Table 4.
Ex vivo cultivation of natural killer (NK) cells with allogeneic feeder cells.
| Protocol features | Starting material/culture system | NK cell expansion rate | NK cell purity | NK cell phenotype | NK cell function | Setting | Reference |
|---|---|---|---|---|---|---|---|
| Irr. allogeneic PBMC activated with ConA + IL-2 | In vivo IL-2 primed PBMC depleted for non-NK cells in flasks | 1–148 (14 days) | 64–98% NK | N/A | Cytotoxic activity against leukemic cell lines | Clinical | (123) |
| Irr. allogeneic PBMC activated with ConA, PHA and ionomycin + IL-2 + IL-15 | PBMC, depleted for CD3, CD4, CD19, and CD33 in bags | 80–200 (15 days) | 91% CD56 0.3% CD3 (day 12) |
Upregulated: CD16, CD25 | Increased cytotoxicity against tumor cell lines in vitro; decreased frequency of INF-g producing cells | In vitro | (118) |
| Irr. allogeneic PBMC + OKT-3 + IL-2 | PBMC, CD3 depleted, and CD56 enriched in plates | 300 (14 days) | 94% NK | Upregulated: CD16, CD56, NKG2D, NKp30, and NKp44 | Increased cytotoxicity against tumor cell lines in vitro | In vitro | (115) |
| Irr. HFWT + IL-2 | PBMC in flasks | 113 (2 weeks) | 86% CD56+/CD16+ | N/A | Cytotoxic against tumor cell lines in vitro | Clinical | (124, 125) |
| Irr. Jurkat/KL-1 + IL-2 | PBMC in flasks | ~130 (2 weeks) | 40–90% NK |
Upregulated: CD54, CD11a, CD48, CD2, CD49d, CD58, NKp30, NKp44, 2B4, DNAM-1, NKG2D, CD25, and CD69 Downregulated: CD16 |
Increased cytotoxicity against tumor cell lines in vitro and antitumor activity in vivo | Preclinical model | (121) |
| Irr. K562 expressing membrane-bound IL-15 and 41BBL + IL-2 | PBMC in plates | 1,089 (3 weeks) | “Virtually pure” | N/A | N/A | In vitro | (126) |
| PBMC in bags | 23, 152, and 277 after 7, 14, and 21 days | 96.8% NK 3.1% T cells (day 21) |
Marked differences of gene expression profile compared to unstimulated or IL-2-stimulated NK cells | Increased cytotoxicity against tumor cell lines in vitro and antitumor activity in vivo | Preclinical model | (127) | |
| PBMC | 447 (days 10–14) | 88% NK 2.2% T cells (day 14) |
Upregulated genes for cytolytic activity, cytokines, chemokines, activating receptors, adhesion molecules, cell cycle regulators, and multiple pathways | Increased cytotoxicity against primary MM cells in vitro and in vivo; high productivity of IFN-γ | Preclinical model | (128) | |
| PBMC in G-Rex, bags | 442—G-Rex 227—bags (10 days) |
70% NK 5–35% T cells |
Upregulated: NKp30, NKp44, NKG2D, CD26, CD70, and CXCR3 Downregulated: CD16, CD62L |
Increased cytotoxicity and ADCC against primary tumor cells in vitro; robust in vivo proliferation post-infusion | Clinical | (129, 130) | |
| Irr. K562 expressing membrane-bound IL-15 and 41BBL + IL-15 | PBMC, CD3 depleted, and CD56 enriched | 1,000 (21 days) | N/A | Upregulated: CD56, NKG2D, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), CD158a, CD158b, and CD158e1 | Increased cytotoxicity in vitro independent of killer cell immunoglobulin-like receptor mismatch; NK infusion contributed to acute graft-versus-host disease in first clinical trial | Clinical | (131, 132) |
| Plasma membrane particles of K562 expressing IL-15 and 41BBL + IL-2 | PBMC in plates and flasks | 1,265 (17 days) | 86% NK cells 9% T cells 2% NK-like T |
Upregulated: NKp30, NKp44, NKp46, NKG2D, 2B4, NKG2A, TRAIL, and Fas ligand (FasL) Downregulated: CD16 |
Increased cytotoxicity against leukemic cell lines and primary acute myeloid leukemia (AML) cells in vitro | In vitro | (133) |
| Irr. K562 expressing membrane-bound IL-21, 41BBL, CD64, CD86, and CD19 + IL-2 | PBMC in flasks | 4.8 × 104 (21 days) | 21.7% T cells | High expression of natural cytotoxicity receptors, CD16, and NKG2D | Cytotoxic against tumor cell lines in vitro; capable of ADCC; increased telomere length | In vitro | (134) |
| 2,363 (14 days) | 83% NK 9.1% T cells |
Upregulated: DNAM-1, NKG2D, CD16, and CD56 | Cytotoxic and capable of ADCC against neuroblastoma cell lines in vitro and in vivo | Preclinical model | (135) | ||
| Plasma membrane particles of K562 expressing membrane-bound IL-21 and 41BBL + IL-2 | PBMC | 825 (14 days) >105 (28 days) |
>90% NK (day 14) | N/A | Increased cytotoxicity against leukemic cell lines and primary AML cells in vitro; enhanced proliferation in vivo | Preclinical model | (136) |
| Irr. allogeneic PBMC; irr. EBV transformed lymphoblastoid cell lines (EBV-LCL) (LAZ 388 cells) + PHA + IL-2 | PBMC depleted for CD3 and monocytes in bags and plates | ~43 (31–21 days) | 90% NK <5% T cells |
N/A | Increased cytotoxicity against tumor cell lines in vitro | Clinical | (137, 138) |
| Irr. EBV-LCL (TM-LCL) + IL-2 | PBMC, CD3 depleted, and CD56 enriched in bags | 800–1,000 (2 weeks) | 98% NK | Upregulated: TRAIL, FasL, NKG2D, NKp30, NKp44, NKp46, CD48, CD25, LTB, MX1, and BAX | Increased cytotoxicity against tumor cell lines in vitro | In vitro | (139, 140) |
| Irr. EBV-LCL (SMI-LCL) + IL-2 | PBMC, CD3 depleted, and CD56 enriched in bags | 3,637 (24–27 days) | 99.7% NK | Clinical | (141) | ||
| PBMC, CD3 depleted, and CD56 enriched in CliniMACS Prodigy | 850 (14 days) | >99% NK | Upregulated: TRAIL, FasL, NKG2D, NKp30, NKp44, and DNAM-1 | Increased cytotoxicity and ADCC against tumor cell lines in vitro | In vitro | (142) | |
| Irr. EBV-LCL (SMI-LCL) + IL-2 + IL-21 | PBMC depleted for non-NK cells (research kit) in plates and flasks | 2,900 (14 days) 2.7 × 1011 (46 days) |
>99% NK | Upregulated: TRAIL, NKG2D, and DNAM-1 | Cytotoxic against tumor cell lines in vitro and in vivo; enhanced and sustained production of IFN-γ and TNF-α | Preclinical model | (96) |
| Lysate of CTV-1 | PBMC, CD3 depleted, and CD56 enriched | N/A (overnight) | 97–98% NK |
Upregulated: CD69 Downregulated: CD16 |
Cytotoxic against NK-resistant leukemia cell lines and primary tumors in vitro | Clinical | (143, 144) |