Use of Natural Killer Cells for Acute Leukemia Patients
Giovanni
F. Torelli
Giovanni F.
Torelli, Hematology, Department of Cellular
Biotechnologies and Hematology, Sapienza University, Rome, Italy
Correspondence to: Giovanni F.
Torelli, MD, PhD, Hematology,
Department of Cellular Biotechnologies and Hematology, Sapienza University, Via
Benevento 6, 00161 Rome, Italy.
Email: torelli@bce.uniroma1.it
Telephone: + 39-49974321
Fax:
+390644241984
Received: March 28,
2015 Revised: April 17, 2015
Accepted: April 22, 2015
Published online: July 6, 2015
ABSTRACT
The management of
acute leukemia has witnessed profound changes in recent years. The
anti-leukemic potential of Natural Killer (NK) cells has raised
considerable interest for the design of new therapeutic approaches based on the
infusion of freshly-isolated or ex vivo manipulated effectors. Several
points need to be addressed for the design of optimal protocols of NK infusion,
including which disease can benefit most, the optimal timing during the course
of the disease, the best preparative regimen and the origin of NK cells. This
review aims at summarizing the biological and clinical data on the role played
by NK cells in patients with acute leukemia; in addition, optimal good
manufacturing practice compliant protocols for ex vivo expansion and
activation of these effector cells are discussed.
© 2015 ACT. All
rights reserved.
Key words: Anti-leukemic activity of NK cells; Ex vivo
manipulation of NK cells; GMP protocols
Torelli GF. Use of Natural
Killer Cells for Acute Leukemia Patients. International
Journal of Hematology Research 2015; 1(2): 57-60 Available from: URL:
http://www.ghrnet.org/index.php/ijhr/article/view/1124
Definition
of Natural Killer cells
Natural Killer
(NK) cells are a subset of peripheral blood lymphocytes immunophenotypically
characterized by the expression of the CD56 surface antigen and the lack of the
CD3 and T cell receptor (TCR) proteins. These effectors play a central role in
the defense against viral infections and tumor growth; they exert their
cytotoxic capacity in a human leukocyte antigen (HLA)-unrestricted fashion, in
addition to the production of various cytokines and chemokines, including
tumor-necrosis-factor and interferon [1-3]. In humans, on the basis of the
intensity of the CD56 antigen expression, it is possible to distinguish two
main circulating NK cell subsets: CD56dim NK cells, which represent
the major subset and mainly express a cytotoxic capacity, and the CD56bright
subset which primarily produces cytokines[4].
The cytolytic activity of
these cells is finely regulated by the balance between inhibitory and
activating signals derived from receptors expressed at the cell surface. In the
late 80s, the “missing self hypothesis” was proposed by Ljunggren and Karre[5], based on the observation
that NK cells could efficiently kill targets who had lost major
histocompatibility complex (MHC)-class I. Specific inhibitory receptors
expressed by NK cells and recognizing allelic forms of HLA-class I were then
identified and collectively termed as killer immunoglobulin-like receptors
(KIRs) (KIR2DL and KIR3DL)[6,7]. Activating KIRs (KIR2DS and KIR3DS)
were also discovered, similar to the corresponding inhibitory KIRs in the
extracellular domain but with substantial differences in the transmembrane and
cytoplasmic portion[8]. Besides
activating KIRs, other receptors involved in tumor recognition include the
natural cytotoxicity receptors (NCRs) (NKp30, NKp44, NKp46), NKG2D and DNAM-1[9-13].
It is the selective engagement of these receptors, in the absence of efficient
inhibitory signals, that render target cells
susceptible to NK-cell mediated lysis. Interestingly, the known ligands for
these receptors are typically over-expressed upon cell stress, such as after
tumor transformation or viral infection, when the expression of HLA-I molecules
is reduced rendering therefore the cell susceptible to NK lysis. While some of
these receptors are still orphan of their ligands, MICA/B and ULBPs have been
discovered to be the ligands for NKG2D[14],
whereas the Poliovirus receptor (PVR, CD155) and Nectin-2 (Nec-2, CD112)
interact with DNAM-1[15,16].
Anti-leukemic
activity of Natural Killer cells
It is from the setting of haploidentical stem cell transplantation
(SCT) that the anti-leukemic capacity of NK cells became evident. In this
context, alloreactive NK cells express inhibitory KIRs specific for HLA-class I
alleles that are missing in the recipient, in addition to activating KIRs recognizing
specific HLA ligands[17,18]; this further underlines the importance
of both inhibitory and activating signals for tumor recognition.
Alloreactive NK cells have been demonstrated to positively affect the
outcome of both adult with acute myeloid leukemia (AML) and children with acute
lymphoid leukemia (ALL) undergoing T-cell depleted haploidentical (SCT)[19,20].
The biological reasons responsible for the different susceptibility of adult
and children ALL blasts to the lytic effect played by alloreactive NK cells are
so far unknown. Nonetheless, we recently reported the significant increased
expression in pediatric B-lineage ALL (B-ALL) of some of the ligands for NKG2D
and DNAM-1, including Nec-2[21]. In addition, these data allowed to
recognize a differential expression of ligands for NK cell activating receptors
in molecularly-defined subgroups of ALL patients. This is the case of BCR-ABL+
ALL blasts, which display, together with the MLL-AF4+
subgroup, the highest intensity of ligand expression within the B-ALL adult
context. Importantly, the high expression of ligands for NK cell activating
receptors correlated with the degree of susceptibility to lysis by expanded
allogeneic NK cells, further supporting the role played by these receptors
during the process of recognition. These results provided for the first time an
explanation to the differential susceptibility of age- or molecularly-defined
subgroups of acute leukemia patients to the lytic action of cytotoxic NK cells.
