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The natural killer cells have been generally

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The last years have seen remarkable advances
in the engineering of immune cells as cancer therapy. Whereas chimeric antigen
receptors (CARs) have been used comprehensively to convey the specificity of
autologous T cells against hematological malignancies with remarkable clinical
results, studies of CAR-modified natural killer cells have been generally in
preclinical phases12. NK cells for adoptive therapy can be
derived from several different sources which is explained in other parts.
Allogeneic NK cells can be generated from the Peripheral blood of healthy
donors or expanded from umbilical cord blood. Regardless of the source, there
are several features of expanded, activated CB, or PB-derived NK cells that
make them useful effectors for gene modification.

with CAR-modified primary human NK cells can
be effector modified immune cells against a number of hematologic and solid
tumor antigens, including CD19, CD20, GD2, and HER-2345. While non-viral expression techniques such
as nucleofection or electroporation can produce robust CAR-mediated killing,
the short-lived nature of these CAR molecules would likely dictate the need for
repeated infusions in the clinical setting. 67

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NKG2D Re

Expanded, activated NK cells generally
express a wide range of activating receptors, including CD16, NKG2D, and the
NCRs (NKp44 and NKp46), in spite of donor-to-donor variability897. These activated NK cells are prepared with
KIRs and are “licensed to kill.” in vivo expansion and persistence capacity of
NK cells is clearly associated with antitumor activity in trials involving
hematologic malignancies such as AML1011. Moreover, ex vivo expanded primary human NK
cells produce a different storms of cytokines more than T cells, including
interferon (IFN)-g, IL-3, and granulocyte macrophage colony-stimulating factor
(GM CSF), which may be associated with a lower risk of CRS(cytokine released
syndrome)

While normal NK cell counts are usually
detected within the first month after alloSCT regardless of the graft source,
several months are required to acquire the immunophenotypic and functional
characteristics of NK cells found in healthy donors. rebuilding NK cells
display a more immature phenotype expressing the inhibitory natural killer
group two A (NKG2A) receptor at around 90% compared to around 50% in healthy
donors . During the NK development and peripheral maturation, the CD56dim NK
cells lose NKG2A expression but up-regulate the expression of the activating
NKG2C receptor, killer cell inhibitory immunoglobulin-like receptors (KIRs) and
CD5712. The allo reactivity of NK cells is determined
by various receptors including the activating CD94/NKG2C and the inhibitory
CD94/NKG2A receptors, which both recognize the non-classical human leukocyte
antigen E (HLA-E. studies  showed that NK
cells expressing the activating CD94/NKG2C receptor are significantly reduced
in patients after alloSCT with severe acute and chronic graft-versus-host
disease (GvHD). Moreover, the ratio of CD94/NKG2C to CD94/NKG2A was reduced in
patients with severe acute and chronic GvHD after receiving an HLA-mismatched
graft. Collectively, these results provide evidence for the first time that
CD94/NKG2C is involved in GvHD prevention13.

Cytokines in NK

IL2 and LAK cells

At the University of Minnesota, reserachers
first confirmed the use of low dose IL-2 daily to expand NK cells after
autologous HSCT in patients with non-Hodgkin lymphoma and breast cancer. Later,
they activated autologous NK cells ex vivo with IL-2 for 24 hours, infused them
into patients and administered daily subcutaneous IL-2 .autologous NK cell
studies showed limited efficacy, they did yield important findings: 1).IL-2 can
be administered safely at daily or 3 times weekly intervals, 2) IL-2 can induce
an increase in circulating cytotoxic lymphocytes with a disproportionate
increase in NK cells14.

In innovative studies at the NCI, Rosenberg
and colleagues infused melanoma and renal cell carcinoma patients with
autologous peripheral blood cells treated ex vivo with IL-2. The product was
enriched with NK cells and named “lymphocyte activated killer” (LAK) cells.
High dose IL-2 was administered to patients after LAK infusions to promote
their in vivo persistence and activity. In a subsequent trial, the NCI group
adoptively transferred in vitro expanded autologous tumor-infiltrating
lymphocytes (TILs) to patients with metastatic melanoma15. These and other studies have contributed
important new knowledge: 1) high-dose IL-2 used in vivo with the goal of
activating NK cells has significant but manageable toxicity owing to severe
capillary leak syndrome, whereas low-dose subcutaneous IL-2 was well tolerated,
2) lymphodepleting chemotherapy using high-dose cyclophosphamide and
fludarabine facilitated in vivo expansion of autologous adoptively transferred
cytotoxic T lymphocytes and lead to enhanced efficacy,3) chemotherapy induces
lymphopenia, changes the competitive balance between transferred lymphocytes
and endogenous lymphocytes, changes the cytokine milieu and depletes inhibitory
cell populations (T regulatory cells Tregs)16.

