# US 2019/0376084 A1 · Methods for transduction and cell processing

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- Number: US 2019/0376084 A1
- Document ID: US20190376084A1
- Status: Published application
- Kind: A1
- Application number: 16/541,083
- Filed: 2019-08-14
- Published: 2019-12-12
- Earliest claimed: 2014-11-05
- Assignees: Juno Therapeutics, Inc.
- Applicants: Juno Therapeutics, Inc.
- Inventors: Ryan L. CRISMAN; Chris RAMSBORG; Travis WOOD
- Web page: https://patentgrep.com/patent/US20190376084A1

## CPC

- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61K 39/0011: {Cancer antigens}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- B04B 7/08: Rotary bowls (centrifugal casting machines B22D)
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C12N 5/0636: {T lymphocytes}
- A61K 2039/5156: {expressing foreign proteins}
- A61K 2039/5158: {Antigen-pulsed cells, e.g. T-cells}
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C12N 15/86: Viral vectors
- C12N 2510/00: Genetically modified cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- B04B: CENTRIFUGES (high-speed drum mills B02C19/11; domestic spin driers D06F; analysing, measuring or monitoring physical or chemical properties of samples during centrifuging, see the relevant subclasses for these procedures, e.g. G01N)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## 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.

## Claims

### Claim 1

A method of transduction, comprising:
a) providing to an internal cavity of a centrifugal chamber that has an internal surface area of at least at or about 1×109 μm2 or at least at or about 1×1010 μm2:
i) an input composition comprising cells and viral particles comprising a recombinant viral vector, wherein:
the number of cells in the input composition is at least 1×107 cells, and
the viral particles are present in the input composition at at least at or about 1 infectious unit (IU) per one of said cells, and
the input composition comprises a liquid volume that is less than the maximum volume of the internal cavity of the centrifugal chamber; and
ii) gas at a volume that is up to the remainder of the maximum volume of the internal cavity of the centrifugal chamber; and
b) incubating the input composition, wherein at least a portion of the incubation is carried out in said internal cavity of said centrifugal chamber while effecting rotation of said centrifugal chamber; and
wherein the method generates an output composition comprising a plurality of the cells transduced with the viral vector.

### Claim 2 (depends on 1)

The method of claim 1, wherein:
the liquid volume of the input composition is less than or equal to 200 mL, less than or equal to 100 mL, less than or equal to 50 mL or less than or equal to 20 mL; and/or
the liquid volume of the input composition is no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the volume of the internal surface area of the cavity during rotation or the maximum internal surface area of the cavity.

### Claim 3 (depends on 1)

The method of claim 1, wherein the volume of gas is up to 200 mL, up to 180 mL, up to 140 mL or up to 100 mL.

### Claim 4 (depends on 1)

The method of claim 1, 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, 1000 g, or 1600 g.

### Claim 5

A method of transduction, comprising incubating an input composition comprising cells and viral particles, the viral particles comprising a recombinant viral vector, at least a portion of said incubating being carried out under rotating conditions, thereby generating an output composition comprising a plurality of the cells transduced with the viral vector, wherein:
said input composition comprises greater than or about 20 mL, greater than at or about 50 mL, at least 100 mL, or at least 150 mL in volume, and/or said input composition comprises at least 1×108 cells; and
said rotating conditions comprise a relative centrifugal force on a surface layer of the cells of greater than about 800 g or greater than about 1000 g or greater than about 1500 g.

### Claim 6 (depends on 5)

The method of claim 5, wherein said incubation is carried out in a cavity of a 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.

### Claim 7

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:
said centrifugal chamber is rotatable around an axis of rotation and comprising an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity;
the centrifugal chamber is rotating around said axis of rotation during at least a portion of the incubation;
the method generates an output composition comprising a plurality of the cells transduced with the viral vector, and
the method further comprises formulating cells transduced by the method in a pharmaceutically acceptable buffer in an internal cavity of a centrifugal chamber, thereby producing a formulated composition, and, optionally effecting expression of the formulated composition to one or a plurality of containers.

