Methods for transduction and cell processing

Patent metadata

Field	Value
Publication number	US-10428351-B2
Application number	US-201514932660-A
Country	US
Kind code	B2
Filing date	Nov 4, 2015
Priority date	Nov 5, 2014
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

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, the 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:
an end wall, a substantially rigid side wall extending from said end wall, and one or more opening, wherein at least a portion of said side wall surrounds said internal cavity and at least one of the one or more opening is capable of permitting intake of liquid or gas into said internal cavity and expression of liquid or gas from said cavity; and
a movable member capable of moving within the chamber to vary the internal volume of the internal cavity, whereby the internal cavity is a cavity of variable volume defined by said end wall, said substantially rigid side wall, and said movable member;

the centrifugal chamber is rotatable around an axis of rotation and is rotating around said axis of rotation during at least a portion of the incubation;
during at least a portion of the incubation in the chamber or during the rotation of the chamber, the liquid volume of the input composition occupies only a portion of the volume of the internal cavity of the chamber, the volume of the cavity during said at least a portion or during said rotation further comprising a gas; wherein prior to or during said incubation, effecting movement of the movable member and effecting intake of the gas into said internal cavity, thereby increasing the volume of the internal cavity of the chamber; and
wherein the method generates an output composition comprising a plurality of the cells transduced with the viral vector.

2. The method of claim 1 , wherein said rotating comprises rotation at a relative centrifugal force at an internal surface of the side wall of the cavity and/or at a surface layer of the cells that is or is at least at or about 600 g.

3. The method of claim 1 , wherein the at least a portion of the incubation during which the chamber is rotating is for a time that is between about 5 minutes and about 60 minutes, inclusive.

4. The method of claim 1 , wherein:
the average liquid volume of said input composition present in said cavity during said incubation is no more than about 5 milliliters (mL) per square inch of the internal surface area of the cavity during said incubation.

5. The method of claim 1 , wherein:
the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells; and/or
the number of said cells in said input composition is no more than 2 times the number of said cells sufficient to form a monolayer on the surface of said cavity during rotation of said centrifugal chamber at a force of at or about 1000 g or at or about 2000 g at an internal surface of the side wall and/or at a surface layer of the cells; and/or
said input composition in the cavity comprises at least at or about 1×10 6 of said cells.

6. The method of claim 1 , wherein said input composition comprises at least at or about 1 infectious unit (IU) of viral particles per one of said cells.

7. The method of claim 1 , wherein:
the internal cavity of the centrifugal chamber has an internal surface area of at least at or about 1×10 9 μm 2 ;
the number of cells in the input composition is at least 1×10 7 cells, and
the viral particles are present in the input composition at, at about, or at least 1 infectious unit (IU) per one of said cells.

8. The method of claim 7 , wherein:
the liquid volume of the input composition is less than or equal to 200 mL; and/or
the liquid volume of the input composition is no more than 50% of the volume of the internal cavity during rotation.

9. The method of claim 7 , wherein the volume of said gas is up to 200 mL.

10. The method of claim 7 , wherein said rotation is at a relative centrifugal force at an internal surface of the side wall of the cavity or at a surface layer of the cells of at least at or about 600 g.

11. The method of claim 1 , wherein:
the input composition comprises greater than or about 20 mL, and/or said input composition comprises at least 1×10 8 cells; and
said rotating conditions comprise a relative centrifugal force on a surface layer of the cells of greater than about 800 g.

12. The method of claim 11 , wherein said incubation is carried out in a cavity of the centrifugal chamber and the number of said cells in said input composition is at or about the number of said cells sufficient to form a monolayer or a bilayer on the inner surface of said cavity during said rotation.

13. The method of claim 1 , wherein a further portion of the incubation of the input composition is carried out outside of the centrifugal chamber and/or without rotation, said further portion carried out subsequent to the at least a portion carried out in the chamber and/or with rotation.

14. The method of claim 13 , wherein:
the further incubation is carried out for a time that is no more than 24 hours;
the cells in the input composition have not been subjected to a temperature of greater than 30° C. for more than 24 hours; and/or
the further incubation is not performed in the presence of a stimulating agent.

15. The method of claim 1 , wherein:
the output composition containing transduced cells comprises at least at or about 1×10 7 cells;
for an input composition comprising a virus at a ratio of about 1 or about 2 IU per cells, said method is capable of producing an output composition in which at least 10% of the cells in said output composition generated by the method comprise said recombinant viral vector and/or express a product of a recombinant nucleic acid comprised within said vector; or
among all the cells in said output composition that contain the recombinant viral vector or into which the viral vector is integrated, the average copy number of said recombinant viral vector is no more than about 10; or among the cells in the output composition, the average copy number of said vector is no more than about 2.

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

17. The method of claim 16 , wherein said at least one container comprises at least one input container comprising said viral vector particles and said cells, a waste container, a product container, and at least one diluent container, and said at least one tubing line comprises a series of tubing lines, wherein each of said containers is connected to said cavity via said series of tubing lines and at least one of the one or more opening.

18. The method of claim 17 , wherein said method further comprises,
prior to and/or during said incubation, effecting intake of said input composition into said cavity, said intake comprising flowing of liquid from said at least

Assignees

• Juno Therapeutics Inc

Inventors

• Crisman Ryan L
• Ramsborg Chris
• Wood Travis

Classifications

• A61P37/06

  Immunosuppressants, e.g. drugs for graft rejection · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title

• C07K16/2896

  against molecules with a "CD"-designation, not provided for elsewhere · CPC title

• C12N15/86 Primary

  Viral vectors · CPC title