CAR a number of hematologic and solid

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modified Natural killer cells

The last several years have seen impressive
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 mostly in preclinical phases. 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-2. 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.( Engineering Natural Killer Cells for Cancer

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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 variability. 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 AML. 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 2,3. 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
CD57.The alloreactivity 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). Here
we analyze the contribution of these receptors to NK cell alloreactivity in 26
patients over the course of the first year after alloSCT due to acute myeloid
leukemia, myelodysplastic syndrome and T cell Non-Hodgkin-Lymphoma. Our results
show 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 prevention.

Moreover, the ratio of CD94/NKG2C to NKG2A/CD94
was reduced in patients with acute or chronic GvHD after receiving an
HLA-mismatched graft. In conclusion, these results provide evidence that the
CD94/NKG2C receptor is associated with alloreactivity of NK cells after
alloSCT, especially regarding acute or chronic GvHD prevention.( The Activating NKG2C Receptor Is
Significantly Reduced in NK Cells after Allogeneic Stem Cell Transplantation in
Patients with Severe Graft-versus-Host Disease)

Cytokines in improving 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 cells.

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 20 patients with metastatic melanoma. Objective responses
were observed in 11 patients. Given the limited persistence of the transferred
tumor-specific T-cells in vivo, a second course of TIL cells was infused
following lympho-depleting chemotherapy combining high dose cyclophosphamide
with fludarabine 88. Objective responses were observed with TILs in patients
with melanoma. 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) 8, 89–91.


Optimizing the proliferation of NK cells
mainly happened by the cytokine IL-15. 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 week. 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 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 study.


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 GVHD.

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 (Casado et al., 2013; Krampera et al.,
2006; Rasmusson et al., 2003; Spaggiari et al., 2008). 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 CD155.

Mesenchymal Stem Cells (MSCs) shows
pleiotropic utilities factors with immunosuppressive activity involved in
cancer progression. We 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

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) signaling.

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 fuzzy. 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 HSCT. Actually,
the adoptive transfer of activated NK cells could potentially kill MSCs if
these are infused shortly before or simultaneously with NK cells. In addition,
MSCs could inhibit NK-cell proliferation and function. In conclusion, our
present study clearly shows that in addition to inhibiting NK-cell
proliferation, MSCs noticeably suppress major NK functions, such as cytolytic
activity and cytokine production. Moreover this could have negative effects on
the NK-mediated GVL, mainly in the haploidentical HSC transplantation setting.
Nevertheless, it is obvious that more confirmation of the relevance of in vitro
findings will need suitable in vivo studies in animal models

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, we have 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 < 1% following CD3-depletion, yielding a final T cell dose of <3 × 105 cells/kg. There was an average of 40-fold less T cells than NK cells. Monocytes (sometimes >50%) comprised the other
major component of the final product. While monocytes express IL-15 receptor
alpha important for trans-presentation of IL-15, we do 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 102. 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 observed passenger lymphocyte syndrome in
2 patients prior to B-depletion 103. We also recognized that transfer of
EBV-transformed B cells leading to donor-derived posttransplant
lymphoproliferative disorder could be prevented.

Future perspectives

6.1 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-genotype 131.

6.2 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 132. Clinically appropriate doses
of UCB-derived NK cells can be generated without the use of feeder cells in
compare to NK cells derived peripheral blood 106, 133. NK cells generated
from UCB contain a mixture of immature and mature cells that produce cytokines
and show cytotoxicity 116. 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 advantages. Yoon, et. al, have tested an approach using
CD34+ cells from adult donors. Fourteen patients received donor-derived NK
cells that were differentiated in vitro in the presence of stem cell factor,
FLT3 ligand, IL7 and hydrocortisone (HDC), followed by IL-7, IL-15 and HDC. The
infusions were given ~ 6–7 weeks after transplant in the outpatient setting
134. Infusions were tolerated and no toxicity was observed except occasional
alanine aminotransferase elevation (grade III) in two patients and development
of grade II skin GVHD in one patient, although the concurrent discontinuation of
immunosuppression suggests that the NK cells were not responsible.

6.3 NK cell lines

Many research teams have explored the use of
cell lines derived from malignant NK cell clones (i.e. NK-92, NKL, KYHG-1, YT,
NKG). 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 melanoma 135. In a phase I dose escalation study
treating 12 patients, investigators reported only transient toxicities and
stable disease in 33% of patients. However, the in vivo activity of the NK-92
cells was difficult to establish 100. Additional data are needed concerning
the in vivo persistence of these infused lines. 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.

6.4 NK cells derived from pluripotent stem

Pluripotent stem cells are available an
additional source of NK cells. These include human embryonic stem cells (hESCs)
and induced pluripotent stem cells (iPSCs) 131, 136. 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 137. 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 89, 137. 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) 138.

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 cells 139. Recently, Schwartz have shown the use of
hESC-derived retinal pigmented epithelial cells to be safe and potentially
effective in treating patients with macular degeneration, thus providing proof
of concept for this cell source type 140

6.5 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 120. 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 reagents. 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) 120, 141, 142. 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). We 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 143–145. Using CD16 ×19 BiKEs and a trispecific CD16
×19 ×22 (TriKE), we have shown 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 platform. We have recently developed
a CD16 × 33 BiKE to target myeloid malignancies (AML and myelodysplastic
syndrome). One of the most remarkable properties of this drug is its potent
signaling. In refractory AML, we found that CD16 × 33 BiKE overcomes inhibitory
KIR signaling, leading to potent killing and production of cytokines by NK
cells 144. Interestingly, ADAM17 inhibition enhances CD16 × 33 BiKE responses
against primary AML targets. 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. Thus, the likelihood
of persistent B cell aplasia, which occurs with CD19 CART cells, is reduced,
decreasing the risk of lifelong 
hypogammaglobulinemia. One limitation 
of this therapeutic approach is the very short half-life of bi and tri
–specific antibodies,which potentially limits trafficking to all tissues.


Clinical applications of NK cells has been
inspired by recognition of their potent anticancer activity. The studies
discussed above provide a solid basis for development of future NK cell trials
for cancer therapy while minimizing risks and toxicities (Figure 1). Important
questions remain to be answered including, most urgently, determination of
minimum in vivo NK cell expansion needed for clinically effective anti-tumor
activity. At present, outcomes involving NK cell expansion interventions remain
unpredictable. Furthermore, NK therapy for solid tumors is limited by uncertain
homing and domination by an immunosuppressive, tumorinduced microenvironment
which may interfere with immune responses. To advance 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 product
characteristics and effective cytokine cocktail proportions will likely vary
for different tumor types and patient populations. Targeting through CD16
remains a powerful and attractive way to increase specificity, rivaling that of
genetically modified T cells. Future clinical trials will be designed to
exploit strategies to overcome the host immune barriers. Similarly, strategies
to explore ex vivo NK cell expansion from blood, lymphoid progenitors, or
pluripotent progenitor cells are being tested. In HCT, prospective studies are
currently evaluating donor NK cell immunogenetics. Strategies to apply CMV-induced
shaping of the immune response to enhance NK cell function are in development.

Categories: Development


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