### Claim 8

A method for selection, the method comprising:
(a) incubating a selection reagent and primary cells in an internal cavity of a centrifugal chamber under mixing conditions, whereby a plurality of the primary cells bind to said selection reagent; and
(b) separating said plurality of said primary cells from another one or more of the primary cells based on binding to the selection reagent,
thereby enriching the primary cells based on binding to the selection reagent, wherein said centrifugal chamber is rotatable around an axis of rotation and said internal cavity has a maximum volume of at least 50, at least 100, or at least 200 mL.

### Claim 9

A method for stimulation of cells, the method comprising incubating a stimulation agent and primary cells under conditions whereby the stimulation agent binds to a molecule expressed by a plurality of the primary cells and said plurality of the cells are activated or stimulated, wherein
at least a portion of the incubation being carried out in an internal cavity of a centrifugal chamber under mixing conditions,
said centrifugal chamber is rotatable around an axis of rotation; and
said internal cavity has a maximum volume of at least 50, at least 100, or at least 200 mL.

### Claim 10

A composition, comprising transduced cells produced by 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:
said centrifugal chamber is rotatable around an axis of rotation and comprising an end wall, a substantially rigid side wall extending from said end wall, and at least one opening, wherein at least a portion of said side wall surrounds said internal cavity and said at least one opening is capable of permitting intake of liquid into said internal cavity and expression of liquid from said cavity;
the centrifugal chamber is rotating around said 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 viral vector.

### Claim 11 (depends on 10)

The composition of claim 10, wherein:
the composition comprises at least at or about 5×107 cells, 1×108 cells, 2×108 cells, 4×108 cells, 6×108, 8×108 cells or 1×109 cells; or
the composition comprises a therapeutically effective number of cells for use in adoptive T cell therapy.

### Claim 12 (depends on 10)

The composition of claim 10, wherein:
the cells are T cells; and
subsequent to transduction, the cells in the composition are not subjected to cell expansion in the presence of a stimulating agent and/or the cells are not incubated at a temperature greater than 30° C. for more than 24 hours or the composition does not contain a cytokine or the composition does not contain a stimulating agent that specifically binds to CD3 or a TCR complex.

### Claim 13

A composition, comprising:
a) at least 1×107 or at least 5×107 cells T cells, at least a plurality of which are transduced with a recombinant viral vector or express a recombinant or engineered antigen receptor, wherein:
subsequent to transduction, the cells in the composition have not been subjected to cell expansion in the presence of a stimulating agent; and/or
subsequent to transduction, the cells have not been incubated at a temperature greater than 30° C. for more than 24 hours; or
b) at least 1×107 or at least 5×107 primary human T cells, at least a plurality of which are transduced with a recombinant viral vector or express a recombinant or engineered antigen receptor, wherein at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells in the composition comprise high expression of CD69 and/or TGF-beta-II.

### Claim 14 (depends on 13)

The composition of claim 13, wherein:
among all the cells in the composition, the average copy number of said recombinant viral vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2; or
among the cells in the composition transduced with the recombinant viral vector, the average copy number of said vector is no more than about 10, no more than 8, no more than 6, no more than 4, or no more than about 2.

### Claim 15

A composition, comprising:
at least 1×107 cells; and
at least at or about 1 infectious unit (IU) per cell of viral particles comprising a recombinant viral vector.

### Claim 16 (depends on 10)

An article of manufacture comprising a container or plurality of containers, the container or the plurality of containers collectively containing a composition of claim 10.

### Claim 17 (depends on 10)

A method of treatment, the method comprising administering to a subject having a disease or condition the composition of claim 10.

### Claim 18 (depends on 15)

A centrifugal chamber rotatable around an axis of rotation, said chamber comprising:
an internal cavity comprising the composition of claim 15, or
an internal cavity comprising: (a) a composition containing at least 5×107 primary T cells transduced with a recombinant viral vector and/or (b) a composition containing at least 5×107 primary T cells and viral particles containing a recombinant viral vector.

### Claim 19 (depends on 18)

A closed system, comprising the centrifugal chamber of claim 18.

### Claim 20 (depends on 19)

The closed system of claim 19, further comprising a multi-way manifold operably connected to one or a plurality of containers.

### Claim 21

A method comprising
(a) washing primary human cells; and
(b) incubating said cells with a selection reagent under agitation conditions whereby at least a plurality of the human cells are specifically bound by the selection reagents,
wherein said washing and incubating are carried out within a closed, sterile system and at least in part in an internal cavity of a centrifugal chamber integral to the closed, sterile system.