unit of viral particles per one of the cells.

11. The method of claim 1 , wherein the titer of viral particles is from about 1×10 6 IU/mL to about 1×10 8 IU/mL.

12. The method of claim 1 , wherein the maximum total liquid volume of the input composition present in the internal cavity at any one time during the incubation is no more than 100 times the total volume of the cells in the input composition.

13. The method of claim 1 , wherein:
the maximum liquid volume of the input composition present in the internal cavity at any one time during the incubation is no more than about 5 milliliters per square inch of the maximum internal surface area of the internal cavity during the incubation; and
the maximum total liquid volume of the input composition present in the internal cavity at any one time during the incubation is no more than 100 times the total volume of the cells in the input composition.

14. The method of claim 1 , wherein the gas is air.

15. The method of claim 1 , wherein the centrifugal chamber comprises a movable member capable of moving within the centrifugal chamber to vary the volume of the internal cavity, and the intake of the gas is effected by movement of the movable member.

16. The method of claim 1 , wherein
the centrifugal chamber is integral to a closed system, the closed system comprising the centrifugal chamber, at least one tubing operably linked to the one or more openings of the centrifugal chamber via at least one connector, and at least one container operably linked to the at least one tubing line, whereby liquid and gas are permitted to move between the internal cavity and the at least one container via the at least one tubing line in at least one configuration of the closed system.

17. The method of claim 16 , wherein:
the at least one container comprises at least one input container comprising the viral particles and the cells, each of the at least one input container comprising one or both of the viral particles and the cells; and
the method further comprises, prior to the incubation, effecting intake of the viral particles and the cells into the internal cavity, the intake of the viral particles and the cells comprising flowing of liquid from the at least one input container into the internal cavity through the at least one opening that is capable of permitting intake and expression of liquid.

18. The method of claim 16 , wherein
the closed system further comprises a microbial filter, the intake of the gas into the internal cavity being effected by flow of the gas under sterile conditions through the microbial filter.

19. The method of claim 1 , wherein the cells in the input composition comprise T cells or NK cells.

20. The method of claim 1 , wherein the input composition further comprises one or more additional agents to promote transduction efficiency.

21. The method of claim 20 , wherein the one or more additional agents is protamine sulfate.

22. The method of claim 1 , wherein the recombinant viral vector encodes a recombinant receptor, which is thereby expressed by cells of the output composition.

23. The method of claim 22 , wherein the recombinant receptor is a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR).

24. The method of claim 1 , wherein the recombinant viral vector is a recombinant lentiviral vector.

25. The method of claim 1 , wherein the rotating is for a time that is greater than about 60 minutes.

26. The method of claim 18 , wherein the intake of the gas into the internal cavity is effected by flow of the gas under sterile conditions through a syringe connected to the microbial filter.

Assignees

• Juno Therapeutics Inc

Inventors

• Crisman Ryan L
• Ramsborg Chris
• Wood Travis

Classifications

• A61K40/11

  T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells · CPC title

• A61K40/32

  T-cell receptors [TCR] · CPC title

• A61K2039/5156

  expressing foreign proteins · CPC title

• A61K2039/5158

  Antigen-pulsed cells, e.g. T-cells · CPC title

• A61K39/0011

  Cancer antigens · CPC title