Process for producing a composition of engineered T cells

Patent metadata

Field	Value
Publication number	US-12577285-B2
Application number	US-201816769971-A
Country	US
Kind code	B2
Filing date	Dec 7, 2018
Priority date	Dec 8, 2017
Publication date	Mar 17, 2026
Grant date	Mar 17, 2026

Abstract

The present disclosure provides methods for genetically engineering T cells, such as CD4+ T cells and/or CD8+ T cells, for use in cell therapy. In some aspects, the provided methods include one or more steps for pooling enriched CD4+ and CD8+ cells, such as at a 1:1 ratio, and then incubating the cells under stimulating conditions, introducing a recombinant polypeptide to the cells through transduction or transfection, and/or cultivating the cells under conditions that promote proliferation and/or expansion. In some aspects, the provided methods are an efficient, reliable means to produce genetically engineered T cells with a high degree of success.

First claim

The invention claimed is:

1 . A method for producing a composition of engineered cells, the method comprising:
(a) incubating an input composition under stimulating conditions, thereby generating a stimulated composition, wherein: the input composition comprises between 100×10 6 and 500×10 6 total CD4+ and CD8+ T cells at a concentration of between 1×10 6 cells/mL and 5×10 6 cells/mL and a ratio of between 3:1 and 1:3 CD4+to CD8+ T cells, wherein the T cells of the input composition are primary T cells obtained from a human subject having a cancer; and the stimulating conditions comprise the presence of a stimulatory reagent comprising a primary agent that specifically binds to CD3 and a secondary agent that specifically binds to CD28; (b) introducing a chimeric antigen receptor (CAR) into T cells from the stimulated composition, thereby generating an engineered cell composition, wherein: the incubation and introducing are each performed in a first serum-free medium comprising 0.5 mM to 5 mM L-glutamine, 0.5 mM to 5 mM L-alanyl-L-glutamine, between 50 IU/mL and 500 IU/mL recombinant IL-2, between 100 IU/mL and 2,000 IU/mL recombinant IL-7, and between 50 IU/mL and 500 IU/mL recombinant IL-15; the introducing is initiated within 2 days after the initiation of the incubation under stimulating conditions; the introducing comprises contacting between 50×10 6 T cells and 200×10 6 T cells from the stimulated composition with an agent comprising a polynucleotide encoding the CAR; and during the introducing, the T cells from the stimulated composition are cultured at a concentration of between 0.5×10 6 cells/mL and 2×10 6 cells/mL; and (c) cultivating the engineered composition under conditions to promote expansion of the engineered T cells, thereby producing an output composition comprising engineered T cells, wherein: the cultivating is performed in a second serum-free medium comprising 0.5 mM to 5 mM L-glutamine, 0.5 mM to 5 mM L-alanyl-L-glutamine, between 50 IU/mL and 500 IU/mL recombinant IL-2, between 100 IU/mL and 2,000 IU/mL recombinant IL-7, and between 50 IU/mL and 500 IU/mL recombinant IL-15; the cultivating is initiated within 3 days after the initiation of the of the incubation under stimulating conditions; the cultivating is performed under steady rocking conditions; at least a portion of the cultivating is performed with perfusion using the second serum-free medium; and the cultivating is performed at least until the engineered composition comprises a threshold number of viable T cells that is at least 2,000×10 6 viable T cells, wherein the threshold number of viable T cells is achieved within 9 days of the initiation of the incubation.

2 . The method of claim 1 , wherein the input composition comprises at or about 300×10 6 total CD4+ and CD8+ T cells.

3 . The method of claim 1 , wherein the input composition comprises a concentration of between 3×10 6 cells/mL and 5×10 6 cells/mL.

4 . The method of claim 1 , wherein the input composition comprises a concentration of or of about 3×10 6 cells/mL.

5 . The method of claim 1 , wherein the input composition comprises a ratio of between 2:1 and 1:2 CD4+to CD8+cells.

6 . The method of claim 1 , wherein the input composition comprises a ratio of or of about 1:1 CD4+to CD8+cells.

7 . The method of claim 1 , wherein the contacting is by transduction with a viral vector.

