Methods for treating cancer with manufactured t cells

A method for the treatment of cancer in a subject in need thereof, comprising:

• a first treatment cycle and one or more additional treatment cycles,
• said first treatment cycle comprising:
• subjecting said subject to a first immune depletion regimen to reduce at least a portion of regulatory T cells and/or end-stage senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and/or end-stage senescent effector T cells;
• each of said one or more additional treatment cycles comprising:
• subjecting said subject to a second immune depletion regimen to reduce at least a portion of regulatory T cells and/or end-stage senescent effector T cells or to reduce at least a portion of the function of regulatory T cells and/or end-stage senescent effector T cells;
• and
• administering to said subject a composition comprising manufactured T cells at a therapeutically effective dose.

A manufactured T cell having one or more of the following properties:

• at least a 50% increase in secretion of IFN-γ relative to T-Rapa cells after one week of incubation using stimulation with anti-CD3/anti-CD28 magnetic beads at bead:T cell ratio of 3:1;
• at least a 50% increase in secretion of TNF-α relative to T-Rapa cells after one week of incubation using stimulation with anti-CD3/anti-CD28 magnetic beads at bead:T cell ratio of 3:1;
• at least a 50% increase in secretion of GM-CSF relative to T-Rapa cells after one week of incubation using stimulation with anti-CD3/anti-CD28 magnetic beads at bead:T cell ratio of 3:1;
• at least a 50% increase in secretion of IL-2 relative to T-Rapa cells after one week of incubation using stimulation with anti-CD3/anti-CD28 magnetic beads at bead:T cell ratio of 3:1;
• an increase in 4EBP1 phosphorylation of no more than 50% relative to a control population of T cells characteristic of the T cells from which the population of T cells was produced;
• at least 50% reduced expression of p70S6K or Raptor relative to a population of T-Rapa cells cultured under the same conditions;
• at least 50% reduced expression of p-STAT5 relative to a population of T-Rapa cells cultured under the same conditions;
• a detectable level of STAT1 and p-STAT1 expression;
• at least 10% increased expression of p70S6K relative to a control population of T cells characteristic of the cells from which the population of manufactured T cells was produced;
• at least 50% reduced expression of CD25 relative to a population of T-Rapa cells;
• an expression level of CD28 within about 20% of a control population of T cells characteristic of T cells from which the population of manufactured T cells was produced;
• an expression level of ICOS within about 20% of a control population of T cells characteristic of T cells from which the population of manufactured T cells was produced;
• an expression level of CD45RA within about 20% of a control population of T cells characteristic of T cells from which the population of manufactured T cells was produced;
• an increase of at least 50% of CD4+ T cells positive for CD45RA as measured by flow cytometry;
• at least a 1.1-fold increase in IL-2 secretion relative to a T-Rapa culture incubated under the same conditions;
• secretion of at least 500 pg/mL/1×106 cells/day of IL-2 after co-stimulation with anti-CD3/anti-CD28 coated magnetic beads at a ratio of between 3:1 and 1:3 beads:T cell;
• at least a 1.1-fold increase in IL-2 secretion relative when incubated in the presence of IL-7, IL-15 or a combination of IL-7 and IL-15, wherein the IL-7 and IL-15, when present, are added at 10 ng/mL each;
• secretion of at least 1000 pg/mL/1×106 cells/day of IL-2 after incubation in the presence of IL-7, IL-15 or a combination of IL-7 and IL-15, wherein the IL-7 and IL-15, when present, are added at 10 ng/mL each;
• at least a 25% reduction in expression of one or more checkpoint inhibitors selected from:
• CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3 and combinations thereof, relative to a corresponding expression level of a population of T-Rapa cells;
• an expression level of one or more checkpoint inhibitors selected from:
• CD39, CD73, GITR, LAG3, PD1, 2B4, LAIR1, CTLA4, KLRG1, TIGIT, TIM3 and combinations thereof, that is within 25% of a corresponding expression level in a control population of T cells characteristic of the cells from which the population of manufactured T cells was produced;
• expressing CD127;
• secretion of low levels of the inflammatory cytokines IFN-γ and TNF-α at the end of manufacturing, as defined by <100 pg/ml per 1×106 cells per 24 hours contained in a culture supernatant after a stimulation procedure using a high level of co-stimulation (3:1 bead-to-T cell ratio);
• an increase in IFN-γ and TNF-α secretion after a 6-day period of expansion in the absence of inhibitors that is at least 5-fold and more preferably 20-fold increased relative to the day 6 secretion levels;
• and
• combinations thereof.