In the last
years, the possibility of using either selected or ex vivo
activated/expanded NK cells in the design of new immunotherapeutic strategies
for the treatment of leukemic patients has gained much interest[22].
Isolated experiences are reported in the literature characterized by the
infusion of NK cells derived from haploidentical donors[23-25] (see
Table 1 for details) or from the hematopoietic stem cell donor when infused
after a transplant[26-28] (see Table 2 for details), mainly for
patient affected by AML, mielodysplastic syndrome or high risk lymphoma.
Altogether these reports demonstrated that the infusion of these cells is safe
and some encouraging results are described. Nonetheless several points need to
be addressed for the design of optimal protocols of NK infusion, including
which disease can benefit most, the optimal timing during the course of the
disease, the best preparative regimen, the origin of NK cells whether
autologous or allogeneic, and finally the ex vivo manipulation protocol.
Ex vivo
manipulation of Natural Killer cells for adoptive immunotherapy
The need of large quantities of highly activated effector cells to
produce an anti-cancer effect translates into the necessity of developing good
manufacturing practice (GMP)-compliant methods for the efficient production of
fully functional NK cells for clinical application. Several protocols for the ex
vivo NK cell expansion and activation have been investigated, including the
long-term culture with cytokines[29,30] and the use of different
sources of allogeneic feeder cells[31-36]; very recently, a feeder-free
particle-based technology which utilizes plasma membrane particles derived from
transfected K562 cell line was also proposed[37]. Despite the
excellent expansion fold, the majority of these protocols are not acceptable
for clinical use in many countries, either because they do not utilize clinical
grade materials or for the potential transmission of infectious diseases due to
the use of allogeneic feeders. In addition, the proportion of contaminating CD3+
T lymphocytes remains high in the majority of these approaches, with an
associated risk of graft-versus-host disease (GVHD) in the allogeneic context[38,39];
the probability of developing such a complication becomes even higher when the
anti-CD3 monoclonal antibody (mAb) is added to the culture system[40,41],
running the risk of expanding and activating residual CD3+ cells.
We recently
developed a new GMP compliant-method for ex-vivo NK cell expansion[42].
Peripheral blood mononuclear cells (PBMCs) are obtained from leukapheretic
procedures and processed in the GMP facility. For NK-cell enrichment, a
two-step immunomagnetic procedure consisting of a CD3+ T-cell
depletion followed by a CD56+ cell positive selection is used.
Isolated NK cells are suspended in serum-free medium containing autologous plasma,
interleukin (IL)-2 and IL-15 in the presence of irradiated autologous feeder
cells and cultured for 14 days at 37℃.
The
application of this method resulted in the efficient production of effector
cells presenting a significantly increased expression of activating receptors
important in tumor cell recognition, including NKG2D, DNAM1, Nkp30 and Nkp44,
when compared to freshly isolated NK cells. The two-step NK cell selection
procedure ensures the highest purity of the final product, as demonstrated by a
minimal contamination by CD3+ cells belonging to the T- and NK-T
compartments; this is important in order to maximally reduce the risk of
inducing a GVHD when the product is used in the allogeneic context.
Furthermore, the high level of B-cell depletion reached with this system is
also an important condition to prevent Epstein-Barr Virus (EBV) reactivation,
which may potentially trigger lymphoproliferative diseases in immunocompromised
patients.
Several other
advantages are also associated with the expansion strategy utilized in this
protocol; these include (1) the lack of in vivo cytokine infusion which
can cause systemic toxicities; (2) the use of clinical grade manufactured IL-2
and IL-15, which play essential roles in NK cell development, expansion,
homeostasis and activation[43,44]; (3) the use of autologous feeder
cells to prevent the unknown effects associated with the use of allogeneic
feeders; and d) the lack of an anti-CD3 mAb in the culture system, thus
reducing the risk of GVHD in the allogeneic setting.
In our model,
both cytokines and feeder cells are necessary to obtain optimal NK cell
proliferation, since a lower expansion fold was observed in the presence of
cytokines or feeder cells alone. Under these conditions, the degree of NK cell
expansion was on average 15.7 fold. When tested in a cytotoxic assay against
the K562 cell line, these expanded effectors proved to be highly active. This
confirms recently published data from our group documenting the ability of
expanded NK cells to recognize and kill primary acute leukemia blast cells[21,45].
Similar results were obtained when the expanded effectors were tested in a
degranulation assay. Interestingly, these cells exerted a comparable cytotoxic
activity when used prior to cryopreservation and after thawing; this is
extremely important since ex vivo expanded cells must be cryopreserved
before in vivo infusion in adoptive cell therapy protocols, in order to
allow the necessary quality tests to be carried out before the batch of cells
can be released for clinical use.
Conlusions
In conclusion, new adoptive immunotherapeutic approaches based on the in
vivo infusion of ex vivo expanded and activated NK cells represent
promising strategies for the treatment of patients affected by acute leukemia.
Several points still need to be addressed for the design of optimal protocols
of NK infusion. The development of GMP methods for efficient production of
fully functional NK cells is mandatory for clinical application. Future
clinical trials are warranted and may solve some of the still debated points.
CONFLICT OF INTERESTS
The authors have no conflicts of interest to declare.
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Peer reviewer: Georgia
D Kaiafa, Aristotle University, AHEPA University Hospital, 1st Propedeutic Dept
of Internal Medicine, Heamatology Unit, Stilponos Kyriakidi 1, PC 546 36,
Thessaloniki, Greece.
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