Process for producing a composition of engineered T cells

A method for producing a composition of engineered cells, the method comprising:

• (a) combining a composition of CD4+ T cells and a composition of CD8+ T cells at a ratio of between 2:1 and 1:2 CD4+to CD8+ T cells, thereby generating an input composition, wherein:
• the input composition comprises between 100×106 and 500×106 total CD4+ and CD8+ T cells at a concentration of between 1×106 cells/mL and 5×106 cells/mL;
• and
• the T cells of the input composition are primary T cells obtained from a human subject having a cancer;
• (b) incubating the input composition under stimulating conditions, thereby generating a stimulated composition, wherein the stimulating conditions comprise a primary agent that specifically binds to CD3 and a secondary agent that specifically binds to CD28;
• (c) 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 50×106 T cells and 200×106 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 0.5×106 cells/mL and 2×106 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 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 2,000×106 viable T cells, wherein the threshold number of viable T cells is achieved within 9 days of the initiation of the incubation.

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 100×106 and 500×106 total CD4+ and CD8+ T cells at a concentration of between 1×106 cells/mL and 5×106 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 50×106 T cells and 200×106 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 0.5×106 cells/mL and 2×106 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 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 2,400×106 viable T cells, wherein the threshold number of viable T cells is achieved between 5 days and 9 days from the initiation of the incubation.