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T regulatory cells, adoptively transferred NK cells were able to eliminate
tumors in these mice and the mice survived significantly longer than mice
treated with NK cells alone in the absence of pretreatment with Ontak
[100]
.
Therefore depleting host T regulatory cells could improve NK cell expan-
sion and function in humans. As IL-15 has recently been made available for
clinical use, it may be preferable to IL-2. IL-15 is critical to NK cell develop-
ment and homeostasis
[48,84]
. Trials testing the safety and toxicity of IL-15
are currently being undertaken.
The alternative is to expand NK cells
ex vivo
and then infuse into the patient.
Several centers are working on this approach. Imai and colleagues engi-
neered the class I-negative cell line K562 to express 41BBL and membrane-
bound IL-15
[101]
. Under GMP conditions they described on average a
1000-fold expansion of NK cells after 3 weeks in culture and retained cyto-
toxic capabilities. A similar approach utilized a GMP-compatible lympho-
blastoid cell line and IL-2 to expand NK cells on average 500-fold in 30 days.
These expanded NK cells were shown to upregulate CD25 (IL-2R), CD48,
NKG2D, and TRAIL compared with resting NK cells. They also increased
secretion of many cytokines and chemokines, including IFN-γ, TNF-α, GM-
CSF, and MIP-1β. Removal of IL-2 resulted in decreased expression of both
NKG2D and TRAIL and multiple cytokines, including IFN-γ. These results
demonstrate that
ex vivo
expanded NK cells may become dependent on
cytokines, and may even become exhausted after prolonged expansion, so
it is unclear how they may function once infused into the patient. Indeed
expanded NK cells have been shown to have shorter telomeres and potential
replicative senescence
[102]
. Recently, it has been demonstrated that K562-
expressing membrane-bound IL-21 and 41BBL expanded NK cells signifi-
cantly more than those expressing IL-15
[103]
. Expanded NK cells did not
reach senescence and had telomere lengths similar to those of resting NK
cells. They also had similar levels of receptor expression compared to those
NK cells expanded with IL-15, but had higher cytokine production. As these
cells had less senescence this approach would be more amenable to use in
the clinic. An alternative source of
ex vivo
-expanded NK cells is the genera-
tion of NK cells from CD34
+
progenitors isolated from UCB
[104]
. Another
promising source of expanded NK cells is from embryonic stem cells
[105]
.
345
Studies have also explored the use of NK cell lines for potential adoptive
therapy. Both irradiated NK-92 and KHYG-1 could prove an unlimited
source of cytotoxic NK cells for adoptive transfer; however, their survival
in vivo
is still unclear
[106,107]
. Irradiated NK-92 cells were used in a phase
I clinical trial for renal cell cancer and melanoma with only mild infusion
toxicities
[108]
.
In addition to the expansion and adoptive transfer of unmanipulated
NK cells, NK cells can also be engineered to express certain receptors to
increase their ability to recognize and eliminate tumors. Chimeric antigen
receptors (CARs) typically involve fusions of single-chain variable frag-
ments derived from monoclonal antibodies such as CD19 and usually
fused to CD3ζ transmembrane and endodomain. Addition of costimula-
tory molecules such as CD28, CD137, and CD134 (OX40) increases the
efficacy of CARs. NK-92 cells have been genetically modified with CARs
that consist of CD20 and Her-2/neu, both fused to CD3ζ, and have shown
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