IL7,15

Optimizing the proliferation of NK cells
mainly happened by the cytokine IL-1517. As serum levels of both IL-15 and IL-7
increases, this depletion allows for inundating levels on the surface of NK
cells and CD8 T cells. Both are populations required for optimal tumor
clearance. It remains to be presented in humans how proliferation can promote a
long-lived population of NK cells. While the non-myeloblative conditioning
regimen results in serum increases of IL-15 and IL-7, the response is limited
and the levels rapidly decrease after 1 week18. Because of side effects and expansion of
Tregs that accompanies systemic IL-2 therapy, alternative cytokines have been
sought to effectively expand lymphocytes in vivo. The most recent advance in
allogeneic NK cell therapy for AML includes an exogenous IL-15 currently being
tested in Phase 1 dose escalation trials at the University of Minnesota (see
ClinicalTrials.gov and search NCT01385423). Patients with refractory AML are
treated with lymphodepleting chemotherapy, allogeneic NK cells and daily
infusion of IL-15 for 10 days. An IL-15 dose has been identified for further
study19.

IL10

Researchers reported that very high
expression of IL-10 in a pattern that reflects the ‘proliferation-induced
conditioning’ observed within murine NK cells and which acts to suppress
adaptive immunity. Importantly, higher numbers of NK cells at 14 days after
transplant are associated with a reduced risk of acute GVHD20.

MSC and NK therapy

Bone-marrow-derived MSCs (BM-MSCs) can
inhibit NK cell proliferation, cytotoxicity, and cytokine production by
secreting IDO1, TGFb, HLA-G, and PGE2 2122.However, they can also be lysed by activated
NK cells, depending on their expression of activating NK receptor ligands,
including MHC class I polypeptide-related sequence (MICA, B), UL16 binding
proteins (ULBPs), CD112, and CD15523.

Mesenchymal Stem Cells (MSCs) shows
pleiotropic utilities factors with immunosuppressive activity involved in
cancer progression24. This is observed that T cell derived MSCs
were more powerfully immunosuppressive than NK-MSCs and affected both NK
function and phenotype by CD56 expression. T-MSCs shifted NK cells toward the
CD56dim phenotype and differentially modulated CD56bright/dim subset functions.
However MSCs affected both degranulation and activating receptor expression in
the CD56dim subset, they mainly inhibited interferon-gama  production in the CD56bright subset.
Pharmacological inhibition of prostaglandin E2 (PGE2) synthesis and, in some
MSCs, interleukin-6 (IL-6) activity restored NK function, whereas NK cell
stimulation by PGE2 alone mirrored T-MSC-mediated immunosuppression. Our
observations provide insight into how stromal responses to cancer reduce NK
cell activity in cancer progression22.

the spectrum of MSC immunosuppressive
activity in humans includes secretion of human leukocyte antigen (HLA-G),
transforming growth factor b (TGFb), prostaglandin E2 (PGE2), tumor necrosis
factor alpha-inducible protein 6 (TNFAIP6/ TSG-6), heme oxygenase 1
(HO-1/HMOX1), IL-10, IL-6, indoleamine 2,3-dioxygenase 1 (IDO1), hepatocyte
growth factor (HGF), and leukemia inhibitory factor (LIF) as well as programmed
death ligand (PD-L1/2) and Fas ligand (FasL) signaling2526.

The finding that MSCs could inhibit the
expression of activating receptors on the surface of NK cells was indicative of
a possible loss of cytotoxic activity known to involve engagement of causing
receptors. To assess a possible MSC-mediated inhibitory effect on the lytic
potential of NK cells, researchres achieved cytolytic assays in different
NK-cell populations from different donors were used as effectors after
short-term culture with 100 U/mL IL-2 either in the presence or in the absence
of MSCs.

MSCs were originally shown to have  strong inhibitory effect on T-cell activation
and function. In recent years, inhibition also has been observed on dendritic
cells (DCs),B cells,and NK cells. In this framework, researchers informed that
MSCs can block the IL-2–induced proliferation of fresh peripheral blood NK
cells. the use of MSCs may become a common approach in BM transplantation not
only for their possible beneficial effect on the engraftment of hematopoietic
stem cells,but also for their immunosuppressive potential.On the other hand, NK
cells have been shown to play a central role in the successful outcome of
haploidentical BM transplantation to treat AML.NK cells derived from the HSCs
of the donor can exert a direct GVL effect, provided they express KIRs that do
not recognize one or more HLA class I alleles of the patient.