8 . The method of claim 7 , wherein the viral vector is a retroviral vector.

9 . The method of claim 1 , wherein at least about 100×10 6 T cells and up to about 200×10 6 T cells of the stimulated composition are contacted with the agent comprising the polynucleotide.

10 . The method of claim 1 , wherein during the introducing, the T cells from the stimulated composition are cultured at a concentration of or of about 1×10 6 cells/mL.

11 . The method of claim 1 , wherein the primary agent comprises an anti-CD3 antibody or an antigen-binding fragment thereof, and the secondary agent comprises an anti-CD28 antibody or an antigen-binding fragment thereof.

12 . The method of claim 11 , wherein the primary agent and secondary agent are present on the surface of a solid support.

13 . The method of claim 12 , wherein the solid support is a bead.

14 . The method of claim 13 , wherein the ratio of beads to cells is from or from about 2:1 to 0.5:1.

15 . The method of claim 13 , wherein the ratio of beads to cells is or is about 1:1.

16 . The method of claim 1 , wherein the input composition is incubated under stimulating conditions for between 12 hours and 36 hours, inclusive.

17 . The method of claim 1 , wherein the contacting is carried out for between 12 hours and 36 hours, inclusive.

18 . The method of claim 1 , wherein at least a portion of the cultivating is performed with perfusion at a rate of at least 500 mL/day.

19 . The method of claim 1 , wherein at least a first portion of the cultivating is performed with a perfusion rate of or of about 750 mL/day, and at least a second portion of the cultivating is performed with a perfusion rate of or of about 1,500 mL/day.

20 . The method of claim 1 , wherein:
the perfusion is initiated at a rate of or of about 750 mL/day when the cells reach a density of or of about 0.6×10 6 cells/mL; and the perfusion is increased to a rate of or of about 1500 mL/day when the cells reach a density of or of about 2.0×10 6 cells/mL.

21 . The method of claim 1 , wherein the threshold number of viable T cells is achieved between about 5 days and about 9 days from the initiation of the incubation.

22 . The method of claim 1 , further comprising formulating cells of the output composition for cryopreservation or administration to a subject.

23 . The method of claim 1 , further comprising isolating the CD4+ and the CD8+ T cells from a biological sample from the subject prior to the incubation.

24 . The method of claim 1 , wherein the CAR is capable of binding to a target antigen that is associated with, specific to, or expressed on a cell or tissue of a disease, disorder or condition.

25 . The method of claim 1 , wherein the recombinant receptor is an anti-B cell maturation antigen (BCMA) CAR.

26 . The method of claim 1 , wherein during at least a portion of the cultivating, the cells are monitored for cell viability, concentration, density, number, or a combination thereof, wherein the monitoring is carried out by differential digital holography microscopy (DDHM).

27 . The method of claim 1 , wherein the input composition comprises at least 80% cells that are CD4+ T cells and CD8+ T cells.

28 . The method of claim 1 , wherein the input composition comprises at least 90% cells that are CD4+ T cells and CD8+ T cells.

29 . The method of claim 1 , wherein the threshold number of viable T cells is achieved within 8 days of the initiation of the incubation.

30 . The method of claim 1 , wherein the threshold number of viable T cells is achieved between about 5 days and about 8 days from the initiation of the incubation.

31 . The method of claim 1 , wherein the cancer is a multiple myeloma.

32 . The method of claim 1 , wherein at least 30% of the cells in the output composition are CCR7+/CD45RA− or CCR7+/CD45RO+.

33 . The method of clai

Assignees

• Juno Therapeutics Inc

Inventors

• Mujacic Mirna
• Rahardjo Ayu
• Beauchesne Pascal
• Khuu-Duong Kien
• Aifuwa Ivie
• Chan Calvin

Classifications

• A61K40/4215

  Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30 · CPC title

• A61K40/31

  Chimeric antigen receptors [CAR] · CPC title

• A61K40/11

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

• A61K2239/38

  characterised by the dose, timing or administration schedule · CPC title

• A61K2239/48

  Blood cells, e.g. leukemia or lymphoma · CPC title