CIML NK cells and Methods Therefor
Cytokine induced memory like (CIML) NK cells with enhanced cytotoxicity are presented. Most typically, the CIML NK cells are derived from a mononuclear cell fraction of peripheral blood or cord blood. In further contemplated aspects, the CIML NK cells are expanded and induced in a contained and automated production environment that substantially reduces operational complexity and production cost.
1 . A method of producing cytokine induced memory like (CIML) NK cells with enhanced cytotoxicity, comprising:
isolating from a biological fluid a mixture of mononuclear cells, and contacting the mixture of the mononuclear cells with an anti-CD16 antibody and N-803 to expand NK cells; and
contacting the expanded NK cells with a stimulatory cytokine composition that includes an IL-18/IL-12-TxM fusion protein complex, a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18 to thereby generate the CIML NK cells with enhanced cytotoxicity.
2 . The method of claim 1 further comprising a step of contacting the CIML NK cells with N-803 after re-stimulating the CIML NK cells.
3 . The method of claim 1 wherein the biological fluid is whole blood or cord blood.
4 . The method of claim 1 wherein the mixture of mononuclear cells is not further processed to enrich NK cells.
5 . The method of claim 1 wherein the anti-CD16 antibody in the step of contacting the mixture is present at a concentration of between 0.05-1.0 mcg/ml, and wherein the N-803 in the step of contacting the mixture is present at a concentration of between 0.1-1.0 nM.
6 . The method of claim 1 wherein the stimulatory cytokine composition includes the IL-18/IL-12-TxM fusion protein complex.
7 . The method of claim 1 wherein the NK cells are expanded to a total cell number of about 0.5-5.0×10 9 cells.
8 . The method of claim 1 wherein the step of contacting the expanded NK cells with a stimulatory cytokine composition is performed in the same container as the step of expanding the NK cells.
9 . A method of activating NK cells to form cytokine induced memory like (CIML) NK cells with enhanced cytotoxicity, comprising:
providing expanded NK cells, wherein the NK cells were expanded from mononuclear cells of whole blood or cord blood; and
contacting the expanded NK cells with a stimulatory cytokine composition that includes an IL-18/IL-12-TxM fusion protein complex, a mixture of IL-12, N-803, and IL-18, or a mixture of IL-12, IL-15, and IL-18 to thereby generate the CIML NK cells with enhanced cytotoxicity.
10 . The method of claim 9 wherein the NK cells are expanded from whole blood or from cord blood.
11 . The method of claim 9 wherein the NK cells are autologous relative to an individual receiving a transfusion comprising the CIML NK cells.
12 . The method of claim 9 wherein the stimulatory cytokine composition includes the IL-18/IL-12-TxM fusion protein complex.
13 . The method of claim 9 wherein the CIML NK cells with enhanced cytotoxicity have cytotoxicity against MS-1 cells.
14 . The method of claim 9 wherein the CIML NK cells with enhanced cytotoxicity have a decreased expression of CD16 as compared to expanded NK cells that are contacted with N-803 alone, or wherein the CIML NK cells with enhanced cytotoxicity have a decreased expression of TIGIT as compared to expanded NK cells that are contacted with N-803 alone.
15 . The method of claim 9 wherein the CIML NK cells with enhanced cytotoxicity have an increased expression of CD25 and/or DNAM1 as compared to expanded NK cells that are contacted with N-803 alone.