Recent studies reported that NK-MSC
interactions not only provided  strong
MSC-mediated anti proliferative effect on NK cells but also verified that
IL-2–activated NK cells can powerfully kill both allogeneic and autologous
MSCs.  Killing reflects the fact that
MSCs are characterized by low levels of HLA class I antigens and also express
several ligands recognized by activating NK receptors.

In the present study, NK cells and MSCs were
derived from different donors (because MSCs were obtained from the BM of
pediatric patients, from whom it was not possible to obtain sufficient numbers
of fresh NK cells). Though, as mentioned above, the results of the interaction
between NK cells and autologous or allogeneic MSCs were fuzzy21. Consequently, it is believable that also in
an autologous MSCs would inhibit NK-cell function for kill cancererous cells.
These data should be taken into account in designing novel protocols of
adoptive immunotherapy in both MSCs and NK cells can be infused into the
patient to improve the clinical outcome of HSCT21.

NK cell production under Good Manufacturing
Practice (GMP) conditions

NK products has changed over the years. Given
the safety of apheresis methods for the donor, it has been replaced replaced a
3-hour apheresis product with a 5-hour product depleted of T cells and B cells
using CD3 and CD19 beads. GMP cell processing resulted in a significant
reduction of T cells in all products, decreasing to 50%)
comprised the other major component of the final product. While monocytes
express IL-15 receptor alpha important for trans-presentation of IL-15, it does
not yet understand their contribution to successful adoptive transfer. Although
5-hour apheresis allows for enhanced NK cell doses up to 20 × 106 cells/kg,
definitive studies need to be done to determine if differences in dose have an
effect. In using ex vivo expanded products, up to 1 × 108 cells/kg have been
infused without major toxicities . Depletion of CD3 cells below 0.1% prevents
transfer of T cells leading to GVHD. Depletion of CD19+ B cells prevents
passenger lymphocyte syndrome and autoimmune phenomena. We also recognized that
transfer of EBV-transformed B cells leading to donor-derived post transplant
lymphoproliferative disorder could be prevented.

Future perspectives

1. Genetic modification and alternative
sources of NK cell products

To overcome restrictions of the donor-derived
NK cell therapies, several groups have investigated alternative donor sources
including UCB, NK cell lines and pluripotent stem cells. If cryopreservation
can be optimized, the quick availability of an off-the-shelf product denotes a
significant step forward. Further advantages include the ability to perform
preclinical testing and to select for donors based on favorable characteristics
including optimal KIR-genotype276 .

2. UCB-derived NK cells

UCB progenitors provide a rich source of
hematopoietic progenitor cells and serve as an important in vitro system for
studying the development of human NK cells. Clinically appropriate doses of
UCB-derived NK cells can be generated without the use of feeder cells in compare
to NK cells derived peripheral blood28 . NK cells generated from UCB contain a
mixture of immature and mature cells that produce cytokines and show
cytotoxicity .Development of functional NK cells (e.g. CD34 isolation, in vitro
expansion) takes up to 4 weeks and requires processing in a GMP facility.
Studies are uncompleted and preliminary data is insufficient to assess
comparative advantages29.

3. NK cell lines

Many research teams have explored the use of
cell lines derived from malignant NK cell clones ( NK-92, NKL, KYHG-1, YT, NKG)26. NK cell lines keep some level of direct
cytotoxic function and usually lack expression of inhibitory KIR. Because they
can be grown in culture, genetic modification with different cytokine genes or
chimeric antigen receptors is easily accomplished. Among the lines, NK-92 cells
remain the most established and have been tested in clinical trials that
include patients with renal cell carcinoma and malignant hematological
malignancies. . Because of their amenability to ex vivo manipulation, these cell
lines may provide an important platform to facilitate whole-body in vivo
imaging of infused cells. Appropriate technology remains to be developed. 30

4. NK cells derived from pluripotent stem
cells

Pluripotent stem cells are available an
additional source of NK cells. These include human embryonic stem cells (hESCs)
and induced pluripotent stem cells (iPSCs). Novel methods of iPSC generation
have approached 100% efficiency, thus bringing closer the day that
hematopoietic-based therapies derived from these lines become available for
clinical use. A defined method for producing NK cells from hESCs and iPSCs
amenable to clinical translation has been recently established 31. By adapting a feeder-free differentiation
system, mature and functional NK cells can be generated in a system agreeable
to clinical scale-up. Significantly, in contrast to UCB-CD34+ derived NK cells
or NK cell lines, the iPSC-derived NK cells maintained high levels of KIR and
CD16 expression. If KIR expression does indeed dictate acquisition of final
effector function, some of the relative advantages of using iPSC-derived NK
cells for anti-cancer therapies are clarified. Using this improved
differentiation method, it is estimated that one 6-well plate of hESCs or iPSCs
could provide enough NK cells to treat several patients at the PB-NK doses
currently used . Other advantages contain:

1) unlimited source of KIR-typed NK cells for
adoptive immunotherapy,

2) high level of function in preclinical
animal models

3) a platform genetically responsive to
modify the therapy based on the patient’s cancer via tumor-specific receptors
(TCRs or CARs)32.

At the present, however, using iPSCs on a
patient-specific basis is impossible. Third party iPSC-derived NK cells are
subject to immune rejection in the recipient. To circumvent this limitation,
specific genetic modulation must be used to decrease immunoreactivity of the
infused cells6.

5. Bi- and Tri-specific antibodies

improvements in recombinant technology and
antibody production have led to a new class of therapeutics which use either
all, or part, of the antibody structure to mediate enhanced effector activity
at the tumor site. These include the fusion of two (bi-specific) or three
(tri-specific) portions of the fragment of antigen-binding (Fab) region of a
traditional antibody. These reagents keep a high level of antigen specificity,
but are derived from a moderately small segment of DNA and therefore offer the
significant flexibility of swapping different reagents33. The reagents serve to crosslink specific
tumor antigens (e.g. CD19, CD20, CD33) with a potent stimulator on the effector
cell (e.g. CD3, CD16, TCR)34 . The major advantage of this technology is
flexibility in selecting from a number of immune effector cells (CD16 on NK
cells, CD3 on T cells) as well as from a variety of tumor antigens (CD19,
EpCAM, Her2/neu, EGFR, CEA, CD33, EphA2, and MCSP)33.there have focused on a platform using
bispecific killer engagers (BiKEs) constructed with a single-chain Fv against
CD16 and a single-chain Fv against a tumor-associated antigen. Using CD16 ×19
BiKEs and a trispecific CD16 ×19 ×22 (TriKE), it has been showed that CD16
signaling is potent and delivers a different signal comparable with natural
recognition of rituximab, especially in regard to cytokine production.
Flexibility and ease of production are important advantages of the BiKE and
TriKE platform35. It s been recently developed a CD16 × 33
BiKE to target myeloid malignancies (AML and myelodysplastic syndrome)5. One of the most remarkable properties of
this drug is its potent signaling. In refractory AML, this is  found that CD16 × 33 BiKE overcomes inhibitory
KIR signaling, leading to potent killing and production of cytokines by NK
cells36. Interestingly, ADAM17 inhibition enhances
CD16 × 33 BiKE responses against primary AML targets37. These immunotherapeutic approaches will be
developed for clinical testing for hematologic malignancies and will allow for
NK cell activation via CD16 while approximating NK cells in direct contact with
targeted tumor cells In contrast to other therapies aimed at redirecting immune
cells, such as chimeric antigen receptor (CARs), the effect of bi-specific
antibodies can be titrated while maintaining specificity. One limitation  of this therapeutic approach is the very
short half-life of bi and tri –specific antibodies,which potentially limits
trafficking to all tissues35.

conclusion

Clinical applications of NK cells has been
inspired by recognition of their potent anticancer activity. The studies
discussed  providing  a solid basis for development of future NK
cell trials for cancer therapy by minimizing risks and toxicities. Important
questions remain to be answered, most urgently, determination of minimum in
vivo NK cell expansion needed for effective anti-tumor activity in clinical. At
present, upshots involving NK cell expansion interventions remain capricious. Also,
NK therapy for solid tumors is limited by uncertain homing and domination by an
immunosuppressive, tumor induced microenvironment that may interfere with
immune responses. To improve and progress NK cell therapies, both further study
of basic NK biology as well as a better understanding of interactions with
other immune cells will be required. NK cell products characteristics and
effective cytokine cocktails with optimal proportions will probably differ from
different tumor types and patients. Targeting CD16 remains an attractive way to
increase specificity, resembling of genetically modified T cells. Future
clinical trials will be designed to exploit strategies to overcome the host
immune barriers. In the same way, strategies to discover ex vivo NK cell expansion
from blood, lymphoid progenitors, or other sources are being tested. In Hematopoetic
stem cell transplantation, future studies are evaluating donor NK cell
immunogenetics. 

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