Large-Scale Generation of Natural Killer Lymphocytes for Clinical Application

Abstract

Natural killer (NK) lymphocytes can be used for adoptive immunotherapeutic strategies. Alternatively, they may be employed as adjuvants for stem cell/bone marrow transplantation, either to re-induce remission, or to purge autografts of contaminating malignant cells. We developed a new protocol that enables the generation of NK cells on a clinical scale in a closed system that enables good manufacturing practice (GMP) conformity. Aside from the initial NK cell inoculum, our protocol includes activated feeder cells [irradiated peripheral blood mononuclear cells (PBMC) and no transformed blasts], cytokines [interleukin-2 (IL-2) and IL-15], human serum, and a complex basic media formulation. During the whole expansion period of approximately 14 days, the cells were handled in PTFE (Teflon) bags, whereby fresh medium was added without opening the system. The use of immortalized or virus-transformed feeder cells, as used in many other current research protocols, was completely avoided. A precise controlling of a number of environmental factors was necessary to achieve reproducible results. Increases in NK cell number ranged between 80- and 200-fold. The resulting NK cells were CD56(+), CD3(-), and CD16(+) (75%). They were highly cytotoxic against different malignant target cells and did not produce significant levels of interferon-gamma. Therefore, they belonged to the cytotoxic rather than the immunoregulatory NK subpopulation. No non-specific activation against normal allogenous lymphocytes occurred. This work might permit the realization of future protocols for evaluating the clinical effect of NK lymphocytes in human disease.

Bringing natural killer cells to the clinic: ex vivo manipulation

“…22,23 Because adoptive NK infusions as tumor immunotherapy remain in a proof-of-concept phase, with allogeneic infusions often being given after immunosuppressive chemotherapy or after an HLA-mismatched transplantation, most investigators have pursued methods to expand highly purified NK cells so that both the efficacy and any toxicities of the infused product can be directly attributable to NK cells themselves. 12,18,[24][25][26][27][28][29] In general, to prevent T-cell and NKT-cell contamination and overgrowth, T cells are usually depleted from PBMCs either before the initiation of NK cell cultures or at the end of the expansion culture. 30 Strategies to activate and/or expand NK cells for adoptive infusion in humans with cancer are summarized in Figure 1.…”

“…2,26,[39][40][41][42][43][44] Typically, these cultures are initiated using an enriched NK cell population isolated from apheresis products that have undergone CD3 ϩ T-cell depletion with or without CD56 ϩ selection to maximize NK cell purity. Utilization of feeder cells in NK cell cultures can dramatically enhance NK cell expansion numbers ex vivo, and this technique has been used recently for the large-scale expansion of clinical-grade NK cells.…”

Combined IL-15 and IL-12 drives the generation of CD34+-derived natural killer cells with superior maturation and alloreactivity potential following adoptive transfer

“…8,9 Although infusions with peripheral blood derived-NK cells appears to be safe, NK cell enrichment from leukapheresis products yields relatively low NK cell numbers with contaminating alloreactive T cells. [11][12][13][14] To overcome these limitations, several methods have been explored to activate and expand NK cells before adoptive transfer. [15][16][17][18][19][20] Alternatively, NK cells can be generated from hematopoietic progenitor cells (HPCs).…”

Expansion of NK cells from PBMCs using immobilized 4-1BBL and interleukin-21

“…Although the safeguards, such as using cultures of irradiated K562 cells and monitoring the cell growth and DNA synthesis rate, were provided, the inability to deplete the feeder cell line provided difficulties for clinical treatment. To overcome the problem of T and NKT cell contamination and overgrowth, the initial NK cells was purified from PBMCs (13,42,43), but such purification procedure was time-consuming, arduous and costly. In this study NK cells in PBMCs without purification were used which certainly is a significant advantage.…”